Free Radical Biology and Medicine 113 (2017) 424–438

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Original article

Acute mental stress induces mitochondrial bioenergetic crisis and hyper- T

fission along with aberrant mitophagy in the gut mucosa in rodent model of
stress-related mucosal disease
Rudranil De, Somnath Mazumder, Souvik Sarkar, Subhashis Debsharma, Asim Azhar Siddiqui,
⁎
Shubhra Jyoti Saha, Chinmoy Banerjee, Shiladitya Nag, Debanjan Saha, Uday Bandyopadhyay
Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India

A R T I C L E I N F O A B S T R A C T

Keywords: Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal
Stress disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to
Mitochondria intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD
Mitophagy model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O2•-),
Superoxide ion
depleted ATP and shifted mitochondrial fission-fusion dynamics towards enhanced fission to induce mucosal
Oxidative stress
injury. Activation of mitophagy to clear damaged and fragmented mitochondria was evident from mitochondrial
translocation of Parkin and PINK1 along with enhanced mitochondrial proteome ubiquitination, depletion of
mitochondrial DNA copy number and TOM 20. However, excess and sustained accumulation of O2•--generating
defective mitochondria overpowered the mitophagic machinery, ultimately triggering Bax-dependent apoptosis
and NF-κB-intervened pro-inflammatory mucosal injury. We further observed that stress-induced enhanced
serum corticosterone stimulated mitochondrial recruitment of glucocorticoid receptor (GR), which contributed
to gut mitochondrial dysfunctions as documented from reduced ETC complex 1 activity, mitochondrial O2•−
accumulation, depolarization and hyper-fission. GR-antagonism by RU486 or specific scavenging of mitochon-
drial O2•− by a mitochondrially targeted antioxidant mitoTEMPO ameliorated stress-induced mucosal damage.
Gut mitopathology and mucosal injury were also averted when the perception of mental stress was blocked by
pre-treatment with a sedative or antipsychotic. Altogether, we suggest the role of mitochondrial GR-O2•−-fission
cohort in brain-mitochondria cross-talk during acute mental stress and advocate the utilization of this pathway
as a potential target to prevent mitochondrial unrest and gastropathy bypassing central nervous system.

1. Introduction response is manifested through ENS more promptly than any other
organ leading to functional gastrointestinal disorders, mucosal
Sustained mental ailments like anxiety and depression significantly bleeding, inflammation, pain, and other bowel symptoms. On the other
affect our health by altering physiological homeostasis [1–3]. Stress- hand poor gut health has been implicated in various psychophysical
related mucosal disease (SRMD) is one such manifestation documented disorders [7].
worldwide in patients experiencing stress [4]. Moderate to acute mu- Being the cellular powerhouse, mitochondrial health, biogenesis
cosal bleeding in critically ill patients of Intensive Care Unit (ICU) is and protein quality control are matters of critical concern; imbalance of
one of the critical stress-associated phenomena and the mortality rate is mitochondrial metabolism is associated with oxidative stress and var-
significantly high (40–50%) [5,6]. Stomach houses a semi-autonomous ious cytopathologies [8,9]. Mitochondrial structural dynamics [10] is
nervous system (enteric nervous system, ENS) consisting of five hun- delicately tuned with outer environment. While mild stress induces
dred million nerves in the lining of the human gut. It is also the source organellar hyperfusion, moderate to acute stress evokes fragmentation
and/or the depository of many neurotransmitters. ENS is sometimes followed by mitophagy in eukaryotes [11,12]. Several quality control
called the “second brain,” and it arises from the same tissue as the proteases like PINK1, PARL and Parkin constantly monitor mitochon-
central nervous system (CNS) during development. CNS and ENS con- drial integrity and ensure timely clearance of damaged organelles
tinue to influence each other lifelong and interestingly the stress [13,14]. Apart from its roles in mitophagy, PINK1 also participates in

⁎
Corresponding author.
E-mail addresses: ubandyo_1964@yahoo.com, udayb@iicb.res.in (U. Bandyopadhyay).

http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.009
Received 21 June 2017; Received in revised form 6 September 2017; Accepted 6 October 2017
Available online 07 October 2017
0891-5849/ © 2017 Elsevier Inc. All rights reserved.
R. De et al. Free Radical Biology and Medicine 113 (2017) 424–438

apoptosis regulation [15] while controlling mitochondrial protein mitochondrial structure [23] during stress, we followed the expression
turnover and suppressing mtDNA damage [16,17]. Interestingly, severe and localisation of mitochondrial dynamics-associated proteins in the
stress pertaining to irreparable cellular damage triggers apoptosis [18]. gastric mucosa from control and stressed rats. Confocal microscopic
In the present study, to mimic an intensive care like ambience of studies documented enhanced expression as well as elevated mi-
stress in the lab we adapted a model of rodent cold restraint stress [19], tochondrial translocation of the predominant fission mediator Drp1
which causes gastric mucosal injury and bleeding. Here we report that (green) in the gastric mucosa of stressed rats compared to control
mental stress induces mitochondrial pathology and shifts mitochondrial (Fig. 1G). Immunoblots further confirmed enhanced Drp1 expression as
structural dynamic balance towards excess fission to damage the gastric well as increased mitochondrial translocation during stress (Fig. 1H and
mucosa by promoting tissue inflammation and apoptosis. Interestingly, I). Moreover, upregulated Drp1 expression was concurrent with en-
trans-activated hypothalamic-pituitary-adrenal (HPA) axis actively hanced phosphorylation at its serine616 residue (p-Drp-S616) (Fig. 1H)
regulates the aforesaid events. Elevated circulating corticosterone with and reduced phosphorylation at serine637 residue (p-Drp-S637) further
concurrent recruitment of glucocorticoid receptor (GR) in mitochondria implying Drp-1-dependent mitochondrial fragmentation during stress
switches on a vicious cycle of enhanced mitochondrial fission-mito- (Fig. 1H). Significant changes in the fusion mediators like Mfn 1 and 2
phagy-bioenergetic crisis, which ultimately triggers cell death to ensure were however indiscernible (Fig. 1H).
tissue injury during stress.
2.2. Activation of mitophagy turns insufficient to control the mitochondrial
2. Results pathology deciding the fate of gastric mucosal cells to undergo apoptosis

