Qin, Et Al. 2022

Signal Transduction and Targeted Therapy www.nature.
com/sigtrans
REVIEW ARTICLE OPEN
Signaling pathways involved in ischemic stroke: molecular

mechanisms and therapeutic interventions
Chuan Qin 1, Sheng Yang1, Yun-Hui Chu1, Hang Zhang1, Xiao-Wei Pang1, Lian Chen1, Luo-Qi Zhou1, Man Chen1, Dai-Shi Tian1 ✉ and
Wei Wang1 ✉
Ischemic stroke is caused primarily by an interruption in cerebral blood flow, which induces severe neural injuries, and is one of the
leading causes of death and disability worldwide. Thus, it is of great necessity to further detailly elucidate the mechanisms of ischemic
stroke and find out new therapies against the disease. In recent years, efforts have been made to understand the pathophysiology of
ischemic stroke, including cellular excitotoxicity, oxidative stress, cell death processes, and neuroinflammation. In the meantime, a
plethora of signaling pathways, either detrimental or neuroprotective, are also highly involved in the forementioned pathophysiology.
These pathways are closely intertwined and form a complex signaling network. Also, these signaling pathways reveal therapeutic
potential, as targeting these signaling pathways could possibly serve as therapeutic approaches against ischemic stroke. In this review,
we describe the signaling pathways involved in ischemic stroke and categorize them based on the pathophysiological processes they
participate in. Therapeutic approaches targeting these signaling pathways, which are associated with the pathophysiology mentioned
above, are also discussed. Meanwhile, clinical trials regarding ischemic stroke, which potentially target the pathophysiology and the
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signaling pathways involved, are summarized in details. Conclusively, this review elucidated potential molecular mechanisms and related
signaling pathways underlying ischemic stroke, and summarize the therapeutic approaches targeted various pathophysiology, with
particular reference to clinical trials and future prospects for treating ischemic stroke.
Signal Transduction and Targeted Therapy (2022)7:215 ; https://doi.org/10.1038/s41392-022-01064-1
INTRODUCTION undergo morphological changes where the cell bodies and axons
Epidemiology, diagnosis, and treatment for ischemic stroke disappear.6,7 Swelling of the cytoplasm and nucleolus disappear-
Ischemic stroke is caused by an interruption in cerebral blood ance are often seen in neurons as well as glial cells. While in the
flow, induced by thrombosis or embolism. It represents the penumbra, neurons, which are referred to as ‘ischemic neurons’
second leading cause of deaths worldwide, with 5.9 million deaths and relatively viable, usually experience several changes such as
and 102 million disability-adjusted life years lost.1,2 Several risk endoplasmic ribosomes and Nissl bodies disintegration.8 Besides
factors have been implicated in the pathogenesis of ischemic neurons, glial cells, including microglia and astrocytes, also
stroke, including diabetes, cigarette smoking, hyperlipidemia, and experience morphological changes after ischemia. Ramified
hypertension.3 Based on the etiology, the cause of ischemic stroke microglia can transform into an “activated state”, characterized
can be traced to embolism from the heart, artery-to-artery by swollen ameboid-like cells, accompanied by the production of
embolism, and in situ small vessel disease.2,4 Typically, stroke pro-inflammatory substances, including cytokines, chemokines,
symptoms include sudden unilateral weakness, numbness, diplo- and reactive oxygen species (ROS),9 while astrocytes usually
pia, slurred speech, ataxia, and non-orthostatic vertigo.5 Various undergo gradual alterations both in molecular expression profiles
efforts have been made to improve outcome after stroke onset. and morphologies, which serves to protect neurons in the
Immediate clinical interventions, such as intravenous thrombolytic ischemic penumbra.10,11 After ischemia, increased blood-brain
treatment and mechanical thrombectomy, contribute to the barrier (BBB) permeability contributes to the infiltration of several
recanalization of cerebral blood vessels.5 While antithrombotic immune cells including leukocytes, monocytes, and macrophages,
therapies, including antiplatelet or anticoagulant agents, are into the ischemic lesions, which release a variety of neurotoxic or
recommended for nearly all patients with no contraindication,3 neurotrophic factors to exert either neuroprotective or detrimen-
pharmacological approaches against ischemic stroke remain tal effects on ischemic brain tissues.12–17
limited, suggesting the need for new treatments. The temporal and spatial alterations in ischemic stroke are
illustrated in Fig. 1.
Morphological changes in ischemic stroke
In the pathogenesis of ischemic stroke, various types of cells in the Experimental models of ischemic stroke
central nervous system experience different morphological Efforts have been made to elucidate the pathophysiological
alterations facing ischemic damages. In the ischemic core, neurons mechanisms and screen potential therapeutical targets of
1
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Correspondence: Dai-Shi Tian (tiands@tjh.tjmu.edu.cn) or Wei Wang (wwang@tjh.tjmu.edu.cn)
These authors contributed equally: Chuan Qin, Sheng Yang.
Received: 21 March 2022 Revised: 1 June 2022 Accepted: 15 June 2022
© The Author(s) 2022, corrected publication 2022

Signaling pathways involved in ischemic stroke: molecular mechanisms and. . .
Qin et al.
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Fig. 1 Spatial and temporal relationships of the pathophysiology in ischemic stroke. BBB Blood-brain barrier, DAMPs Damage-associated
molecular patterns, Th1 T-helper cell 1, Th2 T helper cell 2
ischemic stroke, and models both in vivo and in vitro are utilized oxygen is replaced by N2 and glucose in the medium is omitted.
to mimic ischemic circumstances. The most frequently used Often this model is combined with cell co-cultures to study
experimental ischemic stroke model is middle cerebral artery cellular interactions under ischemic circumstances.23 However, a
occlusion model (MCAO), in which a filament is utilized to block limitation still remains that the in vitro model should be combined
cerebral blood flow from the middle cerebral artery to induce a with in vivo studies to better comprehensively understand
transient occlusion.18,19 This model was mostly used for studying ischemic stroke.18
blood-brain barrier disruption and inflammatory response in
cerebral ischemia.20,21 Besides MCAO model, photothrombosis Pathophysiological mechanisms involved in ischemic stroke
model is also utilized to induce cerebral ischemia in both mice and As a hallmark of ischemic stroke, interrupted cerebral blood flow
rats. In this model, Rose Bengal, a photosensitive dye is injected depletes the brain of oxygen and glucose, which leads to
systematically into the animal, while a 532 nm wavelength laser is disrupted ATP synthesis and energy deficiency, as well as impaired
directly illuminated onto the skull and react with the photo- ion homeostasis and acid-base imbalance.24,25 All these dysfunc-
sensitive dye.22 Advantage of this model include the possibility to tions result in cerebral neuropathological changes, such as brain
select a specific cortical brain region for ischemia and the high edema, neuroinflammation, and neural cell death, eventually
reproducibility with very low mortality.18 Correspondingly, the underpinning severe neurological deficits.26 Progress has been
most frequently used in vitro model to mimic ischemic stroke is made in unveiling the pathogenesis and mechanisms of stroke,
the oxygen and glucose deprivation (OGD) model, in which including cellular excitotoxicity,27 mitochondrial dysfunctions,28
Signal Transduction and Targeted Therapy (2022)7:215

Qin et al.
3
neuroinflammation,29 BBB impairment,30 and cell death pro- K+, and Ca2+ levels, leading to cell depolarization and prompting
cesses.31 Various signaling pathways become activated in these glutamate release.33 The excessive glutamate activates N-methyl-
pathological transitions, and their targeted regulation could serve D-aspartate receptors (NMDARs), inducing toxicity, cell death, and
as a potential therapeutic strategy. Given the complex pathophy- finally severe damage of the central nervous system.34–36 Taken
siology of ischemic stroke, the accompanying injury and signaling together, deficiency in glucose and oxygen may eventually lead to
mechanisms should be first identified and then further elucidated cellular excitotoxicity and mitochondrial dysfunctions, which serve
to develop targeted interventions. as the initiating session of ischemia-induced damage and
The present review describes various signaling pathways subsequently cause other cascade of injuries. In this section, the
associated with ischemic stroke pathophysiology (Fig. 2) and review focuses on the various signaling pathways involved in
categorizes the corresponding therapeutic approaches (Table 1). glutamate and NMDAR-induced cell toxicity, namely, excitotoxi-
Additionally, we summarize evidence from national clinical trials city, as well as oxidative stress and mitochondrial dysfunction in
assessing therapies targeting ischemic stroke (Table 2). ischemic stroke.
Excitotoxicity and related signaling pathways

PATHOPHYSIOLOGY AND SIGNALING PATHWAYS INVOLVED Glucose and oxygen deficiency during cerebral ischemia induces
IN ISCHEMIC STROKE neuronal cell depolarization and glutamate release. The latter then
Energy deficiency due to a lack of glucose and oxygen stimulates Na+/Ca2+ channels coupled with NMDARs.37 Enhanced
Immediately after ischemic stroke, cerebral blood flow is Ca2+ influx perturbs ionic homeostasis, resulting in Ca2+ overload
significantly reduced, which limits the availability of glucose and in both the mitochondria38–40 and cytosol. These changes
oxygen, especially in neurons. Energy disruption leads to stimulate a variety of proteases, lipases, kinases, phosphatases,
mitochondrial dysfunction and oxidative stress-induced damage, endonucleases, and free radicals,41,42 as well as biological
triggered by the production of ROS.32 Concurrently, energy processes causing cell death, such as calpain activation,43
deficiency contributes to an ionic imbalance that affects Na+, oxidative stress, and mitochondrial impairment.44,45 Overall, these
a b
Necroptosis
Autophagosome
Excessive Autophagy
glutamate release
Cell
y
Synaptic and icit dea
Extrasynaptic tox th
sig
to Apoptosis
ci
NMDAR
na
Ex
lin
g
c d
N e u br
ti o n
BBB
ys e s s
r o ea
nc
fl a
in
st
fu
r
m e d
kd ma t i v ROS production
o w tio n a l
n O x i d d ri a
h o n ROS
M it o c
MPTP
Cytc
ROS
BBB breakdown O2-
Immune cells
Ca2+ influx
Cytokines and chemokines
Fig. 2 A brief summary for the pathophysiology involved in ischemic stroke. a Excitotoxicity in ischemic stroke, in which excessive glutamate
are released and both synaptic and extra-synaptic NMDARs are involved; b Cell death signaling pathways, which mainly involves autophagy,
apoptosis and necroptosis in ischemic stroke; c Neuroinflammation and BBB breakdown in ischemic stroke. Here we've presented the
participation of various immune cells and chemokines and cytokines released, which thus contribute to blood-brain barrier breakdown;
d Oxidative stress, which is mainly characterized by ROS production and mitochondrial dysfunction that involves Ca2+ influx into
mitochondria and MPTP in ischemic stroke

Qin et al.
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Table 1. Therapies targeting the related signaling pathways involved in the corresponding pathophysiology in ischemic stroke
Drug/Therapy Targeting signaling Major targeting Authors Citations Applications

pathway pathophysiology
333
NA-1 GluN2B-PSD95-nNOS Excitotoxicity Chen et al. Animals/Neuronal
cultures
426
ZL006 GluN2B-PSD95-nNOS Excitotoxicity Zhou et al. Mice/rat MCAO
27,427
IC87201 GluN2B-PSD95-nNOS Excitotoxicity Lai et al.; Bach et al. In vitro
349
Tat-p53DM DAPK1 Excitotoxicity Pei et al. Animals/Neuron
in vitro
GluN2BCT1292–1304 DAPK1/ GluN2B- Excitotoxicity McQueen et al. 351
Animals/Neuron
PSD95-nNOS in vitro
75
Tat-K13 PTEN Excitotoxicity Zhang et al. Rat focal ischemia
352
Geniposide GluN2A/AKT/ERK Excitotoxicity Huang et al. Rat tMCAO
428
Electroacupuncture PI3K/Akt Excitotoxicity Kim et al. Animals/rat
353
Pseudoginsenoside-F11 Akt-Creb Excitotoxicity Liu et al. Rat MCAO
354
TRPM2 (Gene knockout) Akt/ERK Excitotoxicity Alim et al. Mice MCAO
429
Tat-Panx308 Panx1 Excitotoxicity Weilinger et al. Mice/Rat brain slices
358
tBHQ Nrf2/ARE Oxidative stress Hou et al. Rat MCAO
364
Trans sodium crocetinate (TSC) SIRT3/FOXO3a/SOD2 Oxidative stress Chang et al. Rat MCAO
365
Genipin UCP2-SIRT3 Oxidative stress Zhao et al. Mice MCAO
263,367
CCL2/CCR2 gene knockout CCL2/CCR2 Neuroinflammation Wattananit et al.; Animals/mice MCAO
Dimitrijevic et al.
369,370
Resveratrol TLR4/NF-Kb/STAT3 Neuroinflammation Ghazavi et al.; Rat MCAO
Rahimifard et al.
372
Stevioside TLR/NF-kB Neuroinflammation Zan et al. Rat TBI
373–375
Progesterone TLR4/NF-kB Neuroinflammation Hsieh et al.; Li et al.; Rat MCAO
Wang et al.
430
Tak242 TLR4 Neuroinflammation Abdul et al. Rat/In vitro
431,432
Isoquercetin TLR4 Neuroinflammation Shi et al.; Wang et al. Animals/In vitro
433,434
Propofol TLRs Neuroinflammation Gui et al.; Marik et al. In vitro BV2/Review
376
Dexmedetomidine HMGB1/TLR4/NF-kB Neuroinflammation Zhai et al. Rat MCAO
378
XPro1595 TNFs Neuroinflammation Clausen et al. Mice MCAO
379,380
cTfRMAb-TNFR TNFs Neuroinflammation Zhou et al.; Sumbria et al. Mice MCAO
381
Stnf-Αr1 TNFs Neuroinflammation Liguz-Lecznar et al. Mice MCAO
435
Quercetin Sirt BBB Yang et al. Rat MCAO
396
Minocycline Sirt3/proline BBB Yang et al. HBMECs/Rat
hydroxylase-2/MMP hypobaric hypoxia
397
Hydrogen sulfide MMP9 BBB Liu et al. Mouse MCAO
398
Vagus nerve stimulation MMP2/9 BBB Yang et al. Rat MCAO
399
Hyperbaric oxygen MMP2 BBB Michalski et al. Rat MCAO
104
Metformin AMPK BBB Liu et al. Mice MCAO
400
OPC transplantation Wnt-5a BBB Khan et al. Rat MCAO
436
Patchouli alcohol MAPK BBB Wei et al. Mice MCAO
437
DL-3n-butylphthalide (NBP) MAPK/AQP4/MMP9 BBB Mamtilahun et a l. Rat MCAO
401,402
FTY720 Akt Autophagy Hasegawa et al.; Wei et al. Rat MCAO
438
Hydroxysafflor Akt Autophagy Qi et al. Rat MCAO
404
Selenium PI3K/Mtor/Akt Autophagy? Yang et al. Rat MCAO
439
Electroacupuncture PI3K/Akt Autophagy Wang et al. Rat MCAO
440
DHL PI3K/Mtor/Akt Autophagy Meng et al. In vitro OGD/R
441
Diosgenin STAT2/HIKESHI Autophagy Zhang et al. Rat MCAO
411
Stem cell-secreted vesicles STAT3 Autophagy Xia et al. Rat MCAO/In vitro
442
Melatonin PI3K-Akt Autophagy Yang et al. Rat MCAO
443
MTMR14 PTEN Autophagy Pan et al. Mice MCAO/In vitro
444
Sevoflurane PTEN/AKT1/Mtorc1 Autophagy Xue et al. Rat MCAO
445
Remote ischemic PTEN/AKT1 Autophagy Zhong et al. Mice MCAO
preconditioning
446
Neuroprotectin D1 Wnt/β-catenin Autophagy Mu et al. In vitro OGD/R

Qin et al.
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Table 1. continued
Drug/Therapy Targeting signaling Major targeting Authors Citations Applications
pathway pathophysiology
447
Electropuncture Wnt Autophagy Chen et al. MCAO Rat
405,406
SMXZF AMPK-mTOR Autophagy Guo et al.; Wang et al. Mice MCAO/In vitro
448
Puerarin AMPK/Mtorc/ULK1 Autophagy He et al. Rat MCAO
413
Electroacupuncture SIRT-FOXO1 Autophagy Xu et al. Rat MCAO
414
Luteolin SIRT3/AMPK/Mtor Autophagy Liu et al. Rat MCAO
415
Melatonin SIRT1-BMAL1 Autophagy Liu et al. Mice MCAO
449
Proanthocyanidins ERK Apoptosis Fu et al. Mice MCAO
416
Beta-hydroxybutyrate ERK/CREB/eNOS Apoptosis Li et al. Rat MCAO/In vitro
392
MCC950 NLRP3 Apoptosis/Inflammasome Ye et al. Mice MCAO
393
Genistein NLRP3 Apoptosis Wang et al. Mice MCAO
420
BML-275 AMPK Apoptosis Hu et al. Mice MCAO
421
Glycine AMPK/GSK-3β/HO-1 Apoptosis Chen et al. In vitro OGD/R
450
Apelin-13 AMPK Apoptosis Yang et al. Mice MCAO
451
CTRP3 AMPK/SIRT1-PGC-1α Apoptosis Gao et al. In vitro OGD/R
422
Rosuvastatin Sirt1/NF-kB Apoptosis Yan et al. Rat MCAO
452
Salvianolic acid B SIRT1 Apoptosis Lv et al. Rat MCAO
453
Stem cell therapy SIRT-NFkB Apoptosis Sarmah et al. Rat MCAO
424
MiRNA-29b/SIRT1/ Apoptosis Xu et al. In vitro
PGC-1
454
Tetrahedral frameword TLR2-MyD88-NF- Apoptosis Zhou et al. Rat MCAO/In vitro
nucleic acids kappa B
455
Chinese drugs TLR4/MyD88/MAPK/ Apoptosis Gu et al. Rat MCAO
NF-kappaB
cellular dysfunctions are termed excitotoxicity and involve downstream targets, including glycogen synthase kinase 3 beta
NMDARs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (GSK3β), pro-apoptotic B-cell lymphoma 2 (Bcl2)-associated BAD,60
receptors, and kainite receptors.1,46 c-Jun N-terminal kinase (JNK)/p38 activator ASK1,61 and apoptotic
Despite their involvement in ischemic stroke-related excitotoxi- p53.54 These effects not only exist in neurons, but also in other
city, NMDARs act as a double-edged sword. Functional and neural cell types, which are possibly related to the inhibition of
structural studies have revealed that activation of NMDARs synaptic excitotoxicity and thus exert neuroprotective effects in
containing the GluN2B subunit triggers excitotoxicity during ischemic stroke.
ischemic stroke and subsequent neuronal apoptosis, whereas
activation of NMDARs containing the GluN2A subunit exerts a Brain-derived neurotrophic factor (BDNF) and cAMP-response
neuroprotective effect.33,47 Similarly, it has been hypothesized that element-binding protein (CREB)-related gene products. Synaptic
synaptic NMDARs promote neuronal survival, whereas extra- NMDAR activation and Ca2+ influx activate the Ras/extracellular
synaptic NMDARs play detrimental roles in neuronal activity.48 signal-regulated kinase (ERK) signaling pathway and nuclear Ca2+/
The analogy between synaptic vs. extra-synaptic NMDARs and calmodulin-dependent protein kinases, which in turn phosphor-
GluN2A-containing NMDARs vs. GluN2B-containing NMDARs ylate and activate CREB.62,63 Together with NMDAR and BDNF,
demonstrates the dual effect of NMDARs and their regulation of CREB promotes the expression of numerous pro-neuronal survival
signaling pathways with neuroprotective or detrimental effects on genes.64–67 BDNF production in the brain relies on Ca2+ influx
ischemic stroke (Fig. 3). through NMDAR.64,68,69 Synaptic NMDARs promote BDNF gene
expression,70 whereas extra-synaptic NMDARs block CREB-
Phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Stimu- mediated BDNF expression.71 In experimental ischemic stroke
lation of synaptic NMDARs activates the pro-survival PI3K/Akt models, BDNF is secreted into the brain and protects against
signaling pathway, thereby exerting a neuroprotective effect. PI3K ischemia-induced injury via neuronal GluN2A-NMDAR activa-
is an intracellular kinase classified into three categories (I, II, and III) tion.72,73 Together, these results show that BDNF and, to some
based on structure and substrate specificity. In neurons, activation extent, the upstream CREB signaling pathway contribute to the
of the PI3K/Akt signaling pathway by NMDAR occurs via Ca2+ and neuroprotective effect associated with synaptic excitotoxicity in
calmodulin, which recruit phosphoinositide-dependent protein cerebral ischemia.
kinase 1. At the same time, Ca2+ triggers tyrosine phosphorylation
of insulin receptor substrate 1, reinforcing NMDAR-induced Akt Phosphatase and tensin homolog (PTEN) signaling pathway. Extra-
activation.49–51 The protective effect of the PI3K/Akt signaling synaptic NMDARs are closely linked to signaling pathways
pathway on ischemic stroke has been reported both in in vitro associated with cell death and often contradict the effects
neurons during hypoxia52–54 and in vivo against ischemic triggered by synaptic NMDARs. Upon activation by Ca2+ influx
neuronal death,52,55–57, and PI3K/Akt signaling inhibition aggra- through NMDARs, PTEN is recruited to GluN2B-NMDARs. The
vates ischemia-induced neuronal death in experimental stroke direct interaction between PTEN and the GluN1 subunit of
animals.55,56,58,59 Mechanistically, the neuroprotective effect of Akt GluN2B-NMDARs enhances current flow through the channel,
is related to the phosphorylation and inactivation of various tightening the junctions between PTEN and the neuronal death

6
Table 2. Clinical trials targeting the related signaling pathways involved in corresponding pathophysiology in ischemic stroke
Trial number Trial name Current Status Study start Duration[y] Phase Sponsor No.of Type Dose Route Time form Description (Signaling
and participants stroke onset pathways)
end date
NCT00591084 Safety and Compl 2005–2006 1 2 Hospital 199 ginsenoside-Rd 10 or IV <72 h Ca2+channel blocker
Pharmacokinetic Study 20 mg/d
of Carbamylated
Erythropoietin (CEPO) to
Treat Patients With Acute
Ischemic Stroke
NCT00815763 Efficacy and Safety of Compl 2006–2008 2 3 Hospital 390 ginsenoside-Rd 20 mg/d IV <72 h Ca2+channel blocker
Ginsenoside-Rd for
Acute Ischemic Stroke
NCT02446977 Administration of Compl 2015–2015 1 2 Hospital 50 CBG000592 20 mg/d IV <3 h FMN,FAD
CBG000592 (Riboflavin/
Vitamin B2) in Patients
With Acute
Ischemic Stroke
NCT02930018 Safety and Efficacy of Compl 2017–2019 2 3 Industry 1105 NA-1 2.6 mg/kg IV <12 h GluN2B-PSD95–nNOS
Nerinetide (NA-1) in interaction
Subjects Undergoing
Endovascular
Thrombectomy
for Stroke
NCT04462536 Efficacy and Safety of Recru 2020–2022 2 3 Industry 1020 NA-1 2.6 mg/kg IV <12 h GluN2B-PSD95–nNOS
Nerinetide in interaction
Participants With Acute
Ischemic Stroke
Undergoing
Qin et al.
Endovascular
Thrombectomy
Excluding Thrombolysis
NCT00728182 Evaluating Compl 2008–2011 3 2 Industry 185 NA-1 2.6 mg/kg IV <72 h GluN2B-PSD95–nNOS
Neuroprotection in interaction
Aneurysm Coiling
Therapy
NCT02315443 Field Randomization of Recru 2015–2022 7 3 Industry 558 NA-1 2.6 mg/kg IV <3 h GluN2B-PSD95–nNOS
NA-1 Therapy in Early interaction
Responders
NCT02549846 AdminiStration of Statin Compl 2015–2017 2 4 University 270 Atorvastatin 20 mg/d PO <24 h HMG-CoA Reductase
On Acute Ischemic Pitavastain 4 mg/d Inhibitors
stRoke patienT Trial Rosuvastatin 5 mg/d
NCT04834388 Studying Anakinra to Not Recru 2021–2022 1 2 University Hospital 75 anakinra 100 or IV <8 h IL-1 system
Reduce Secondary Brain 500 mg
Damage After
Spontaneous
Haemorrhagic Stroke
NCT03737344 BLOC-ICH: Interleukin-1 Compl 2019–2021 2 2 University/College 25 IL-1Ra Kineret® 100 mg SC <8 h IL-1 system
Receptor Antagonist in
Intracerebral
Haemorrhage
NCT02002390 Efficacy and Safety of Compl 2012–2014 2 2 Hospital 22 Fingolimod 0.5 mg PO <72 h Inflammation
FTY720 for Acute Stroke
NCT04629872 Fingolimod in Recruiting 2020–2021 1 2 University Hospital 30 Fingolimod 0.5 mg PO <6–24 h Inflammation
Endovascular Treatment
of Ischemic Stroke
NCT04675762 Combinating Fingolimod Recruiting 2021–2022 1 2 University Hospital 118 Fingolimod 0.5 mg PO <24 h Inflammation

