molecules
Article
Myricitrin, a Glycosyloxyflavone in Myrica esculenta Bark
Ameliorates Diabetic Nephropathy via Improving Glycemic
Status, Reducing Oxidative Stress, and Suppressing Inflammation
Tarun K. Dua 1,2,† , Swarnalata Joardar 1,† , Pratik Chakraborty 1 , Shovonlal Bhowmick 3 , Achintya Saha 3 ,
Vincenzo De Feo 4, * and Saikat Dewanjee 1, *
1
2
3
4
*
†
Citation: Dua, T.K.; Joardar, S.;
Chakraborty, P.; Bhowmick, S.;
Saha, A.; De Feo, V.; Dewanjee, S.
Myricitrin, a Glycosyloxyflavone in
Myrica esculenta Bark Ameliorates
Diabetic Nephropathy via Improving
Glycemic Status, Reducing Oxidative
Stress, and Suppressing Inflammation.
Molecules 2021, 26, 258. https://
doi.org/10.3390/molecules26020258
Academic Editor: Maria Atanassova
Received: 7 December 2020
Accepted: 1 January 2021
Published: 6 January 2021
Advanced Pharmacognosy Research Laboratory, Department of Pharmaceutical Technology,
Jadavpur University, Kolkata 700032, India; tarunkduaju@gmail.com (T.K.D.);
swarnalatajoardar@yahoo.in (S.J.); pratik.chakraborty88@yahoo.com (P.C.)
Department of Pharmaceutical Technology, University of North Bengal, Darjeeling 734013, India
Department of Chemical Technology, University of Calcutta, Kolkata 700009, India;
sovonlal@gmail.com (S.B.); achintya_saha@yahoo.com (A.S.)
Department of Pharmacy, University of Salerno, 84084 Fisciano, Italy
Correspondence: defeo@unisa.it (V.D.F.); saikat.dewanjee@jadavpuruniversity.in (S.D.);
Tel.: +39-089-969-751 (V.D.F.); +91-33-2457-2043 (S.D.)
These authors contributed equally to this work.
Abstract: The present study evaluated the therapeutic potential of myricitrin (Myr), a glycosyloxyflavone extracted from Myrica esculenta bark, against diabetic nephropathy. Myr exhibited a
significant hypoglycemic effect in high fat-fed and a single low-dose streptozotocin-induced type
2 diabetic (T2D) rats. Myr was found to improve glucose uptake by the skeletal muscle via activating
IRS-1/PI3K/Akt/GLUT4 signaling in vitro and in vivo. Myr significantly attenuated high glucose
(HG)-induced toxicity in NRK cells and in the kidneys of T2D rats. In this study, hyperglycemia
caused nephrotoxicity via endorsing oxidative stress and inflammation resulting in the induction
of apoptosis, fibrosis, and inflammatory damages. Myr was found to attenuate oxidative stress via
scavenging/neutralizing oxidative radicals and improving endogenous redox defense through Nrf-2
activation in both in vitro and in vivo systems. Myr was also found to attenuate diabetes-triggered
renal inflammation via suppressing NF-κB activation. Myr inhibited hyperglycemia-induced apoptosis and fibrosis in renal cells evidenced by the changes in the expressions of the apoptotic and
fibrotic factors. The molecular docking predicted the interactions between Myr and different signal
proteins. An in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) study
predicted the drug-likeness character of Myr. Results suggested the possibility of Myr to be a potential
therapeutic agent for diabetic nephropathy in the future.
Keywords: diabetic nephropathy; glucose utilization; inflammation; oxidative stress; Myrica esculenta;
myricitrin; type 2 diabetes mellitus
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Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Type 2 diabetes mellitus (T2D) is a chronic metabolic syndrome accounting for 90–95%
of the total diagnosed cases of diabetes mellitus [1]. The number of T2D cases is increasing
steadily around the world [2]. It is characterized by hyperglycemia which is caused due
to the establishment of insulin resistance, decrease in insulin production, and eventually
loss of β-cell function [3]. Persistent hyperglycemia, hypertension, and dyslipidemia in
T2D collectively impart gluco-lipo toxicity resulting in a number of slowly or rapidly
growing pathological occurrences to the critical organs, which are the major causes of
mortality in T2D [4]. Diabetic nephropathy or diabetic kidney disease is the most common
T2D complication affecting around 40% of T2D patients [5]. Diabetic nephropathy is a
multifunctional degenerative syndrome characterized by albuminuria, glomerular lesions,
Molecules 2021, 26, 258. https://doi.org/10.3390/molecules26020258
https://www.mdpi.com/journal/molecules
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tubulointerstitial fibrosis, and loss of renal filtration rate. Emerging evidence revealed
that high glucose-provoked oxidative stress and inflammation play crucial roles in the
development and progression of diabetic nephropathy via endorsing apoptosis, fibrosis, and other pathological changes [6]. Since hyperglycemia is the primary contributor
to diabetic nephropathy, glycemic control would remain the major therapeutic strategy
against diabetic nephropathy [7]. In addition, several anti-inflammatory agents, such as indomethacin, naproxen, mycophenolate mofetil, and retinoic acid conferred protective roles
in attenuating diabetes-provoked renal injury [2,8]. Moreover, several antioxidants such as
silymarin, bardoxolone methyl, and vitamin C in combination with vitamin E, and pyridoxamine, were found to be clinically successful in alleviating diabetic nephropathy [9,10].
Thus, it would be hypothesized that an antidiabetic agent simultaneously possessing
anti-inflammatory and antioxidant effects would serve as an effective therapeutic agent in
diabetic nephropathy. Naturally occurring plant-derived secondary metabolites have been
known to possess a broad spectrum of pharmacological effect. Thus, there is a scope to
develop potential therapeutic agents from plant-derived molecules to be effective against
T2D and T2D-associated complications.