2.1. Acute mental stress prevents ATP formation by affecting electron To check the plausible elimination of damaged mitochondria we
transport chain, induces mitochondrial pathology and severe fragmentation next evaluated mitochondrial content in mucosal tissue by measuring
in gastric mucosa in rodent model of stress-related mucosal disease (SRMD) the level of mitochondrial marker TOM 20. Interestingly, it was found
to be lower in stress compared to control indicating mitophagic clear-
The exposure of rats to cold-restraint stress developed significant ance (Fig. 2A). Moreover evaluation of mitochondrial DNA (mtDNA)
(P < 0.001) gastric mucosal erosions and bleeding whereas control copy number revealed depletion of mitochondrial content in gastric
animals did not show any gastric bleeding (Fig. 1A). Time-course stu- mucosa of rats exposed to stress (Fig. 2B) and significant induction of
dies revealed that stress-exposed animals showed highest injury at 3.5 h mitophagic cascade was evident from Parkin upregulation (Fig. 2C).
(Fig. S1). This time point was chosen for the rest of the experiments Further higher magnification documented a considerable enhancement
unless mentioned otherwise. Interestingly, impairment of stress per- of mitochondrial localisation of Parkin during “stress” (Fig. 2C). To get
ception by pre-treatment with diazepam (typical sedative) drastically a deeper insight into the kinetics of mitochondrial remodelling, we
reduced gastric mucosal injury and bleeding. Furthermore, anti- evaluated the mitochondrial translocation of key fission-mitophagy
psychotic drug, olanzapine that only interfered with the perception of markers in a time resolved manner following induction of stress
stress, while keeping the animals alert, offered even better protection (Fig. 2D). Immunoblot data revealed that mitochondrial translocation
under same experimental setup (Fig. 1A). It is worth mentioning that of Drp1 initiated early within 30 min of stress induction. Within 1 h of
none of the aforesaid drugs offered any protection against in- stress initiation, mitochondrial inner membrane potential sensor PARL
domethacin (a typical NSAID)-induced gastric injury, where brain is started degradation, leading to stabilisation of PINK1 on mitochondrial
apparently not involved (Fig. S2A). Stress-induced gastric mucosal outer membrane. This was succeeded by increase in mitochondrial
bleeding was evaluated by measuring haemoglobin release in mucosal translocation of Parkin, which peaked at 2 h after stress exposure. Ul-
washing in different experimental sets (Fig. 1A, Fig. S2B). timately, ubiquitination of the damaged mitochondria started and
Next, in order to check whether the manifestation of mucosal da- eventually peaked at 3.5th h of stress. Moreover, mitochondrial se-
mage in stress involved any underlying bioenergetic crisis, we mon- questration, that started 1 h after stress exposure, gradually increased
itored mitochondrial function. Gastric mucosal ATP content was sig- and peaked at 165 min (2.75 h) as evident from upregulation of p62
nificantly reduced (P < 0.05) in rats exposed to stress compared to (sequestosome 1) in the mitochondrial fraction.
control (Fig. 1B). Mitochondrial respiration (measured by respiratory To check the fate of mucosal cells that was decided by mitochon-
control ratio, RCR) and ETC complex I activity were also compromised drial fission and mitophagy, we next investigated the extent of mucosal
in the gastric mucosa of rats under stress (Fig. 1C, Fig. S3A). Next, in cell death by following caspase activity in a time resolved fashion fol-
order to check mitochondrial functional integrity during stress, we lowing induction of stress. Data indicated a temporal increase in cas-
evaluated the status of mitochondrial fatty acid oxidation and dehy- pase 9 (Fig. 2E) followed by caspase 3 activities in the gastric mucosa
drogenase activities. Data revealed significantly reduced (P < 0.01) (Fig. 2F). Kinetic assessment of mitophagy revealed that mitophagic
fatty acid oxidation (Fig. 1D) and dehydrogenase activity (Fig. S3B) in clearance of damaged mitochondria followed a regressive pattern at the
stress. In addition, significant (P < 0.01) depolarization of ΔΨm further later hours of stress as evident from reduced mitochondrial Parkin re-
confirmed mitochondrial pathology during stress (Fig. S3C). Depo- cruitment and rate of mitochondrial protein ubiquitination. Compara-
larised mitochondria with compromised ETC complex I generate O2•− tive kinetic analysis of mitophagy and apoptosis (Fig. 2G-H) therefore
[20] that serves as a progenitor molecule for various other reactive clearly indicated the failure of mitochondrial quality control machinery
oxidants [21] ultimately causing damage by macromolecular perox- to eliminate damaged organelles and consequent caspase activation in
idation and carbonylation [22]. Therefore, we directly measured mi- the later phase of the stress. Further, we also observed mitochondrial
tochondrial reactive oxidants accumulation in gastric mucosal cells translocation of Bax (Fig. S5A) along with a significant increase
from “control” and stressed rats. Data indicated heavy accumulation of (P < 0.01) in cytochrome C in the cytosolic fractions of gastric mucosa
mitochondrial O2•− during stress (Fig. 1E). Further, DCFDA-based flow isolated from stressed rats (Fig. S5B and C) compared to control. Al-
cytometric analysis was performed wherein cells isolated from stressed- tered cellular redox status differentially modulates NF-κB signalling and
rat stomach exhibited high DCFDA-fluorescence revealing the probable tissue integrity [24,25]. In corroboration with previous reports of NF-κB
occurrence of Fe-catalysed redox reactions (Fig. S4A). A direct impact activation and proinflammatory tissue damage [26], we found that
of elevated mitochondrial reactive pro-oxidants (during stress) was stress-induced mitochondrial pathology was concurrent with NF-κB
observed from significant decrease in cardiolipin (Fig. 1F) and other activation. Immunoblots revealed a significant elevation and nuclear
biomarkers of oxidative stress [22] including mitochondrial macro- translocation of NF-κB p65 during stress (Fig. 3A). Higher level of IκBα
molecule oxidation and protein carbonylation (Fig. S4B and C). in the control rats (Fig. 3A) further implied its degradation during
Next, to check the probable effects of functional derangement on stress. Interestingly, stress-induced IκBα degradation paralleled with

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Fig. 1. Mental stress induces acute gastric mucosal injury, mitochondrial dysfunction, redox imbalance and mitochondrial fragmentation. (A) Gastric mucosal morphology and histology
(by haematoxylin-eosin staining) of control, stress, “diazepam + stress”, “olanzapine + stress”-exposed rats. II represents injury index corresponding to the experimental conditions. The
extreme right panel contain the data obtained from Soret spectroscopy of the gastric mucosal washing to estimate gastric mucosal bleeding. (B) Tissue ATP content. (C) Mitochondrial
respiratory control ratio (RCR). (D) β-oxidation of fatty acids. (E) Flow cytometric analysis to estimate mitochondrial O2.-. Values represent percentage of cells giving MitoSOX-positive
signal in every 10,000 events scanned. (F) Cardiolipin content. (G) Immunohistochemical analysis for mitochondrial recruitment of Drp1. Mitochondria (red) were stained by COX-IV
primary and alexa fluor 555-conjugated secondary antibodies. Drp1 (green) was immunostained by anti-Drp1 primary and alexa fluor 488-tagged secondary antibodies; nucleus (blue)
was stained by DAPI. A representative image was presented (20X); further magnification (40X) to the extent of cellular level and has been presented with the corresponding samples; the
region of interest (ROI) of the micrographs were enlarged for clear depiction. Cyan and white scale bars correspond to 50 µm and 5 µm respectively. Pearson's correlation coefficient, ‘‘r”
and rate of colocalization in % for each experimental group has been presented. (H) Immunoblot analysis of mitochondrial dynamics mediators; Drp1, p-Drp1-S616, p-Drp1-S637, Mfn1
and Mfn2 in the total tissue lysate; β-actin was used as the loading control. Numerical values presented below the bands represent the corresponding densitometry values. (I) Immunoblot
analysis for detecting differential localisation of Drp1 in the mitochondrial and cytosolic fractions of the ‘control’ and ‘stress’ tissue samples; TOM 20 and β-actin were used as the loading
controls for the mitochondrial and cytosolic fractions respectively. For all animal experiments n = 6–8 and experiments were performed thrice. A detail of each method is described under
Section 4. Error bars, mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001 versus control; ##P < 0.01; ###P < 0.001 versus stress calculated by Mann-Whitney U test. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.).