With Alteplase Bridging
With Thrombectomy in
NCT04419337 Pioglitazone and SGLT2 Recruiting 2021–2023 2 3 University Hospital 550 Metformin / PO <3 months Inflammation
Inhibitors vs. DPP4
Inhibitors in Patients
With Stroke
Table 2. continued
end date
NCT04069546 The Efficacy of Remote Compl 2019–2020 1 Not applicable University 46 Remote Physical Physical strategy <48 h Inflammation
Ischemic Conditioning ischemic strategy
on Stroke-induced conditioning
Immunodeficiency
NCT00376207 Physical Activity After Compl 2006–2007 1 Not applicable Hospital 200 Physical exercise / Physical strategy / Inflammation
Stroke: How Does it
Effect Chronical
Inflammation and Insulin
Sensitivity
NCT02225834 Atorvastatin in Acute Compl 2011–2014 3 4 University 50 Atorvastatin 80 mg PO <48 h Inflammation
Stroke Treatment
NCT00097318 Safety Study of Compl 2004–2011 7 1 NIH Clinical center 60 rh IFN-Beta 1a 11 mcg/ PO <24 h Inflammation/BBB
Interferon Beta 1a to for 22 mcg/
Acute Stroke 44 mcg/
66 mcg/
88 mcg
NCT02878772 Vinpocetine Inhibits NF- Compl 2014–2015 1 2,3 University Hospital 60 Vinpocetine 30 mg PO <48 h Inflammation/NF-Kb

κB-dependent
Inflammation in Acute
Ischemic Stroke Patients
NCT01831011 Mildronate for Acute Compl 2008–2010 2 2 Hospital 227 mildronate 500 mg/d IV <7 days inhibitor of carnitine-
Ischemic Stroke injection dependent metabolism
NCT04479449 Efficacy and Safety of SP- Compl 2019–2020 1 2 Industry 178 SP-8203 80 mg/d IV <4.5 h Matrix metalloprotease
8203 in Patients With pathway
Ischemic Stroke
Requiring rtPA
NCT02787278 Safety and Efficacy of Compl 2016–2017 1 2a Industry 80 SP-8203 80 or IV <4.5 h Matrix metalloprotease
Two Doses of SP-8203 in 160 mg/d pathway
Patients With Ischemic
Stroke Requiring rtPA
NCT00901381 Granulocyte-colony Compl 2007–2009 2 2 Research Institute 20 Filgrastim 10 µg/kg IH <48 h Multiple
Qin et al.
Stimulating Factor for mechanisms(activation of

Stem Cells Therapy for endogenous bone marrow
Acute Ischemic Stroke cells and neuroprotection)
NCT03394950 Butyphthalide in Compl 2018–2021 3 4 Hospital 120 Butyphthalide 25 mg IV <4.5 h Multiple
Combination With mechanisms(PMCA,SERCA)
Recombinant Tissue
Plasminogen Activator
for Acute
Ischemic Stroke
NCT00796887 Randomized, Controlled Compl 2009–2012 3 2 Research Institute 28 Extended- 500 or PO 3–7days Multiple mechanisms(TNF-
Trial of Extended-Release Release Niacin 1000 mg/d α,TGF-β,cAMP,HDL,LDL)
Niacin (Niaspan®) to
Augment Subacute
Ischemic Stroke
Recovery
NCT03686163 Effects of Intranasal Compl 2016–2020 4 4 Hospital 106 Nerve Growth 20 ug/d IN <72 h Multiple mechanisms(TrkA)
Nerve Growth Factor for Factors
NCT02828540 Clinical Trial to Assess Compl 2016–2018 2 2 University 78 HT047 1500 or PO <14days Multiple mechanisms (herbal
the Efficacy and to 2250 mg/d extracts)
Evaluate Safety of HT047
in Patients With Acute
Ischemic Stroke
NCT01762163 Efficacy and Safety of Compl 2013–2016 3 4 University/College 622 Qizhitongluo 12 PO 15–28 days Multiple mechanisms
Qizhitongluo Capsule in Capsule granules/d (traditional Chinese medicine)
the Recovery Phase of Naoxintong 12
Ischemic Stroke Capsule granules/d
7
8
Table 2. continued
end date
NCT01958957 A Safety Study of Compl 2013–2014 1 4 Industry 6300 Ginkgolides 25 mg/d IV 0.5–6 months Multiple mechanisms
Ginkgolides Meglumine Meglumine (traditional Chinese medicine)
Injection in the Injection
Treatment of Ischemic
Stroke.
NCT01919671 Tongxinluo Capsule in Compl 2014–2016 2 4 University Hospital 2007 Tongxinluo 12 PO <72 h Multiple mechanisms
Ischemic Stroke capsule granules/d (traditional Chinese medicine,
Patients (TISS) mainly vasodilator)
NCT04649398 Cerebral Nimodipine Recruiting 2020–2023 3 / University 30 Nimodipine 60 mg PO/IV / Neuroinflammation/BBB
Concentrations
Following Oral, Intra-
venous, and Intra-arterial
Administration
NCT04734548 Phase Ib/IIa Clinical Compl 2020–2022 2 1, 2 Ministry 151 ApTOLL 0.025 mg/ IV <6 h Neuroinflammation/TLR
Study of ApTOLL for the kg–0.2 mg/
Treatment of Acute kg
Ischemic Stroke
NCT04453800 The Efficacy and Safety Compl 2016–2018 2 2 University Hospital 236 Sofadil 500, 750 or IV <6 h NMDA
of Sofadil for Injection in 1500 mg
the Treatment of Acute
Ischemic Stroke
NCT04486430 Efficacy and Safety Study Compl 2017–2019 2 2 Hospital 236 Neu2000 500 mg/ IV <6 h NMDAR
of Neu2000KWL for 750 mg/
Acute Ischemic Stroke 1500 mg
Patients Within 6 h
Qin et al.
of Onset
NCT04453800 The Efficacy and Safety Compl 2016–2018 2 2 Hospital 236 Neu2000 500 mg/ IV <6 h NMDAR
of Sofadil for Injection in 750 mg/
the Treatment of Acute 1501 mg
Ischemic Stroke
NCT00059332 Field Administration of Compl 2005–2013 8 3 Research Institute 1700 Magnesium 4g IV <2 h N-type Ca2+channel blocker
Stroke Therapy - sulfate
Magnesium (FAST-
MAG) Trial
NCT01502761 Intra-arterial Magnesium Termi 2012–2016 4 1,2 University 4 Magnesium 0.75 or 1.5 g IA N-type Ca2+channel blocker
Administration for sulfate
Acute Stroke
NCT02912663 Magnesium And Compl 2017–2020 3 1 University 10 Magnesium 1 g 10 mg IA N-type Ca2+channel blocker
Verapamil After sulfate
Recanalization in Verapamil
Ischemia of the
Cerebrum: a Clinical and
Translational Study.
NCT05032781 Intra-Arterial Recru 2021–2022 1 1 Industry 24 Magnesium 2 or 4 g 2, 4 IA N-type Ca2+channel blocker
Neuroprotective Agents sulfate or 6 mg/kg
and Cold Saline in Minocycline
Ischemic Stroke
Intervention
NCT02505295 Selenium and Ischemic Compl 2015–2018 3 Not applicable University 44 Selenium 1000 mg PO <72 h Oxidative stress
Stroke Outcome
NCT03945526 Effect of Astaxanthin Compl Mar, 2010- 3 months(0.25) 1 University 24 Astaxanthine 2*8 mg PO <48 h Oxidative stress
Supplementation on Jun, 2010
Plasma Malondialdehyde

Levels and NIHSS of
Stroke Patients
Table 2. continued
end date
NCT03402204 Efficacy of High and Compl 2014–2015 1 3 University 64 Simvastatin 10 mg/ PO <24 h Oxidative stress
Low-Dose Simvastatin 40 mg
on Vascular Oxidative
Stress and Neurological
Outcome in Patients
With AIS
NCT04931628 Efficacy and Safety of Not Recru 2022–2023 1 Not applicable University Hospital 190 Salvianolic acid 100 mg IV <72 h Oxidative stress
Salvianolic Acid on AIS
NCT03539445 Efficacy and Safety of Recruiting 2018–2022 4 3 Hospital 1200 Butylphthalide 0.2 g IV / Oxidative stress
Butylphthalide for Acute
Ischemic Stroke Patients
Receiving Intravenous
Thrombolysis or
Endovascular Treatment
NCT02222714 Safety Evaluation of Compl 2014–2017 3 2 Industry 110 3K3A-APC 120, 240, IV <4.5 h PAR1
3K3A-APC in 360 or
Ischemic Stroke 540 ug/kg

NCT04950920 Phase III Clinical Trial of Compl 2020–2022 2 / University Hospital 900 Y-2 sublingual Edaravone PO <48 h ROS/Neuroinflammation
Y-2 Sublingual Tablets in tablets 30 mg and
the Treatment of Acute d-borneol
Ischemic Stroke 6 mg
NCT01949948 Study of Tenecteplase Compl 2012–2016 4 3 Hospital 1050 Tenecteplase 0.4 mg/kg IV <4.5 h tPA
Versus Alteplase for
Thrombolysis (Clot
Dissolving) in Acute
Ischemic Stroke
NCT01675115 Efficacy of BNG-1 to Treat Compl 2012–2014 2 3 Hospital 129 BNG-1 9 g/d PO <10 days Unknown
NCT01436487 Study to Examine the Compl 2011–2015 4 2 Industry 134 MultiStem 400 or 1200 IV 24–48 h Unknown(Immunotherapy)
Effects of MultiStem in million
Ischemic Stroke
Qin et al.
NCT02963376 A Phase Ib/II in Patients Compl 2017–2018 1 1 University 24 DDFPe 0.05, 0.10 or IV <12 h Unknown(lactate)
With Acute 0.17 ml/kg
Ischemic Stroke
NCT00756249 Safety Study of Compl 2007–2008 1 1 Industry 16 Lu AA24493 0.005, 0.05, IV 12–48 h Unknown(SHH /Patched/
Carbamylated 0.5, 5 or Smoothened,Mash1,frataxin)
Erythropoietin (CEPO) to 50 mcg/kg
Ischemic Stroke
NCT00870844 Safety and Compl 2009–2011 2 1 Industry 24 Lu AA24493 0.5, 5 or IV <48 h Unknown(SHH /Patched/
Pharmacokinetic Study 50 mcg/kg Smoothened,Mash1,frataxin)
of Carbamylated
Erythropoietin (CEPO) to
Ischemic Stroke
NCT01678534 Reparative Therapy in Compl 2014–2017 3 2 Hospital 19 Allogenic 1 million IV 12 h Cell therapy
Acute Ischemic Stroke mesenchymal units/kg
With Allogenic stem cells from
Mesenchymal Stem Cells adipose tissue
From Adipose Tissue,
Safety Assessment, a
Randomised, Double
Blind Placebo Controlled
Single Center Pilot
Clinical Trial (AMASCIS-01)
NCT01501773 Intravenous Autologous Compl 2008–2011 3 2 Industry 120 Autologous 30–500 IV Sudden onset Cell therapy
Bone Marrow-derived bone marrow million
Stem Cells Therapy for stem cell
Patients With Acute
Ischemic Stroke
9
Qin et al.
10
signaling complex. Concurrently, the excitotoxic stimulation of
NMDARs initiates PTEN nuclear translocation, thus significantly
lowering the phosphorylation of phosphatidylinositol-
Description (Signaling
trisphosphate and Akt and consequently blocking PI3K/Akt

signaling.74,75 Thus, contrary to the protective effect of PI3K/
Akt, PTEN signaling may decrease cell survival and induce
Cell therapy
Cell therapy
pathways)
neuronal death.76 In agreement with this hypothesis, down-

regulating PTEN expression reportedly inhibits extra-synaptic
NMDAR currents and protects neurons from experimental
ischemic injury.74 The above evidence hints at the detrimental
Within 7 days
3–12 months
stroke onset
role of PTEN in ischemic stroke, which is largely mediated by

Time form
regulation of extra-synaptic NMDAR activities.
Death-associated protein kinase 1 (DAPK1) signaling pathway.

DAPK1 is a Ca2+/calmodulin-dependent serine/threonine-protein
Intracathecal
kinase, whose phosphorylation contributes to apoptotic cell

death.77,78 DAPK1 participates in excitotoxicity in ischemic stroke.
Route
During ischemia, NMDAR overactivation promotes Ca2+ influx,

IV
activates Ca2+/calmodulin, and stimulates calcineurin phospha-

tase, which subsequently dephosphorylates and activates
cells per kg
applicalble
2.5 million
DAPK1.79 The latter is then transferred to the GluN2B subunit of

NMDARs, aggravating ischemic injury.80 Preventing the interac-
Dose
M2 macrophage Not
tion between GluN2B and DAPK1 attenuated neuronal excito-

toxicity in mouse ischemic stroke models and downregulated the
transplantation
transplantation
NMDAR current in vitro.80 In addition, NMDAR-regulated calci-

introduction
Autologous
Autologous
Genetic: IV
infusion of
neurin activation contributes to DAPK1 activation, whereas

Genetic:
Genetic:
placebo
BMSCs
NMDAR or calcineurin inhibition prevents DAPK1 dephosphoryla-

Type
EPCs
tion. DAPK1 inhibition protects against ischemic injury both in

cultured neurons and in vivo, suggesting that potential treat-
participants
ments for ischemic stroke could be based on inhibiting DAPK1.81

It is interesting to note that the pro-survival signaling factor ERK
No.of
13
20
serves as a downstream effector of DAPK1, and the DAPK1-ERK

interaction could block the neuroprotective effect of ERK on
experimental ischemic stroke, possibly by retaining ERK in
neuronal cytoplasm.82
University
University
Sponsor
Postsynaptic density protein-95 (PSD95)/neuronal nitric oxide

synthase (nNOS) signaling pathways and excitotoxicity-induced cell
death. Neuronal NMDARs contribute to nitric oxide production,
which is associated with calcium/calmodulin and is regulated by
nNOS.83 NMDAR subunits bind directly to PSD95, which is
Phase
composed of three PDZ domains.84–86 The binding of PSD95 to

1/2
1
NMDAR and nNOS enhances Ca2+ influx, a hallmark of excitotoxi-

city.87,88 PSD95/nNOS signaling may play a pivotal role in ischemic
Duration[y]
stroke, as evidenced by the amelioration of neurological deficits in

animals suffering from cerebral ischemia and whose nNOS activity
was inhibited by either pharmacological or genetic means.89
3
Cerebral ischemia has been shown to enhance NMDAR/PSD95/

Current Status Study start
2013–2016
2011–2015
nNOS interactions in neurons, thus further aggravating brain

end date
injuries after experimental ischemic stroke.90 All these results

and
show that signaling through the PSD95/nNOS complex is crucial

for excitotoxicity in ischemic stroke and contributes to the
neurotoxic effects of extra-synaptic NMDARs.
NCT01845350 Safety of Autologous M2 Compl
Compl
Mitochondrial dysfunction, oxidative stress, and related signaling

pathways
Marrow Stromal Cell and
Ischemic Stroke (AMETIS)
Mitochondria are essential for maintaining energy homeostasis.

Treatment of Non-Acute
Cell Transplantation in
Endothelial Progenitor
When ATP synthesis and energy balance are disrupted by a lack of

NCT01468064 Autologous Bone
glucose and oxygen, the status and function of mitochondria

Macrophage in
Stroke Patients
become substantially altered. Ca2+ influx leads to mitochondrial

Trial name
permeability transition pore (MPTP) opening and cytochrome c

Table 2. continued
release.91,92 At the same time, insufficient ATP supply triggers

mitochondrial membrane depolarization, which is characterized
by the influx of Na+ and efflux of K+.93–95 Besides mitochondrial
Trial number
dysfunction, energy deficiency in cerebral ischemia leads to

oxidative stress, which severely damages cells and brain tissues.96
Oxidative stress accompanies several pathological processes and
results from increased ROS production,97 mostly via oxidative

Qin et al.
11
Fig. 3 Excitotoxicity and signaling pathways involved in ischemic stroke. NMDAR N-methyl-D-aspartate receptors, PI3K Phosphatidylinositol 3
kinase, BDNF Brain-derived neurotrophic factor, CREB cAMP-response element-binding protein PTEN Phosphate and tension homology
deleted on chromosome ten, PIP3 plasma membrane intrinsic protein 3, DAPK1 Death-associated protein kinase 1, PSD95 Postsynaptic
density protein 95, nNOS Neuronal nitric oxide synthase
phosphorylation in the mitochondria.98 Considering the intimate deprivation.110 In summary, HIF signaling may be closely
link between ROS and mitochondrial metabolism, mitochondrial associated with oxidative stress. Although there is still debate
dysfunction is often related to oxidative stress pathologies. During whether HIF-1α signaling enhances oxidative stress or not,
ischemia, oxidative damage and excessive Ca2+ levels contribute activation of HIF-1α may be closely associated with production
to MPTP induction, which further promotes succinate release and of ROS and oxidative stress, which consequently affects cellular
mitochondrial damage-associated molecular patterns including redox equilibrium and biological activities.
the activation of downstream inflammatory responses.99–102
Consequently, all these damaging factors lead to neurotoxic and Nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Nrf2
cell death processes, in which a plethora of signaling pathways are regulates cellular redox homeostasis and counteracts oxidative
involved (Fig. 4). stress. Nrf2 activation protects individuals against cerebral
ischemic damage. In the resting state, Nrf2 is coupled to Keap1,
Hypoxia-inducible factor (HIF) signaling pathway. HIF-1, a key its specific cytoplasmic receptor. The structure of Keap1 changes
transcription factor activated during cerebral ischemia and upon electrophilic or oxidative stress. As Nrf2 is phosphorylated
hypoxia, comprises two subunits: HIF-1α and HIF-1β.103–105 HIF-1 through the protein kinase C pathway, it becomes uncoupled
enhances the expression of several glycolysis-associated genes from Keap1, leading to enhanced expression of various anti-
under hypoxic conditions, thus helping cells and tissues become inflammatory proteins, antioxidant enzymes, and growth fac-
accustomed to hypoxia.106 Also, HIF-1α expression strongly tors.111,112 In ischemic stroke, oxidative stress caused by elevated
correlates with ROS levels, with HIF-1α chains stabilized by the ROS levels induces Nrf2 accumulation in the nucleus, where it
large quantities of ROS generated under hypoxia.28,107 In a binds to antioxidant response elements (ARE) and maintains
positive feedback loop, lack of oxygen and glucose due to normal mitochondrial function.113 In contrast, insufficient Nrf2
ischemia may enhance HIF-1 expression, thereby causing oxida- contributes to neuronal mitochondrial depolarization, ATP deple-
tive stress and further stimulating HIF-1 activity. tion, and respiratory function impairment. suggested the bene-
Conversely, it has been reported by other studies that HIF-1α ficial role of Nrf2 in mitochondria.114
may also play protective roles in the regulation of energy A variety of downstream signaling pathways, including PI3K/
metabolism, especially in neurons. Consequently, HIF-1α depletion Akt, ERK/mitogen-activated protein kinase (MAPK), and nuclear
in mouse embryo fibroblasts results in excessive ROS, reduced factor kappa beta (NF-κB), potentially mediate the antioxidant
glycolytic metabolism, and cell death.108 Besides controlling ROS effect of Nrf2 during ischemia. The neuroprotective PI3K/Akt
production, the activation of HIF-1α may benefit cellular home- pathway induces the nuclear translocation of Nrf2, which in turn
ostasis by maintaining the redox equilibrium.109 Knockout of HIF- stimulates the production of various antioxidants.115,116 Like-
1α has been shown to disrupt redox homeostasis and glucose wise, ERK/MAPK signaling pathway during ischemia is associated
metabolism, such as pentose phosphate pathway and glucose with a variety of neuroprotective biological processes, such as
transportation in SHSY5Y cell lines cultured under oxygen-glucose preventing apoptosis or enhancing Nrf2 phosphorylation and

Qin et al.
12
Oxidative stress
NOX2
ROS P P
CK2
IKK IkBα
P P PARP-1
IkBα IkBα
AIF Keap1
NF-kB NF-kB f2
Nr Keap1
ROS NF-kB
f2
Nr
MPTP HIF-1α
Cytc NF-kB HIF-1α

ROS
HIF-1β
ROS
Nucleus
O2-
Nrf2
MAF
ARE
Ca2+ influx
Mitochondria dysfunction
Fig. 4 Oxidative stress and mitochondrial dysfunctions and signaling pathways involved in ischemic stroke. MPTP mitochondrial permeability
transition pore, ROS Reactive oxygen species, ATP Adenosine triphosphate, HIF-1 Hypoxia-induced factor, Nrf2 Nuclear factor E2-related factor
2, ARE Antioxidant response element, CK2 Casein kinase 2, PARP-1 Poly ADP-ribose polymerase 1, AIF Apoptosis-inducing factor, PINK1 PTEN
induced putative kinase 1, NF-kB Necrosis factor-kB
translocation.117,118 Also, NRF2 and NF-κB signaling pathways Notably, CK2 was shown to activate ROS-generating NADPH
closely interact with each other under a variety of circumstances. oxidase isoform 2 in an experimental ischemic stroke model, which
On the one hand, deletion of NRF2 results in increased induced AIF release into the mitochondria and subsequent DNA
inflammation, as well as high levels of NF-κB; on the other damage and apoptosis.128 Moreover, studies have shown that
hand, the elevated expression of NRF2 inhibits NF-κB-regulated cyclin dependent kinase 5 and AKT/GSK3β are activated by CK2 in
pro-inflammatory and immune responses.119 This show the ischemia/reperfusion injuries.129 Given that inhibition of cyclin
neuroprotective effects of NRF2 against NF-kB-induced inflam- dependent kinase 5 reportedly alleviates cerebral ischemic stroke-
matory responses in cerebral ischemia. induced damage, CK2 may do more harm than good.130,131 Taken
In summary, Nrf2 is a crucial player against oxidative stress and together, the CK2 signaling pathway and related molecules play
mitochondrial dysfunction in ischemic brain injuries, possibly via either protective or detrimental roles in ischemic stroke, especially
the regulation of various downstream signaling pathways. in relation to oxidative stress. Importantly, downstream effectors of
CK2 may function as potential targets against ischemic stroke.
Casein kinase 2 (CK2) signaling pathway. CK2, an important
oncogenic kinase, is crucial for counteracting ROS accumula- Mitophagy and related signaling pathways. Mitophagy describes
tion.120 First, it exerts a protective effect by inhibiting NADPH the process whereby mitochondrial content is taken up by
oxidase via regulating Rac1, a GTPase which significantly activate mitochondria-derived vesicles and then transferred to lysosomes
NADPH oxidase, possibly through interactions with other subunits or peroxisomes for degradation. Mitophagy is essential for
and link the cytosolic subunits with the cell membrane.121–123 maintaining cellular homeostasis and serves as a protective
Second, CK2 reportedly phosphorylates Janus kinase and signal strategy in various central nervous system diseases.132 Signaling
transducer and activator of transcription 3 (STAT3), enabling ROS pathways, such as PTEN induced kinase 1 (PINK1)/Parkin, Bcl2-
detoxification by superoxide dismutase 2 (SOD2).124,125 Third, CK2 interacting protein 3 (Bnip3), BNIP3-like, and FUN14 domain
activates HIF-1α and phosphorylates NF-κB to promote the release containing 1 pathways, are reportedly involved in mitophagy
of vascular endothelial growth factor (VEGF) and angiogenic during ischemia–reperfusion. In the reperfusion stage, the levels of
proteins under in vitro hypoxic conditions.126,127 Conversely, CK2 the free radical ONOO− are increased, which leads to dynamin
inhibition in the ischemic region contributes to poly (ADP-ribose) related protein 1 recruitment to the mitochondria and PINK1/
polymerase 1 accumulation, which leads to the release of Parkin-associated mitophagy.133 Meanwhile, elevated ROS levels
mitochondrial cytochrome c and apoptosis-inducing factor (AIF), upregulate the levels of Parkin RBR E3 ubiquitin protein ligase,
with subsequent activation of downstream apoptotic events.120 which is recruited by PINK1, further exacerbating mitophagy.134
These findings reveal the protective effect of CK2 against oxidative Interestingly, PINK1-regulated mitophagy is mechanistically asso-
stress and inflammation, while promoting the release of ciated with MPTP opening, whereas Bnip3-induced mitophagy is
angiogenic factors under hypoxia. independent of MPTP.135,136

Qin et al.
13
Fig. 5 Cell death signaling pathways involved in ischemic stroke. GSK3β Glycogen synthase kinase-3β; Bcl-2 B-cell lymphoma-2; ERK Ras/
extracellular signal-regulated kinase; CAMKs Ca2+/calmodulin-dependent protein kinases; MAPK Mitogen-activated protein kinase; TNF Tumor
necrosis factor; mTOR mammalian target of rapamycin; AMPK 5′-AMP-activated protein kinase; FADD Fas-associating protein with a novel
death domain; TRADD TNFRSF1A Associated Via Death Domain; RIPK Receptor-interacting protein kinase; MLKL Mixed lineage kinase domain-
like protein; RIP1 Receptor interaction protein 1; RIP3 Receptor interaction protein 3; PGAM5 Phosphoglycerate Mutase Family Member 5;
MLKL mixed lineage kinase domain like pseudokinase; Atg5 Autophagy related 5; Atg12 Autophagy related 12; TFEB Transcription factor EB;
ULK1 Unc-51 Like Autophagy Activating Kinase 1; AMPK 5′-AMP-activated protein kinase; mTOR mammalian target of rapamycin; Apaf-1
Apoptotic peptidase activating factor 1
The activated mitophagy pathway may alleviate oxidative stress- long-lived proteins, misfolded and aggregated proteins, and
induced cell injuries by promoting the degradation of damaged damaged organelles to obtain energy or in response to cellular
mitochondria.137 Enhanced mitophagy has been shown to possibly stress.142 Subsequently, autolysosomes are newly formed to
ameliorate ROS accumulation in cerebral ischemic stroke.138 In cleave the cargo for subsequent recycling.143 Emerging evidence
conclusion, mitophagy is significantly involved in the pathophysiol- indicates that autophagy is activated in various cell types
ogy of ischemic stroke, along with the activation of various following ischemic stroke, including neurons, glial cells, and
signaling pathways. Targeting these signals could potentially endothelial cells. Autophagy can exert either beneficial or
ameliorate the pathological changes and symptoms of ischemic detrimental effects on cerebral ischemic injuries, as moderate
stroke; however, the mechanisms remain to be elucidated. autophagy may help degrade aggregated proteins,144–146
whereas inadequate or excessive autophagy may eventually lead
Cell death signaling pathways in ischemic stroke to cell death.147 The dual role of autophagy in ischemic stroke
Damage caused by excitotoxicity, oxidative stress, and mitochon- may be explained by the involvement of multiple signaling
drial dysfunctions in ischemic stroke may induce a variety of cellular pathways, such as mammalian target of rapamycin (mTOR), 5′-
signaling cascades, which lead neural cells to undergo either AMP-activated protein kinase (AMPK), MAPK, NF-κB, p53, HIF-1,
programmed or unprogrammed death.139 Usually, programmed cell and Bcl2 pathways.148
death includes apoptosis and autophagy, which are normal cellular
functions,140 whereas unprogrammed cell death involves necrosis mTOR-related signaling pathways. mTOR is a serine/threonine
and is likely caused by external stimuli.141 Lack of oxygen and protein kinase that comes in two major forms: mTORC1 (rapamycin-
glucose in the ischemic core often leads to irreversible necrosis; in sensitive) and mTORC2 (rapamycin-insensitive). The former is
contrast, relatively minor damage in the penumbra is responsible for responsible for cell growth and cell cycle progression, whereas
reversible death processes, such as apoptosis and autophagy.40 A the latter contributes to cellular skeleton formation. mTOR is a key
variety of signaling pathways are highly involved in cell death, and regulator of the initial phase of autophagy, as it senses changes in
they could either enhance or inhibit the process (Fig. 5). signaling within the cell. Usually, mTOR limits autophagy by
inhibiting phosphorylation of the Atg1/ULK1 protease complex.149
Signaling pathways related to autophagy in stroke During ischemic stroke, mTOR interacts with multiple signaling
Autophagy is a self-protective pathway that maintains cell pathway components that regulate autophagy,150 including PI3K/
homeostasis and promotes cell survival by degrading circulating Akt, AMPK, and MAPK.