Myrica esculenta Buch.-Ham. ex D. Don. (Myricaceae) is an evergreen tree growing in
hilly regions of north eastern India and Nepal. Different parts of M. esculenta are widely
used in Ayurveda against several ailments including diabetes, inflammatory diseases, and
infections [11]. M. esculenta bark extract is known for anti-inflammatory, antimicrobial,
wound healing, and antioxidant effects [11–13]. Myricitrin (Myr) (Figure 1), a glycosyloxyflavone, was isolated from the bark extract of M. esculenta. The present investigation
was undertaken to evaluate the protective effect of Myr against diabetic nephropathy.
Special attention was given to reveal the molecular mechanism behind the protective effect
of Myr using suitable in vitro and in vivo preclinical assays. Finally, in silico absorption,
distribution, metabolism, excretion, and toxicity (ADMET) and molecular docking analyses were executed to predict the drug likeliness and the interactions between Myr and
signal proteins.
Figure 1. Structure of Myr.
2. Results
2.1. In Vitro Assays
2.1.1. Effect on Glucose Uptake In Vitro
Skeletal muscle plays a key role in glucose metabolism. In this study, the effect of
Myr treatment on the D-glucose uptake by rat skeletal (L6) myoblast was measured. The
effect of Myr on the signal proteins involved in glucose metabolism in the L6 myoblast
was estimated. A set of L6 cells cultured with 5.5 mM glucose served as a control (HG−),
and the glucose utilization of the HG− set was assigned to 100%. High glucose (HG)
−
caused a ~40% reduction in glucose uptake
(p < 0.01) by L6 myoblast, while Myr (20, 30,
−
and 50 µM)
⁓ treatment significantly reversed (p < 0.05–0.01) the suppression of glucose
utilization by the L6 cells (Figure 2a). The optimum effect of Myr was observed at the dose
of 30 µM, at which Myr exhibited glucose utilization of ~87% (p < 0.01) by HG-exposed
L6 myotubes. Based on the observed effect in the glucose uptake study, Myr at 30 µM
was chosen as the optimum dose for studying the effect on the signal proteins involved in
Molecules 2021, 26, 258
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glucose uptake by the⁓skeletal muscle cells (Figure 2b,c). In this study, HG treatment caused
suppression (~0.4-fold, p < 0.01) of phosphoinositide 3-kinases (PI3K) (p 85) expression in
murine skeletal muscle cells (Figure 2c). Phosphorylation of insulin receptor substrate 1
(IRS-1) and protein kinase B (Akt) proteins was downregulated in HG-exposed L6 myoblast
⁓
(Figure 2c). Thus, considerably low P-IRS-1/total IRS (~0.4-fold, p < 0.01) and P-Akt/total
⁓
Akt (~0.6-fold, p < 0.01) ratios were observed in the HG-treated L6 myoblast (Figure 2c).
⁓
In addition, HG treatment caused downregulation (~0.5-fold, p < 0.01) in the expression of
glucose transporter type 4 (GLUT4) in the membrane in murine myoblast, which signified
the suppression of GLUT4 translocation to the membrane (Figure 2c). In contrast, Myr
(30 µM) reciprocated (p < 0.01) HG-provoked reduction in the expression of PI3K, P-IRS-1,
P-Akt, and GLUT4 (in the membrane) in L6 myoblast (Figure 2c). No major change was
observed in the expression of either of the signal proteins in Myr-treated murine skeletal
muscle cells cultured in HG-condition.
Figure 2. Effect of Myr on glucose uptake events by the skeletal muscle cells. (a) Effect of Myr on glucose uptake by L6 cells.
(b) The immunoblot images showing effects of Myr in the expressions of signal proteins involved in glucose uptake by L6
cells. (c) The graphs showing the densitometric analysis of immunoblots. Myr was able to improve glucose uptake by L6
cells by activating PI3K expression, IRS1 phosphorylation, Akt phosphorylation, and GLUT4 translocation to membrane.
β
The intensity of normal control band was assigned 1. β-actin served as the loading control for normalization. Data are
−
represented as the mean ± SD, n (number of plates) = 3. # Values significantly (p < 0.01) differed from the control (HG−)
group. * Values significantly (p < 0.05) differed from the HG-treated (HG+) group. ** Values significantly (p < 0.01) differed
from the HG-treated (HG+) group.
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2.1.2. Effect of Myr on HG-induced Cytotoxicity in Kidney Cells
In this study, the concentration and time-dependent cytotoxic effects of D-glucose on
murine kidney cells were estimated to optimize the glucose concentration and exposure
time for an in vitro diabetic nephropathy model. The normal rat kidney (NRK) cells were
exposed to different concentrations of D-glucose (5.5, 10, 20, 30, 40 mM) for 72 h. D-glucose
− −), and the cell viability was assigned to 100%.
at 5.5 mM served as the control (HG
D-glucose caused the loss of cell viability in a concentration-dependent manner with a
⁓ of ~46% (p < 0.01) at 40 mM (Figure 3a). To optimize the exposure
maximum reduction
time, NRK cells were incubated with 30 mM of D-glucose for 6, 12, 24, 48, and 72 h.