enhanced DNA binding of activated NF-κB as confirmed by electro- Because corticosterone exerts multiple effects upon binding to gluco-
phoretic mobility shift assay (EMSA) (Fig. 3B). Significantly high corticoid receptor (GR), we checked the in situ expressional profile of
complex formation due to active NF-κB-DNA interaction was observed GR in the rat stomach tissues. It is worth mentioning that im-
during stress (lane 3), as evident from an intense band compared to munohistochemical microscopy of GR revealed a 1.5 fold elevation
control (lane 2). Specificity of DNA-protein interaction in stress was during stress as evident from the mean intensity value of fluorescent
evident from significantly low band intensity (lane 4) where an 80 fold signal (green) corresponding to GR (Fig. 5B). Upregulated expression
excess unlabelled probe was used for competition (Fig. 3B). Histo- was accompanied by enhanced mitochondrial translocation (r = 0.74)
chemical data also reconfirmed increased nuclear localisation of NF-κB of GR during stress as evident from the co-localisation of the red (mi-
in stress (r = 0.653) compared to control (r = 0.305) (Fig. 3D). The tochondria) and green (GR) signals (Fig. 5B). To verify if it is indeed the
consequence of NF-κB activation was next studied by gene expression mental stress that activated GR in the present SRMD model, we checked
profiling and ELISA array of typical proinflammatory cytokines and mitochondrial recruitment of GR in control, stress and in diazepam/
chemokines (Fig. 3E and F). Gene expressions of IL-1α, IL-6, CCL-17 olanzapine pre-treated-stress exposed rats. Western blot data confirmed
and CXCL-3 were found to be significantly upregulated in stress. Si- enhanced mitochondrial translocation of GR during stress and this
milarly, elevated levels of IL-1β, IL-2, IL-12, IFN-γ, TNF-α, GM-CSF, elevated mitochondrial GR level was significantly rectified in the dia-
RANTES, LIX (Fig. 3F) as documented by ELISA array further confirmed zepam/olanzapine pre-treated rats (Fig. 5C). Finally to verify the in-
the establishment of a pro-inflammatory milieu in the gastric mucosa volvement of stress-induced GR activation on gastric mucosal pa-
during stress. thology, we pre-treated the rats with mifepristone (RU486, a
competitive inhibitor of GR), and found that it significantly prevented
2.3. Scavenging of mitochondrial O2.- or prevention of mitochondrial stress-induced mucosal injury (Fig. 5D).
structural and functional anomalies prevents gut mucosal injury in stress
2.5. Prevention of stress perception by sedative or atypical antipsychotic
From the previous experiments, it was apparent that mitochondrial also inhibits gastric mitochondrial pathology and mucosal cell apoptosis
redox imbalance and unregulated fission coupled with anomalous mi-
tophagic clearance catalysed the gastro-damaging signalling during Prevention of stress-induced gastric injury and associated HPA ac-
stress. Therefore, we asked whether scavenging of mitochondrial O2•− tivation by diazepam/olanzapine in the previous data next directed us
or inhibition of excess fission could ameliorate the detrimental mani- to check their effect on mitochondrial integrity. Data indicated ≈70%
festations of stress. We treated the rats intravenously with mitoTEMPO retention in ΔΨm compared to control (Fig. 6A). Fatty acid oxidation
(a hybrid antioxidant molecule of piperidine nitroxide and lipophilic and mitochondrial dehydrogenase activity in the “Diazepam + Stress”
cation triphenylphosphonium) or Mdivi-1 (a molecule known to rectify group were almost 80% of the values as in control (Fig. 6B and C).
several mitochondrial structural and functional abnormalities) at a dose Further almost 80% of β-oxidation and mitochondrial dehydrogenase
of 10 mg/kg or 2 mg/kg b.w. respectively 1 h prior to stress. Data in- activity along with 70% of membrane potential was preserved in
dicated that both mitoTEMPO and Mdivi-1 prevented mucosal injury “Olanzapine + Stress” (Fig. 6B and C). Diazepam or olanzapine pre-
and bleeding (Fig. 4A-B) besides averting stress-induced mitochondrial treatment also significantly prevented stress-induced oxidative damage
damage as evident from rectification of ΔѰm depolarization (Fig. 4C), (P < 0.01) (Fig. 6D) and associated bio-energetic crisis as evident from
perturbed fatty acid oxidation (Fig. 4D), loss of dehydrogenase activity prevention of ATP depletion, mitochondrial respiration failure and loss
(Fig. 4E), tissue ATP depletion (Fig. 4F) and mitochondrial macro- of ETC complex I activity (Fig. 6E, F and G). Finally, a close evaluation
molecular oxidation (Fig. 4G). Western blot data demonstrated mito- of the effects of diazepam/olanzapine on stress-induced excess mi-
TEMPO/Mdivi-1 also reduced mitophagic activation and apoptosis as tochondrial fission and aberrant mitophagy revealed that both the
evident from the decreased levels of mitochondrial Parkin and Bax drugs significantly reduced the phosphorylation of Drp1 at serine 616
(Fig. 4H). (p-Drp-S616) and mitochondrial protein ubiquitination along with
lowering of mitochondrial translocation of Parkin, PINK1 (Fig. 6H) and
2.4. GR translocation to gastric mucosal mitochondria is an essential Bax (Fig. 6I). Interestingly, the level of anti-apoptotic Bcl2 was main-
outcome during mental stress-induced mitochondrial pathology and acute tained at a level comparable to control (Fig. 6I) and caspase 3 activity
gastropathy was significantly (P < 0.01) down regulated (Fig. 6J) indicating at-
tenuation of stress-induced gastric mucosal apoptosis.
Next we were interested to figure out the molecule(s) that acted as
stress-responsive cargo from mind to gut mitochondria for evoking the 3. Discussion
pathology. Hence we first checked the serum level of stress-associated
biogenic amines and neurotransmitters (Fig. S6). Data indicated that Here we report that excess mitochondrial oxidative damage and
corticosterone was significantly (P < 0.05) elevated during stress fragmentation along with aberrant protein quality control play a sig-
(Fig. 5A). Interestingly, pre-treatment with diazepam or olanzapine nificant role in mental stress-induced gastric mucosal injury. Stress-
normalized stress-induced elevated corticosterone level (Fig. 5A). mediated common responses are apparent in critically ill patients,

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Fig. 2. Aberrant mitophagy and stress-induced activation of gastric mucosal apoptosis. (A) Mitochondrial content measurement by immunoblot of TOM 20 in total tissue lysate; Actin was
used as loading control. (B) Mitochondrial content in gastric mucosa of control and stress-exposed rats in terms of mitochondrial DNA (mtDNA) copy number assessment represented as 2-
ΔΔCq
values normalized with nuclear DNA. (C) Immunohistochemical analysis for mitochondrial recruitment of Parkin during stress. Mitochondria (red) were stained by COX-IV primary
and alexa fluor 555-conjugated secondary antibodies; Parkin (green) was immunostained by anti-Parkin primary and alexa fluor 488-tagged secondary antibodies. Nucleus (blue) was
stained by DAPI. Representative images of 20X magnification were presented; further cell-level magnified (40X) images have been presented with the corresponding samples; en-
largement of the region of interest (ROI) was done for clear depiction. Cyan and white scale bars correspond to 50 µm and 5 µm respectively. Pearson's correlation coefficient, ‘‘r” and rate
of colocalization in % for each experimental group has been presented. (D) Immunoblot analysis to detect the expressions of Drp1, PARL, PINK1, Parkin, ubiquitination and p62 in
mitochondrial fractions obtained in a time dependent manner; TOM 20 was used as the loading control for the mitochondrial fractions. (E) Kinetic analysis of caspase 9 activity. (F)
Kinetics of caspase 3 activation. For all animal experiments n = 6–8 and experiments were performed thrice. (G) Comparative kinetics of death pathway activation (in terms of Caspase 3
activity) and status of functional mitophagy (in terms of mitochondrial translocation of Parkin) with propagation of stress; “II” adjacent to each time point represents gastric injury index
at that time point (H) Comparative kinetics of death pathway activation (in terms of Caspase 3 activity) and status of mitochondrial protein ubiquitination; “II” adjacent to each time point
represents gastric injury index at that time point. A detail of each method is described under Section 4. Error bars, mean ± S.E.M. *P < 0.05, **P < 0.01 versus control calculated by
Mann-Whitney U test; ns = non-significant difference relative to control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.).