Qin et al.
14
Akt, which is involved in various biological processes, can affect Under ischemic conditions, the accumulation of misfolded
cellular autophagy through multiple signaling pathways, of which proteins and disruption of Ca2+ homeostasis lead to self-
PI3K/Akt/mTOR is the most important one.151–153 The PI3K/Akt protective events in the unfolded protein response (UPR) path-
signaling pathway was suggested to exert a neuroprotective effect way.151 The UPR can promote autophagy by stimulating the PERK/
on ischemic stroke, possibly by regulating mTORC and, hence, eIF2 and Ire1/TRAF2/JNK pathways.169 The UPR signaling pathway
autophagy in both mice MCAO models and OGD-treated primary mediator, activating transcription factor 6, can also affect
neurons in vitro.154 Another study found that inhibition of mTOR by autophagy in stroke.105 Rab7, a lysosome-associated small Rab
rapamycin activated the PI3K/Akt signaling pathway and, in turn, GTPase, regulates autophagy during cerebral ischemia and
autophagy, thus protecting neonatal rats against hypoxia.155 provides neuroprotection against ischemic brain injury.169 Speci-
Interestingly, homocysteine exerts a neurotoxic effect, possibly fically, Rab7 enables the fusion of autophagosomes with
owing to excessive autophagy following downregulation of PI3K/ lysosomes, thus affecting autophagosome maturation, lysosome
Akt/mTOR signaling in neural stem cells, suggesting the bi-faceted formation, and maintenance of lysosomal function.164 However,
role of autophagy in ischemic stroke.156 the actual mechanisms of UPR signaling and that of Rab7 in
AMPK is a member of the serine/threonine kinase family and ischemic stroke require further investigation.
serves as an important endogenous defense factor against cerebral
ischemia.151 During cerebral ischemia or hypoxia, energy deficiency Signaling pathways related to apoptosis in stroke
and the consequent elevated AMP/ATP ratio contribute to AMPK Apoptosis is a highly regulated, energy-dependent form of cell
phosphorylation, which activates autophagy to enhance energy death characterized by distinct morphological changes, such as
production.157 Several studies on animal experimental ischemic cell shrinkage, cytoplasmic condensation, nuclear membrane
stroke models have found that protective autophagy can be breakdown, and apoptotic body formation.170 Apoptosis, espe-
induced by regulating the AMPK/mTOR signaling pathway, thereby cially neuronal apoptosis, is involved in the pathology of post-
alleviating cerebral ischemic injury.105,158 A variety of downstream ischemic stroke. Cerebral ischemia leads to a decrease in ATP,
and upstream factors contribute to AMPK activity in both in vivo which causes cellular apoptosis in the ischemic penumbra. Anti-
experimental ischemic stroke models and in vitro. Mechanistically, apoptotic signals enable the potential recovery of dysfunctional
AMPK inhibits mTORC1 activity by phosphorylating and stimulating neurons, while pro-apoptotic signals contribute to neuronal death,
the TSC1/TSC2 complex during ischemia, thereby promoting thus modulating the balance between pro-apoptotic and anti-
autophagy.24 Furthermore, during ischemic stroke, Ca2+ overload apoptotic signals serve as potential therapeutic targets.171 Stroke
can activate AMPK via calcium/calmodulin-dependent protein triggers two principal apoptotic pathways: the extrinsic (or death
kinase beta and thus activate autophagy via the AMPK/mTOR receptor) pathway and the intrinsic (or mitochondrial) pathway.
pathway.159 Meanwhile, cytosolic p53 has been shown to directly Initiated by a variety of both external and internal damaging
inhibit autophagosome formation, whereas activated p53 functions stimuli, apoptosis eventually triggers a caspase cascade, which
to promote AMPKβ expression and inhibits mTOR expression to leads to the cellular injuries experienced during ischemic stroke.
promote autophagy.160 These molecules contribute to the function
of AMPK in autophagy in ischemic stroke. Apoptosis by the extrinsic/death receptor pathway. The extrinsic
MAPK is another important regulator of autophagy associated apoptotic pathway is triggered by the combination of ligands,
with ischemic stroke.161 MAPKs act as upstream regulators of including TNF-α, FasL, and TRAIL, and the corresponding death
mTORC1 and modulate autophagy through the MAPK/mTOR receptors (TNF-α receptor 1, Fas/CD95/APO1, and TRAIL-R,
signaling pathway in ischemic stroke.162 Wang et al. found that respectively) on the cell surface.172 In the event of an ischemic
autophagy protected against animal experimental cerebral stroke, the receptor recruits the death domain adaptor proteins
ischemic injury through induction of an Akt-independent MAPK/ FADD and TRADD, which form a complex by binding to
mTOR signaling pathway, wherein ERK negatively regulated procaspase-8.173 This complex induces a variety of downstream
mTORC1.163 In contrast, Zhang et al. found that ERK negatively damaging processes and eventually leads to activation of caspase-
controlled autophagy by activating mTOR, which contributed to 8.174 Once activated, caspase-8 triggers downstream effector
neuronal survival after experimental ischemic stroke injuries.164 caspases, either directly via proteolytic cleavage or indirectly by
Furthermore, an in vitro OGD/R study revealed that ERK could cleaving BH3-interacting domain (BID) to its truncated form, which
modulate autophagy by regulating mTOR in oxygen-glucose mediates apoptotic cell death via the mitochondria-dependent
deprivation models.165 Therefore, the MAPK/ERK signaling pathway pathway.175,176 Furthermore, during ischemic injury, neurons and
family could exert either a positive or negative regulation over glial cells release TNF-α, increasing Fas mRNA and protein levels.
mTOR in ischemic stroke; however, the exact mechanisms will These could function as stimuli for the extrinsic apoptotic pathway
require further investigation. and ultimately lead to neuronal death.31
Beclin1/Bcl2 signaling pathway. Beclin1 plays a significant role in Apoptosis by the intrinsic/mitochondrial pathway. The intrinsic
the early stage of autophagy. Local cerebral ischemia can pathway, also called the mitochondrial pathway, is a receptor-
upregulate Beclin1 expression and induce autophagy-like cell independent signaling cascade that affects mitochondrial energy
death, suggesting the involvement of Beclin1/Bcl2 signaling in metabolism. Apoptotic stimuli, such as excessive Ca2+ accumula-
the regulation of autophagy in ischemic stroke.166 Qi et al. found tion and oxidative stress, mediate mitochondrial cell death.177,178
that Bcl2 phosphorylation after cerebral ischemia in rats perturbed Lack of ATP due to oxygen and glucose deficiency results in
the Beclin1-Bcl2 complex and triggered distal ischemic conditional cellular depolarization and excessive glutamate release, both of
autophagy, thereby alleviating mitochondrial damage.167 Moreover, which further enhance Ca2+ influx.179–183 Ca2+ overload triggers
peroxisome proliferator-activated receptor γ (PPAR-γ) expression calpain activation, which mediates the cleavage of Bcl2-
increases during experimental cerebral ischemic injury. Activated interacting BID into its truncated active form, together with
PPAR-γ inhibits Beclin1-mediated autophagy, possibly by upregu- caspase-8 in the death receptor pathway.177,184 Truncated BID
lating the expression of Bcl2/BclXL.168 Thus, either detrimental or interacts with pro-apoptotic Bcl2 family members, forming a
neuroprotective factors impact on Beclin1-Bcl2 signal activities, dimer and causing MPTP opening.185 These changes trigger the
subsequently affecting autophagy in ischemic stroke. release of various pro-apoptotic factors, including cytochrome c,
endonuclease G, and AIF,186 which ultimately lead to apopto-
Other autophagy-related pathways. Several other signaling path- some formation by binding to apoptotic protease activating
ways are also involved in autophagy during ischemic stroke. factor-1.187 Upon apoptosome formation, procaspase-9 becomes

Qin et al.
15
activated into caspase-9, which triggers downstream effector caused mainly by decreased ATP in ischemia.31 Recent studies
caspases (caspase-3, caspase-6, and caspase-7) that promote have reported necrosis to be a highly regulated process involving
neuronal cell apoptosis.31 various signaling pathways.200 The major downstream signaling
pathways controlled by TNF-α include receptor-interacting protein
p53-mediated apoptotic pathway. Besides the extrinsic and kinase (RIPK1 and RIPK3) and mixed lineage kinase domain-like
intrinsic apoptotic pathways, another programmed cell death pathways.201
process activated by ischemic stroke depends primarily on p53. Facing cerebral ischemic damage, a complex containing
The tumor suppressor p53 becomes activated in ischemic areas of TRADD, RIPK1, and ubiquitin 3 ligases is recruited by the
the brain, whereby it contributes to neuronal apoptosis. By combination of TNF-α and its TNFR1120 receptor. Complex IIb is
translocating to the nucleus and binding to its specific DNA site, subsequently activated in both ischemia and hypoxia, contribut-
p53 induces apoptosis in ischemic brain cells.188 A plethora of ing to the phosphorylation and association of RIPK1 and
detrimental signals could stimulate p53. One is DNA damage, RIPK3.202–204 Within the complex formed by this association,
which can activate the apoptotic pathway via p53 phosphoryla- mixed lineage kinase domain-like is further activated by RIPK3,
tion.189 Another is represented by hypoxia and oxidative stress, which eventually leads to cell death.205 Concurrently, a cascade
which can also upregulate p53 protein levels.190 Concurrently and of inflammatory reactions, including secretion of pro-
mechanistically, some upstream cascade proteins, including JNKs, inflammatory cytokines, favors necrosis damage and exacer-
p38, DAPK, ASK1, and Notch may also lead to p53 activation.31 All bates ischemic brain injuries.206
these factors stimulate p53 activity and lead to cellular apoptosis
in ischemic stroke. Pyroptosis and ferroptosis in ischemic stroke. Majorly observed in
p53-induced apoptosis involves a variety of downtream genes ischemic penumbra, pyroptosis potentially induces pro-
and molecules, such as the pro-apoptotic genes Bax, Noxa, inflammatory pathways in ischemic stroke.207 During the process
p53AIP1, and PUMA, all of which act directly on mitochondria to of pyroptosis, cells get swollen and cellular organelles are released
induce apoptosis.190 Subsequently, p53 leads to the intrinsic to induce inflammation, in which caspase-1 is activated and form
apoptotic pathway, releasing pro-apoptotic factors, forming an inflammasomes.208 All these contribute to pyroptotic cell death and
apoptosome, activating effector caspases, and inducing neuronal secretion of inflammatory factors, such as IL-1β and IL-18.208,209
apoptosis.191 In addition, p53 mediates apoptosis by inducing the Another less mentioned but important cell death pathway is
expression of paternally expressed 3 and blocking cell survival ferroptosis. Ferroptosis is regulated by peroxidation, which requires
signaling.190 All these processes contribute to the onset and sufficient accessible iron.210 In ischemic brain regions, enhanced
progress of p53-mediated apoptosis. cellular excitotoxicity leads to the decrease in activity of GPX4 and
reduction in GSH production,211 which accumulates excessive ferric
Notch signaling pathways in apoptosis. Notch signaling path- ion and subsequently induces ferroptotic cell death. Also, damaged
ways, the most important component of which is Notch1, plays blood-brain barrier induces the iron to be transferred into neuronal
pivotal roles in a variety of biological processes in the central cells, which further enhances ferroptosis.212 From another perspec-
nervous system. Activation of Notch1, as well as other signaling tive, ferroptosis is also closely associated with oxidative stress, in
pathways, including NF-κB, p53, contributes to neuronal death which signaling pathways such as calcium-related signals, ATF4 and
processes. It has been reported that p53 and Pin1 are highly Keap1-Nrf2 signaling pathways play a role.213 Despite being less
associated with Notch and NICD in ischemic stroke. As an frequently discussed, ferroptosis may also be greatly involved in the
important mediator of apoptosis, p53 is activated by damages pathogenesis of ischemic stroke,with a variety of signaling pathways
such as hypoxia.192 The combination of Notch with p53 is crucial potentially participating in.
for neuronal apoptosis during ischemic stroke, which majorly
involves stabilization of p53 and transcriptional regulation of p53 Neuroinflammation, BBB disintegration, and related signaling
and NICD target genes.193 Besides, Pin1, an isomerase that pathways in ischemic stroke
regulates p53 transactivation, is deemed to be involved in the Inflammation is a key component of ischemic stroke pathologies.
pathogenesis of ischemic stroke, which is also related with Notch Existing in nearly all stages of ischemic stroke, neuroinflammation
signaling and is responsible for ischemic stroke-induced neuronal is initiated by the release of DAMPs from injured or dead cells.
death and neurological deficits.194 These DAMPs, including adenosine, heat shock proteins, high
In the meantime, studies have shown that Notch plays mobility group box 1, and interleukin-33, are subsequently
significant roles in modulating NF-κB-related cell death pathways. recognized by corresponding immune cells, and then trigger a
For instance, γ-secretase inhibitors down-regulate levels of NICD variety of downstream signaling pathways.214,215 During the
and protect against ischemic stroke damages. This protection whole process of inflammation, various immune cells including
effect is possibly via regulating NF-κB-related signals.195 Mean- microglia, macrophages, and T lymphocytes are activated.216,217
while, γ-secretase inhibitors block Notch signals and alleviates Also, the production of inflammation-related cytokines are
microglial activation.196 All these reveal the interactions between stimulated, as well as interferons or chemokines including
Notch and NF-kB pathways in both neurons and microglia in monocyte chemoattractant protein-1 (MCP-1).218 Upregulation of
cerebral ischemia. levels of several adhesion molecules assists leukocytes in adhering
Besides, it has also been reported that ischemic stroke to vascular surfaces,219 which facilitates the infiltration of immune
increases HIF-1α expression levels, which could directly bind cells. An abundance of pro-inflammatory cytokines leads to BBB
with NICD and NF-κB.197,198 Inhibition of both γ-secretase/Notch disintegration via activation of endothelial cells and peri-
and HIF-1α significantly reduced cell apoptosis, while enhanced cytes,220,221 along with the release of specific markers, such as
expression of NICD and HIF-1α increased NF-kB levels. All these von Willebrand factor and nerve growth factor.222,223 BBB leakage
show the close interactions among NICD, p53, HIF-1α and NF-kB, results in cerebral edema, as well as astrocytic aquaporin 4
which are highly associated with neuronal death processes, expression.224,225 All these factors, including MCP-1, von Will-
especially neuronal apoptosis in ischemic stroke. ebrand factor, nerve growth factor, and aquaporin4, could induce
immune cell adhesion to the vascular wall and then infiltrate into
Necrosis or necroptosis in cerebral ischemia. Following the onset the central nervous system, consequently contribute to BBB
of stroke, cerebral blood flow in the infarct area becomes disintegration and cellular edema.
significantly reduced, which induces necrotic death of resident Several signaling pathways are involved in neuroinflammatory
neurons.199 Necrosis is an unprogrammed cell death process processes and BBB breakdown in ischemic stroke; they are

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Fig. 6 Neuroinflammation, BBB breakdown and related signaling pathways involved in ischemic stroke. DAMPs Damage-associated molecular
patterns; AQP4 Aquaporin 4; HMGB1 High-mobility group box protein 1, TLR2 Toll-like receptor 2; TLR4 Toll-like receptor 4; MAPK Mitogen-
activated protein kinase; NF-kB Necrosis factor-kB; NLRP3 Nod-like receptor protein-3; MCP-1 monocyte chemoattractant protein-1; MIP
Macrophage inflammatory protein; CCL2 Chemokine-chemokine ligand 2; IL-1β Interleukin-1β; IL-6 Interleukin-6; TNF Tumor necrosis factor;
BBB Blood-brain barrier; S1PRs Sphingosine-1-phosphate receptor; VCAM Vascular cell adhesion molecule; LFA Lymphocyte Function-
associated Antigen; ICAM Intercellular cell adhesion molecule; DC Dendritic cells; MMP Matrix metalloproteinase
strongly associated with each other and determine the patho- studies have identified a polymorphism in the TNF gene, which
physiology of cerebral ischemia (Fig. 6). enhances stroke susceptibility, suggesting a pivotal role of TNF/
TNFR1 in the etiopathogenesis of stroke.232 Moreover, TNF levels
Cytokine- and chemokine-induced signaling pathways in are significantly upregulated upon cerebral ischemia, whereby
neuroinflammation they mediate neuronal plasticity.233 As previously mentioned, TNF
During ischemic stroke, microglia, which represent the main is secreted mainly by microglia, which protect against cerebral
resident immune cells in the brain, are the first cells recruited to ischemia. Specific myeloid cell-TNF-knockout mice have been
infarct lesions. They secrete both pro-inflammatory cytokines, such found to have larger infarct volumes and more severe neurolo-
as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), gical deficits than control mice.227,234 Removal of solTNF in mice
and anti-inflammatory cytokines, including IL-1R antagonist (IL-1Ra) reportedly alleviates the symptoms and pathology of cerebral
and IL-10.15,226–228 Together, these cytokines form a complex ischemia, suggesting that elimination of solTNF and retention of
signaling network in ischemic stroke-induced neuroinflammation. tmTNF ameliorate cerebral ischemic injuries.235 Thus, different
forms of TNF impact ischemic stroke, corroborating the important
Cytokines. TNF is the most studied cytokine in ischemic stroke; it role of TNF in this disease.
comprises a secreted form (solTNF) and a transmembrane form The IL-1 family constitutes a huge and complex network of
(tmTNF).229 The signal from both types of TNF is transferred via pleiotropic pro-inflammatory cytokines closely involved in reg-
two different receptors, TNFR1 and TNFR2, respectively.230 The ulating immune cells and inflammatory processes.236 Among IL-1
solTNF-TNFR1 signal is deemed responsible for pro-inflammatory family members, IL-1α, IL-1β, and IL-1Rα have been studied in
effects of TNF, which trigger cell death signaling pathways. detail in relation to ischemic stroke. A polymorphism in the IL-1A
Instead, TNFR2 promotes cell growth and regeneration.204,230,231 gene has been associated with increased susceptibility to
Given the important regulatory role of TNF signals in inflammation stroke;237 conversely, a polymorphism in the IL-1B gene lowers
and other neurological processes, TNFs are likely involved in the stroke risk.238 IL-1α expression is significantly increased in cerebral
pathophysiology of ischemic stroke. Genome-wide association ischemia.228 Platelet-derived IL-1α contributes to neurovascular

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inflammation and causes the infiltration of neutrophils to ischemic experimental ischemic stroke models.265 Consistently, external
lesions.239 Primarily secreted by microglia and macrophages,15,240 administration of CCL3 to brain ventricles exacerbated ischemia-
IL-1β affects neurons, glial cells, and the vasculature.241 IL-1β levels induced injuries.266 Meanwhile, another chemokine CCL5 has
are significantly increased in the cerebrospinal fluid at days 2 and been found to regulate ischemia/reperfusion (I/R) injuries in
3 post-stroke, suggesting a predictive value in stroke pathophy- experimental ischemic stroke models.267 Clinical studies have
siology.233,242 The IL-1 family has been shown to exacerbate stroke also shown that plasma CCL5 levels were increased in sympto-
pathology, as revealed by reduced infarct volumes in experimental matic patients in comparison with asymptomatic ones.268
ischemic stroke models of IL-1α/β knockout mice.243 Conversely, Besides the CC chemokine family, the CXC chemokines, also
IL-1β administration worsens the outcomes of mice subjected to plays crucial roles in ischemic stroke pathogenesis. Among them,
ischemic stroke.244 Overall, the IL-1 family plays a detrimental role those ELR+ CXC chemokines, including CXCL1, CXCL2, and
in the pathophysiology of cerebral ischemic stroke and could CXCL8, directly function to neutrophils toward ischemic brain
serve as a potential therapeutic target. regions; however, those ELR− CXC chemokines, including
Another vital member among pro-inflammatory interleukins is CXCL10, CXCL12, and CXCL16, mainly induce Th1-cell infiltration
IL-6, which is secreted by a variety of cells, including monocytes, in postischemic inflammation.269
neurons, and glial cells.245,246 The IL-6 signaling pathways can
be classified into classic signaling, which requires IL-6R and High-mobility group box protein 1 (HMGB1)/Toll-like receptor
gp130, and trans-signaling, whereby IL-6 is linked to sIL-6R.247 (TLR) and NF-κB signaling pathways in neuroinflammation
Reportedly, the former is deemed to be neuroprotective and Various immune cells, as well as the corresponding cellular
helps maintain neuronal homeostasis,248 whereas the latter products, are associated with oxidative stress and necrosis activate
contributes to IL-6-induced pro-inflammatory outcomes.249,250 the innate immune system, probably via the TLR signaling
IL-6 levels are upregulated during cerebral ischemia, which pathway. TLRs, which are expressed on both the cell surface and
correlates with infarct volumes and survival rates.251,252 Inter- in the intracellular space, regulate the status and function of
estingly, IL-6 levels are seemingly upregulated by IL-1β.253 The numerous immune cells.270–275 TLR signaling can be categorized
fact that brain-derived IL-6 promotes neurogenesis after stroke, based on two major downstream adaptor proteins: myeloid
and thus contributes to long-term functional recovery, points to differentiation primary response 88 (MyD88)-dependent and
its potential neuroprotective effect following cerebral ische- adapter-inducing interferon-β-dependent pathways.276 Both TLR
mia.254 Even though only a few studies have focused on the role signaling pathways activate NF-κB, which subsequently triggers
of IL-6 in ischemic stroke, its pleiotropic effects are worth further the release of pro-inflammatory cytokines.277–279
investigation. Interestingly, TLRs may act as another double-edged sword in
Contrary to the aforementioned pro-inflammatory cytokines, ischemic stroke. In case of relatively moderate ischemic injury,
IL-10 is released primarily by type-2 helper T cells and serves as TLR2 and TLR4/NF-κB signaling pathways are inhibited, whereas
an anti-inflammatory cytokine, reducing inflammation and interferon regulatory factor 3 signaling is enhanced. Both of these
limiting cellular apoptosis.255 IL-10 gene polymorphism is processes exert neuroprotective effects on ischemia.280 Pretreat-
associated with the risk of stroke subtypes.256 In experimental ment with TLR2, TLR3, TLR4, TLR7, or TLR9 agonists alleviates the
ischemic stroke models, transgenic mice with enhanced IL-10 symptoms and pathological damage in various ischemic stroke
expression showed reduced infarct volumes and cellular models.281,282 Administration of lipopolysaccharide prior to
apoptosis.257 Likewise, clinical studies have shown that low IL- ischemic insult protects against cerebral ischemia, possibly by
10 levels correlate with poor stroke outcomes, worse neurolo- modulating the TLR4 signaling pathway and inhibiting NF-κB after
gical deficits, and extravagated inflammatory reactions.258–260 ischemic stroke attack.283 In contrast, elevated levels of plasma
These results indicate that the anti-inflammatory properties of lipopolysaccharide appear to promote the expression of TLR4,
IL-10 serve as a potential clue for the diagnosis and prognosis of causing the release of inflammatory cytokines, larger infarct
ischemic stroke. volumes, and more severe functional deficits in rat cerebral
ischemia models.284 These seemingly contradictory results sug-
Chemokines. In addition to cytokines, chemokines represent gest that LPS modulation of TLR4 response possibly depends on
another group of small signaling proteins that contributes to the whether activation occurs before or after ischemic insult.
inflammatory processes in ischemic stroke. Immediately after One key component in the TLR-related signaling pathway is
cerebral ischemia, pro-inflammatory cytokines, such as TNF-α and HMGB1, which triggers downstream neuroinflammatory responses
IL-1β, induce the secretion of chemokines, such as MCP-1, during stroke.285 HMGB1 levels are significantly elevated in the
fractalkine, macrophage inflammatory protein 1, microglial brain, specifically in microglia, astrocytes, and blood vessel cells,
response factor-1, and cytokine-induced neutrophil chemoattrac- which are closely associated with neuroinflammation and cellular
tant.261 Chemokine-chemokine ligand 2 (CCL2) and its corre- stress such as stroke.286–289 As one of the major ligands for TLRs,
sponding receptor, CCR2, are involved in regulating the extracellular HMGB1 interacts with TLR2 or TLR4 and in turn NF-κB
inflammatory response in ischemia, possibly via immune cell to elicit pro-inflammatory reactions.280,290,291 Moreover, the
recruitment and adhesion to cerebral endothelial cells.151,262 CCL2 release of HMGB1 activates TLR4 and enhances IL-1β production
expression becomes enhanced in the ischemic penumbra, through Nod-like receptor protein 3 (NLRP3) inflammasome
cerebrospinal fluid, and serum after ischemia or ischemia- activation.292 Furthermore, HMGB1 enhances the secretion of
reperfusion.153,263 Moreover, CCL2/CCR2 expression correlates several pro-inflammatory cytokines, including inducible NOS,
positively with infarct area and lesion enlargement,151,262 and cytochrome c oxidase subunit 2, IL-1β, and TNF-α, promoting
enhanced CCL2 expression further aggravates ischemic injury in neuronal cell death during ischemia.293 These results suggest that
mice.153 Ischemic damage significantly increased MCP-1 mRNA both inflammatory reactions and cell death signaling pathways
(CCL2) expression, which further exacerbated ischemic brain are induced by HMGB1/TLR signals in ischemic stroke, possibly
injury, together with abundant infiltration of inflammatory cells aggravating ischemic injury.
in an experimental ischemic stroke model.264 All these findings
suggest the detrimental role that CCL2/CCR2 signaling pathways MAPK signaling pathway in inflammation and BBB dysfunction
play in ischemic stroke. MAPK comprises three main effectors: ERK1/2, JNK, and p38.294
Besides the most frequently discussed CCL2, other chemokines Stress-activated protein kinases, JNK, p38 MAPK, and ERK exert
are also involved in the pathogenesis of ischemic stroke. detrimental effects during cerebral ischemia.295 Specifically, the
For instance, CCL3 has been reported to be upregulated in MAPK signaling pathway is activated soon after the onset of