A gradual decrease in the viability of NRK cells was observed over time, with substantial
⁓
(p < 0.05–0.01) differences between 24 and 72 h and a maximum reduction
of ~39% (p < 0.01)
at 72 h (Figure 3b). On the basis of the observed effects, the concentration of D-glucose of
30 mM and an incubation time of 48 h were chosen as optimum dose and exposure time,
respectively, for in vitro model of diabetic nephropathy. To observe the effect of Myr (alone)
on the viability of murine kidney cells, NRK cells were exposed to different concentrations
of Myr (10, 20, 30, and 50 µM). No substantial change was observed in the viability of NRK
cells (Figure 3c). To investigate the effect of Myr on HG-induced renal cell injury, NRK
cells were incubated D-glucose (30 mM) with or without Myr (30 µM) for 48 h. The cell
viability in the control set was assigned 100% (Figure 3d). HG caused a reduction (~40%,
p⁓ < 0.01) in the viability of NRK cells. In contrast, Myr treatment rescued (p < 0.01) the
HG-induced decrease in the cell viability of NRK cells (Figure 3d). Hoechst nuclear staining
was executed to visualize the protective effect of Myr in HG-induced cytotoxicity in NRK
cells (Figure 3e). In this study, HG (30 mM) caused a substantial reduction in the number of
visible nuclei and the presence of apoptotic nuclei characterized by nuclear fragmentation,
condensation, and shrinkage (Figure 3e). In contrast, Myr (30 µM) treatment attenuated
a HG-induced decrease in the nuclear count and restored nuclear morphology to near
normal status (Figure 3e).
Figure 3. Cont.
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Figure 3. Cytotoxic effect of D-glucose on murine renal cells and protective effect of Myr. (a) Concentration dependent cytotoxic effect
of D-glucose on NRK cells. (b) Time dependent cytotoxic effect of D-glucose on NRK cells. (c) Effect of Myr on the viability of NRK
− HG-induced toxicity in NRK cells as observed in cell viability
cells cultured in controlled (HG−) condition. (d) Effect of Myr against
assay. (e) Effect of Myr against HG-induced toxicity in NRK cells as observed in Hoechst nuclear staining. HG caused loss of cell
viability of NRK cells in both concentration and time dependent manner. Myr culture in HG-condition did not affect cell viability. Myr
reciprocated HG-induced loss of cell viability as seen in cell viability assay and Hoechst nuclear staining. Data are represented as the
mean ± SD, n (number of plates) = 3. $ Values significantly (p < 0.05) differed from the control (HG−) group. # Values significantly
−
(p < 0.01) differed
from the control (HG−) group. ** Values significantly (p < 0.01) differed from the HG-treated (HG+) group.
−
2.1.3. Effect on Redox Status in Kidney Cells
HG (30 mM) caused a substantial (p < 0.01) increase in reactive oxygen species (ROS)
accumulation in NRK cells evidenced by an increase in 2’,7’-dichlorodihydrofluorescein
(DCF) fluorescence as compared to the control (Figure 4a). The respective fluorescence
was quantified to estimate the quantity of intracellular ROS level (Figure 4b). In this study,
HG-treated NRK cells exhibited a ~9.1-fold
(p < 0.01) increase in ROS accumulation as
⁓
compared to the control (Figure 4b). The HG also caused a⁓ ~8.6- (p < 0.01) and
⁓ ~2.5-fold
(p < 0.01) increase in the levels of nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase and nitric oxide (NO) in NRK cells, respectively (Figure 4c,d). Consequently, the
degree of lipid peroxidation and the extent of protein carbonylation were increased to ~1.6(p
(p < 0.01), respectively, in HG-exposed NRK cells (Figure 4e,f). In
⁓ < 0.01) and ~2.1-fold
⁓
contrast, Myr (30 µM) treatment could attenuate (p < 0.01) HG-induced increase in the
levels of ROS, NADPH oxidase, NO, thiobarbituric acid reactive substances (TBARS), and
carbonylated proteins in NRK cells (Figure 4a–f). HG treatment also reduced (p < 0.01) the
levels of endogenous antioxidant enzymes, such as catalase (CAT), superoxide dismutase
(SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) in murine renal
cells (Figure 4g–j). On the other hand, Myr (30 µM) treatment improved (p < 0.01) the
levels of endogenous antioxidant enzymes in HG-exposed NRK cells to near normal status
(Figure 4g–j). In addition, HG treatment caused ~0.7- (p < 0.01) and ~0.3-fold (p < 0.01)
⁓
⁓
reductions in reduced glutathione (GSH) level and redox ratio (GSH/GSSG) in NRK cells,
respectively (Figure 4k,l). However, Myr (30 µM) treatment could rescue (p < 0.01) the HGinduced decrease in GSH level and redox ratio in NRK cells to near normal status (Figure 4k,l).
The cells incubated with Myr (30 µM) under the control (HG−) condition did not show any
substantial change in either of the aforementioned redox parameters (Figure 4).−
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Figure 4. The effects of Myr on HG-induced redox imbalance on NRK cells. (a) The level of intracellular ROS was observed
in cells by DCF fluorescence. (b) The graph showing the quantity of intracellular ROS. (c–l) The graphs showing the effects
on NADPH oxidase (c), NO (d), TBARS (e), protein carbonyl (f), CAT (g), SOD (h), GPx (i), GST (j), GSH (k), and redox
ratio (l). Myr was effective in reducing HG-induced increase in intracellular ROS, NADPH oxidase, NO, TBARS, and
protein carbonyl levels, while it increased CAT, SOD, GPx, GST, GSH, and redox ration in HG-exposed NRK cells. Data
−
are represented as the mean ± SD, n (number of plates) = 3. # Values significantly (p < 0.01) differed from the control
(HG− group. ** Values significantly (p < 0.01) differed from the HG-treated (HG+) group. SOD unit, “U”, is defined as
inhibition (µ-moles) of nitro blue tetrazolium (NBT)-reduction/min. CAT unit, “U”, is defined as H2 O2 consumption/min.