including those with cardiothoracic failure (requiring mechanical ven- due to partial unconsciousness offered by mild sedation. Furthermore it
tilation), acute hepatic and renal failure, sepsis and severe burn, head was equally necessary to inquire whether the resultant protection was
or spinal cord injury [27]. Interestingly, gut is the most frequently af- an attribute of mind dissociation (by tranquilization) or neutralisation
fected in response to stress which may be an attribute of the ENS-CNS of physical unrest. To address the issue, an atypical anxiolytic, olan-
crosstalk [28]. Even though, stress-associated detrimental factors sys- zapine was selected which can interfere with the stress response
temically affect other organs, harsh luminal gastric microenvironment pathway without affecting consciousness. It is often used to treat psy-
renders stomach increasingly susceptible [29,30]. chotic diseases including depressive disorders [31]. Interestingly,
Rodent cold restraint-induced gastric mucosal injury is a suitable olanzapine offered better protection than diazepam. Moreover, specific
experimental model to study the involvement of CNS in SRMD. involvement of CNS in SRMD and consequent gastropathy was con-
Interestingly, sedation by a tranquilizer (diazepam) showed drastic firmed from the observation that both diazepam and olanzapine failed
reduction in mucosal damage. However, complete protection was to offer protection against indomethacin-induced gastric damage where
lacking probably because of the leaky signals transmitted to the brain apparently no mental component was involved.

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Fig. 3. Mitochondrial pathology is associated with

activation of NF-κB-mediated proinflammatory ma-
chinery during stress. (A) Immunoblot analysis to
follow NF-κB (p65) and IκBα in the total tissue lysate
from ‘stress’ and ‘control’ rats and also nuclear
translocation of NF-κB (p65) in the nuclear extract;
GAPDH and histone 3 A were used as the loading
control for total protein and nuclear extract respec-
tively. (B) Translocation to nucleus and DNA-binding
activity of NF-κB as documented by EMSA. Lane 1,
only labeled DNA probe; Lane 2, nuclear extract of
control rat stomach with labeled probe; Lane 3, nu-
clear extract of rat stomach exposed to stress with
labeled probe; Lane 4, nuclear extract of rat stomach
exposed to stress with labeled probe along with
competition with 80-fold excess of cold probe. (C)
Densitometric analysis of EMSA. (D)
Immunohistochemical analysis for detecting nuclear
translocation of NF-κB (p65) in situ during stress;
nucleus was stained by DAPI and NF-κB (p65) (red)
was immunostained by anti- NF-κB (p65) primary
and alexa fluor 647-tagged secondary antibody;
white arrows indicate cells with intra-nuclear NF-κB
and insets show magnified images of the selected
portions of mucosa; size bars indicate 50 µm (E)
Gene expression analysis for some selected NF-κB-
inducible proinflammatory markers by quantitative
RT-PCR. Bar graphs indicate fold change in gene
expression relative to control (after normalization
with gapdh) (F) ELISA array of some selected NF-κB-
regulated proinflammatory markers; Bar graphs in-
dicate fold change in protein levels in the gastric
mucosal tissue lysate relative to control. For all an-
imal experiments n = 6–8 and experiments were
performed thrice. Details of each method are de-
scribed under Section 4. Error bars, mean ± S.E.M.
*P < 0.05, **P < 0.01 versus control calculated by
Mann-Whitney U test. (For interpretation of the re-
ferences to color in this figure legend, the reader is
referred to the web version of this article.).

The concurrence of mitochondrial pathology as documented by the major supplier of reducing equivalents needed for oxidative phos-
battery of mitochondrial metabolic assays including mitochondrial re- phorylation. Mitochondrial ETC complex activity was checked pri-
spiration, ETC complex assay, fatty acid oxidation and dehydrogenase marily focussing the complex I. It is worth mentioning that although
activity strongly pointed towards the underlying bioenergetic deficit other ETC complex activities were also measured including complex III
during stress. Depletion of tissue ATP and depolarization of mi- (data not shown), the most prominent change was observed in complex
tochondria supported the above claim and justified the fact that de- I activity during stress. It is the major centre for electron leakage and
crease in ATP is especially detrimental to gastric mucosal integrity that complex I dysfunction is evident in several cases associated with ATP
thrives on energy dependent cellular turnover. Deviation in normal depletion, reactive oxygen or nitrogen species formation [33] that
respiratory chain function is frequently associated with free radical sometimes cause damage with mitophagic involvement [34]. Oxidative
generation. O2•− can attack Fe-S cluster containing proteins to release stress-associated apoptotic tissue damage is linked with active build up
free iron [32]. Dismutation of O2•− also generates H2O2 either spon- of inflammatory milieu which is characterised by initial surge of neu-
taneously or by superoxide dismutase (SOD)-induced catalysis. More- trophilic infiltration [26,35]. Basal level of nuclear NF-κB in the im-
over the released free iron in presence of H2O2 can generate •OH munoblot and EMSA indicate the inherent proliferative nature of the
through Fenton's reaction. Thus O2•− may be considered as the pro- gastric mucosa. However stress-induced enhancement of pro-in-
genitor reactive oxygen species that can sequentially produce several flammatory NF-κB activation and nuclear translocation followed by
damaging reactive pro-oxidants. All these reactive oxidants (originating elevation of inflammatory cytokines and chemokines contribute to the
from O2•− accumulation) collectively damage the mitochondrial mac- operation of a self-catalysing gastro-damaging reaction. Redox im-
romolecules by oxidation, peroxidation and carbonylation. These det- balance, accumulation of mitochondrial pro-oxidants and their intricate
rimental outcomes were evident in stress. It can be postulated that, association with the pathogenesis of stress-induced mucosal injury were
metabolic crisis due to reduced fatty acid oxidation and ETC function firmly supported by the observation that a mitochondrially targeted
might have occurred because of the detrimental oxidative modifications small molecule antioxidant, mitoTEMPO successfully neutralized mi-
of the protein complexes. Moreover compromised ETC machinery tochondrial oxidative damage, rectified mitochondrial pathologies and
might not demand much of reducing equivalents thereby causing a prevented tissue injury. MitoTEMPO has a wide range of protective
functional alteration that slows down β-oxidation, which is otherwise a applications in various oxidative stress-associated disease models and

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Fig. 4. MitoTEMPO and Mdivi-1 prevent gastric injury and correct mitochondrial dysfunction induced in stress. (A) Gastric mucosal morphology and histology (by haematoxylin-eosin
staining) of ‘control’, ‘stress’, ‘MitoTEMPO + stress’ and ‘Mdivi-1 + stress’-exposed rats. II represents injury index corresponding to the experimental conditions; n = 6–8 and ex-
periments were performed thrice. (B) Soret spectroscopy of the gastric mucosal washing to estimate the content of haemoglobin resulting from gastric mucosal bleeding in the Control and
treated samples. (C) Mitochondrial trans-membrane potential, ΔѰm. (D) β-oxidation of 14C palmitate. (E) Mitochondrial dehydrogenase activity. (F) Gastric mucosal tissue ATP content
(G) Mitochondrial macromolecule oxidation products. (H) Immunoblot analysis to detect Parkin and Bax in mitochondrial fractions; TOM 20 was used as the loading control. Numerical
values presented below the bands represent the corresponding densitometry values after normalization and fold calculation relative to control. For all animal experiments n = 6–8 and
experiments were performed thrice. Details of each method are described under Section 4. Error bars, mean ± S.E.M. **P < 0.01, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01,
###
P < 0.001 versus stress calculated by ANOVA followed by post hoc test.