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ischemic injury, and p38 MAPK regulates the expression of various stroke and could provide a therapeutic target against cerebral
pro-inflammatory cytokines.296 Activation of the p38/MAPK/AR- ischemia.
related signaling pathway has been shown to promote the
microglial pro-inflammatory phenotype in cerebral ischemia.297 Microglial phagocytosis and complement activation
Activation of MAPK/ERK signaling and consequent stimulation of Microglia functions as the major phagocyte in the central nervous
metalloproteinase (MMP) expression could exacerbate BBB system, which is responsible for myelin debris clearance and
damage in ischemic stroke, further enhancing the expression of pruning synapsis.318 It has been reported that microglia phago-
pro-inflammatory factors.298 Similarly, BBB damage in cerebral cytose tissue debris in experimental ischemic stroke model, which
ischemia induced by a high-salt diet, has been associated with the contribute to tissue repair and neuronal network reconstruc-
p38/MAPK/SGK1 signaling pathway.299 These results suggest that tion.319,320 However, other studies have also pointed out that over-
MAPK-related signaling pathways exacerbate ischemic brain enhanced microglia engulfment exacerbates cerebral ischemia-
injury, possibly by enhancing neuroinflammatory processes and induced brain injuries.321,322 Hence, microglial phagocytosis may
BBB dysfunction. play both beneficial or detrimental roles in ischemic stroke.
Microglia could engulf a variety of dying cells and debris, in which
MMPs and BBB dysfunction in ischemic stroke a plethora of signaling pathways are involved. TMEM16F is
MMPs are crucial for the function and structure of the BBB in both expressed by stressed neurons in ischemic stroke, which induces
human and animal stroke models.300,301 The elevated production neurons to expose phospholipid phosphatidylserine (PS), an ‘eat-
of MMPs and myeloperoxidase in ischemic stroke favors BBB me’ signal. Consistently, knockdown of TMEM16F hindered
breakdown.302 In particular, MMP9 induces proteolysis of the BBB microglial phagocytosing viable neurons in the penumbra after
basal lamina.300,301 Clinical studies have shown that baseline experimental ischemic stroke.323 Besides, triggering receptor
MMP9 serves as an important indicator of BBB disruption in expressed on myeloid cells (TREM2) signaling pathways are
ischemic stroke and is related to the hyperintense acute deemed to be greatly involved in microglial phagocytosis in
reperfusion injury marker used in magnetic resonance imaging.303 ischemic stroke. TREM2 deficiency dampens microglial phagocy-
Hypothermia followed by rapid rewarming enhances the perme- tosis of neurons, which further exacerbates ischemic brain
ability of the BBB in ischemic stroke, along with elevated MMP9 injuries,319 indicated the neuroprotective role of Trem2 in
expression levels and damage to tight junctions.304 MMP12 levels ischemic stroke.324
have been found to be elevated in rat cerebral ischemic stroke Another part of phagocytosis is the complement system,
models, whereas suppressing MMP12 alleviates the symptoms including C1q and C3. Upon activation, C3 is cleaved into C3a
induced by ischemia.305 Concurrently, MMP2 may participate in and C3b, of which C3b as well as its receptor, CR3, function
the pathophysiology of ischemic stroke, together with VEGF together to regulate dying cells clearance.325,326 Meanwhile, C1q,
signaling. The latter is likely involved in the initial stages of the biggest component of the C1 complex, has been reported to
ischemic stroke, whereby hypoxic preconditioning exacerbates strengthen microglial clearance of apoptotic cells in ischemic
BBB injury and brain edema.306 Furthermore, it has been shown stroke.327 After ischemic stroke, microglial phagocytosis of both
that recovery from BBB damage is associated with both the MMP2 synapses and neurons was directed by activation of complement,
and VEGF pathways in acute cerebral ischemia, suggesting a close which eventually contributes to cognitive decline.328,329 Thus, with
link between MMP2 and VEGF.307 a variety of signaling pathways involved, activation of the
complement system may also be closely interacted with microglial
Sphingosine-1-phosphate receptor (S1PR)-related signaling phagocytosis, which possibly, greatly influence the pathologies of
pathways during neuroinflammation in ischemic stroke ischemic stroke.
S1PRs form a group of G protein-coupled receptors abundant in
microglia and are thought to regulate inflammatory responses in Therapeutic approaches targeting pathophysiological signaling
ischemic stroke.308 In vitro studies have shown that the addition of pathways involved in ischemic stroke
S1P to microglia subjected to oxygen-glucose deprivation/ So far, the only drug approved by FDA for treating ischemic
reperfusion exacerbates hypoxia-induced neuronal apoptosis.309 stroke is tissue plasminogen activator (tPA), which breaks down
In experimental ischemic stroke models, sphingosine kinase 1 the blood vessel clot.8 This therapy has several limitations, such
phosphorylates sphingosine to S1P, which binds to S1PR3 and as the therapeutic window is only 4.5 h, and treatment outside
confers microglia a pro-inflammatory phenotype. Sphingosine the therapeutic window could possibly result in cerebral
kinase 1 enlarges the brain infarct volume and exacerbates hemorrhage.330 Progress have been made in discovering new
neurological symptoms by upregulating the expression of pro- therapeutic approaches against ischemic stroke. Current studies
inflammatory cytokines.310 Intriguingly, the S1PR agonist fingoli- have shed lights on micro-RNA therapies, in which expression
mod has been recently reported to switch microglia from a pro- levels of miRNA are changed and apoptosis-related genes are
inflammatory to an alternatively activated phenotype in a chronic subsequently mediated.331 Another potential treatment is cell
hypo-perfused ischemic stroke model in mice.311 Thus, the pro- therapy, which utilizes stem cells to differentiate.332 However,
inflammatory mechanism of S1PRs in ischemic stroke requires therapeutic approach is quite limited, and more research are
further exploration. need to discover new potential therapeutic strategy for
ischemic stroke.
Inflammasome activation in ischemic stroke Given the pivotal roles the pathophysiology and signaling
Inflammasomes are large multiprotein complexes,312,313 which pathways play in ischemic stroke, numerous therapeutic
can mediate neuroinflammation and contribute to neural cell approaches have been explored in both experimental and clinical
death in ischemic stroke.314 Both in vivo and in vitro model studies, and several of them have been demonstrated to be
studies suggest that the NLRP3 inflammasome plays a pivotal effective in treatment of ischemic stroke (Table 2).
role in microglia-associated neuroinflammation in ischemic
stroke, possibly through alterations to the microglial pheno- Therapeutic approaches targeting excitotoxicity and related
type.315 These effects may be linked to activation of the NF-κB signaling pathways in cerebral ischemic stroke
signaling pathway.316 Additionally, NLRP1 is related to cerebral Targeting the GluN2B-PSD95-nNOS complex. The GluN2B-PSD95-
ischemic injuries, and its inhibition alleviates neuroinflamma- nNOS complex plays a central role in regulating NMDAR activity
tion in ischemia.317 Thus, inflammasome activation, either via and related signaling pathways; therefore, it could potentially serve
NLRP1 or NLRP3, contributes to the pathogenesis of ischemic as a therapeutic target for cerebral ischemic stroke. The Tat-NR2B9c

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peptide, which binds to either PSD95 or nNOS, was shown to ischemic lesions, altered expression of GluN2A and GluN2B, and
prevent downstream neurotoxic pathways and superoxide produc- stimulation of pro-survival Akt and ERK signaling in an experi-
tion.333 Furthermore, Tat-NR2B9c administration reportedly mental ischemic stroke model.354 Overall, therapeutic approaches
improved behavioral deficits, reduced infarct volumes, and retained involving drugs, physical treatment, or gene modifications
the gene transcription profiles in animal ischemic stroke mod- enhancing AKT-related signaling pathways and NMDAR activities
els.334,335 Another study reported that TAT-NR2B9c alleviated could reinforce synaptic NMDAR activities and their neuroprotec-
neuronal death and p38-induced damage in ischemic injury,336 tive effects in ischemic stroke.
while a clinical study found that it significantly decreased infarcts in
ischemic stroke patients.337 Another small molecule called ZL006 Targeting the Panx1 signaling pathway. During ischemia, NMDAR
has been found to disrupt the interaction between PSD95 and activates Src kinases, which subsequently phosphorylate residue
nNOS in ischemia, without affecting the normal functions of Y308 in the C-terminus of pannexin 1 (Panx1), leading to
NMDARs and nNOS.90,338 Similarly, IC87201 has been found to secondary ischemic currents.355,356 Preventing Panx1 phosphor-
disrupt pathogenic interactions between PSD95 and nNOS but ylation may alleviate the symptoms and pathologies of ischemic
without impairing normal nNOS activities.27 Finally, a study has stroke. Indeed, use of the interfering peptide Tat-Panx308 helped
shed light on Neu2000, a sulfasalazine derivative and GluN2B reduce infarct lesion size and alleviate sensorimotor deficit
antagonist that selectively blocks NMDARs and scavenges free symptoms in middle cerebral artery occlusion (MCAO) rats,
radicals, which exerted a neuroprotective effect in ischemic suggesting its effectiveness in treating ischemic stroke.356 In
stroke.339,340 All this experimental evidence highlights the potential spite of the limited number of studies, regulation of Panx1 in
of treating ischemic stroke by targeting the GluN2B-PSD95-nNOS excitotoxicity could represent a promising strategy for ischemic
complex and preventing its participation in excitotoxicity. However, stroke treatment.
several shortcomings still exist. Although overactivation of NMDARs
is acknowledged to be important in the etiology of cerebrovascular Therapeutic approaches targeting signaling pathways to alleviate
insults, the importance in physiological function has made the symptoms and damage caused by oxidative stress in ischemic
current NMDAR antagonists ‘undruggable’ for clinical application in stroke
ischemic stroke.27,341 Also, the therapeutic time window is relatively Nrf2/ARE signaling pathway. The Nrf2/ARE signaling pathway
short, and safety issues including nausea, vomiting, cardiovascular contributes to the generation of numerous protective factors, such
and psychomimetic effects, remain to be considered.342–348 as anti-inflammatory proteins, antioxidant enzymes, and growth
factors. Its antioxidant target genes include those encoding for
Targeting the DAPK1 signaling pathway. DAPK1 phosphorylates heme oxygenase 1 (HO1), NADP(H) quinone dehydrogenase 1
p53, a tumor suppressor that serves as one of its substrates. The (NQO1), and glutathione S-transferase (GST).357 Thus, regulation of
interfering peptide Tat-p53DM241–281 inhibits specifically the the Nrf2/ARE signaling pathway could potentially protect against
downstream targets of DAPK1, such as the pro-apoptotic genes oxidative stress-induced damage in ischemic stroke. It has been
Bax, Puma, and caspase-3, which are also regulated by p53.349 The reported that injection of tBHQ, an Nrf2 inducer, alleviates the
administration of Tat-p53DM241–281 was observed to significantly symptoms of experimental cerebral ischemic stroke.358 Similarly,
reduce infarct area and alleviate behavioral deficits in experi- administration of metformin in cerebral ischemic stroke models
mental ischemic stroke models.350 Another drug, alleviated oxidative stress-induced BBB damage, possibly through
GluN2BCT1292–1304, dissociates DAPK1 from the GluN2B subunit activation of the NRF2/ARE signaling pathway.359 In contrast,
and protects neurons from ischemic injury.351 However, it still higher vulnerability and exacerbated brain damage were
remains controversial that McQueen et al. have found that genetic observed in cerebral ischemic stroke models of Nrf2-knockout
depletion of DAPK1 could not alleviate excitotoxic and ischemic mice.360 Generally, activating the Nrf2/ARE signaling pathway may
injuries in neurons.351 With possible uncertainties, these results confer a neuroprotective effect in cerebral ischemic stroke, which
indicate that DAPK1 inhibition could potentially alleviate ischemic is associated with mitigation of oxidative stress.
brain damage through decreasing cellular excitotoxicity.
Sirtuin (SIRT)/forkhead box O (FOXO) signaling pathway. SIRT1–7
Targeting the PTEN-induced signaling pathway. Based on the play important roles in oxidative stress during ischemic stroke.
function of PTEN in inhibiting the PI3K/Akt signaling pathway and The SIRT/FOXO signaling pathway has been shown to prevent
inducing apoptotic cell death via excitotoxicity, regulating PTEN oxidative stress in cerebral ischemia-reperfusion. SIRT1 exerts
could possibly help ameliorate excitotoxicity and, in turn, an antioxidant effect by activating either the FOXO family or
neurological deficits in ischemic stroke. Genetic knockdown of PPAR-γ coactivator-1 and, as such, could serve as a potential
PTEN was found to retain PI3K/Akt signaling while downregulating therapeutic target.361,362 SIRT3 has been reported to enhance
the extra-synaptic NMDAR current, which exerted a neuroprotec- SOD2 activity and decrease ROS levels.363 Moreover, transso-
tive effect on an experimental ischemic stroke model.74 Pharma- dium crocetinate protected animals from oxidative stress
cologically, an interfering peptide, Tat-K13, was utilized to disrupt induced by cerebral ischemia–reperfusion injury, probably by
the cell death signaling pathway activated by PTEN.75 Tat-K13 activating the SIRT3/FOXO3a/SOD2 signaling pathway.364 Simi-
exerted a neuroprotective effect in rats suffering from experi- larly, genipin was found to regulate the UCP2/SIRT3 signaling
mental ischemic stroke by reducing the size of the infarct pathway and alleviate oxidative stress induced by cerebral
lesion.33,75 These findings suggest that, owing to its link to PI3K/ ischemia.365 These findings reveal the potential of SIRT signaling
Akt signaling, the PTEN-related pathway could serve as a potential pathways in therapeutic approaches against oxidative stress and
therapeutic target in the treatment of ischemic stroke. ischemic stroke.
Targeting the AKT signaling pathway. The iridoid glycoside Therapies targeting neuroinflammation-related signaling
geniposide has been reported to protect neurons from ischemic pathways
damage by activating the GluN2A/AKT/ERK signaling pathway.352 Chemokine-related signaling pathways. Therapeutic approaches
Accordingly, pseudoginsenoside-F11 prevents calpain1 activation regulating CCL2/CCR2 expression may alleviate the symptoms and
while promoting the GluN2A-mediated AKT/CREB pathway.353 pathologies of ischemic stroke. Whereas CCL2 gene disruption
Genes involved in the modulation of NMDAR expression along the reduced infarct volume, CCR2 deletion reduced infarct size, while
Akt/ERK pathway could also potentially serve as therapeutic also improving locomotor ability of mice in an experimental
targets. TRPM2 knockout mice showed significantly smaller ischemic stroke model.263,366 CCR knockout reduced infarct

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volumes and mortality of mice in experimental ischemic stroke effective and persistent therapeutic concentration.388,389 Also,
models. However, it should be mentioned that hindering there’re studies showing that IL-1Ra increased the possibility of
monocyte infiltration using an anti-CCR2 antibody delayed long- poor mRS outcomes.390 Though that, IL-1Ra still has good
term behavioral recovery, along with decreased expression of anti- perspectives in cerebral ischemic stroke treatment owing to its
inflammatory genes in MCAO mice, suggesting a double-edged anti-inflammatory properties.
role of CCL2/CCR2 in ischemic stroke.367 Infarct size in rat MCAO
models has been reduced also via inhibition of another NLRP3 inflammasome. NLRP3 inflammasome regulation has been
chemokine, CCL23 (also known as MIP3α).265 Taken together, acknowledged as a potential therapeutic approach for ischemic
regulating chemokine expression, especially the CCL2/ stroke.391 Brilliant blue G, a P2X7R purinergic receptor antagonist,
CCR2 signaling pathway, may serve as a potential therapeutic or MCC950, an NLRP3 inhibitor, not only attenuated cerebral
approach against cerebral ischemic stroke, although the harmful infarct areas and neurological impairments but also inhibited
effects of such an intervention should be carefully considered. caspase-3-associated neuronal apoptosis.392 Similarly, genistein, a
natural phytoestrogen, has been reported to alleviate cerebral
TLR-associated signaling pathways. Considering the important ischemia-induced injury in senescent mice by inhibiting NLRP3
role played by TLRs in neuroinflammation, several studies have inflammasome formation.393 An in vitro study revealed that
demonstrated that TLR signaling could serve as a treatment target. treatment modulating the immunoproteasome/NF-κB/NLRP3
Overexpression of miR-18a-5p downregulates the levels of TLR4 inflammasome signaling axis could work against hypoxia and
and TLR7, exerting a protective effect against ischemic injury ischemia, as well as prevent apoptosis.394 Therefore, inhibition of
in vitro.368 Resveratrol modulates microglial activity and improves NLRP3 inflammasome formation could possibly attenuate
ischemia-induced neurological symptoms by regulating the TLR4/ ischemic stroke inflammatory processes and limit cell death.
NF-κB/STAT3 signaling pathway.369–371 Stevioside, a natural glyco-
side, protects against cerebral ischemia by inhibiting TLR/NF-κB Therapeutic approaches targeting the BBB in ischemic stroke
pathway-mediated neuroinflammation.372 Moreover, treatment Sirt signaling pathways. Protecting the BBB could help alleviate
with progesterone and its metabolites has been shown to ischemic stroke. In an experimental rat model of stroke, hyperbaric
alleviate the symptoms of various cerebrovascular diseases by oxygen treatment helped protect the BBB, potentially by regulating
regulating the TLR4/NF-κB signaling pathway and inhibiting the ATP/NAD+/Sirt1 signaling pathway.164 Similarly, quercetin has
neuroinflammation.373–375 Similarly, dexmedetomidine has been been shown to protect the BBB and alleviate ischemia–reperfusion-
proven effective against inflammatory reactions, oxidative stress, induced injuries via activation of Sirt1 signals in rats.395 Minocycline
increased infarct volume, and brain edema in MCAO rats by has also been shown to ameliorate hypoxia-induced BBB disrup-
inhibiting the HMGB1/TLR4/NF-κB signaling pathway.376 Interest- tion. This effect was mediated by the Sirt3/proline hydroxylase-2
ingly, one study reported that activating TLR7 reduced infarct degradation pathway, together with decreased levels of MMP2,
volume and neurological deficits by enhancing interferon expres- MMP9, and VEGF, as well as upregulation of tight junction
sion.377 This observation is possibly associated with the dual effect proteins.396
of TLRs on neuroinflammation and ischemic stroke. In conclusion,
regulation of TLR signaling has been revealed to attenuate MMP inhibition for BBB protection. Given the indispensable role of
neuroinflammation and, thus, protect against ischemic stroke. This MMPs, inhibition of the MMP signaling pathway may be beneficial
therapeutic effect is possibly related to a variety of downstream in anti-stroke therapy. Administration of hydrogen sulfide donors
molecules, including NF-κB and STAT3, whose modulation could may help ameliorate cerebral BBB damage, most likely via MMP9
promote the beneficial effects of TLRs in ischemic stroke. inhibition.397 In addition, vagus nerve stimulation could help
protect the BBB in ischemic damage by inhibiting MMP2/9-
Cytokine-related signaling pathways. Regulation of IL-1 and TNF mediated tight junction protein disruption.398 Similarly, hyperbaric
cytokine families could also help attenuate ischemic stroke oxygen has been reported to stabilize the BBB in an experimental
injuries. A study using a single intravenous dose of XPro1595 or ischemic stroke model, possibly by blocking MMP2 activation.399
etanercept, which targets TNFs, found that both compounds Finally, intra-arterial norcantharidin alleviated cerebral BBB
alleviated inflammatory reactions and enhanced locomotor damage by decreasing MMP9 expression in an experimental
abilities in a mouse model of focal cerebral ischemia; however, ischemic stroke model.400 These results suggest that regulation of
they did not decrease infarct volume.378 Another modified MMP-related signaling pathways protects the BBB from ischemic
therapy, cTfRMAb-TNFR, which transfers TNFR across the BBB, stroke injuries.
has been reported to successfully reduce the infarct area and
ameliorate neurological deficits.379,380 Similarly, a preclinical study Cell death-related signaling pathways as targets for ischemic
demonstrated that sTNF-αR1 retained axonal plasticity in the stroke treatment
cerebral cortex after stroke,381 which is in agreement with the Autophagy-related signaling pathways. A variety of signaling
results of another study showing that injection of solTNFR1 in pathways related with autophagy, including Akt, AMPK, and
dendritic cells alleviated infarct injury and inflammation after others, has been shown to be potential therapeutic approaches
experimental stroke.382 However, it’s still worth mentioning that against ischemic stroke. Fingolimod, a well-established
targeting both solTNF and tmTNF may concurrently raise the risk sphingosine-1-phosphate receptor agonist, alleviates neurological
of cardiovascular and demyelinating disease.383 Due to the deficits and reduces infarct areas by enhancing Akt signaling and
possible side effects of the anti-TNF therapies, more efforts should ameliorating neuronal apoptosis,401,402 as well as regulating the
be made for more specific anti-TNF therapeutics. mTOR/p70S6K autophagy signaling pathway in ischemic stroke
IL-1Ra is the only therapeutic agent against IL-1-associated models.403 Studies have also reported that selenium protects the
inflammation.226 Preclinical studies have shown that recombinant BBB from ischemia-reperfusion injuries associated with PI3K/
IL-1Ra protects against ischemia-induced injuries in rats384,385 and mTOR/AKT signaling pathway activation, which is possibly related
mice.386 Concomitantly, the first randomized, double-blind, to autophagy inhibition.152,404 As for the AMPK signaling pathway,
placebo-controlled trial utilizing recombinant human IL-1Ra SMXZF, a combination of Rb1, Rg1, schizandrin, and DT-13 (6:9:5:4),
showed that patients receiving rhIL-1Ra displayed milder inflam- exerts a neuroprotective effect on cerebral ischemia-reperfusion
matory reactions and nearly no disability 3 months after stroke.387 injury, possibly by suppressing autophagy through regulation of
There’re several shortcomings that rhIL-1Ra crosses the BBB slowly the AMPK/mTOR and JNK signaling pathways, both in animals and
and has relatively short half-life in the circulation to achieve oxygen-glucose deprivation/reperfusion models.405,406 Likewise, by