2.1.4. Effect on Signal Transduction in Kidney Cells
In this study, HG (30 mM) treatment induced apoptosis to murine renal cells in vitro,
evidenced by the activation (p < 0.01) of protein expressions of B-cell lymphoma 2-associated
death promoter (Bad) in the mitochondria, cytochrome C (Cyt C) in the cytosol, cleaved
caspase 9, and cleaved caspase 3 with concomitant downregulation (p < 0.01) in the expression
of B-cell lymphoma 2 (Bcl-2) protein (Figure 5a,b). On the other hand, Myr (30 µM) treatment
Molecules 2021, 26, 258
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κ α
could suppress HG-induced apoptosis in NRK cells by reciprocating (p < 0.01) the expression
⁓
of the aforementioned signal proteins involved in the apoptosis pathway (Figure 5a,b). HG
κ
⁓
(30 mM) treatment enhanced the expressions of phosphorylated nuclear factor of kappa light
polypeptide gene enhancer in B-cells inhibitor (P-IκBα) in the cytosol to ~3.1 folds (p < 0.01)
and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (P-NF-κB)
κ α
κ
to ~2.7 folds (p < 0.01) in the nucleus, which signified the induction of inflammation in HGexposed NRK cells (Figure 5c,d). In contrast, Myr (30 µM) treatment could reduce HG-induced
⁓
inflammation by reducing the expressions of P-IκBα (p < 0.01) in the cytosol and P-NF-κB
⁓ suppressed nuclear factor erythroid
(p < 0.01) in the nucleus (Figure 5c,d). In addition, HG
2-related factor 2 (Nrf-2) signaling of endogenous redox defense evidenced by a ~0.4-fold
(p < 0.01) reduction in phosphorylated-Nrf-2 (P-Nrf-2) expression in the nucleus and ~2.7-fold
(p < 0.01) activation of Kelch-like ECH-associated protein 1 (Keap1) expression in the cytosol
β
(Figure 5e,f). In contrast, Myr (30 µM) treatment could endorse redox defense in NRK cells
by upregulating (p < 0.01) Nrf-2 signaling (Figure 5e,f). In this study, HG (30 mM) endorsed
fibrosis to NRK cells, evidenced by the activation (p < 0.01) of transforming growth factor-beta
1 (TGF-β1), phosphorylated mothers against decapentaplegic homolog 3 (P-Smad3), and
collagen-IV and suppression (p < 0.01) of mothers against decapentaplegic homolog 7 (Smad7)
−
protein (Figure 5g,h). On the other hand, Myr (30 µM) treatment could attenuate HG-induced
fibrosis in NRK cells by reciprocating the expressions of the aforementioned fibrotic factors
(Figure 5g,h). The cells incubated with Myr (30 µM) under the control (HG−) condition did
not show any considerable change in either of the aforementioned signaling events (Figure 5).
Figure 5. Cont.
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Figure 5. The effects of Myr on HG-induced signaling on NRK cells. (a) The immunoblot images showing the effects on the expressions
of Bad (in mitochondria), Bcl-2, Cyt C (in cytosol), cleaved caspase 9, and cleaved caspase 3 involved in apoptosis. (b) The graphs
showing the densitometric analysis of the bands of apoptotic factors. (c) The immunoblot images showing the effects on the expressions
α
of P-IκBα (in cytosol), and P-NF-κB (in nucleus) κinvolved
in inflammation. κ(d) The graphs showing the densitometric analysis of
the bands of inflammatory factors. (e) The immunoblot images showing the effects on the expressions of Keap1 (in cytosol), and
P-Nrf-2 (in nucleus) involved in redox defense. (f) The graphs showing the densitometric analysis of the bands of redox defense
factors. (g) The immunoblot
β images showing the effects on the expressions of TGF-β1, P-Smad3, Smad7, and collagen IV involved in
fibrosis. (h) The graphs showing the densitometric analysis of the bands of fibrotic factors. Myr was effective in reducing HG-induced
increase in the expressions of Bad (in mitochondria), Cyt C (in cytosol), cleaved caspase
(in cytosol), and
κ α 9, cleaved caspase 3, P-IκBα
κ
P-NF-κB (in nucleus), Keap1 β(in cytosol), TGF-β1, P-Smad3, and collagen IV, while it activated the expressions of Bcl-2, P-Nrf-2 (in
β
nucleus), and Smad7. The intensity of normal control band was assigned
1. β-actin served as the loading control for normalization.
Data are represented as the mean ± SD, n (number of plates) = 3. # Values significantly (p < 0.01) differed from the control−(HG−)
group. ** Values significantly (p < 0.01) differed from the HG-treated (HG+) group.
2.2. In Vivo Assays
2.2.1. Effect on Fasting Blood Glucose Level, Body Mass Gain, Foods, and Water Intake
In this study, T2D rats exhibited significantly high (p < 0.01) fasting blood glucose
levels when compared with normal rats (Figure 6a). In contrast, Myr (300 mg/kg) treatment
⁓reduced (p < 0.05–0.01) fasting blood glucose level with a maximum decrease of ~35.1%
(p < 0.01) on day 28 (Figure 6a). The T2D rats also exhibited an increase (p < 0.05–0.01) in
body mass gain when compared to normal rats (Figure 6b). On day 28, T2D rats exhibited
⁓
a ~2-fold increase in body mass gain when compared to normal rats. In contrast, Myr
(300 mg/kg) treatment reciprocated (p < 0.05–0.01) body mass gain on day 14th after the
therapeutic schedule as compared to T2D rats (Figure 6b). In this study, T2D rats exhibited
the signs of polyphagia and polydipsia evidenced by the elevation (p < 0.01) in the food and
Molecules 2021, 26, 258
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water intakes when compared with normal rats (Figure 6c,d). However, Myr (300 mg/kg)
treatment reversed polyphagia (p < 0.05–0.01) and polydipsia (p < 0.01) on day 14th after
the therapeutic schedule compared to T2D rats (Figure 6c,d). The non-diabetic rats that
received Myr (300 mg/kg) did not show any significant change in fasting blood glucose
level, body weight, food consumption, or water intake as compared to vehicle-treated
non-diabetic rats (normal control) (Figure 6).