considered to be a safe molecule with no reported negative health ef- however it is still a topic of active research whether Mdivi-1 offered
fects [36,37]. The observations were further substantiated by testing gastroprotection in a Drp1-dependent or Drp1-independent manner.
the effect of another small molecule, Mdivi-1, which reportedly influ- Stress-induced persistent oxidative damage to mitochondria is ex-
ences various aspects of mitochondrial function [38] and blocks pro- pected to demand segregation and elimination of the malfunctioning
apoptotic Bax-intervened cytochrome c release from mitochondria pool of mitochondria for restoring physiological homeostasis.
[39]. Putative fission blocking and associated therapeutic effects of Activation of mitochondrial fission coupled with concurrent initiation
Mdivi-1 has been already documented in several other models [40]. The of mitophagy, upon exposure to stress, indicated the activation of en-
involvement of aberrant mitochondrial dynamics and metabolic crisis dogenous quality control machinery to eliminate the damaged orga-
therefore justified the consideration of Mdivi-1 in this study. Interest- nelles. The predominance of the fission mediators in regulating mi-
ingly Mdivi-1 significantly prevented stress-induced gastropathy; tochondrial dynamics in the present study indicated that acute mental

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Fig. 5. Translocation of glucocorticoid receptor to gastric mucosal mitochondria during stress. (A) Serum corticosterone level under different experimental conditions. (B)
Immunohistochemical analysis of GR expression and mitochondrial localisation in gastric mucosa of rats in control and during stress exposure. GR (green) was immunostained by anti-GR
primary and alexa fluor 488-tagged secondary antibodies. Mitochondria (red) were stained by COX-IV primary and alexa fluor 555-conjugated secondary antibodies. A representative
image was presented; a portion of the micrograph was magnified to the extent of cellular level and has been presented below the corresponding samples. Cyan and white scale bars
correspond to 50 µm and 5 µm respectively. Numerical values presented in green fonts represent the intensity values of GR. Numerical values presented in yellow fonts represent the
Pearson's correlation coefficient (r) indicating the overlap of mitochondrial signal (red) and GR signal (green). (C) Immunoblot analysis to detect mitochondrial glucocorticoid receptor
(GR) under different experimental conditions; TOM 20 was used as the loading control. (D) Gastric mucosal morphology and histology (by haematoxylin-eosin staining) of ‘control’,
‘stress’ and ‘RU486 + stress’-exposed rats. II represents injury index corresponding to the experimental conditions. For all animal experiments n = 6–8 and experiments were performed
thrice. Details of each method are described under Section 4. Error bars, mean ± S.E.M. **P < 0.01, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01 versus stress calculated by
ANOVA followed by post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

stress probably operates through modulation of fission GTPases more insufficiency in subverting the damage beyond control. Indeed, kinetic
pronouncedly compared to the fusion GTPases as also evident in evaluation of intrinsic apoptotic mediators (including mitochondrial
myocardial ischemia-reperfusion injury where Drp1 acts as a key player Bax translocation and activation of caspase 9 followed by caspase 3)
[41]. However further studies are advocated for better understanding of revealed a progressive trend subsequent to stress exposure. Although,
the impact of stress on mitochondrial fusion mediators. Uncontrolled mitophagy is considered to be the ultimate mitochondrial surveillance
fission often triggers the apoptotic cascade to mediate disease pro- mechanism to save cells from activation of death pathways, recent re-
gression [23,42,43]. In this context, where severe fission is triggered, ports suggest that the role of Parkin/PINK1 system may be more
the fate of the cells containing a large amount of dysfunctional and complex in deciding cell fate [44]. They may act as key factors scruti-
fragmented mitochondria is expected to depend upon the quality con- nising cellular condition before deciding whether to destroy dysfunc-
trol system. Time resolved analysis of ubiquitination and spatial dis- tional mitochondria or in case of irreversible severe damage, to sensi-
tribution of Parkin, PINK1, PARL and p62 revealed the activation and tise cells towards apoptosis. Moreover, excessive mitochondrial
progression of mitophagy. Interestingly, in the later time points of stress clearance by mitophagy in some cases, as in chronic obstructive pul-
following 2 h, Parkin started showing a decreasing trend of mitochon- monary disease, is also shown to promote cell death [45]. Therefore
drial localisation along with plateauing of mitochondrial proteome either ways, the activation of mitophagic signalling provides an evi-
ubiquitination. This observation was probably indicative of mitophagic dence of significant mitochondrial pathology and also proves that

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Fig. 6. Diazepam or olanzapine prevents stress-induced gastric mitochondrial pathology and mucosal cell apoptosis. (A) Mitochondrial trans-membrane potential, ΔѰm. (B) β-oxidation
of 14C palmitate. (C) Mitochondrial dehydrogenase activity. (D) Mitochondrial macromolecule oxidation products. (E) Gastric mucosal ATP content. (F) Mitochondrial respiratory control
ratio (RCR). (G) ETC complex I activity. (H) Immunoblot analysis for detecting mitochondrial localisation of p-Drp1-S616, Parkin, PINK1 as well as protein ubiquitination. TOM 20 was
used as the loading control. (I) Bcl2 as well as Bax localisation to mitochondria under different experimental sets; TOM 20 was used as the loading control for the mitochondrial fractions.
Numerical values presented below the bands represent the corresponding densitometry values after normalization and fold calculation relative to control. (J) Caspase 3 activity. Details of
each method are described under Section 4. Error bars, mean ± S.E.M. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus stress calculated by ANOVA followed by post
hoc test.

mitochondrial dysfunction is indeed the cause and not the consequence and GC not only induce mitochondrial pathology; they are also reported
of apoptosis in the present model. to induce expression of several genes of the extrinsic and intrinsic death
Glucocorticoids (GC) are released in response to a plethora of en- pathways in sensitive lymphoid tissues [51]. In fact, GR-induced
vironmental and physiological stress stimuli affecting both develop- apoptosis is evident in many studies [52,53]. Apart from the classical
ment and metabolism. In different physiological contexts, manifesta- genomic pathways, GR also exerts its effect via non-genomic pathways
tions of stress response may differ but the correlation between by intercalating (at high concentrations) within cell as well as mi-
molecular players involved in stress and HPA axis are evident [46]. In tochondrial membrane resulting in increased proton leakage from mi-
rodents, corticosterone exerts multiple effects by binding to gluco- tochondria [54]. Another notable effect of mitochondrial GR is its as-
corticoid receptor (GR) and increase in corticosterone level has been sociation with some of the Bcl-2 family of proteins that in turn results in
positively associated with GR activation [47]. GR translocation from mitochondrial Bax translocation, elevated cytosolic cytochrome C and
cytosol to mitochondria directly influences mtDNA transcription and consequent caspase activation [55]. In this context, if corticosterone-
mitochondrial physiology [48,49] where it can generate genomic and bound GR initiates the damage then ectopic administration of corti-
non-genomic mitochondrial dysfunctions [50]. In hippocampus, at the costerone should mimic the same situation. However, it is extremely
time of immobilization stress, association of GR to GC response ele- difficult to determine the optimum titre of the corticosteroids that
ments (GREs) within mtDNA negatively affects the expression of NADH reaches the cell. Hence we adapted an alternative approach of blocking
dehydrogenase 1, 3, and 6 (ND-1, ND-3, ND-6) and ATP synthase 6 the GR by an established competitive inhibitor, RU486. The sig-
(ATP-6) genes [49], which can further explain compromised complex 1 nificance of GR activation in the detrimental SRMD is successfully va-
activity and reduced ATP turnover in gastric mucosa during stress. GR lidated by prevention of the injury by GR antagonist RU486. However,