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activating AMPK-induced autophagy, ezetimibe ameliorates neu- significant therapeutic effects on stroke patients.425 Various
ronal apoptosis and infarct volume, while improving neurological types of cells, including allogenic mesenchymal stem cells from
deficits in MCAO rat models.407 Finally, physical exercise induces adipose tissue(NCT01678534), bone-marrow-derived stem cell
AMPK activation and mTORC1 inhibition, thereby promoting (NCT01501773), endothelial progenitor cells (NCT01468064), and
autophagy, which consequently improves cerebral ischemia autologous M2 macrophages (NCT018453500) have been tested
outcomes.408–410 in clinical trials as a reparative therapy for acute ischemic stroke.
Besides these two main target signals, additional autophagy- All these reveal prospects for targeting the pathophysiology and
associated signaling pathways, related mainly to STAT, and SIRT, related signaling pathways in treating ischemic stroke.
could also serve as targets for ischemic stroke therapies.
Extracellular vesicles secreted by stem cells help mitigate ischemic Concluding remarks and future perspectives
brain damage, possibly by modulating STAT3-dependent autop- Ischemic stroke is characterized by the blockade of cerebral blood
hagy, both in vivo and in vitro.411 In an experimental rat cerebral flow caused by the presence of thrombi in the blood vessels and
ischemia-reperfusion injury model, electroacupuncture mitigated has an overwhelming effect on people’s health and their quality of
neurological symptoms and related pathologies through inhibi- life. In recent years, studies have sought to further elucidate the
tion of maladaptive autophagy and activation of the SIRT/ mechanisms of ischemic stroke. Nevertheless, the complex
FOXO1 signaling pathway.412,413 In addition, other signaling pathogenesis of ischemic stroke means that the participating
pathways involving SIRT, including SIRT3/AMPK/mTOR and signaling pathways need further comprehensive exploration. In
SIRT1/BMAL1, are activated by luteolin and melatonin, respec- this review, we summarized the signaling pathways involved in
tively, and help protect against cerebral ischemia–reperfusion- ischemic stroke and categorized them based on their specific
induced injuries.414,415 pathophysiological roles in excitotoxicity, mitochondrial dysfunc-
tion, oxidative stress, neuroinflammation, and cell death. Because
Apoptosis-associated signaling pathways. Likewise, regulation of these signaling pathways are interconnected, combined thera-
several signaling pathways, such as ERK/MAPK, AMPK and SIRT peutic targets against ischemic stroke may be elucidated.
signaling pathways, are shown to mediate apoptosis in ischemic At present, recanalization of blood vessels via intravenous
stroke. Beta-hydroxybutyrate ameliorates cerebral ischemic stroke thrombolytic treatment or mechanical thrombectomy represents
injuries by suppressing apoptosis induced via oxidative stress and the major therapeutic approach for ischemic stroke. However, this
mitochondrial dysfunction, both in vivo and in vitro. The curative is underscored by the lack of suitable pharmacological treatments,
effects on apoptosis are probably associated with ERK/CREB/eNOS calling for the discovery of new therapeutic targets against
signaling pathway activation.416 Modulation of other ERK/MAPK ischemic stroke. In this review, we combed through existing
signaling axes, including the MAPK/ERK/EGR1, CXCL13/ERK/MEK, therapeutic approaches and classified them according to their
and DAPK1/ERK signaling pathways, has also been shown to target signaling pathways. In conclusion, our review comprehen-
protect against ischemia-induced injuries both in vitro and sively elucidates the signaling pathways involved in the patho-
in vivo.417–419 With respect to the AMPK signaling pathways, physiology of ischemic stroke and also points out potential
BML-275, an AMPK inhibitor, exerts a neuroprotective effect on therapeutic approaches against ischemic stroke associated with
cerebral ischemic stroke by downregulating cytochrome c and AIF those key signaling pathways.
expression, consequently blocking apoptosis.420 In addition,
glycine was shown to attenuate cellular apoptosis and improve
ischemic stroke damage by suppressing the AMPK/GSK3β/ ACKNOWLEDGEMENTS
HO1 signaling pathway.421 SIRT signals are also possibly involved, This work was funded by National Natural Science Foundation of China (Grants:
as Rosuvastatin may exert protective effects on cerebral ischemia 82071380, 81873743, 81801223), and Tongji Hospital (HUST) Foundation for Excellent
in rats through the Sirt1/NF-κB signaling pathway and inhibition of Young Scientist (Grant No. 2020YQ06).
apoptosis.422 Stem cell therapies also attenuate ischemia-induced
injuries, potentially through the SIRT/NF-κB signaling pathway.423
Finally, an in vitro study revealed that regulation of the miRNA- AUTHOR CONTRIBUTIONS
All authors have read and approved the article. Concept and design: C.Q., D.S.T., and
29b/SIRT1/PPAR-γ coactivator 1 alpha axis ameliorated oxygen-
W.W. Drafting of the manuscript: C.Q., S.Y., Y.H.C., H.Z., X.W.P., L.C., L.Q.Z., and M.C.
glucose deprivation-induced cell apoptosis, thus protecting cells Critical revision of the manuscript for important intellectual content: D.S.T. and W.W.
from ischemia.424 All these reveal the potential of therapeutics Obtained funding: C.Q. and D.S.T. Supervision: D.S.T. and W.W.
against cellular apoptosis in ischemic stroke.
National clinical trials of therapeutic approaches targeting ADDITIONAL INFORMATION

ischemic stroke and signaling pathways Competing interests: The author declares no competing interests.
Clinical trials targeting the pathophysiology and the related
signaling pathways mentioned above have been implemented
with respect to ischemic stroke. For instance, the value of REFERENCES
targeting cellular excitotoxicity in ischemic stroke has been 1. Moskowitz, M. A., Lo, E. H. & Iadecola, C. The science of stroke: mechanisms in
recognized by investigators pursuing clinical trials with search of treatments. Neuron 67, 181–198 (2010).
nerinetine (NA-1), the inhibitor of GluN2B-PSD95-nNOS complex 2. Feigin, V. L. et al. Global and regional burden of stroke during 1990–2010:
(NCT02930018, NCT04462536, NCT00728182, NCT02315443), findings from the Global Burden of Disease Study 2010. Lancet 383, 245–255
Neu2000 (NCT04486430), and sofadil (NCT04453800). In addition, (2014).
several clinical trials focused on neuroinflammation during 3. Kleindorfer, D. O. et al. 2021 Guideline for the Prevention of Stroke in Patients
ischemic stroke have also been implemented, including those With Stroke and Transient Ischemic Attack: A Guideline From the American
Heart Association/American Stroke Association. Stroke 52, E364–E467 (2021).
targeted IL-1 (NCT04834388, NCT03737344), S1P receptors
4. Adams, H. P. Jr. et al. Classification of subtype of acute ischemic stroke. Defi-
(NCT02002390), and Toll-like receptors (TLRs) (NCT04734548). nitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute
Furthermore, therapeutic approaches targeting oxidative stress Stroke Treatment. Stroke 24, 35–41 (1993).
in ischemic stroke have also been tested in clinical trials, such as 5. Hankey, G. J. Stroke. Lancet 389, 641–654 (2017).
selenium (NCT02505295), astaxanthine (NCT03945526), and sima- 6. Nguyen, T.-V. V. et al. Multiplex immunoassay characterization and species
vastatin (NCT03402204). Concurrently, stem cell therapy is comparison of inflammation in acute and non-acute ischemic infarcts in human
attracting much attention due to its potential for exerting and mouse brain tissue. Acta Neuropathol. Comm. 4, (2016).

Qin et al.
22
7. Park, J. Y. et al. Neuroprotective effect of human placental extract on hypoxic- 39. Barone, F. C. & Feuerstein, G. Z. Inflammatory mediators and stroke: New
ischemic brain injury in neonatal rats. Brain Dev. 35, 68–74 (2013). opportunities for novel therapeutics. J. Cereb. Blood Flow. Metab. 19, 819–834
8. Barthels, D. & Das, H. Current advances in ischemic stroke research and thera- (1999).
pies. Biochimica Et Biophysica Acta-Mol. Basis Dis. 1866, (2020). 40. Lipton, P. Ischemic cell death in brain neurons. Physiol. Rev. 79, 1431–1568
9. Yenari, M. A., Kauppinen, T. M. & Swanson, R. A. Microglial activation in stroke: (1999).
therapeutic targets. Neurotherapeutics 7, 378–391 (2010). 41. Mazala, D. A. G., Grange, R. W. & Chin, E. R. The role of proteases in excitation-
10. Koyama, Y. Signaling molecules regulating phenotypic conversions of astrocytes contraction coupling failure in muscular dystrophy. Am. J. Physiol. Cell. Physiol.
and glial scar formation in damaged nerve tissues. Neurochem. Int. 78, 35–42 308, C33–C40 (2015).
(2014). 42. Weber, J. T. Altered calcium signaling following traumatic brain injury. Front.
11. Sims, N. R. & Yew, W. P. Reactive astrogliosis in stroke: Contributions of astro- Pharmacol. 3, 60 (2012).
cytes to recovery of neurological function. Neurochem. Int. 107, 88–103 (2017). 43. Xu, J. et al. Extrasynaptic NMDA receptors couple preferentially to excitotoxicity
12. Slujitoru, A.-S. et al. Clinical and morphological correlations in acute ischemic via calpain-mediated cleavage of STEP. J. Neurosci. 29, 9330–9343 (2009).
stroke. Rom. J. Morphol. Embryol. 53, 917–926 (2012). 44. Casas, A. I. et al. Calcium-dependent blood-brain barrier breakdown by NOX5
13. Lakhan, S. E., Kirchgessner, A., Tepper, D. & Leonard, A. Matrix metalloprotei- limits postreperfusion benefit in stroke. J. Clin. Invest. 129, 1772–1778 (2019).
nases and blood-brain barrier disruption in acute ischemic stroke. Front. Neurol. 45. Liu, F. et al. Mitochondria in Ischemic Stroke: New Insight and Implications.
4, (2013). Aging Dis. 9, 924–937 (2018).
14. Jin, R. et al. Role of inflammation and its mediators in acute ischemic stroke. J. 46. Dirnagl, U., Iadecola, C. & Moskowitz, M. A. Pathobiology of ischaemic stroke: an
Cardiovasc. Transl. Res. 6, 834–851 (2013). integrated view. Trends Neurosci. 22, 391–397 (1999).
15. Clausen, B. H. et al. Interleukin-1 beta and tumor necrosis factor-alpha are 47. Zhu, J. et al. Up-regulation of GluN2A-containing NMDA receptor protects cul-
expressed by different subsets of microglia and macrophages after ischemic tured cortical neuron cells from oxidative stress. Heliyon 4, e00976 (2018).
stroke in mice. J. Neuroinflammation. 5, (2008). 48. Benveniste, H., Drejer, J., Schousboe, A. & Diemer, N. H. Elevation of the extra-
16. Gronberg, N. V., Johansen, F. F., Kristiansen, U. & Hasseldam, H. Leukocyte cellular concentrations of glutamate and aspartate in rat hippocampus during
infiltration in experimental stroke. J. Neuroinflammation. 10, (2013). transient cerebral ischemia monitored by intracerebral microdialysis. J. Neu-
17. Ritzel, R. M. et al. Functional differences between microglia and monocytes after rochem. 43, 1369–1374 (1984).
ischemic stroke. J. Neuroinflammation. 12, (2015). 49. Joyal, J. L. et al. Communication - Calmodulin activates phosphatidylinositol
18. Sommer, C. J. Ischemic stroke: experimental models and reality. Acta Neuro- 3-kinase. J. Biol. Chem. 272, 28183–28186 (1997).
pathol. 133, 245–261 (2017). 50. Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependent protein
19. Smith, H. K., Russell, J. M., Granger, D. N. & Gavins, F. N. E. Critical differences kinase which phosphorylates and activates protein kinase B alpha. Curr. Biol. 7,
between two classical surgical approaches for middle cerebral artery occlusion- 261–269 (1997).
induced stroke in mice. J. Neurosci. Methods 249, 99–105 (2015). 51. Zhang, F. X., Rubin, R. & Rooney, T. A. N-methyl-D-aspartate inhibits apoptosis
20. Knowland, D. et al. Stepwise Recruitment Of Transcellular And Paracellular through activation of phosphatidylinositol 3-kinase in cerebellar granule
Pathways Underlies Blood-brain Barrier Breakdown In Stroke. Neuron 82, neurons - A role for insulin receptor substrate-1 in the neurotrophic action of
603–617 (2014). N-methyl-D-aspartate and its inhibition by ethanol. J. Biol. Chem. 273,
21. Huang, L. et al. Human neural stem cells rapidly ameliorate symptomatic 26596–26602 (1998).
inflammation in early-stage ischemic-reperfusion cerebral injury. Stem Cell. Res. 52. Jo, H. et al. Small molecule-induced cytosolic activation of protein kinase Akt
Ther. 5, 129 (2014). rescues ischemia-elicited neuronal death. Proc. Natl Acad. Sci. USA 109,
22. Qian, C. et al. Precise characterization of the penumbra revealed by MRI: a 10581–10586 (2012).
modified photothrombotic stroke model study. PLoS One 11, e153756 (2016). 53. Soriano, F. X. et al. Preconditioning doses of NMDA promote neuroprotection by
23. Yang, L., Shah, K. K. & Abbruscato, T. J. In Astrocytes: Methods and Protocols Vol. enhancing neuronal excitability. J. Neurosci. 26, 4509–4518 (2006).
814 Methods in Molecular Biology (ed R. Milner) 451–466 (2012). 54. Yamaguchi, A. et al. Akt activation protects hippocampal neurons from apop-
24. Li, D. et al. mTORC1 pathway disruption ameliorates brain inflammation fol- tosis by inhibiting transcriptional activity of p53. J. Biol. Chem. 276, 5256–5264
lowing stroke via a shift in microglia phenotype from M1 type to M2 type. FASEB (2001).
J. 30, 3388–3399 (2016). 55. Endo, H. et al. Activation of the Akt/GSK3 beta signaling pathway mediates
25. Li, K. et al. Middle East Respiratory Syndrome Coronavirus causes multiple organ survival of vulnerable hippocampal neurons after transient global cerebral
damage and lethal disease in mice transgenic for human dipeptidyl Peptidase 4. ischemia in rats. J. Cereb. Blood Flow. Metab. 26, 1479–1489 (2006).
J. Infect. Dis. 213, 712–722 (2016). 56. Kawano, T. et al. Neuroprotective effect of sodium orthovanadate on delayed
26. Xiong, X.-Y., Liu, L. & Yang, Q.-W. Refocusing neuroprotection in cerebral neuronal death after transient forebrain ischemia in gerbil hippocampus. J.
reperfusion era: new challenges and strategies. Front. Neurol. 9, 249 (2018). Cereb. Blood Flow. Metab. 21, 1268–1280 (2001).
27. Lai, T. W., Zhang, S. & Wang, Y. T. Excitotoxicity and stroke: Identifying novel 57. Noshita, N. et al. Copper-zinc superoxide dismutase affects Akt activation after
targets for neuroprotection. Prog. Neurobiol. 115, 157–188 (2014). transient focal cerebral ischemia in mice. Stroke 34, 1513–1518 (2003).
28. Jia, J. et al. New insights into targeting mitochondria in ischemic injury. Apop- 58. Noshita, N., Lewen, A., Sugawara, T. & Chan, P. H. Evidence of phosphorylation of
tosis 26, 163–183 (2021). Akt and neuronal survival after transient focal cerebral ischemia in mice. J.
29. Macrez, R. et al. Stroke and the immune system: from pathophysiology to new Cereb. Blood Flow. Metab. 21, 1442–1450 (2001).
therapeutic strategies. Lancet Neurol. 10, 471–480 (2011). 59. Yano, S. et al. Activation of Akt/protein kinase B contributes to induction of
30. Steliga, A. et al. Neurovascular unit as a source of ischemic stroke biomarkers- ischemic tolerance in the CA1 subfield of gerbil hippocampus. J. Cereb. Blood
limitations of experimental studies and perspectives for clinical application. Flow. Metab. 21, 351–360 (2001).
Transl. Stroke Res. 11, 553–579 (2020). 60. Downward, J. & How, B. A. D. phosphorylation is good for survival. Nat. Cell Biol.
31. Datta, A. et al. Cell Death Pathways In Ischemic Stroke And Targeted Pharma- 1, E33–E35 (1999).
cotherapy. Transl. Stroke Res. 11, 1185–1202 (2020). 61. Kim, A. H. et al. Akt phosphorylates and negatively regulates apoptosis signal-
32. Andrabi, S. S., Parvez, S. & Tabassum, H. Ischemic stroke and mitochondria: regulating kinase 1. Mol. Cell. Biol. 21, 893–901 (2001).
mechanisms and targets. Protoplasma 257, 335–343 (2020). 62. Wu, G. Y., Deisseroth, K. & Tsien, R. W. Activity-dependent CREB phosphorylation:
33. Wu, Q. J. & Tymianski, M. Targeting NMDA receptors in stroke: new hope in Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less
neuroprotection. Mol. Brain. 11, 15 (2018). sensitive mitogen-activated protein kinase pathway. Proc. Natl Acad. Sci. USA 98,
34. Olney, J. W. Brain lesions, obesity, and other disturbances in mice treated with 2808–2813 (2001).
monosodium glutamate. Science 164, 719–721 (1969). 63. Impey, S. et al. Phosphorylation of CBP mediates transcriptional activation by
35. Garthwaite, G., Williams, G. D. & Garthwaite, J. Glutamate toxicity: an experi- neural activity and CaM kinase IV. Neuron 34, 235–244 (2002).
mental and theoretical analysis. Eur. J. Neurosci. 4, 353–360 (1992). 64. Favaron, M. et al. NMDA-stimulated expression of BDNF mRNA in cultured
36. Choi, D. W., Koh, J. Y. & Peters, S. Pharmacology of glutamate neurotoxicity in cerebellar granule neurones. Neuroreport 4, 1171–1174 (1993).
cortical cell culture: attenuation by NMDA antagonists. J. Neurosci. 8, 185–196 65. Hansen, H. H. et al. Mechanisms leading to disseminated apoptosis following
(1988). NMDA receptor blockade in the developing rat brain. Neurobiol. Dis. 16,
37. Willard, S. S. & Koochekpour, S. Glutamate, glutamate receptors, and down- 440–453 (2004).
stream signaling pathways. Int. J. Biol. Sci. 9, 948–959 (2013). 66. Shieh, P. B. et al. Identification of a signaling pathway involved in calcium
38. George, P. M. & Steinberg, G. K. Novel Stroke Therapeutics: Unraveling Stroke regulation of BDNF expression. Neuron 20, 727–740 (1998).
Pathophysiology And Its Impact On Clinical Treatments. Neuron 87, 297–309 67. Tao, X. et al. Ca2+ influx regulates BDNF transcription by a CREB family tran-
(2015). scription factor-dependent mechanism. Neuron 20, 709–726 (1998).

Qin et al.
23
68. Ghosh, A., Carnahan, J. & Greenberg, M. E. Requirement for BDNF in activity- 98. Buendia, I. et al. Nrf2-ARE pathway: An emerging target against oxidative stress
dependent survival of cortical neurons. Science 263, 1618–1623 (1994). and neuroinflammation in neurodegenerative diseases. Pharmacol. Ther. 157,
69. Zafra, F., Castren, E., Thoenen, H. & Lindholm, D. Interplay between glutamate 84–104 (2016).
and gamma-aminobutyric acid transmitter systems in the physiological 99. Schinzel, A. C. et al. Cyclophilin D is a component of mitochondrial permeability
regulation of brain-derived neurotrophic factor and nerve growth factor transition and mediates neuronal cell death after focal cerebral ischemia. Proc.
synthesis in hippocampal neurons. Proc. Natl Acad. Sci. USA 88, 10037–10041 Natl Acad. Sci. USA 102, 12005–12010 (2005).
(1991). 100. Nakagawa, T. et al. Cyclophilin D-dependent mitochondrial permeability tran-
70. Chen, Q. et al. Differential roles of NR2A- and NR2B-containing NMDA receptors sition regulates some necrotic but not apoptotic cell death. Nature 434,
in activity-dependent brain-derived neurotrophic factor gene regulation and 652–658 (2005).
limbic epileptogenesis. J. Neurosci. 27, 542–552 (2007). 101. Baines, C. P. et al. Loss of cyclophilin D reveals a critical role for mitochondrial
71. Hardingham, G. E., Fukunaga, Y. & Bading, H. Extrasynaptic NMDARs oppose permeability transition in cell death. Nature 434, 658–662 (2005).
synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. 102. Lutz, J., Thuermel, K. & Heemann, U. Anti-inflammatory treatment strategies for
Neurosci. 5, 405–414 (2002). ischemia/reperfusion injury in transplantation. J. Inflamm. 7, 27 (2010).
72. Comelli, M. C. et al. Photochemical stroke and brain-derived neurotrophic factor 103. Hou, Y., Wang, J. & Feng, J. The neuroprotective effects of curcumin are asso-
(BDNF) mRNA expression. Neuroreport 3, 473–476 (1992). ciated with the regulation of the reciprocal function between autophagy and
73. Chen, M. et al. Differential roles of NMDA Receptor Subtypes In Ischemic Neu- HIF-1α in cerebral ischemia-reperfusion injury. Drug Des., Dev. Ther. 13,
ronal Cell Death And Ischemic Tolerance. Stroke 39, 3042–3048 (2008). 1135–1144 (2019).
74. Ning, K. et al. Dual neuroprotective signaling mediated by downregulating 104. Liu, Y. Q. et al. Metformin attenuates blood-brain barrier disruption in mice
two distinct phosphatase activities of PTEN. J. Neurosci. 24, 4052–4060 following middle cerebral artery occlusion. J. Neuroinflammation. 11, (2014).
(2004). 105. Dai, S. H. et al. Sirt3 confers protection against neuronal ischemia by inducing
75. Zhang, S. et al. Critical role of increased PTEN nuclear translocation in excitotoxic autophagy: Involvement of the AMPK-mTOR pathway. Free Radic. Biol. Med. 108,
and ischemic neuronal injuries. J. Neurosci. 33, 7997–8008 (2013). 345–353 (2017).
76. Kyrylenko, S., Roschier, M., Korhonen, P. & Salminen, A. Regulation of PTEN 106. Semenza, G. L. Regulation of oxygen homeostasis by hypoxia-inducible factor 1.
expression in neuronal apoptosis. Mol. Brain Res. 73, 198–202 (1999). Physiology 24, 97–106 (2009).
77. Deiss, L. P. et al. Identification of a novel serine/threonine kinase and a novel 15- 107. Ziello, J. E., Jovin, I. S. & Huang, Y. Hypoxia-Inducible Factor (HIF)-1 regulatory
kD protein as potential mediators of the gamma interferon-induced cell death. pathway and its potential for therapeutic intervention in malignancy and
Genes Dev. 9, 15–30 (1995). ischemia. Yale J. Biol. Med. 80, 51–60 (2007).
78. Kissil, J. L. et al. DAP-kinase loss of expression in various carcinoma and B-cell 108. Kim, J. W., Tchernyshyov, I., Semenza, G. L. & Dang, C. V. HIF-1-mediated
lymphoma cell lines: possible implications for role as tumor suppressor gene. expression of pyruvate dehydrogenase kinase: A metabolic switch required for
Oncogene 15, 403–407 (1997). cellular adaptation to hypoxia. Cell Metab. 3, 177–185 (2006).
79. Marshall, J. et al. Calcium channel and NMDA receptor activities differentially 109. Hu, C. J. et al. Differential regulation of the transcriptional activities of hypoxia-
regulate nuclear C/EBP beta levels to control neuronal survival. Neuron 39, inducible factor 1 alpha (HIF-1 alpha) and HIF-2 alpha in stem cells. Mol. Cell.
625–639 (2003). Biol. 26, 3514–3526 (2006).
80. Duval, X. et al. Effect of early cerebral magnetic resonance imaging on clinical 110. Guo, S., Miyake, M., Liu, K. J. & Shi, H. Specific inhibition of hypoxia inducible
decisions in infective endocarditis: a prospective study. Ann. Intern. Med. 152, factor 1 exaggerates cell injury induced by in vitro ischemia through deterior-
497–504 (2010). W175. ating cellular redox environment. J. Neurochem. 108, 1309–1321 (2009).
81. Shamloo, M. et al. Death-associated protein kinase is activated by depho- 111. Itoh, K. et al. Emerging functional cross-talk between the Keap1-Nrf2 system and
sphorylation in response to cerebral ischemia. J. Biol. Chem. 280, 42290–42299 mitochondria. J. Clin. Biochem. Nutr. 56, 91–97 (2015).
(2005). 112. Malhotra, D. et al. Global mapping of binding sites for Nrf2 identifies novel
82. Chen, C. H. et al. Bidirectional signals transduced by DAPK-ERK interaction targets in cell survival response through ChIP-Seq profiling and network ana-
promote the apoptotic effect of DAPK. EMBO J. 24, 294–304 (2005). lysis. Nucleic Acids Res. 38, 5718–5734 (2010).
83. Garthwaite, J., Charles, S. L. & Chess-Williams, R. Endothelium-derived relaxing 113. Dinkova-Kostova, A. T. & Abramov, A. Y. The emerging role of Nrf2 in mito-
factor release on activation of NMDA receptors suggests role as intercellular chondrial function. Free Radic. Biol. Med. 88, 179–188 (2015).
messenger in the brain. Nature 336, 385–388 (1988). 114. Holmstrom, K. M. et al. Nrf2 impacts cellular bioenergetics by controlling sub-
84. Brenman, J. E. et al. Cloning and characterization of postsynaptic density 93, a strate availability for mitochondrial respiration. Biol. Open. 2, 761–770 (2013).
nitric oxide synthase interacting protein. J. Neurosci. 16, 7407–7415 (1996). 115. Xu, Y.-P., Han, F. & Tan, J. Edaravone protects the retina against ischemia/
85. Kornau, H.-C., Schenker, L. T., Kennedy, M. B. & Seeburg, P. H. Domain interaction reperfusion-induced oxidative injury through the PI3K/Akt/Nrf2 pathway. Mol.
between NMDA receptor subunits and the postsynaptic density protein PSD-95. Med. Report. 16, 9210–9216 (2017).
Science 269, 1737–1740 (1995). 116. Kitagawa, H. et al. Immunoreactive Akt, PI3-K and ERK protein kinase expression
86. Muller, B. M. et al. SAP102, a novel postsynaptic protein that interacts with in ischemic rat brain. Neurosci. Lett. 274, 45–48 (1999).
NMDA receptor complexes in vivo. Neuron 17, 255–265 (1996). 117. Cen, J. et al. Polyamine analogue QMA attenuated ischemic injury in MCAO rats
87. Sattler, R. et al. Specific coupling of NMDA receptor activation to nitric oxide via ERK and Akt activated Nrf2/HO-1 signaling pathway. Eur. J. Pharmacol. 844,
neurotoxicity by PSD-95. protein Sci. 284, 1845–1848 (1999). 165–174 (2019).
88. Christopherson, K. S., Hillier, B. J., Lim, W. A. & Bredt, D. S. PSD-95 assembles a 118. Chang, C.-Y. et al. Tetramethylpyrazine inhibits neutrophil activation following
ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent permanent cerebral ischemia in rats. Biochem. Biophys. Res. Commun. 463,
neuronal NO synthase PDZ domain. J. Biol. Chem. 274, 27467–27473 (1999). 421–427 (2015).
89. Huang, Z. et al. Effects of cerebral ischemia in mice deficient in neuronal nitric 119. Innamorato, N. G. et al. The transcription factor Nrf2 is a therapeutic target
oxide synthase. Science 265, 1883–1885 (1994). against brain inflammation. J. Immunol. 181, 680–689 (2008).
90. Zhou, L. et al. Treatment of cerebral ischemia by disrupting ischemia-induced 120. Kim, G. S. et al. Release of mitochondrial apoptogenic factors and cell death are
interaction of nNOS with PSD-95. Nat. Med. 16, 1439–U1123 (2010). mediated by CK2 and NADPH oxidase. J. Cereb. Blood Flow. Metab. 32, 720–730
91. Liu, X. et al. Induction of apoptotic program in cell-free extracts: requirement for (2012).
dATP and Cytochrome c. Cell 86, 147–157 (1996). 121. Diekmann, D. et al. Interaction of Rac with p67-phox and regulation of pha-
92. Murphy, A. N., Fiskum, G. & Flint Beal, M. Mitochondria in neurodegeneration: gocytic NADPH oxidase activity. Sci. (Wash. D. C). 265, 531–533 (1994).
Bioenergetic function in cell life and death. J. Cereb. Blood Flow. Metab. 19, 122. Sarfstein, R. et al. Dual role of Rac in the assembly of NADPH oxidase, tethering
231–245 (1999). to the membrane and activation of p67(phox) - A study based on mutagenesis
93. Hofmeijer, J. & van Putten, M. J. A. M. Ischemic cerebral damage an appraisal of of p67(phox)-Rac1 chimeras. J. Biol. Chem. 279, 16007–16016 (2004).
synaptic failure. Stroke 43, 607–615 (2012). 123. Zimmer, S. et al. Inhibition of Rac1 GTPase decreases vascular oxidative stress,
94. Lee, J. M., Grabb, M. C., Zipfel, G. J. & Choi, D. W. Brain tissue responses to improves endothelial function, and attenuates atherosclerosis development in
ischemia. J. Clin. Invest. 106, 723–731 (2000). mice. Front. Cardiovasc. Med. 8, 680775 (2021).
95. Dharmasaroja, P. A. Fluid intake related to brain edema in acute middle cerebral 124. Mandal, T. et al. Reduced phosphorylation of Stat3 at Ser-727 mediated by
artery infarction. Transl. Stroke Res. 7, 49–53 (2016). casein kinase 2-Protein phosphatase 2A enhances Stat3 Tyr-705 induced
96. Huang, Y., Li, W., Su, Z.-Y. & Kong, A.-N. T. The complexity of the Nrf2 pathway: tumorigenic potential of glioma cells. Cell. Signal. 26, 1725–1734 (2014).
beyond the antioxidant response. J. Nutr. Biochem. 26, 1401–1413 (2015). 125. Jung, J. E., Kim, G. S. & Chan, P. H. Neuroprotection by Interleukin-6 is mediated
97. Xu, J. et al. Luteolin provides neuroprotection in models of traumatic brain by signal transducer and activator of Transcription 3 and antioxidative signaling
injury via the Nrf2-ARE pathway. Free Radic. Biol. Med. 71, 186–195 (2014). in ischemic stroke. Stroke 42, 3574–U3371 (2011).