Figure 6. Effects of Myr on fasting blood glucose level (a), body mass gain (b), food intake (c), and water intake (d) in T2D
rats. Myr was effective in reducing fasting blood glucose level, body mass gain, food intake, and water intake in T2D rats.
Data are expressed as mean ± SD (n = 6). $ Values significantly (p < 0.05) differed from the normal control (T2D−) group.
−
# Values significantly (p < 0.01) differed from the normal control (T2D−) group. * Values significantly (p < 0.05) differed
−
from the diabetic control (T2D+) group. ** Values significantly (p < 0.01) differed from the diabetic control (T2D+) group.
2.2.2. Effects on Serum Insulin Level, HOMA-IR, and HOMA-β β
In this study, T2D rats exhibited a ~2.6-fold (p < 0.01) increase in fasting blood glucose
⁓
level with concomitant ~0.7-fold reduction (p < 0.01) in serum insulin level on day 29 when
⁓
compared with normal rats (Figure 7a,b). In contrast, Myr (300 mg/kg) treatment caused
the reversal (p < 0.01) of hyperglycemia and hypoinsulinemia in T2D rats (Figure 7a,b). In
this study, the homeostatic model assessment (HOMA) was executed to evaluate β-cell
function (HOMA-β) and insulin resistance (HOMA-IR). Almost a 1.8-fold (p < 0.01) increaseβ
in HOMA-IR score
β in T2D rats compared to normal rats signified the establishment of
insulin resistance in T2D rats (Figure 7c). In this study, ~0.3-fold reduction in HOMA-β
score in T2D rats compared to normal rats suggested the reduction
in β-cell functions
⁓
(Figure 7d). On the other hand, Myr (300 mg/kg) treatment could reduce (p < 0.01) HOMAβ
β
IR and improved (p < 0.01) HOMA-β scores in T2D rats, which inferred that Myr could
β
β
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reciprocate insulin resistance and restored β-cell functions in T2D rats (Figure 7c,d). The
non-diabetic rats that received Myr (300 mg/kg) did not show any substantial change in
fasting blood glucose (day 29), serum insulin, HOMA-IR, and HOMA-β levels as compared
to normal control rats (Figure 7).
Figure 7. Effect of Myr on fasting blood glucose level (mmol/L) (a), serum insulin (U/L) (b), HOMA-IR, (c) score and
HOMA-β score (d) on day 29 of post-treatment in T2D rats. Myr was effective in reducing blood glucose level and HOMA-IR
score in T2D rats, while it improved serum insulin
and HOMA-β score. Data are expressed as mean ± SD (n = 6). # Values
β
significantly (p < 0.01) differed
− from the normal control (T2D−) group. ** Values significantly (p < 0.01) differed from
the diabetic control (T2D+) group. HOMA-IR = (Fasting serum insulin in U/L × Fasting blood
glucose inmmol/L)/22.5;
β
HOMA- β = (Fasting serum insulin in U/L × 20/Fasting blood glucose in mmol/L)—3.5.
β
2.2.3. Effects on Serum Biochemical Parameters
In this study, considerably (p < 0.01) high levels of total cholesterol, triglycerides, and
low-density lipoprotein (LDL)-cholesterol was observed in the sera of T2D rats (Table 1).
T2D rats also showed a⁓~0.6-fold (p < 0.01) decrease in high-density lipoprotein (HDL)cholesterol level (Table 1). These changes in serum lipid profile indicated the establishment
of dyslipidemia in T2D rats. In contrast, Myr (300 mg/kg) treatment reversed total cholesterol (p < 0.05), triglycerides (p < 0.01), HDL-cholesterol (p < 0.05), and LDL-cholesterol
(p < 0.01) levels in the sera of T2D rats as compared to T2D control animals (Table 1).
In addition, T2D rats exhibited high (p < 0.01) levels of glycosylated-hemoglobin, lactate
dehydrogenase (LDH), creatine kinase (CK), urea, uric acid, creatinine, C-reactive protein,
and advanced glycation end products (AGEs) in the sera as compared to the normal control
group (Table 1). However, Myr (300 mg/kg) treatment reversed glycosylated-hemoglobin
(p < 0.05), LDH (p < 0.05), CK (p < 0.01), urea (p < 0.01), uric acid (p < 0.01), creatinine
(p < 0.01), C-reactive protein (p < 0.01), and AGEs (p < 0.05) levels in the sera of T2D
Molecules 2021, 26, 258
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rats as compared to T2D control animals (Table 1). The non-diabetic rats that received
Myr (300 mg/kg) did not show any major change in either of the aforementioned serum
biochemical parameters as compared to vehicle-treated non-diabetic rats (Table 1).
Table 1. Effects on serum biochemical parameters of experimental rats.