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lack of complete protection was probably due to the inability of sys- mitochondrial fission in conjunction with mitophagic insufficiency
temically administered RU486 to completely block all the receptors leads to cell death and inflammation that culminates into tissue injury
present in the gastric system. A higher RU486 concentration might have Fig. 7. From therapeutic point of view, this mind-gut-mitochondria
solved the issue but again the constraint of maximum limit of the de- crosstalk opens a scope for new generation mitochondrial tranquilizers,
liverable suspension of RU486 in CM-cellulose suspension prevented those can improve mitochondrial fitness by blocking excess fission or
the usage of higher dose. In this regard targeted blocking of GR either scavenging mitochondrial O2•− thereby non-addictively averting stress-
by intra-gastric GR antagonists or any gastric peptide conjugated hybrid related disorders.
GR blocker might solve this issue. Serotonergic (5-HT) system and the
HPA axis crosstalk in many circumstances [56] and 5-HT2CRs are ne- 4. Materials and methods
cessary for 5-HT-induced HPA axis activation [57]. Co-incidentally,
olanzapine is a known antagonist of 5-HT2AR and 5-HT2CR [58]. Thus 4.1. Reagents and antibodies
in this model, we speculated the involvement of 5-HTRs and their
crosstalk with GR-GC mediated HPA axis. Data in this report clearly 3, 3’-diaminobenzidine (DAB), ubiquinone, decylubiquinone, so-
suggest the activation of HPA axis as evident from corticosterone re- dium azide, fatty acid free BSA, NADH, rotenone, Mdivi-1, MitoTEMPO,
lease in circulation during stress. RU486, secondary anti-rabbit and anti-mouse HRP-conjugated anti-
Thus it is concluded that mental stress activates HPA axis, which bodies were procured from Sigma (St Louis, MO, USA). PBS (HyClone)
mediates mitochondrial translocation of GR that in turn down regulates and mitochondria isolation kit were purchased from Biochain. Mito
mitochondrial oxidative phosphorylation and metabolism causing tracker red CMXRos, DAPI-antifade, alexa fluor 555-tagged secondary
bioenergetic crisis with concurrent oxidative damage. Finally, excess antibody, alexa fluor 488-tagged secondary antibody, ATP

Fig. 7. Schematic representation of how mental

stress affects gut mitochondria. Mental stress acti-
vates stress response pathway in which hypotha-
lamic-pituitary-adrenal (HPA) axis-mediated upre-
gulation of corticosterone in the circulation causes
GR translocation to mitochondria. Under this situa-
tion, mitochondrial respiratory chain complex ac-
tivity goes down which favours mitochondrial su-
peroxide generation and accumulation that
ultimately causes oxidative damage to the organelle
and the tissue as well. This condition disrupts the
mitochondrial structural dynamic balance by indu-
cing excessive fission and triggering mitophagic
cascade which even fails to rescue the mitochondrial
pathology. Finally an uncontrolled state of mi-
tochondrial damage beyond the rescue capacity of
mitochondrial quality control system sensitizes the
cellular machinery towards apoptotic death.
Concurrent activation of NF-κB signalling triggers
pro-inflammatory cytokine and chemokines release
that perpetuates the inflammation along with the
active cross-talk with mental stress-mitochondria
axis to further worsen the tissue damage. The present
hypothesis of detrimental activation of mitochon-
drial fission and mitophagy followed by inflamma-
tion during acute mental stress was pharmacologi-
cally validated at different tiers by use of diazepam
and olanzapine (drugs that block stress perception),
a mitochondrially targeted antioxidant mitoTEMPO
and finally a competitive inhibitor/blocker of GR
(RU486). In all cases the dreadful manifestation of
SRMD was averted. Abbreviations used; FAO, Fatty
acid oxidation; RCR, respiratory control ratio; ETC,
electron transport chain; ΔΨm mitochondrial trans-
membrane potential; GR, glucocorticoid receptor;
HPA, hypothalamic-pituitary-adrenal axis; 5HT, 5-
hydroxy tryptamine.

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R. De et al. Free Radical Biology and Medicine 113 (2017) 424–438

determination kit and Super Signal West Pico/ femto, 4.4. Histological study of gastric mucosal tissue sections and Soret
Tetramethylrhodamine Methyl Ester (TMRM) were purchased from spectroscopy to detect mucosal bleeding
Invitrogen, Life Technologies. Protease inhibitor cocktail (Set V) was
purchased from Calbiochem. MitoSOX Red, nonyl acridine orange Gastric mucosal tissues from rats were fixed in 10% buffered for-
(NAO), DCFDA were procured from Molecular Probes (Eugene, OR, malin (pH 7.4) for 12 h, subjected to ethanol washing and subsequently
Thermo Fisher Scientific Inc.). Sulfosalicylic acid was obtained from embedded in paraffin. Next, semi-thin (5 µM) sections were collected
SRL (Mumbai, India). All other reagents were also of analytical grade on poly-L-lysine-coated glass slides for dual-staining with eosin-hae-
purity. Diazepam was procured from Ranbaxy Laboratories, Gurgaon, matoxylin and observed under microscope (Leica DM-2500, Germany).
India and olanzapine was purchased from Intas Biopharmaceuticals, Haemoglobin release in gastric mucosal washing was quantified by
Ahmedabad, India. Soret spectroscopy to monitor gastric mucosal bleeding as mentioned
earlier [62] in UV–VIS spectrophotometer (Shimadzu, UV-1700 Phar-
maSpec).
4.2. Animals and SRMD model of gastric damage induced by cold-restraint
stress and drug treatments
4.5. Measurement of tissue ATP content
The guidelines of the animal ethical committee were strictly fol-
ATP was measured with an ATP determination kit (Invitrogen Corp.,
lowed while performing all the in vivo experiments. Sprague-Dawley
Carlsbad, CA, USA) following manufacturer's instructions. Briefly, the
rats (200–250 g weight) used for the experiments were maintained at
scraped gastric mucosa from control and cold restrained rats were
24 ± 2 °C with a light and dark cycle (12 h). Rats were starved for 24 h
minced, homogenized in 3% sulfosalicylic acid, and subsequently cen-
prior to the experiment (with ad libitum access to water). Cold-restraint
trifuged at 12,000× g. The clear supernatant was used for the mea-
stress was given as mentioned in the established literature [59]. Gastric
surement of ATP in a luciferase-based luminometric assay. Intensity
mucosal damage was induced by immobilizing the animals in a dorsal
readings for the above mixtures were measured with BioTEK lumin-
supine position under light ether anaesthesia and keeping them at
ometer (at emission maximum ~ 560 nm). The ATP contents were
4 ± 1 °C for 3.5 h. This experimental set was termed “Stress”. Time
expressed as Relative Light Units (RLU).
course analysis for stress-induced gastric injury was followed by sub-
jecting rats to aforementioned stress for different time points including
4.6. Isolation of mitochondria
0.5 h, 1 h, 2 h, 2.75 h and 3.5 h. In the “Control” set, fasted animals
were kept at room temperature in a normo-thermic non-stressed con-
Gastric mucosal mitochondria and mitochondria-free cytosolic ex-
dition for a similar period of time. Indomethacin induced gastric mu-
tract were isolated using commercially available kit (Biochain) as per
cosal injury was induced by oral administration of indomethacin (at
manufacturer's protocol. For mitochondrial respiration analysis the
48 mg/kg b.w.) and incubating for 4 h. Diazepam (5 mg/kg b.w.) was
isolated mitochondria were further purified by Percoll density gradient
administered 30 min prior to stress treatment by intra-muscular injec-
centrifugation as mentioned earlier [62,66].
tion. Olanzapine (10 mg/kg b.w.), suspended in sterile 0.9% saline, was
administered 1 h prior to stress by intra-peritoneal injection. Mito-
4.7. Quantitative estimation of mitochondrial respiration
TEMPO (10 mg/kg b.w.), dissolved in sterile saline, was administered
1 h prior to stress by intra-venous injection. Mdivi-1 (2 mg/kg b.w.)
Mitochondrial respiration was evaluated in terms of oxygen con-
was administered 1 h prior to stress by intra-venous injection. The stock
sumption by Clark-type electrode (Oxytherm System, Hansatech
solution of Mdivi-1 (dissolved in DMSO) was diluted in sterile saline
Instruments Ltd. United Kingdom) in liquid-phase as mentioned before
during injection so as to keep the final concentration of DMSO at 0.1%.
[67]. Respiratory control ratio (RCR) was obtained from the ratio of
At this concentration DMSO did not affect gastric injury induced by
State 3 and State 4 respiration measured in nanomoles of O2 consumed.
stress. RU486 (20 mg/kg b.w.), suspended in a solution of 0.5% CMC
O2 consumed in presence of 1 mM of ADP was represented as state 3
containing 0.05% Tween-20, was administered 40 min prior to stress
respiration while state 4 respiration was recorded after addition of
treatment by oral gavage. Vehicles were administered separately each
15 µM oligomycin, inhibitor of the ATP synthase.
time to check any effect on gastric injury. The dosages of the aforesaid
molecules were decided based on previous reports [60–64]. Animals
4.8. Mitochondrial fatty acid oxidation measurement
were euthanatized and sacrificed after 31/2 h for collection of the sto-
machs. All the experiments were carried out with six to eight animals (n 14
C-labeled palmitate oxidation to 14CO2 was performed as de-
= 6–8) in each group. The quantitative estimation of mucosal lesions
scribed in other studies [68]. Briefly, equal amount of stomach tissue
was scored by an individual unaware of the treatment and expressed as
(45 mg) was taken to perform the assay. The assay mixture contained
“injury index” as described earlier [65]: 0 = no pathology; 1 = 1
1µCi of [1-14C] palmitate pre-incubated in 5% BSA. 14CO2 was trapped
pinhead lesion (1–2 mm); 2 = a medium lesion (3– 4 mm); 4 = a large
in Whatman chromatography paper soaked in 3 M NaOH. The papers
lesion (5–6 mm); and 8 = larger lesion (> 6 mm). The sum of the total
were dried overnight and 14C levels were measured in a scintillation
scores in each set divided by the number of the animals was represented
counter (Perkin-Elmer, U.S.A). The assays were carried out in triplicate
as the mean injury index.
and normalized by tissue weight.