Qin et al.
24
126. Chao, C. C., Ma, Y. L. & Lee, E. H. Y. Brain-derived neurotrophic factor enhances 156. Wang, M. Y. et al. Homocysteine enhances neural stem cell autophagy in in vivo
Bcl-xL expression through protein kinase Casein Kinase 2-activated and nuclear and in vitro model of ischemic stroke. Cell Death Dis. 10, 561 (2019).
factor Kappa B-mediated pathway in Rat Hippocampus. Brain Pathol. 21, 157. Jiang, T. et al. Acute metformin preconditioning confers neuroprotection
150–162 (2011). against focal cerebral ischaemia by pre-activation of AMPK-dependent autop-
127. Afonyushkin, T., Oskolkova, O. V., Binder, B. R. & Bochkov, V. N. Involvement of hagy. Br. J. Pharmacol. 171, 3146–3157 (2014).
CK2 in activation of electrophilic genes in endothelial cells by oxidized phos- 158. Yu, Z. H. et al. Neuroprotective effects of Tongxinluo on focal cerebral ischemia
pholipids. J. Lipid Res. 52, 98–103 (2011). and reperfusion injury in rats associated with the activation of the MEK1/2/
128. Baltan, S. et al. CK2 inhibition protects white matter from ischemic injury. ERK1/2/p90RSK signaling pathway. Brain Res. 1685, 9–18 (2018).
Neurosci. Lett. 687, 37–42 (2018). 159. Bootman, M. D. et al. The regulation of autophagy by calcium signals: Do we
129. Bastian, C. et al. CK2 inhibition confers functional protection to young and aging have a consensus? Cell Calcium 70, 32–46 (2018).
axons against ischemia by differentially regulating the CDK5 and AKT signaling 160. Morselli, E. et al. Mutant p53 protein localized in the cytoplasm inhibits
pathways. Neurobiol. Dis. 126, 47–61 (2019). autophagy. Cell Cycle 7, 3056–3061 (2008).
130. Meyer, D. A. et al. Ischemic stroke injury is mediated by Aberrant Cdk5. J. 161. Cuenda, A. & Rousseau, S. p38 MAP-kinases pathway regulation, function and
Neurosci. 34, 8259–8267 (2014). role in human diseases. Biochim Biophys. Acta 1773, 1358–1375 (2007).
131. Tan, X. et al. The inhibition of CDK5 activity after hypoxia/ischemia injury 162. Kalra, P., Khan, H., Kaur, A. & Singh, T. G. Mechanistic insight on autophagy
reduces infarct size and promotes functional recovery in neonatal rats. Neu- modulated molecular pathways in cerebral ischemic injury: from preclinical to
roscience 290, 552–560 (2015). clinical perspective. Neurochem. Res., (2022).
132. Palikaras, K. & Tavernarakis, N. Mitochondrial homeostasis: The interplay 163. Cui, D. R. et al. Propofol prevents cerebral ischemia-triggered autophagy acti-
between mitophagy and mitochondrial biogenesis. Exp. Gerontol. 56, 182–188 vation and cell death in the rat hippocampus through the NF-kappaB/
(2014). p53 signaling pathway. Neuroscience 246, 117–132 (2013).
133. Feng, J. et al. Inhibition of peroxynitrite-induced mitophagy activation 164. Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated
attenuates cerebral ischemia-reperfusion injury. Mol. Neurobiol. 55, microglia. Nature 541, 481–487 (2017).
6369–6386 (2018). 165. Xu, D. et al. Orexin-A protects against cerebral ischemia-reperfusion injury by
134. Di Sante, G. et al. Loss of Sirt1 promotes prostatic intraepithelial neoplasia, inhibiting excessive autophagy through OX1R-mediated MAPK/ERK/mTOR
reduces mitophagy, and delays Park2 translocation to mitochondria. Am. J. pathway. Cell. Signal. 79, 109839 (2021).
Pathol. 185, 266–279 (2015). 166. Rami, A. Upregulation of Beclin 1 in the ischemic penumbra. Autophagy 4,
135. Gustafsson, A. B. Bnip3 as a dual regulator of mitochondrial turnover and cell 227–229 (2008).
death in the myocardium. Pediatr. Cardiol. 32, 267–274 (2011). 167. Qi, Z. et al. Bcl-2 phosphorylation triggers autophagy switch and reduces
136. Cui, T. et al. Silencing of PINK1 induces mitophagy via mitochondrial perme- mitochondrial damage in limb remote ischemic conditioned rats after ischemic
ability transition in dopaminergic MN9D cells. Brain Res. 1394, 1–13 (2011). stroke. Transl. Stroke Res. 6, 198–206 (2015).
137. Livingston, M. J. et al. Clearance of damaged mitochondria via mitophagy is 168. Xu, F. et al. Peroxisome proliferator-activated receptor-gamma agonist 15d-
important to the protective effect of ischemic preconditioning in kidneys. prostaglandin J2 mediates neuronal autophagy after cerebral ischemia-
Autophagy 15, 2142–2162 (2019). reperfusion injury. PLoS One 8, e55080 (2013).
138. Fan, P. et al. Molecular regulation mechanisms and interactions between reac- 169. Qi, J. et al. Rab7b overexpression-ameliorated ischemic brain damage following
tive oxygen species and mitophagy. DNA Cell Biol. 38, 10–22 (2019). tMCAO involves suppression of TLR4 and NF-kappaB p65. J. Mol. Neurosci. 68,
139. Bursch, W. et al. Active cell death induced by the anti-estrogens tamoxifen and 163–170 (2019).
ICI 164384 in human mammary carcinoma cells (MCF-7) in culture: The role of 170. Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic biological phenomenon
autophagy. Carcinogenesis 17, 1595–1607 (1996). with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257
140. Clarke, P. G. Developmental cell death: morphological diversity and multiple (1972).
mechanisms. Anat. Embryol. 181, 195–213 (1990). 171. Uzdensky, A. B. Apoptosis regulation in the penumbra after ischemic stroke:
141. Majno, G. & Joris, I. Apoptosis, oncosis, and necrosis: An overview of cell death. expression of pro- and antiapoptotic proteins. Apoptosis 24, 687–702 (2019).
Am. J. Pathol. 146, 3–15 (1995). 172. Benn, S. C. & Woolf, C. J. Adult neuron survival strategies-slamming on the
142. He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of brakes. Nat. Rev. Neurosci. 5, 686–700 (2004).
autophagy. Annu. Rev. Genet. 43, 67–93 (2009). 173. Velier, J. J. et al. Caspase-8 and caspase-3 are expressed by different populations
143. Parzych, K. R. & Klionsky, D. J. An overview of autophagy: morphology, of cortical neurons undergoing delayed cell death after focal stroke in the rat. J.
mechanism, and regulation. Antioxid. Redox Signal. 20, 460–473 (2014). Neurosci. 19, 5932–5941 (1999).
144. Balin, B., Abrams, J. T. & Schrogie, J. Toward a unifying hypothesis in the 174. Love, S. Apoptosis and brain ischaemia. Prog. Neuro-Psychopharmacol. Biol.
development of Alzheimer’s disease. CNS Neurosci. Ther. 17, 587–589 (2011). Psychiatry 27, 267–282 (2003).
145. Berezniuk, I. & Fricker, L. D. A defect in cytosolic carboxypeptidase 1 (Nna1) 175. Plesnila, N. et al. BID mediates neuronal cell death after oxygen/ glucose
causes autophagy in Purkinje cell degeneration mouse brain. Autophagy 6, deprivation and focal cerebral ischemia. Proc. Natl Acad. Sci. USA 98,
558–559 (2010). 15318–15323 (2001).
146. Medeiros, R., Baglietto-Vargas, D. & LaFerla, F. M. The role of Tau in Alzheimer’s 176. Yin, X. M. et al. Bid-mediated mitochondrial pathway is critical to ischemic
disease and related disorders. CNS Neurosci. Ther. 17, 514–524 (2011). neuronal apoptosis and focal cerebral ischemia. J. Biol. Chem. 277, 42074–42081
147. Chu, C. T. Eaten alive - Autophagy and neuronal cell death after hypoxia- (2002).
ischemia. Am. J. Pathol. 172, 284–287 (2008). 177. Sarmah, D. et al. Mitochondrial Dysfunction in Stroke: Implications of Stem Cell
148. Wang, X. et al. An updated review of autophagy in ischemic stroke: From Therapy. Transl. Stroke Res., (2018).
mechanisms to therapies. Exp. Neurol. 340, 113684 (2021). 178. Sekerdag, E., Solaroglu, I. & Gursoy-Ozdemir, Y. Cell death mechanisms in stroke
149. Kim, Y. C. & Guan, K.-L. mTOR: a pharmacologic target for autophagy regulation. and novel molecular and cellular treatment options. Curr. Neuropharmacol. 16,
J. Clin. Investig. 125, 25–32 (2015). 1396–1415 (2018).
150. Perez-Alvarez, M. J., Villa Gonzalez, M., Benito-Cuesta, I. & Wandosell, F. G. Role of 179. Scheinberg, P. Survival of the ischemic brain: a progress report. Circulation 60,
mTORC1 controlling Proteostasis after Brain Ischemia. Front. Neurosci. 12, 60 1600–1605 (1979).
(2018). 180. Leng, T., Shi, Y., Xiong, Z. G. & Sun, D. Proton-sensitive cation channels and ion
151. Fang, W. et al. CCR2-dependent monocytes/macrophages exacerbate acute exchangers in ischemic brain injury: new therapeutic targets for stroke? Prog.
brain injury but promote functional recovery after ischemic stroke in mice. Neurobiol. 115, 189–209 (2014).
Theranostics 8, 3530–3543 (2018). 181. Chamorro, Á., Dirnagl, U., Urra, X. & Planas, A. M. Neuroprotection in acute
152. Otxoa-de-Amezaga, A. et al. Microglial cell loss after ischemic stroke favors brain stroke: targeting excitotoxicity, oxidative and nitrosative stress, and inflamma-
neutrophil accumulation. Acta Neuropathol. 137, 321–341 (2019). tion. Lancet Neurol. 15, 869–881 (2016).
153. Guo, Y.-Q. et al. Expression of CCL2 and CCR2 in the hippocampus and the 182. Collingridge, G. L., Isaac, J. T. & Wang, Y. T. Receptor trafficking and synaptic
interventional roles of propofol in rat cerebral ischemia/reperfusion. Exp. Ther. plasticity. Nat. Rev. Neurosci. 5, 952–962 (2004).
Med. 8, 657–661 (2014). 183. Vats, K. et al. Inflammasomes in stroke: a triggering role for acid-sensing ion
154. Wei, H. P. et al. cPKC gamma-modulated autophagy in neurons alleviates channels. Ann. N. Y. Acad. Sci. 1431, 14–24 (2018).
ischemic injury in brain of mice with ischemic stroke through Akt-mTOR path- 184. Li, H., Zhu, H., Xu, C. J. & Yuan, J. Cleavage of BID by caspase 8 mediates the
way. Transl. Stroke Res. 7, 497–511 (2016). mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491–501 (1998).
155. Carloni, S., Buonocore, G. & Balduini, W. Protective role of autophagy in neonatal 185. Bauer, T. M. & Murphy, E. Role of mitochondrial calcium and the permeability
hypoxia-ischemia induced brain injury. Neurobiol. Dis. 32, 329–339 (2008). transition pore in regulating cell death. Circ. Res. 126, 280–293 (2020).

Qin et al.
25
186. Fricker, M. et al. Neuronal cell death. Physiol. Rev. 98, 813–880 (2018). 218. Huang, J., Upadhyay, U. M. & Tamargo, R. J. Inflammation in stroke and focal
187. Hollville, E., Romero, S. E. & Deshmukh, M. Apoptotic cell death regulation in cerebral ischemia. Surg. Neurol. 66, 232–245 (2006).
neurons. FEBS J. 286, 3276–3298 (2019). 219. Zhang, R. L. et al. The expression of P- and E-selectins in three models of middle
188. Leker, R. R., Aharonowiz, M., Greig, N. H. & Ovadia, H. The role of p53-induced cerebral artery occlusion. Brain Res. 785, 207–214 (1998).
apoptosis in cerebral ischemia: effects of the p53 inhibitor pifithrin alpha. Exp. 220. Liebner, S. et al. Functional morphology of the blood-brain barrier in health and
Neurol. 187, 478–486 (2004). disease. Acta Neuropathol. 135, 311–336 (2018).
189. Culmsee, C. & Krieglstein, J. Ischaemic brain damage after stroke: new insights 221. Stanimirovic, D. & Satoh, K. Inflammatory mediators of cerebral endothelium: A
into efficient therapeutic strategies. International Symposium on Neurodegen- role in ischemic brain inflammation. Brain Pathol. 10, 113–126 (2000).
eration and Neuroprotection. EMBO Rep. 8, 129–133 (2007). 222. Gragnano, F. et al. The Role of von Willebrand Factor in vascular inflammation:
190. Morrison, R. S. et al. p53-dependent cell death signaling in neurons. Neurochem. from pathogenesis to targeted therapy. Mediators Inflamm. 2017, 5620314
Res. 28, 15–27 (2003). (2017).
191. Cregan, S. P. et al. p53 activation domain 1 is essential for PUMA upregulation 223. Ishitsuka, K. et al. Neurotrophin production in brain pericytes during hypoxia: A
and p53-mediated neuronal cell death. J. Neurosci. 24, 10003–10012 (2004). role of pericytes for neuroprotection. Microvasc. Res. 83, 352–359 (2012).
192. Norbury, C. J. & Zhivotovsky, B. DNA damage-induced apoptosis. Oncogene 23, 224. Badaut, T., Lasbennes, T., Magistretti, P. J. & Regli, L. Aquaporins in brain: Dis-
2797–2808 (2004). tribution, physiology, and pathophysiology. J. Cereb. Blood Flow. Metab. 22,
193. Balaganapathy, P. et al. Interplay between Notch and p53 promotes neuronal 367–378 (2002).
cell death in ischemic stroke. J. Cereb. Blood Flow. Metab. 38, 1781–1795 (2018). 225. Badaut, J. et al. Temporal and regional evolution of aquaporin-4 expression and
194. Baik, S.-H. et al. Pin1 promotes neuronal death in stroke by stabilizing notch magnetic resonance imaging in a rat pup model of neonatal stroke. Pediatr. Res.
intracellular domain. Ann. Neurol. 77, 504–516 (2015). 62, 248–254 (2007).
195. Arumugam, T. V. et al. Evidence that gamma-Secretase-mediated notch sig- 226. Lambertsen, K. L., Finsen, B. & Clausen, B. H. Post-stroke inflammation-target or
naling induces neuronal cell death via the nuclear Factor-kappa B-Bcl-2- tool for therapy? Acta Neuropathol. 137, 693–714 (2019).
interacting mediator of cell death pathway in ischemic stroke. Mol. Pharmacol. 227. Lambertsen, K. L. et al. Microglia protect neurons against ischemia by synthesis
80, 23–31 (2011). of tumor necrosis factor. J. Neurosci. 29, 1319–1330 (2009).
196. Wei, Z. et al. Notch activation enhances the microglia-mediated inflammatory 228. Clausen, B. H. et al. Cell therapy centered on IL-1Ra is neuroprotective in
response associated with focal cerebral ischemia. Stroke 42, 2589–U2344 (2011). experimental stroke. Acta Neuropathol. 131, 775–791 (2016).
197. Cheng, Y.-L. et al. Evidence that neuronal Notch-1 promotes JNK/c-Jun activa- 229. Kirchner, S. et al. LPS resistance in monocytic cells caused by reverse signaling
tion and cell death following ischemic stress. Brain Res. 1586, 193–202 (2014). through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway. J.
198. Cheng, Y.-L. et al. Evidence that collaboration between HIF-1 alpha and Notch-1 Leukoc. Biol. 75, 324–331 (2004).
promotes neuronal cell death in ischemic stroke. Neurobiol. Dis. 62, 286–295 230. Grell, M., Wajant, H., Zimmermann, G. & Scheurich, P. The type 1 receptor
(2014). (CD120a) is the high-affinity receptor for soluble tumor necrosis factor. Proc. Natl
199. Deb, P., Sharma, S. & Hassan, K. M. Pathophysiologic mechanisms of acute Acad. Sci. USA 95, 570–575 (1998).
ischemic stroke: An overview with emphasis on therapeutic significance beyond 231. Grell, M. et al. The transmembrane form of tumor necrosis factor is the prime
thrombolysis. Pathophysiol.: Off. J. Int. Soc. Pathophysiol. 17, 197–218 (2010). activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 83, 793–802
200. Festjens, N., Vanden Berghe, T. & Vandenabeele, P. Necrosis, a well-orchestrated (1995).
form of cell demise: Signalling cascades, important mediators and concomitant 232. Um, J. Y., An, N. H. & Kim, H. M. TNF-alpha and TNF-beta gene polyrnorphisms in
immune response. Biochimica Et. Biophysica Acta-Bioenerg. 1757, 1371–1387 cerebral infarction. J. Mol. Neurosci. 21, 167–171 (2003).
(2006). 233. Lambertsen, K. L., Biber, K. & Finsen, B. Inflammatory cytokines in experimental
201. Weber, K. et al. Nuclear RIPK3 and MLKL contribute to cytosolic necrosome and human stroke. J. Cereb. Blood Flow. Metab. 32, 1677–1698 (2012).
formation and necroptosis. Commun. Biol 1, 6 (2018). 234. Taoufik, E. et al. TNF receptor I sensitizes neurons to erythropoietin- and VEGF-
202. Vieira, M. et al. Ischemic insults induce necroptotic cell death in hippocampal mediated neuroprotection after ischemic and excitotoxic injury. Proc. Natl Acad.
neurons through the up-regulation of endogenous RIP3. Neurobiol. Dis. 68, Sci. USA 105, 6185–6190 (2008).
26–36 (2014). 235. Madsen, P. M. et al. Genetic ablation of soluble tumor necrosis factor with
203. Sun, L. et al. Mixed Lineage Kinase Domain-like Protein mediates necrosis sig- preservation of membrane tumor necrosis factor is associated with neuropro-
naling downstream of RIP3 Kinase. Cell 148, 213–227 (2012). tection after focal cerebral ischemia. J. Cereb. Blood Flow. Metab. 36, 1553–1569
204. Vanden Berghe, T. et al. Regulated necrosis: the expanding network of non- (2016).
apoptotic cell death pathways. Nat. Rev. Mol. Cell Biol. 15, 134–146 (2014). 236. Dinarello, C. A. Interleukin-1 in the pathogenesis and treatment of inflammatory
205. Dondelinger, Y. et al. MLKL compromises plasma membrane integrity by diseases. Blood 117, 3720–3732 (2011).
binding to phosphatidylinositol phosphates. Cell Rep. 7, 971–981 (2014). 237. Zou, L. et al. The association between three promoter polymorphisms of IL-1
206. Galluzzi, L., Kepp, O. & Kroemer, G. MLKL regulates necrotic plasma membrane and stroke: A meta-analysis. Gene 567, 36–44 (2015).
permeabilization. Cell Res. 24, 139–140 (2014). 238. Bis, J. C. et al. Variation in inflammation-related genes and risk of incident
207. Dong, Z. et al. The possibility and molecular mechanisms of cell pyroptosis after nonfatal myocardial infarction or ischemic stroke. Atherosclerosis 198, 166–173
cerebral ischemia. Neurosci. Bull. 34, 1131–1136 (2018). (2008).
208. Kono, H., Kimura, Y. & Latz, E. Inflammasome activation in response to dead cells 239. Thornton, P. et al. Platelet interleukin-1 alpha drives cerebrovascular inflam-
and their metabolites. Curr. Opin. Immunol. 30, 91–98 (2014). mation. Blood 115, 3632–3639 (2010).
209. Poh, L. et al. Evidence that NLRC4 inflammasome mediates apoptotic and 240. Davies, C. A. et al. The progression and topographic distribution of interleukin-1
pyroptotic microglial death following ischemic stroke. Brain Behav. Immun. 75, beta expression after permanent middle cerebral artery occlusion in the rat. J.
34–47 (2019). Cereb. Blood Flow. Metab. 19, 87–98 (1999).
210. Cao, J. Y. & Dixon, S. J. Mechanisms of ferroptosis. Cell. Mol. Life Sci. 73, 241. Spulber, S., Bartfai, T. & Schultzberg, M. IL-1/IL-1ra balance in the brain revisited -
2195–2209 (2016). Evidence from transgenic mouse models. Brain Behav. Immun. 23, 573–579
211. Tan, S. L. et al. The regulation of reactive oxygen species production during (2009).
programmed cell death. J. Cell Biol. 141, 1423–1432 (1998). 242. Sobowale, O. A. et al. Interleukin-1 in stroke from bench to bedside. Stroke 47,
212. Kenny, E. M. et al. Ferroptosis contributes to neuronal death and functional 2160–2167 (2016).
outcome after traumatic brain injury. Crit. Care Med. 47, 410–418 (2019). 243. Boutin, H. et al. Role of IL-1 alpha and IL-1 beta in ischemic brain damage. J.
213. Ren, J. X. et al. Crosstalk between oxidative stress and ferroptosis/oxytosis in Neurosci. 21, 5528–5534 (2001).
ischemic stroke: possible targets and molecular mechanisms. Oxid. Med. Cell. 244. McColl, B. W., Rothwell, N. J. & Allan, S. M. Systemic inflammatory stimulus
Longev. 2021, 6643382 (2021). potentiates the acute phase and CXC chemokine responses to experimental
214. Shichita, T. et al. MAFB prevents excess inflammation after ischemic stroke by stroke and exacerbates brain damage via interleukin-1- and neutrophil-
accelerating clearance of damage signals through MSR1. Nat. Med. 23, 723 dependent mechanisms. J. Neurosci. 27, 4403–4412 (2007).
(2017). 245. Erta, M., Quintana, A. & Hidalgo, J. Interleukin-6, a major cytokine in the central
215. Gadani, S. P., Walsh, J. T., Lukens, J. R. & Kipnis, J. Dealing with danger in the CNS: nervous system. Int. J. Biol. Sci. 8, 1254–1266 (2012).
the response of the immune system to injury. Neuron 87, 47–62 (2015). 246. Gronhoj, M. H. et al. Beneficial potential of intravenously administered IL-6 in
216. Gelderblom, M. et al. Temporal and spatial dynamics of cerebral immune cell improving outcome after murine experimental stroke. Brain Behav. Immun. 65,
accumulation in stroke. Stroke 40, 1849–1857 (2009). 296–311 (2017).
217. Iadecola, C. & Anrather, J. The immunology of stroke: from mechanisms to 247. Riethmueller, S. et al. Cleavage site localization differentially controls interleukin-
translation. Nat. Med. 17, 796–808 (2011). 6 receptor proteolysis by ADAM10 and ADAM17. Sci. Rep. 6, 25550 (2016).