Parameters
Group I
Group II
Group III
Group IV
Total cholesterol (mg/dL)
HDL cholesterol (mg/dL)
LDL cholesterol (mg/dL)
Triglycerides (mg/dL)
Glyco-haemoglobin (mg/g haemoglobin)
LDH (U/L)
CK (IU/mg of protein)
Urea (mg/dL)
Uric acid (mg/dL)
Creatinine (mg/dL)
C-reactive protein (mg/dL)
AGEs (µg/mL)
84.32 ± 7.54
35.43 ± 2.89
25.04 ± 2.34
118.67 ± 9.24
0.28 ± 0.03
164.22 ± 14.27
10.45 ± 1.15
18.79 ± 1.91
2.11 ± 0.23
0.42 ± 0.05
1.32 ± 0.17
438.17 ± 37.28
83.21 ± 8.02
37.17 ± 3.87
21.79 ± 2.11
121.22 ± 10.22
0.27 ± 0.02
159.87 ± 13.48
9.87 ± 1.01
19.63 ± 1.91
2.04 ± 0.17
0.43 ± 0.03
1.26 ± 0.13
416.22 ± 40.24
156.33 ± 14.45 #
20.22 ± 2.43 #
98.67± 7.91 #
187.22 ± 16.43 #
0.71 ± 0.08 #
256.69 ± 24.75 #
19.22 ± 2.07 #
71.25 ± 6.62 #
3.67 ± 0.42 #
0.73 ± 0.08 #
2.57 ± 0.29 #
769.37 ± 80.72 #
137.67 ± 10.98 *
29.50 ± 1.67 *
76.82 ± 6.82 **
156.74 ± 14.87 **
0.62 ± 0.05 *
221.24 ± 20.11 *
14.55 ± 1.49 **
56.29 ± 5.18 **
2.87 ± 0.31 **
0.56 ± 0.05 **
1.78 ± 0.20 **
659.37 ± 71.26 *
Data are expressed as mean ± SD (n = 6). # p < 0.01 compared with Group I; * p< 0.05 compared with Group II; ** p< 0.01 compared with
Group II. Group I: normal control; Group II: normal rats treated with Myr (300 mg/kg, p.o); Group III: T2D control rats; Group IV: T2D rats
treated with Myr (300 mg/kg, p.o).
2.2.4. Effects on Signal Proteins in Skeletal Muscle
The immunoblot analyses were performed to study the effect on the signal proteins
involved in glucose metabolism in the skeletal muscle of rats (Figure 8a,b). In this study,
T2D caused ~0.5-fold (p < 0.01) suppression of PI3K (p 85) expression in the skeletal muscle
of rats (Figure 8b). The extents of IRS-1 and Akt phosphorylation were also decreased,
resulting in considerably low P-IRS-1/total IRS (~0.4-fold, p < 0.01) and P-Akt/total Akt
(~0.5-fold, p < 0.01) ratios in the skeletal muscle of T2D rats. In addition, ~0.6-fold (p < 0.01)
downregulation in the expression of GLUT4 was observed in the membrane fraction of the
skeletal muscle of T2D rats (Figure 8b). In contrast, Myr (300 mg/kg) treatment rescued
(p < 0.01) the reduction in PI3K, P-IRS-1, P-Akt, and GLUT4 (in the membrane) expressions
in the skeletal muscle of T2D rats (Figure 8b). The non-diabetic rats that received Myr
(300 mg/kg) did not show any considerable change in either of the aforementioned signal
proteins involved in glucose metabolism as compared to vehicle-treated non-diabetic
rats (Figure 8a,b).
2.2.5. Effects on Kidney Mass and Urine Parameters
In this study, T2D rats exhibited ~38% (p < 0.01) increase in the kidney mass as
measured on day 29 (Table 2). On the other hand, Myr (300 mg/kg) treatment reinstated
(p < 0.05) the kidney mass in T2D rats to near normal status (Table 2). T2D rats exhibited a
significantly (p < 0.01) high level of albumin in urine with simultaneous reduction (p < 0.01)
in urinary creatinine level (Table 2). In contrast, Myr (300 mg/kg) treatment reversed
(p < 0.01) albumin and creatinine levels in the urine of T2D rats as compared to T2D control
animals (Table 2).
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Figure 8. Effect of Myr on glucose uptake events by the skeletal muscle in T2D rats. (a) The immunoblot images showing
effects of Myr in the expressions of signal proteins involved in glucose uptake by rat soleus muscle. (b) The graphs showing
the densitometric analysis of immunoblots. Myr was able to improve glucose uptake by skeletal muscle by activating
of PI3K expression, IRS1 phosphorylation, Akt phosphorylation, and GLUT4 translocation to membrane. The intensity
β
of normal control band was assigned 1. β-actin served as the loading control for normalization. Data are expressed as
−
mean ± SD (n = 6). # Values significantly (p < 0.01) differed from the normal control (T2D−) group. ** Values significantly
(p < 0.01) differed from the diabetic control (T2D+) group.
Table 2. Effect on kidney mass and renal function-related urine parameters.
Parameters
Group I
Kidney mass (g)
Urinary creatinine (mg/dL)
Urinary albumin (mg/dL)
0.98 ± 0.13
56.87 ± 6.12
2.74 ± 0.33
Group II
0.96 ± 0.09
57.83 ± 5.89
2.79 ± 0.28
Group III
Group IV
#
1.35 ± 0.17
21.32 ± 2.37 #
9.84 ± 1.13 #
1.12 ± 0.14 *
38.14 ± 4.26 **
6.87 ± 0.67 **
Data are expressed as mean ± SD (n = 6). # p < 0.01 compared with Group I; * p < 0.05 compared with Group II; ** p < 0.01 compared with
Group II. Group I: Normal control; Group II: Normal rats treated with Myr (300 mg/kg, p.o); Group III: T2D control rats; Group IV: T2D
rats treated with Myr (300 mg/kg, p.o).
2.2.6. Effects on Renal Polyol Enzymes⁓
⁓
⁓ in
In this study, T2D rats exhibited ~3- (p < 0.01) and ~1.8-fold (p < 0.01) increases
the levels of aldose reductase (Figure 9a) and sorbitol dehydrogenase (Figure 9b) with a
~0.8-fold (p < 0.01) depletion in glyoxalase-I level (Figure 9c) in the renal tissue as compared
to normal rats. In contrast, Myr (300 mg/kg) treatment reduced aldose reductase (p < 0.01)
and sorbitol dehydrogenase (p < 0.05) levels and improved glyoxalase-I level (p < 0.05) in
the renal tissue homogenate of T2D rats as compared to T2D control animals (Figure 9a–c).