4.3. Ethics statement 4.9. Assay of mitochondrial electron transport chain complex

All animals were housed in the general animal centre of the institute The enzyme assay was performed as described [69]. Mitochondria
(CSIR-Indian Institute of Chemical Biology, Kolkata). Procedures in- were isolated as described above. Subsequently the isolated mi-
volving animals were conducted in accordance with the guidelines of tochondrial fraction was resuspended in the storage buffer and an ali-
Institutional Animal Ethics Committee (IAEC) and Committee for the quot was used for protein estimation by Lowry method. For the complex
Purpose of Control and Supervision of Experiments on Animals I (NADH: ubiquinone oxidoreductase) activity assay isolated mi-
(CPCSEA). The protocols we adapted were approved by the Animal tochondrial samples were freeze thawed in hypotonic buffer and added
Ethics Committee of the institute (CSIR-IICB) registered with the (containing 10 µg of protein) to 700 μl of distilled water in a 1-ml
CPCSEA, India (Permit Number: 147/1999/CPCSEA). Animals were cuvette followed by addition of 100 μl of potassium phosphate buffer
handled with care and efforts were made to minimize their suffering. (0.5 M, pH 7.5), 60 μl of fatty acid–free BSA (50 mg ml−1), 30 μl of

434
R. De et al. Free Radical Biology and Medicine 113 (2017) 424–438

sodium azide (10 mM) and 10 μl of NADH(10 mM). The volume was 0.9% normal saline and incubated with double volume of TBA-TCA
adjusted to 994 μl with distilled water and was mixed by inverting. mixture (0.375% w/v and 15% w/v, respectively) in 0.25 N HCl. The
Reaction was started by addition of 6 μl of ubiquinone (10 mM), mixed reaction mixture was incubated in boiling water bath for 15 min. After
by inverting and the decrease of absorbance at 340 nm was followed for cooling the solution was clarified by high speed centrifugation and
2 min. In parallel, a separate cuvette containing the same quantity of analyzed spectrophotometrically at 535 nm. Tetraethoxypropane was
reagents and sample in presence of 10 μl of 1 mM rotenone solution was used as standard [72].
prepared to measure the decrease of absorbance at 340 nm for 2 min. Isolated mitochondria were quantitated and equal amount of mi-
Specific complex I activity is the rotenone-sensitive activity. tochondrial protein was precipitated with 25% tricholoroacetic acid
and derivatized by incubation with 0.5 ml of 10 mM 2, 4- dini-
4.10. Mitochondrial dehydrogenase assay trophenylhydrazine for 1 h. The protein was subsequently washed with
ethanol: ethylacetate (1:1) solvent for three times and dissolved in
Condition of mitochondrial metabolism was evaluated in terms of 0.6 ml of a solution containing 6 M guanidine-HCl in 20 mM potassium
mitochondrial dehydrogenase activity. Mitochondrial dehydrogenases phosphate buffer (adjusted to pH 2.3 with trifluoroacetic acid) and
are capable of reducing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- centrifuged The clarified supernatant was used for measuring carbonyl
Diphenyltetrazolium Bromide) into purple coloured formazan which formation by spectrophotometric measurement at 362 nm.
can be estimated colorimetrically at 570 nm. Briefly, protein con-
centrations of isolated mitochondria were estimated by Lowry method 4.15. Measurement of cardiolipin content
and equal amounts of mitochondrial protein were incubated with MTT
in PBS at 37 °C in 5% CO2 for 4 h. The samples were then centrifuged at The content of mitochondrial cardiolipin was measured using nonyl
maximum speed and the precipitates were dissolved in equal amounts acridine orange (NAO). Briefly, equal amounts of mitochondria (25 µg)
of anhydrous DMSO. The absorbance of the purple solution was mea- isolated from gastric tissues were used for the assay. The fluorescence of
sured at 570 nm in ELISA reader (BioTek Instruments, Inc., Winooski, each sample was measured in a Biotek fluorimeter (excitation 495 nm;
VT, USA). emission 519 nm, gain set at 50%) after incubation with NAO. Mean
fluorescence of gastric mitochondrial fractions were calculated per
4.11. Measurement of mitochondrial transmembrane potential (ΔΨm) by gram of mitochondrial protein.
tetramethylrhodamine methyl ester (TMRM)
4.16. Confocal immunofluorescence microscopy
Mitochondria were isolated as described earlier [70]. Equal amount
of mitochondria (25 µg) were taken in 100 μl of PBS and were in- Mitochondrial translocation of Drp1, Parkin, GR and nuclear
cubated with TMRM (100 nM) in dark for 15 min at 25 °C. The fluor- translocation of NF-κB were followed by confocal fluorescent im-
escence of each sample was measured in a Hitachi F-7000 fluorescence munohistochemical analyses. Fixed-paraffinized tissue sections were
spectrophotometer (excitation 548 nm, emission 574 nm). processed for antibody staining as mentioned earlier [73]. Blocking was
done in 10% goat serum containing 1% BSA for 2 h at room tempera-
4.12. Isolation of gastric mucosal cells ture. The tissue sections were stained with respective antibodies men-
tioned in Table S1. Nucleus was counter stained with DAPI. The slides
Gastric mucosal cells were isolated as mentioned elsewhere [71] were next observed under 40X oil immersion or 20X objective lens in
with minor modifications. Briefly, mucosal scrapings from control and the Leica TCS-SP8 Confocal microscope (Leica Microsystems, Wetzlar,
drug-treated stomachs were washed and minced in pre-warmed Hank's Germany) as required. All experiments were performed thrice and
balanced salt solution (HBSS) (pH 7.4) containing 100 units/ml peni- confocal micrographs presented were representatives of randomly
cillin and 100 μg/ml streptomycin and suspended in the pre-aerated chosen portion of the gastric mucosal sections. Digital zooming of the
enzyme solution [HBSS containing 0.05% hyaluronidase and 0.1% obtained micrographs was performed as and when required. Post cap-
collagenase] for 80 min, at 37 °C in 5% CO2 under shaking conditions. ture image analysis for colocalization and intensity calculation was
The suspension was then filtered through a sterile nylon mesh. The done in Leica Application Suite X (LAS X) software. Images were
filtrate was centrifuged at 600×g for 10 min, and the cell pellet was cropped and processed globally for enhancement of brightness and