Qin et al.
26
248. Wolf, J., Rose-John, S. & Garbers, C. Interleukin-6 and its receptors: A highly 278. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immu-
regulated and dynamic system. Cytokine 70, 11–20 (2014). nity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 (2010).
249. Rothaug, M., Becker-Pauly, C. & Rose-John, S. The role of interleukin-6 signaling 279. Brien, J. D. et al. Interferon Regulatory Factor-1 (IRF-1) shapes both innate and
in nervous tissue. Biochimica Et. Biophysica Acta-Mol. Cell Res. 1863, 1218–1227 CD8(+) T cell immune responses against West Nile virus infection. Plos Pathog.
(2016). 7, e1002230 (2011).
250. Scheller, J., Chalaris, A., Schmidt-Arras, D. & Rose-John, S. The pro- and anti- 280. Takahashi, H. & Nishibori, M. Current status and future prospects in HMGB1 and
inflammatory properties of the cytokine interleukin-6. Biochimica Et. Biophysica receptor researches. Nihon Rinsho. 74, 703–711 (2016).
Acta-Mol. Cell Res. 1813, 878–888 (2011). 281. Marsh, B. et al. Systemic Lipopolysaccharide protects the brain from ischemic
251. Smith, C. J. et al. Peak plasma interleukin-6 and other peripheral markers of injury by reprogramming the response of the brain to stroke: a critical role for
inflammation in the first week of ischaemic stroke correlate with brain infarct IRF3. J. Neurosci. 29, 9839–9849 (2009).
volume, stroke severity and long-term outcome. BMC Neurol. 4, 2 (2004). 282. Gesuete, R., Kohama, S. G. & Stenzel-Poore, M. P. Toll-like receptors and ischemic
252. Beridze, M. et al. Selected acute phase CSF factors in ischemic stroke: findings brain injury. J. Neuropathol. Exp. Neurol. 73, 378–386 (2014).
and prognostic value. BMC Neurol. 11, 41 (2011). 283. Vartanian, K. B. et al. LPS preconditioning redirects TLR signaling following
253. Singh, N. et al. The effect of intravenous interleukin-1 receptor antagonist on stroke: TRIF-IRF3 plays a seminal role in mediating tolerance to ischemic injury.
inflammatory mediators in cerebrospinal fluid after subarachnoid haemorrhage: J. Neuroinflammation. 8, 140 (2011).
a phase II randomised controlled trial. J. Neuroinflammation. 11, 1 (2014). 284. Kurita, N. et al. Metabolic endotoxemia promotes neuroinflammation after focal
254. Meng, C. et al. Inhibition of interleukin-6 abolishes the promoting effects of pair cerebral ischemia. J. Cereb. Blood Flow. Metab. 40, 2505–2520 (2020).
housing on post-stroke neurogenesis. Neuroscience 307, 160–170 (2015). 285. Singh, V., Roth, S., Veltkamp, R. & Liesz, A. HMGB1 as a key mediator of immune
255. Sabat, R. et al. Biology of interleukin-10. Cytokine Growth Factor Rev. 21, 331–344 mechanisms in ischemic stroke. Antioxid. Redox Signal. 24, 635–651 (2016).
(2010). 286. Magna, M. & Pisetsky, D. S. The role of HMGB1 in the pathogenesis of inflam-
256. Xu, L. et al. Astrocyte targeted overexpression of Hsp72 or SOD2 reduces matory and autoimmune diseases. Mol. Med. 20, 138–146 (2014).
neuronal vulnerability to forebrain ischemia. Glia 58, 1042–1049 (2010). 287. Yang, H. & Tracey, K. J. Targeting HMGB1 in inflammation. Biochimica Et. Bio-
257. de Bilbao, F. et al. In vivo over-expression of interleukin-10 increases resistance physica Acta-Gene Regul. Mech. 1799, 149–156 (2010).
to focal brain ischemia in mice. J. Neurochem. 110, 12–22 (2009). 288. Yang, H., Wang, H. C., Czura, C. J. & Tracey, K. J. The cytokine activity of HMGB1. J.
258. Perez-de Puig, I. et al. IL-10 deficiency exacerbates the brain inflammatory Leukoc. Biol. 78, 1–8 (2005).
response to permanent ischemia without preventing resolution of the lesion. J. 289. Qiu, J. et al. Early release of HMGB-1 from neurons after the onset of brain
Cereb. Blood Flow. Metab. 33, 1955–1966 (2013). ischemia. J. Cereb. Blood Flow. Metab. 28, 927–938 (2008).
259. Vila, N. et al. Levels of anti-inflammatory cytokines and neurological worsening 290. Deng, W. et al. Transcriptomic characterization of microglia activation in a rat
in acute ischemic stroke. Stroke 34, 671–675 (2003). model of ischemic stroke. J. Cereb. Blood Flow. Metab. 40, S34–S48 (2020).
260. Protti, G. G., Gagliardi, R. J., Forte, W. C. N. & Sprovieri, S. R. S. Interleukin-10 may 291. Yang, H. et al. MD-2 is required for disulfide HMGB1-dependent TLR4 signaling.
protect against progressing injury during the acute phase of ischemic stroke. J. Exp. Med. 212, 5–14 (2015).
Arq. Neuropsiquiatr. 71, 846–851 (2013). 292. Kim, E. J. et al. HMGB1 increases IL-1 beta production in vascular smooth muscle
261. Mennicken, F., Maki, R., de Souza, E. B. & Quirion, R. Chemokines and chemokine cells via NLRP3 inflammasome. Front. Physiol. 9, 313 (2018).
receptors in the CNS: a possible role in neuroinflammation and patterning. 293. Faraco, G. et al. High mobility group box I protein is released by neural cells
Trends Pharmacol. Sci. 20, 73–78 (1999). upon different stresses and worsens ischemic neurodegeneration in vitro and
262. Hughes, P. M. et al. Monocyte chemoattractant protein-1 deficiency is protective in vivo. J. Neurochem. 103, 590–603 (2007).
in a murine stroke model. J. Cereb. Blood Flow. Metab. 22, 308–317 (2002). 294. Kim, E. K. & Choi, E.-J. Compromised MAPK signaling in human diseases: an
263. Dimitrijevic, O. B., Stamatovic, S. M., Keep, R. F. & Andjelkovic, A. V. Absence of update. Arch. Toxicol. 89, 867–882 (2015).
the chemokine receptor CCR2 protects against cerebral Ischemia/reperfusion 295. Sun, J. & Nan, G. The Mitogen-Activated Protein Kinase (MAPK) signaling
injury in mice. Stroke 38, 1345–1353 (2007). pathway as a discovery target in stroke. J. Mol. Neurosci. 59, 90–98 (2016).
264. Chen, Y. et al. Overexpression of monocyte chemoattractant protein 1 in the 296. Choudhury, G. R. et al. Involvement of p38 MAPK in reactive astrogliosis induced
brain exacerbates ischemic brain injury and is associated with recruitment of by ischemic stroke. Brain Res. 1551, 45–58 (2014).
inflammatory cells. J. Cereb. Blood Flow. Metab. 23, 748–755 (2003). 297. Zhang, T. S. et al. Excess salt intake promotes M1 microglia polarization via a
265. Kim, J. S. et al. Expression of monocyte chemoattractant protein-1 and macro- p38/MAPK/AR-dependent pathway after cerebral ischemia in mice. Int. Immu-
phage inflammatory protein-1 after focal cerebral ischemia in the rat. J. Neu- nopharmacol. 81, 106176 (2020).
roimmunol. 56, 127–134 (1995). 298. Maddahi, A. & Edvinsson, L. Cerebral ischemia induces microvascular pro-
266. Takami, S. et al. Chemokine receptor antagonist peptide, viral MIP-II, protects inflammatory cytokine expression via the MEK/ERK pathway. J. Neuroin-
the brain against focal cerebral ischemia in mice. J. Cereb. Blood Flow. Metab. 21, flammation. 7, 14 (2010).
1430–1435 (2001). 299. Zhang, T. S. et al. Excess salt exacerbates blood-brain barrier disruption via a
267. Terao, S. et al. Blood cell-derived RANTES mediates cerebral microvascular p38/MAPK/SGK1-dependent pathway in permanent cerebral ischemia. Sci. Rep.
dysfunction, inflammation, and tissue injury after focal ischemia-reperfusion. 5, 16548 (2015).
Stroke 39, 2560–2570 (2008). 300. Rosenberg, G. A. & Yang, Y. Vasogenic edema due to tight junction disruption
268. Montecucco, F. et al. Systemic and intraplaque mediators of inflammation are by matrix metalloproteinases in cerebral ischemia. Neurosurg. Focus. 22, E4–E4
increased in patients symptomatic for ischemic stroke. Stroke 41, 1394–1404 (2007).
(2010). 301. Pfefferkorn, T. & Rosenberg, G. A. Closure of the blood-brain barrier by matrix
269. Chen, C. et al. Chemokines play complex roles in cerebral ischemia. Neurochem. metalloproteinase inhibition reduces rtPA-mediated mortality in cerebral
Int. 112, 146–158 (2018). ischemia with delayed reperfusion. Stroke 34, 2025–2030 (2003).
270. Glezer, I., Simard, A. R. & Rivest, S. Neuroprotective role of the innate immune 302. Bao Dang, Q. et al. High-density lipoproteins limit neutrophil-induced damage
system by microglia. Neuroscience 147, 867–883 (2007). to the blood-brain barrier in vitro. J. Cereb. Blood Flow. Metab. 33, 575–582
271. McGettrick, A. F. & O’Neill, L. A. J. Toll-like receptors: key activators of leucocytes (2013).
and regulator of haematopoiesis. Br. J. Haematol. 139, 185–193 (2007). 303. Barr, T. L. et al. Blood-brain barrier disruption in humans is independently
272. Arancibia, S. A. et al. Toll-like receptors are key participants in innate immune associated with increased matrix metalloproteinase-9. Stroke 41, E123–E128
responses. Biol. Res. 40, 97–112 (2007). (2010).
273. Manicassamy, S. & Pulendran, B. Modulation of adaptive immunity with Toll-like 304. Zhu, S. Z. et al. Hypothermia followed by rapid rewarming exacerbates
receptors. Semin. Immunol. 21, 185–193 (2009). ischemia-induced brain injury and augments inflammatory response in rats.
274. Wu, Y. & Zhou, B. P. TNF-alpha/NF-kappa B/Snail pathway in cancer cell Biochem. Biophys. Res. Commun. 474, 175–181 (2016).
migration and invasion. Br. J. Cancer 102, 639–644 (2010). 305. Chelluboina, B. et al. Matrix metalloproteinase-12 induces blood-brain barrier
275. Chaturvedi, A. & Pierce, S. K. How location governs toll-like receptor signaling. damage after focal cerebral ischemia. Stroke 46, 3523–3531 (2015).
Traffic 10, 621–628 (2009). 306. Chi, O. Z. et al. Hypoxic preconditioning increases blood-brain barrier disruption
276. Balch, M. H. H., Nimjee, S. M., Rink, C. & Hannawi, Y. Beyond the brain: the in the early stages of cerebral ischemia. Curr. Neurovasc. Res. 14, 26–31 (2017).
systemic pathophysiological response to acute ischemic stroke. J. Stroke 22, 159 307. Shen, Y. et al. Inhibition of HIF-1 alpha reduced blood brain barrier damage by
(2020). regulating MMP-2 and VEGF during acute cerebral ischemia. Front. Cell. Neurosci.
277. Florez-Alvarez, L., Ruiz-Perez, L., Taborda, N. & Hernandez, J. C. Toll-like receptors 12, 288 (2018).
as a therapeutic target in cancer, infections and inflammatory diseases. Immu- 308. Spiegel, S. & Milstien, S. Sphingosine-1-phosphate: An enigmatic signalling lipid.
notherapy 12, 311–322 (2020). Nat. Rev. Mol. Cell Biol. 4, 397–407 (2003).

Qin et al.
27
309. Lv, M. et al. Sphingosine kinase 1/sphingosine-1-phosphate regulates the 339. Hong, J. M. et al. Safety and optimal neuroprotection of neu2000 in acute
expression of interleukin-17A in activated microglia in cerebral ischemia/ Ischemic stroke with reCanalization: study protocol for a randomized, double-
reperfusion. Inflamm. Res. 65, 551–562 (2016). blinded, placebo-controlled, phase-II trial. Trials 19, 375 (2018).
310. Hyakkoku, K. et al. Toll-like receptor 4 (TLR4), but not TLR3 or TLR9, knock-out 340. Cho, S. I., Park, U. J., Chung, J.-M. & Gwag, B. J. NEU2000, AN NR2B-Selective,
mice have neuroprotective effects against focal cerebral ischemia. Neuroscience moderate nmda receptor antagonist and potent spin trapping molecule for
171, 258–267 (2010). stroke. Drug N. Perspect. 23, 549–556 (2010).
311. Qin, C. et al. Fingolimod protects against ischemic white matter damage by 341. Ginsberg, M. D. Neuroprotection for ischemic stroke: Past, present and future.
modulating microglia toward M2 polarization via STAT3 pathway. Stroke 48, Neuropharmacology 55, 363–389 (2008).
3336–3346 (2017). 342. Takaoka, S. et al. Neuroprotective effect of NMDA receptor glycine recognition
312. Schroder, K. & Tschopp, J. The Inflammasomes. Cell 140, 821–832 (2010). site antagonism persists when brain temperature is controlled. J. Cereb. Blood
313. Mohamed, I. N., Ishrat, T., Fagan, S. C. & El-Remessy, A. B. Role of inflammasome Flow. Metab. 17, 161–167 (1997).
activation in the pathophysiology of vascular diseases of the neurovascular unit. 343. Muir, K. W. & Lees, K. R. Clinical experience with excitatory amino acid
Antioxid. Redox Signal. 22, 1188–1206 (2015). antagonist drugs. Stroke 26, 503–513 (1995).
314. Fann, D. Y.-W. et al. Pathogenesis of acute stroke and the role of inflamma- 344. Lai, T. W., Shyu, W.-C. & Wang, Y. T. Stroke intervention pathways: NMDA
somes. Ageing Res. Rev. 12, 941–966 (2013). receptors and beyond. Trends Mol. Med. 17, 266–275 (2011).
315. Yang, F. et al. NLRP3 deficiency ameliorates neurovascular damage in experi- 345. Wood, P. L. & Hawkinson, J. E. N-methyl-D-aspartate antagonists for stroke and
mental ischemic stroke. J. Cereb. Blood Flow. Metab. 34, 660–667 (2014). head trauma. Expert Opin. Investig. Drugs 6, 389–397 (1997).
316. Chi, W. et al. HMGB1 promotes the activation of NLRP3 and caspase-8 inflam- 346. Albers, G. W., Atkinson, R. P., Kelley, R. E. & Rosenbaum, D. M. Safety, tolerability,
masomes via NF-kappa B pathway in acute glaucoma. J. Neuroinflammation. 12, and pharmacokinetics of the N-methyl-D-aspartate antagonist dextrorphan in
137 (2015). patients with acute stroke. Stroke 26, 254–258 (1995).
317. Fann, D. Y. W. et al. Intravenous immunoglobulin suppresses NLRP1 and NLRP3 347. Grotta, J. et al. Safety and tolerability of the glutamate antagonist CGS 19755
inflammasome-mediated neuronal death in ischemic stroke. Cell Death Dis. 4, (Selfotel) in patients with acute ischemic stroke: Results of a phase IIa rando-
e790 (2013). mized trial. Stroke 26, 602–605 (1995).
318. Wang, K. et al. Central nervous system diseases related to pathological micro- 348. Diener, H. C. et al. Treatment of acute ischaemic stroke with the low-affinity, use-
glial phagocytosis. CNS Neurosci. Ther. 27, 528–539 (2021). dependent NMDA antagonist AR-R15896AR - A safety and tolerability study. J.
319. Kawabori, M. et al. Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Neurol. 249, 561–568 (2002).
deficiency attenuates phagocytic activities of microglia and exacerbates 349. Pei, L. et al. DAPK1-p53 interaction converges necrotic and apoptotic pathways
ischemic damage in experimental stroke. J. Neurosci. 35, 3384–3396 (2015). of ischemic neuronal death. J. Neurosci. 34, 6546–6556 (2014).
320. Ting, S.-M. et al. Brain cleanup as a potential target for poststroke recovery the 350. Wang, X. et al. Intervention of death-associated protein Kinase 1-p53 interaction
role of RXR (Retinoic X Receptor) in phagocytes. Stroke 51, 958–966 (2020). exerts the therapeutic effects against stroke. Stroke 45, 3089 (2014).
321. Brown, G. C. & Neher, J. J. Microglial phagocytosis of live neurons. Nat. Rev. 351. McQueen, J. et al. Pro-death NMDA receptor signaling is promoted by the
Neurosci. 15, 209–216 (2014). GIuN2B C-terminus independently of Dapk1. Elife 6, e17161 (2017).
322. Neher, J. J. et al. Phagocytosis executes delayed neuronal death after focal brain 352. Huang, B. S. et al. Geniposide attenuates post-ischaemic neurovascular damage
ischemia. Proc. Natl Acad. Sci. USA 110, E4098–E4107 (2013). via GluN2A/AKT/ERK-dependent mechanism. Cell. Physiol. Biochem. 43, 705–716
323. Zhang, Y. et al. TMEM16F aggravates neuronal loss by mediating microglial (2017).
phagocytosis of neurons in a rat experimental cerebral ischemia and reperfu- 353. Liu, Y. et al. Neuroprotective effect of pseudoginsenoside-F11 on permanent
sion model. Front. Immunol. 11, 1144 (2020). cerebral ischemia in rats by regulating calpain activity and NR2A submit-
324. Gervois, P. & Lambrichts, I. The emerging role of triggering receptor expressed mediated AKT-CREB signaling pathways. Phytomed: Int. J. Phytother. Phyto-
on myeloid Cells 2 as a target for immunomodulation in ischemic stroke. Front. pharmacol. 96, 153847 (1800).
Immunol. 10, 1668 (2019). 354. Alim, I. et al. Modulation of NMDAR subunit expression by TRPM2 channels
325. Campagne, M. V. L., Wiesmann, C. & Brown, E. J. Macrophage complement regulates neuronal vulnerability to ischemic cell death. J. Neurosci. 33,
receptors and pathogen clearance. Cell. Microbiol. 9, 2095–2102 (2007). 17264–17277 (2013).
326. Ma, Y., Liu, Y., Zhang, Z. & Yang, G.-Y. Significance of complement system in 355. Thompson, R. J. Pannexin channels and ischaemia. J. Physiol. -Lond. 593,
ischemic stroke: a comprehensive review. Aging Dis. 10, 429–462 (2019). 3463–3470 (2015).
327. Fraser, D. A., Pisalyaput, K. & Tenner, A. J. C1q enhances microglial clearance of 356. Weilinger, N. L. et al. Metabotropic NMDA receptor signaling couples Src family
apoptotic neurons and neuronal blebs, and modulates subsequent inflamma- kinases to pannexin-1 during excitotoxicity. Nat. Neurosci. 19, 432 (2016).
tory cytokine production. J. Neurochem. 112, 733–743 (2010). 357. Alfieri, A. et al. Sulforaphane preconditioning of the Nrf2/HO-1 defense
328. Alawieh, A., Langley, E. F. & Tomlinson, S. Targeted complement inhibition pathway protects the cerebral vasculature against blood-brain barrier dis-
salvages stressed neurons and inhibits neuroinflammation after stroke in mice. ruption and neurological deficits in stroke. Free Radic. Biol. Med. 65, 1012–1022
Sci. Transl. Med. 10, eaao6459 (2018). (2013).
329. Alawieh, A. M. et al. Complement-dependent synaptic uptake and cognitive 358. Hou, Y. et al. Nrf2 inhibits NLRP3 inflammasome activation through regulating
decline after stroke and reperfusion therapy. J. Neurosci. 40, 4042–4058 Trx1/TXNIP complex in cerebral ischemia reperfusion injury. Behav. Brain Res.
(2020). 336, 32–39 (2018).
330. Fukuta, T. et al. Combination therapy with liposomal neuroprotectants and 359. Prasad, S. et al. Role of Nrf2 and protective effects of Metformin against tobacco
tissue plasminogen activator for treatment of ischemic stroke. FASEB J. 31, smoke-induced cerebrovascular toxicity. Redox Biol. 12, 58–69 (2017).
1879–1890 (2017). 360. Zhang, W. et al. Sirtuin 6 Protects the brain from cerebral ischemia/reperfusion
331. Rink, C. & Khanna, S. MicroRNA in ischemic stroke etiology and pathology. injury through NRF2 activation. Neuroscience 366, 95–104 (2017).
Physiol. Genomics. 43, 521–528 (2011). 361. Hou, J., Chong, Z. Z., Shang, Y. C. & Maiese, K. Early apoptotic vascular signaling
332. Trounson, A. & McDonald, C. Stem cell therapies in clinical trials: progress and is determined by Sirt1 through nuclear shuttling, forkhead trafficking, bad, and
challenges. Cell. Stem Cell. 17, 11–22 (2015). mitochondrial caspase activation. Curr. Neurovasc. Res. 7, 95–112 (2010).
333. Chen, Y. et al. Tat-NR2B9c prevents excitotoxic neuronal superoxide production. 362. Morris, K. C., Lin, H. W., Thompson, J. W. & Perez-Pinzon, M. A. Pathways for
J. Cereb. Blood Flow. Metab. 35, 739–742 (2015). ischemic cytoprotection: Role of sirtuins in caloric restriction, resveratrol, and
334. Cook, D. J., Teves, L. & Tymianski, M. Treatment of stroke with a PSD-95 inhibitor ischemic preconditioning. J. Cereb. Blood Flow. Metab. 31, 1003–1019 (2011).
in the gyrencephalic primate brain. Nature 483, 213–U112 (2012). 363. Wang, R. et al. Curcumin attenuates IR-induced myocardial injury by activating
335. Teves, L. M., Cui, H. & Tymianski, M. Efficacy of the PSD95 inhibitor Tat-NR2B9c in SIRT3. Eur. Rev. Med. Pharmacol. Sci. 22, 1150–1160 (2018).
mice requires dose translation between species. J. Cereb. Blood Flow. Metab. 36, 364. Chang, G., Chen, Y., Zhang, H. & Zhou, W. Trans sodium crocetinate alleviates
555–561 (2016). ischemia/reperfusion-induced myocardial oxidative stress and apoptosis via the
336. Soriano, F. X. et al. Specific targeting of pro-death NMDA receptor signals SIRT3/FOXO3a/SOD2 signaling pathway. Int. Immunopharmacol. 71, 361–371
with differing reliance on the NR2B PDZ Ligand. J. Neurosci. 28, 10696–10710 (2019).
(2008). 365. Zhao, B. et al. Genipin protects against cerebral ischemia-reperfusion injury by
337. Hill, M. D. et al. Safety and efficacy of NA-1 in patients with iatrogenic stroke regulating the UCP2-SIRT3 signaling pathway. Eur. J. Pharmacol. 845, 56–64
after endovascular aneurysm repair (ENACT): a phase 2, randomised, double- (2019).
blind, placebo-controlled trial. Lancet Neurol. 11, 942–950 (2012). 366. Hammond, M. D. et al. CCR2(+)Ly6C(hi) inflammatory monocyte recruitment
338. Luo, C.-X. et al. Interaction of nNOS with PSD-95 negatively controls regen- exacerbates acute disability following intracerebral hemorrhage. J. Neurosci. 34,
erative repair after stroke. J. Neurosci. 34, 13535–13548 (2014). 3901–3909 (2014).