However, the non-diabetic rats that received Myr (300 mg/kg) did not show any major
change in either of the aforementioned polyol enzymes as compared to vehicle-treated
non-diabetic rats (Figure 9).
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Figure 9. Effect of Myr on polyol enzymes, such as aldose reductase (a), sorbitol dehydrogenase (b), and glyoxalase-I (c) in the
kidneys of T2D rats. Myr was able to decrease polyol activation in the kidneys of T2D rats. Data are expressed as mean ± SD
−
(n = 6). # Values significantly (p < 0.01) differed from the normal control (T2D−) group. * Values
significantly (p < 0.05) differed
from the diabetic control (T2D+) group. ** Values significantly (p < 0.01) differed from the−diabetic control (T2D+) group.
2.2.7. Effect on Redox Status in Kidney
In this study, a ~1.8-fold
(p < 0.01) high level of intercellular ROS was observed in the
⁓
kidneys of T2D rats (Figure 10a). The levels of intracellular NADPH oxidase (Figure 10b)
⁓
and NO (Figure 10c) were amplified to ⁓~6.1- (p < 0.01) and
⁓ ~2.4-fold (p < 0.01), respectively,
in the renal tissue of T2D rats. Consequently, the degree of lipid peroxidation (TBRAS
⁓
⁓
levels) and the extent of protein carbonylation were enhanced
⁓ to ~2.4- (p < 0.01)
⁓ and
~1.6-fold (p < 0.01), respectively, in the renal tissue of T2D rats (Figure 10d,e). In contrast,
⁓
⁓
Myr (300 mg/kg) treatment significantly reduced T2D-induced augmentation of ROS
(p < 0.01), NO (p < 0.01), NADPH oxidase (p < 0.01), TBRAS (p < 0.05), and carbonylated
protein (p < 0.05) levels in the renal tissue of T2D rats as compared to T2D control rats
(Figure 10a–e). The levels of the endogenous antioxidant enzymes, such as CAT, SOD,
GPx, and GST, were decreased (p < 0.01) in the renal tissues of T2D rats as compared to
the normal rats (Figure 10f–i). On the other hand, Myr (300 mg/kg) treatment improved
(p < 0.01) the levels of the aforementioned antioxidant enzymes in the renal tissue of
T2D rats as compared to the T2D control⁓ group (Figure 10f–i). A ~0.6-fold (p < 0.01)
⁓ which resulted in a ~0.5-fold
reduction in GSH was observed in the renal⁓tissue of T2D rats,
(p < 0.01) decrease in redox ratio as compared to non-diabetic
⁓ rats (Figure 10j,k). However,
Myr (300 mg/kg) treatment increased in renal GSH (p < 0.01) and redox ratio (p < 0.01)
⁓
in T2D rats as compared to T2D control animals
(Figure 10j,k). A ~2.3-fold (p < 0.01)
increment in the extent of DNA oxidation was
⁓ observed in the renal tissue of T2D rats;
however, Myr (300 mg/kg) treatment reduced the extent of DNA oxidation in the renal
tissue of T2D rats as compared to the T2D control group (Figure 10l). Interestingly, Myr
(300 mg/kg) treatment to the non-diabetic rats did not show any substantial change in
either of the aforementioned redox status-associated parameters as compared to normal
control rats (Figure 10).
Figure 10. Cont.
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Figure 10. The effects of Myr on redox imbalance in the kidneys of T2D rats. The graphs showing the effect on intracellular ROS
(a), NADPH oxidase (b), NO (c), TBARS (d), protein carbonyl (e), CAT (f), SOD (g), GPx (h), GST (i), GSH (j), redox ratio (k), and
DNA oxidation (l). Myr was effective in reducing T2D-induced increase in the levels of intracellular ROS, NADPH oxidase, NO, lipid
peroxidation, protein carbonylation, and DNA oxidation, while it increased CAT, SOD, GPx, GST, GSH, and redox ration. Data are
expressed as mean ± SD (n =−6). # Values significantly (p < 0.01) differed from the normal control (T2D−) group. * Values significantly
(p < 0.05) differed from the diabetic control (T2D+) group. ** Values significantly (p < 0.01) differed from the diabetic control (T2D+) group.
SOD unit, “U”, is defined as inhibition (µ-moles) of nitro blue tetrazolium (NBT)-reduction/min. CAT unit, “U”, is defined as H2 O2
consumption/min.
2.2.8. Effects on Renal Inflammation and Fibrosis
α
β
To study the effect
of Myr on βpro-inflammatory
markers, the levels of tumor necro⁓
⁓
⁓
sis factor-alpha (TNF-α), interleukin-1β (IL-1β), and
interleukin-6 (IL-6)
were estimated
α
β
(Figure 11a). In this study, T2D caused ~1.5- (p < 0.01), ~1.8- (p < 0.01), and ~1.7-fold
(p < 0.01) increases in the levels of TNF-α, IL-1β, and IL-6, respectively, in the kidneys
α
β
of the experimental rats as compared to non-diabetic rats (Figure 11a). In contrast, Myr
(300 mg/kg) treatment reciprocated diabetes-provoked increase in the levels of TNF-α
(p < 0.01), IL-1β (p < 0.01), and IL-6 (p < 0.05) in the renal tissue homogenate of T2D
β
rats as compared to T2D control animals (Figure
11a). In search
of the effect of Myr
on
⁓
⁓
⁓
fibrotic markers, the levels of TGF-β1, collagen
IV,
and
hydroxyproline
in
the
renal
tissue
β
homogenates were estimated (Figure 11b). In this study, T2D caused ~1.5- (p < 0.01),
~2.9- (p < 0.01), and ~2.0-fold (p < 0.01) increases in the levels of TGF-β1, collagen IV, and
hydroxyproline,
respectively, in the renal tissue homogenate of T2D rats as compared to
β
non-diabetic rats (Figure 11b). In contrast, Myr (300 mg/kg) treatment caused a significant
reduction in the levels of TGF- β1 (p < 0.01), collagen IV (p < 0.01), and hydroxyproline
(p < 0.05) in the renal tissue homogenate of T2D rats as compared to T2D control animals
(Figure 11b). Myr (300 mg/kg) treatment to the non-diabetic rats did not show any major
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change in either of the aforementioned pro-inflammatory or fibrotic markers as compared
to normal control rats (Figure 11).