washed with HBSS thrice to get rid of unwanted tissue debris. The re- contrast using Adobe Photoshop CS6. Images were assembled in Cor-
sulting mucosal cells were checked for viability using Trypan blue ex- elDRAW X7.
clusion test and subsequently used for different downstream assays.
4.17. Western blotting
4.13. Measurement of mitochondrial superoxide anion (O2.–) and other
reactive oxidants Total protein was isolated from homogenized gastric mucosal tis-
sues in lysis buffer supplemented with protease and phosphatase in-
Mitochondrial O2.– and other reactive oxidants accumulation in hibitor (Sigma). 80 µg of protein was loaded to 10% polyacrylamide-
isolated gastric mucosal cells were followed by MitoSox staining. SDS gels and subjected to electrophoresis and immunoblotting as de-
DCFDA staining was performed to detect free iron-catalysed formation scribed previously [73]. Immunoblotting of mitochondrial protein was
of reactive oxidants. The cells were analyzed in fluorescence-activated done by incubating the mitochondrial pellets in lysis buffer provided in
cell sorter (FACS) LSR Fortessa, BD; using FACS DIVA software under the isolation kit (Biochain). List of antibodies used were provided in
standard parameters. Table S1. Immuno-reactive bands were detected in Biorad chemidoc
system or by staining with DAB and hydrogen peroxide solution. Den-
4.14. Measurement of oxidation products in mitochondria sitometric analyses of the developed bands were done by ImageJ soft-
ware and values were presented after normalization and fold calcula-
Mitochondrial oxidative damage was measured as described earlier tion relative to control.
by assessing mitochondrial macromolecular oxidation and protein
carbonyl formation [62]. Mitochondrial macromolecule oxidation was 4.18. Analysis of mitochondrial copy number
assayed by measuring thiobarbituric acid reactive substances (TBARS)
formed due to oxidative modification of lipids, proteins and nucleic Measurement of mitochondrial content was assessed in terms of
acids. Equal amount of mitochondrial fraction was homogenized in mtDNA content. Analysis of mtDNA from DNA samples isolated from

435
R. De et al. Free Radical Biology and Medicine 113 (2017) 424–438

rat gastric mucosa was performed as reported elsewhere [74]. Briefly, (R & D systems). Pro-inflammatory cytokines in the gastric tissue lysates
DNA was isolated using Blood & Cell Culture DNA Mini kit (Qiagen) and were estimated by ELISA array kit from Raybiotech and Qiagen.
amplified by quantitative RT-PCR using mitochondrial gene specific
primers. The data were represented as 2-ΔΔCq values normalized with 4.23. Statistical analyses
nuclear gene segment amplification by nuclear gene specific primers.
All primers are listed in Table S2. All experiments were done in triplicates with n = 6–8. Data ob-
tained from experiments were expressed as mean ± Standard Error of
4.19. Assay of caspase-9 and caspase-3 activities Mean (S.E.M.). Calculations of the levels of significance were done with
Mann-Whitney U test when comparing two groups and ANOVA fol-
Caspase activity assays were performed using commercially avail- lowed by Bonferroni's Multiple-Comparisons procedure as a post hoc
able Caspase 9 and Caspase 3 (Calbiochem; Merck, Germany) assay kits test when comparing 3 or more groups. P value less than 0.05
following the manufacturer's protocol. Equal amount of tissue from (P < 0.05) was considered as statistically significant. GraphPad Prism 6
different experimental sets was used for preparing respective gastric and Microsoft Office Excel 2007 software were used for the statistical
mucosal homogenates which were subsequently quantified and used for analysis of the data.
the assay. The mixture was incubated at 37 °C for 2 h; absorbance was
taken at 405 nm for caspase 9 and fluorescence was measured at Acknowledgments
460 nm for caspase 3.
We are grateful to Council of Scientific and Industrial Research,
4.20. Electrophoretic mobility shift assay (EMSA) India for providing fund (BEnD, BSC0206) and offering fellowship to
Rudranil De to carry out this work. We also acknowledge J. C. Bose
EMSA was performed with double-stranded consensus oligonu- National Fellowship (DST), India.
cleotide probes specific to NF-κB with the sequences 5’-
AGTTGAGGGGACTTTCCCAGGC-3’ and 3’-TCAACTCCCCTGAAAGGG Conflict of interest
TCCG-5’ [75]. The probes were 5’ end labeled using [γ-32P] ATP (Per-
kinElmer Life Sciences) as mentioned earlier [76]. Un-reacted label was The authors declare that they have no conflicts of interest with the
washed and removed. Nuclear protein (10 µg) was used for DNA-pro- contents of this article.
tein binding reaction. Complexes were resolved by 5% native PAGE and
the gel was fixed in a solution containing 30% methanol and 10% Author contributions
glacial acetic acid and dried in gel drying apparatus. Dried gel was
visualized by autoradiography. A competition assay was also done by All contributing authors have agreed for the submission of this
incubating the sample for 30 min with cold probe (80-fold excess) on manuscript for publication. U.B. and R.D. conceived the study, per-
ice before adding 32P-labeled probe. Densitometric quantification was formed experiments, analysed data and interpreted results. Animal
performed by ImageJ software. experiments were done by R.D., S.M., S.S., S.J.S. and S.D.S. qPCR were
performed by C.B., and S.N. Confocal microscopy was done by S.M.,
4.21. RNA isolation and real-time RT-PCR A.A.S and D.S. The figures were prepared by R.D. and S.S. manuscript
was written by R.D., S.M. and U.B.
Total RNA from different experimental sets was isolated using the
TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer's in- Appendix A. Supplementary material
structions and estimated in MaestroNano Micro Volume
Spectrophotometer (Life Teb Gen co, Tehran-Iran). Subsequently, after Supplementary data associated with this article can be found in the
rDNase treatment, reverse transcription of the obtained total RNA online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.
(2 µg) was done with oligo-dT18 primer using RevertAid First Strand 009.
cDNA synthesis kit (Thermo Fisher Scientific Inc.). Finally, the obtained
cDNA was used for qPCR using Primers (Table S2) obtained from In- References
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