Qin et al.
28
367. Wattananit, S. et al. Monocyte-derived macrophages contribute to spontaneous 393. Wang, S. Q. et al. Genistein attenuates acute cerebral ischemic damage by
long-term functional recovery after stroke in mice. J. Neurosci. 36, 4182–4195 inhibiting the NLRP3 inflammasome in reproductively senescent mice. Front.
(2016). Aging Neurosci. 12, (2020).
368. Lu, Y.-Y., Ma, X.-J. & Yang, Y.-N. MicroRNA-18a-5p mitigates oxygen-glucose- 394. Chen, X. Y., Wang, Y. Z., Yao, N. N. & Lin, Z. J. Immunoproteasome modulates
deprivation/reoxygenation-induced injury through suppression of TLRs/NF- NLRP3 inflammasome-mediated neuroinflammation under cerebral ischaemia
kappa B signaling by targeting TLR8 in PC12 cells. Biosci., Biotechnol. Biochem. and reperfusion conditions. J. Cell. Mol. Med. 26, 462–474 (2022).
84, 2476–2483 (2020). 395. Yang, R. et al. Quercetin attenuates ischemia reperfusion injury by protecting
369. Ghazavi, H. et al. The role of resveratrol as a natural modulator in glia activation the blood-brain barrier through Sirt1 in MCAO rats. J. Asian Nat. Prod. Res. 24,
in experimental models of stroke. Avicenna J. Phytomed. 10, 557–573 (2020). 278–289 (2022).
370. Rahimifard, M. et al. Targeting the TLR4 signaling pathway by polyphenols: A 396. Yang, F. et al. Minocycline ameliorates hypoxia-induced blood-brain barrier
novel therapeutic strategy for neuroinflammation. Ageing Res. Rev. 36, 11–19 damage by inhibition of HIF-1 alpha through sirt-3/PHD-2 degradation pathway.
(2017). Neuroscience 304, 250–259 (2015).
371. Zhang, X.-S. et al. Resveratrol attenuates acute inflammatory injury in experi- 397. Liu, H. et al. Hydrogen sulfide attenuates tissue plasminogen activator-induced
mental subarachnoid hemorrhage in rats via inhibition of TLR4 pathway. Int. J. cerebral hemorrhage following experimental stroke. Transl. Stroke Res. 7,
Mol. Sci. 17, 1331 (2016). 209–219 (2016).
372. Zan, J. et al. Isosteviol sodium injection improves outcomes by modulating 398. Yang, Y. et al. Non-invasive vagus nerve stimulation reduces blood-brain barrier
TLRs/NF-B-dependent inflammatory responses following experimental trau- disruption in a rat model of ischemic stroke. Brain Stimul. 11, 689–698 (2018).
matic brain injury in rats. Neuroreport 29, 794–803 (2018). 399. Michalski, D. et al. Early outcome and blood-brain barrier integrity after co-
373. Hsieh, J. T. et al. Sex-specific effects of progesterone on early outcome of administered thrombolysis and hyperbaric oxygenation in experimental stroke.
intracerebral hemorrhage. Neuroendocrinology 103, 518–530 (2016). Exp. Transl. Stroke Med. 3, 5–5 (2011).
374. Li, X. et al. Progesterone reduces inflammation and apoptosis in neonatal rats 400. Khan, I. S. et al. Intraarterial administration of norcantharidin attenuates
with hypoxic ischemic brain damage through the PI3K/Akt pathway. Int. J. Clin. ischemic stroke damage in rodents when given at the time of reperfusion: novel
Exp. Med. 8, 8197–8203 (2015). uses of endovascular capabilities. J. Neurosurg. 125, 152–159 (2016).
375. Wang, Z. et al. Progesterone administration modulates cortical TLR4/NF-kappa B 401. Hasegawa, Y. et al. Activation of Sphingosine 1-Phosphate Receptor-1 by FTY720 is
signaling pathway after subarachnoid hemorrhage in male rats. Mediators neuroprotective after ischemic stroke in rats. Stroke 41, 368–374 (2010).
Inflamm. 2011, 848309 (2011). 402. Wei, Y. et al. Fingolimod provides long-term protection in rodent models of
376. Zhai, Y. et al. Dexmedetomidine post-conditioning alleviates cerebral ischemia- cerebral ischemia. Ann. Neurol. 69, 119–129 (2011).
reperfusion injury in rats by inhibiting high mobility group Protein B1 Group 403. Li, X. et al. Fingolimod suppresses neuronal autophagy through the mTOR/
(HMGB1)/Toll-Like Receptor 4 (TLR4)/Nuclear Factor kappa B (NF-kappa B) Sig- p70S6K pathway and alleviates ischemic brain damage in mice. PLoS One. 12,
naling Pathway. Med. Sci. Monit. 26, e918617 (2020). (2017).
377. Leung, P. Y. et al. Toll-like Receptor 7 preconditioning induces robust neuro- 404. Yang, B. A. et al. Selenium attenuates ischemia/reperfusion injury-induced
protection against stroke by a novel Type I interferon-mediated mechanism. damage to the blood-brain barrier in hyperglycemia through PI3K/AKT/mTOR
Stroke 43, 1383–1389 (2012). pathway-mediated autophagy inhibition. Int. J. Mol. Med. 48, (2021).
378. Clausen, B. H. et al. Systemically administered anti-TNF therapy ameliorates 405. Guo, Z. et al. A combination of four active compounds alleviates cerebral ischemia-
functional outcomes after focal cerebral ischemia. J. Neuroinflammation. 11, 203 reperfusion injury in correlation with inhibition of autophagy and modulation of
(2014). AMPK/mTOR and JNK pathways. J. Neurosci. Res. 92, 1295–1306 (2014).
379. Zhou, Q.-H. et al. Brain-penetrating tumor necrosis factor decoy receptor in the 406. Wang, G. et al. Schizandrin protects against OGD/R-induced neuronal injury by
mouse. Drug Metab. Disposition. 39, 71–76 (2011). suppressing autophagy: involvement of the AMPK/mTOR pathway. Molecules.
380. Sumbria, R. K., Boado, R. J. & Pardridge, W. M. Brain protection from stroke with 24, (2019).
intravenous TNF alpha decoy receptor-Trojan horse fusion protein. J. Cereb. 407. Yu, J. et al. Ezetimibe, a NPC1L1 inhibitor, attenuates neuronal apoptosis
Blood Flow. Metab. 32, 1933–1938 (2012). through AMPK dependent autophagy activation after MCAO in rats. Exp. Neurol.
381. Liguz-Lecznar, M., Zakrzewska, R. & Kossut, M. Inhibition of Tnf-alpha 307, 12–23 (2018).
R1 signaling can rescue functional cortical plasticity impaired in early post- 408. Schwalm, C. et al. Activation of autophagy in human skeletal muscle is
stroke period. Neurobiol. Aging 36, 2877–2884 (2015). dependent on exercise intensity and AMPK activation. Faseb. J. 29, 3515–3526
382. Works, M. G., Koenig, J. B. & Sapolsky, R. M. Soluble TNF receptor 1-secreting ex (2015).
vivo-derived dendritic cells reduce injury after stroke. J. Cereb. Blood Flow. 409. Batatinha, H. A. P. et al. Regulation of autophagy as a therapy for
Metab. 33, 1376–1385 (2013). immunosenescence-driven cancer and neurodegenerative diseases: The
383. Scheinfeld, N. A comprehensive review and evaluation of the side effects of the role of exercise. J. Cell. Physiol. 234, 14883–14895 (2019).
tumor necrosis factor-alpha blockers etanercept, infliximab, and adalimumab. J. 410. Pan, G. et al. Treadmill exercise improves neurological function by inhibiting
Dermatol. Treat. 15, 280–294 (2004). autophagy and the binding of HMGB1 to Beclin1 in MCAO juvenile rats. Life Sci.
384. Relton, J. K., Martin, D., Thompson, R. C. & Russell, D. A. Peripheral administration 243, 117279 (2020).
of interleukin-1 receptor antagonist inhibits brain damage after focal cerebral 411. Xia, Y. et al. Small extracellular vesicles secreted by human iPSC-derived MSC
ischemia in the rat. Exp. Neurol. 138, 206–213 (1996). enhance angiogenesis through inhibiting STAT3-dependent autophagy in
385. Loddick, S. A. & Rothwell, N. J. Neuroprotective effects of human recombinant ischemic stroke. Stem Cell. Res. Ther. 11, (2020).
interleukin-1 receptor antagonist in focal cerebral ischaemia in the rat. J. Cereb. 412. Mei, Z.-G. et al. Electroacupuncture ameliorates cerebral ischemia/reperfusion
Blood Flow. Metab. 16, 932–940 (1996). injury by suppressing autophagy via the SIRT1-FOXO1 signaling pathway. Aging-
386. Nawashiro, H., Martin, D. & Hallenbeck, J. M. Neuroprotective effects of TNF Us. 12, 13187–13205 (2020).
binding protein in focal cerebral ischemia. Brain Res. 778, 265–271 (1997). 413. Xu, S. Y. et al. Electroacupuncture alleviates cerebral ischemia/reperfusion injury
387. Emsley, H. C. A. et al. A randomised phase II study of interleukin-1 receptor in rats by Histone H4 Lysine 16 acetylation-mediated autophagy. Front. Psy-
antagonist in acute stroke patients. J. Neurol. Neurosurg. Psychiatry 76, chiatry 11, 576539 (2020).
1366–1372 (2005). 414. Liu, S. et al. Luteolin protects against CIRI, potentially via regulation of the SIRT3/
388. Gueorguieva, I. et al. Pharmacokinetic modelling of interleukin-1 receptor AMPK/mTOR signaling pathway. Neurochem. Res. 45, 2499–2515 (2020).
antagonist in plasma and cerebrospinal fluid of patients following subarachnoid 415. Liu, L. et al. Melatonin ameliorates cerebral ischemia-reperfusion injury in dia-
haemorrhage. Br. J. Clin. Pharmacol. 65, 317–325 (2008). betic mice by enhancing autophagy via the SIRT1-BMAL1 pathway. FASEB J. 35,
389. Granowitz, E. V. et al. Pharmacokinetics, safety and immunomodulatory effects e22040 (2021).
of human recombinant interleukin-1 receptor antagonist in healthy humans. 416. Li, Y., Zhang, X. P., Ma, A. J. & Kang, Y. Rational application of beta-
Cytokine 4, 353–360 (1992). Hydroxybutyrate attenuates ischemic stroke by suppressing oxidative stress
390. Smith, C. J. et al. SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist and mitochondrial-dependent apoptosis via activation of the Erk/CREB/eNOS
in Ischemic Stroke) a randomized controlled Phase 2 Trial. Stroke 49, 1210 pathway. ACS Chem. Neurosci. 12, 1219–1227 (2021).
(2018). 417. Yong, Y.-X. et al. Up-regulated microRNA-199b-3p represses the apoptosis of
391. Hong, P. et al. NLRP3 inflammasome as a potential treatment in ischemic stroke cerebral microvascular endothelial cells in ischemic stroke through down-
concomitant with diabetes. J. Neuroinflammation. 16, (2019). regulation of MAPK/ERK/EGR1 axis. Cell Cycle 18, 1868–1881 (2019).
392. Ye, X. C. et al. Purinergic 2X7 receptor/NLRP3 pathway triggers neuronal 418. Xiong, W. et al. DAPK1-ERK signal mediates oxygen glucose deprivation reper-
apoptosis after ischemic stroke in the mouse. Exp. Neurol. 292, 46–55 (2017). fusion induced apoptosis in mouse N2a cells. J. Neurol. Sci. 387, 210–219 (2018).

Qin et al.
29
419. Guo, T. et al. Promoting role of long non-coding RNA small nucleolar RNA Host of apoptosis and autophagy by activating the PI3K/AKT/mTOR pathway. Eur. J.
Gene 15 (SNHG15) in neuronal injury following ischemic stroke via the Micro- Pharmacol. 911, 174554 (2021).
RNA-18a/CXC Chemokine Ligand 13 (CXCL13)/ERK/MEK Axis. Med. Sci. Monit. 26, 441. Zhang, X. et al. Alterations of brain quantitative proteomics profiling revealed
e923610 (2020). the molecular mechanisms of diosgenin against cerebral lschemia reperfusion
420. Hu, Y. et al. AMPK inhibitor BML-275 induces neuroprotection through effects. J. Proteome Res. 19, 1154–1168 (2020).
decreasing cyt c and AIF expression after transient brain ischemia. Biorg. Med. 442. Yang, B. et al. Melatonin plays a protective role by regulating miR-26a-5p-NRSF and
Chem. 52, 116522 (2021). JAK2-STAT3 pathway to improve autophagy, inflammation and oxidative stress of
421. Chen, Z. J. et al. Glycine improves ischemic stroke through miR-19a-3p/AMPK/ cerebral ischemia-reperfusion injury. Drug Des. Dev. Ther. 14, 3177–3188 (2020).
GSK-3 beta/HO-1 pathway. Drug Des. Devel. Ther. 14, 2021–2031 (2020). 443. Pan, Q. et al. MTMR14 protects against cerebral stroke through suppressing
422. Yan, L. & Zhu, T. Effects of rosuvastatin on neuronal apoptosis in cerebral PTEN-regulated autophagy. Biochem. Biophy. Res. Commun. 529, 1045–1052
ischemic stroke rats via Sirt1/NF-kappa B signaling pathway. Eur. Rev. Med. (2020).
Pharmacol. Sci. 23, 5449–5455 (2019). 444. Xue, H. et al. Sevoflurane post-conditioning alleviates neonatal rat hypoxic-
423. Sarmah, D. et al. Sirtuin-1 - Mediated NF-κB pathway modulation to mitigate ischemic cerebral injury via Ezh2-regulated autophagy. Drug Des. Dev. Ther. 13,
inflammasome signaling and cellular apoptosis is one of the neuroprotective 1691–1706 (2019).
effects of intra-arterial mesenchymal stem cell therapy following ischemic 445. Zhong, S.-J. et al. MicroRNA-144 promotes remote limb ischemic
stroke. Stem. Cell. Rev. Rep. 18, 821–838 (2022). preconditioning-mediated neuroprotection against ischemic stroke via PTEN/
424. Xu, J., Wang, C., Meng, F. & Xu, P. Long non-coding RNA H19 inhibition ame- Akt pathway. Acta Neurol. Belg. 121, 95–106 (2021).
liorates oxygen-glucose deprivation-induced cell apoptosis and inflammatory 446. Mu, Q. et al. NPD1 inhibits excessive autophagy by targeting RNF146 and wnt/
cytokine expression by regulating the microRNA-29b/SIRT1/PGC-1 alpha axis. beta-catenin pathway in cerebral ischemia-reperfusion injury. J. Recept. Signal
Mol. Med. Rep. 23, 131 (2021). Transduct. 40, 456–463 (2020).
425. Kawabori, M., Shichinohe, H., Kuroda, S. & Houkin, K. Clinical trials of stem cell 447. Chen, C. et al. Electroacupuncture pretreatment prevents ischemic stroke and
therapy for cerebral ischemic stroke. Int. J. Mol. Sci. 21, 7380 (2020). inhibits Wnt signaling-mediated autophagy through the regulation of GSK-3
426. Zhou, L. et al. Treatment of cerebral ischemia by disrupting ischemia-induced beta phosphorylation. Brain Res. Bull. 158, 90–98 (2020).
interaction of nNOS with PSD-95. Nat. Med. 16, 1439–1443 (2010). 448. He, H. et al. Puerarin provides a neuroprotection against transient cerebral
427. Bach, A. et al. Biochemical investigations of the mechanism of action of small ischemia by attenuating autophagy at the ischemic penumbra in neurons but
molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95- not in astrocytes. Neurosci. Lett. 643, 45–51 (2017).
PDZ interactions. Sci. Rep. 5, 12157 (2015). 449. Fu, K. et al. Grape seed proanthocyanidins attenuate apoptosis in ischemic
428. Kim, Y. R. et al. Electroacupuncture confers beneficial effects through ionotropic stroke. Acta Neurol. Belg. 121, 357–364 (2021).
glutamate receptors involving phosphatidylinositol-3 kinase/Akt signaling 450. Yang, Y. et al. Apelin-13 protects against apoptosis by activating AMP-activated
pathway in focal cerebral ischemia in rats. Eur. J. Int. Med. 4, E413–E420 (2012). protein kinase pathway in ischemia stroke. Peptides 75, 96–100 (2016).
429. Weilinger, N. L., Tang, P. L. & Thompson, R. J. Anoxia-induced NMDA receptor 451. Gao, J. F., Qian, T. T. & Wang, W. CTRP3 Activates the AMPK/SIRT1-PGC-1 alpha
activation opens pannexin channels via Src Family Kinases. J. Neurosci. 32, pathway to protect mitochondrial biogenesis and functions in cerebral ischemic
12579–12588 (2012). stroke. Neurochem. Res. 45, 3045–3058 (2020).
430. Abdul, Y. et al. Inhibition of Toll-Like Receptor-4 (TLR-4) improves neurobeha- 452. Lv, H. et al. Salvianolic acid B attenuates apoptosis and inflammation via SIRT1
vioral outcomes after acute ischemic stroke in diabetic rats: possible role of activation in experimental stroke rats. Brain Res. Bull. 115, 30–36 (2015).
vascular endothelial TLR-4. Mol. Neurobiol. 56, 1607–1617 (2019). 453. Sarmah, D. et al. Sirtuin-1-mediated NF-kappa B pathway modulation to miti-
431. Shi, H. et al. Role of Toll-like receptor-mediated signaling in traumatic brain gate inflammasome signaling and cellular apoptosis is one of the neuropro-
injury. Neuropharmacology 145, 259–267 (2019). tective effects of intra-arterial mesenchymal stem cell therapy following
432. Wang, Y., Ge, P. & Zhu, Y. TLR2 and TLR4 in the brain injury caused by cerebral ischemic stroke. Stem. Cell. Rev. Rep. 18, 821–838 (2022).
ischemia and reperfusion. Mediators Inflamm. 2013, 124614 (2013). 454. Zhou, M. et al. A DNA nanostructure-based neuroprotectant against neuronal
433. Gui, B. et al. Neuroprotective effects of pretreatment with Propofol in LPS- apoptosis via inhibiting toll-like Receptor 2 signaling pathway in acute ischemic
induced BV-2 microglia cells: role of TLR4 and GSK-3 beta. Inflammation 35, stroke. ACS Nano. 16, 1456–1470 (2022).
1632–1640 (2012). 455. Gu, J. et al. Anti-inflammatory and anti-apoptotic effects of the combination of
434. Marik, P. E. Propofol: An immunomodulating agent. Pharmacotherapy 25, Ligusticum chuanxiong and Radix Paeoniae against focal cerebral ischaemia via
28S–33S (2005). TLR4/MyD88/MAPK/NF-kappa B signalling pathway in MCAO rats. J. Pharm.
435. Yang, R. et al. Quercetin attenuates ischemia reperfusion injury by protecting Pharmacol. 70, 268–277 (2018).
the blood-brain barrier through Sirt1 in MCAO rats. J. Asian Nat. Prod. Res. 24,
278–289 (2021).
436. Wei, L. L. et al. Patchouli alcohol protects against ischemia/reperfusion-induced Open Access This article is licensed under a Creative Commons
brain injury via inhibiting neuroinflammation in normal and obese mice. Brain Attribution 4.0 International License, which permits use, sharing,
Res. 1682, 61–70 (2018). adaptation, distribution and reproduction in any medium or format, as long as you give
437. Mamtilahun, M. et al. DL-3n-Butylphthalide improves blood-brain barrier appropriate credit to the original author(s) and the source, provide a link to the Creative
integrity in rat after middle cerebral artery occlusion. Front. Cell. Neurosci. 14, Commons license, and indicate if changes were made. The images or other third party
610714 (2021). material in this article are included in the article’s Creative Commons license, unless
438. Qi, Z. F. et al. AKT-related autophagy contributes to the neuroprotective efficacy indicated otherwise in a credit line to the material. If material is not included in the
of Hydroxysafflor Yellow A against ischemic stroke in rats. Transl. Stroke Res. 5, article’s Creative Commons license and your intended use is not permitted by statutory
501–509 (2014). regulation or exceeds the permitted use, you will need to obtain permission directly
439. Wang, M. M. et al. Electroacupuncture inhibits neuronal autophagy and apop- from the copyright holder. To view a copy of this license, visit http://
tosis via the PI3K/AKT pathway following ischemic stroke. Front. Cell. Neurosci. creativecommons.org/licenses/by/4.0/.
14, 134 (2020).
440. Meng, J., Ma, H., Zhu, Y. & Zhao, Q. Dehydrocostuslactone attenuated oxygen
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Signal Transduction and Targeted Therapy www.nature.

REVIEW ARTICLE OPEN

Signaling pathways involved in ischemic stroke: molecular

Signal Transduction and Targeted Therapy (2022)7:215 ; https://doi.org/10.1038/s41392-022-01064-1

Received: 21 March 2022 Revised: 1 June 2022 Accepted: 15 June 2022

© The Author(s) 2022, corrected publication 2022

Signal Transduction and Targeted Therapy (2022)7:215

Excitotoxicity and related signaling pathways

BBB breakdown O2-

Signal Transduction and Targeted Therapy (2022)7:215

Drug/Therapy Targeting signaling Major targeting Authors Citations Applications

Signal Transduction and Targeted Therapy (2022)7:215

Signal Transduction and Targeted Therapy (2022)7:215

Signal Transduction and Targeted Therapy (2022)7:215

Signal Transduction and Targeted Therapy (2022)7:215

Stimulating Factor for mechanisms(activation of

Signal Transduction and Targeted Therapy (2022)7:215

Signal Transduction and Targeted Therapy (2022)7:215

trisphosphate and Akt and consequently blocking PI3K/Akt

neuronal death.76 In agreement with this hypothesis, down-

role of PTEN in ischemic stroke, which is largely mediated by

regulation of extra-synaptic NMDAR activities.

Death-associated protein kinase 1 (DAPK1) signaling pathway.

kinase, whose phosphorylation contributes to apoptotic cell

During ischemia, NMDAR overactivation promotes Ca2+ inﬂux,

activates Ca2+/calmodulin, and stimulates calcineurin phospha-

DAPK1.79 The latter is then transferred to the GluN2B subunit of

tion between GluN2B and DAPK1 attenuated neuronal excito-

NMDAR current in vitro.80 In addition, NMDAR-regulated calci-

neurin activation contributes to DAPK1 activation, whereas

NMDAR or calcineurin inhibition prevents DAPK1 dephosphoryla-

tion. DAPK1 inhibition protects against ischemic injury both in

ments for ischemic stroke could be based on inhibiting DAPK1.81

serves as a downstream effector of DAPK1, and the DAPK1-ERK

Postsynaptic density protein-95 (PSD95)/neuronal nitric oxide

composed of three PDZ domains.84–86 The binding of PSD95 to

NMDAR and nNOS enhances Ca2+ inﬂux, a hallmark of excitotoxi-

stroke, as evidenced by the amelioration of neurological deﬁcits in

Cerebral ischemia has been shown to enhance NMDAR/PSD95/

nNOS interactions in neurons, thus further aggravating brain

injuries after experimental ischemic stroke.90 All these results

show that signaling through the PSD95/nNOS complex is crucial

Mitochondrial dysfunction, oxidative stress, and related signaling

Ischemic Stroke (AMETIS)

Mitochondria are essential for maintaining energy homeostasis.

When ATP synthesis and energy balance are disrupted by a lack of

glucose and oxygen, the status and function of mitochondria

become substantially altered. Ca2+ inﬂux leads to mitochondrial

permeability transition pore (MPTP) opening and cytochrome c

release.91,92 At the same time, insufﬁcient ATP supply triggers

dysfunction, energy deﬁciency in cerebral ischemia leads to

Signal Transduction and Targeted Therapy (2022)7:215

Signal Transduction and Targeted Therapy (2022)7:215

Cytc NF-kB HIF-1α

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