Figure 11. The effects of Myr on renal inflammation and fibrosis in T2D rats. (a) The graphs showing the effects on TNF-α,
α
β collagen IV, and hydroxyproline. Data are expressed as
IL-1β, βand IL-6. (b) The graphs showing the effects on TGF-β1,
− −) group. * Values significantly
mean ± SD (n = 6). # Values significantly (p < 0.01) differed from the normal control (T2D
(p < 0.05) differed from the diabetic control (T2D+) group. ** Values significantly (p < 0.01) differed from the diabetic control
(T2D+) group.
2.2.9. Effect on Signal Transduction in Kidney of T2D Rats
In this study, T2D endorsed apoptosis to the renal cells of rats evidenced by the activation
of pro-apoptotic Bad protein and suppression
⁓ of anti-apoptotic Bcl-2 protein in the kidney
cells of T2D rats resulting in a ~3.2-fold (p < 0.01) increase in mitochondrial
Bad/Bcl-2
ratio
⁓
⁓
(Figure 12a,b).
In
addition,
T2D
rats
exhibited
~2.9(p
<
0.01),
~2.9(p
<
0.01),
and
~2.2-fold
⁓
(p < 0.01) increases in cytosolic Cyt C, cleaved caspase 9, and cleaved caspase 3 expressions
in the renal cells, respectively (Figure 12a,b). In contrast, Myr (300 mg/kg) treatment could
attenuate (p < 0.01) the activation of Bad, Cyt C, caspase 9, and caspase 3 in the renal cells
of T2D rats (Figure 12a,b). In addition, Myr (300 mg/kg) treatment activated (p < 0.01)
Bcl-2 expression in the kidneys of T2D rats (Figure 12a,b). Thus, experimental observation
signified that Myr could attenuate T2D-induced apoptosis in the renal cells of experimental
rats. T2D can also endorse renal inflammation.
In this study,⁓T2D caused activation of P-IκBα
κ α
κ
(in the cytosol) to ~2.7-fold
(p
<
0.01)
and
P-NF-κB
(in the nucleus) to ~2.2-fold (p < 0.01)
⁓
in kidneys of experimental rats, which signified the induction of renal inflammation in the
diabetic milieu (Figure 12c,d). In contrast, Myr (300 mg/kg) treatment could reduce renal
inflammation
κ α by reducing the expressions
κ of cytosolic P-IκBα (p < 0.01) and nuclear P-NF-κB
(p < 0.01) in the kidneys of T2D rats (Figure 12c,d). The experimental observation signified
that Myr could attenuate T2D-induced inflammation in the kidneys of experimental rats.
In addition, T2D caused suppression of Nrf-2 signaling of endogenous redox defense in
⁓
the renal cells evidenced by a ~0.6-fold (p < 0.01) reduction in P-Nrf-2 expression in the
⁓
nucleus and ~2.6-fold (p < 0.01) activation of Keap1 expression in the cytosol (Figure 12e,f).
In contrast, Myr (300 mg/kg) treatment significantly (p < 0.01) triggered redox defense in
the renal cells of T2D rats by upregulating Nrf-2 signaling evidenced by enhancement of
nuclear P-Nrf-2 expression in the kidneys of T2D rats (Figure 12e,f). In this study, T2D
significantly endorsed fibrosis to renal cells of experimental rats. T2D rats exhibited ~2.7⁓
⁓
⁓
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(p < 0.01), ~3.1- (p < 0.01), and ~2.2-fold (p < 0.01) increases in TGF-β1, P-Smad3, and
β in renal cells, respectively (Figure 12g,h). In addition, a ~0.5collagen IV expressions
⁓
fold (p < 0.01) reduction in Smad7
expression was observed in the renal cells of T2D rats
(Figure 12g,h). In contrast, Myr (300 mg/kg) treatment reciprocated TGF-β1 (p < 0.01),
P-Smad3 (p < 0.01),β Smad7 (p < 0.05), and collagen IV (p < 0.05) expressions in the kidneys
of T2D rats (Figure 12g,h). Myr (300 mg/kg) treatment to the non-diabetic rats did not
show any major change in either of the aforementioned signal proteins as compared to
normal control rats (Figure 12).
Figure 12. Cont.
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Figure 12. The effects of Myr on different signal proteins in the kidneys of T2D rats. (a) The immunoblot images showing the effects on
the expressions of Bad (in mitochondria), Bcl-2, Cyt C (in cytosol), cleaved caspase 9, and cleaved caspase 3 involved in apoptosis.
(b) The graphs showing the densitometric analysis of the bands of apoptotic factors. (c) The immunoblot images showing the effects on
κ α
κ
the expressions of P-IκBα (in cytosol) and P-NF-κB (in nucleus) involved
in inflammation. (d) The graphs
showing the densitometric
analysis of the bands of inflammatory factors. (e) The immunoblot images showing the effects on the expressions of Keap1 (in cytosol)
and P-Nrf-2 (in nucleus) involved in redox defense. (f) The graphs showing the densitometric analysis of the bands of redox defense
factors. (g) The immunoblot images showing the effects on the expressions of TGF-β1, P-Smad3, Smad7, and collagen IV involved in
β
fibrosis. (h) The graphs showing the densitometric analysis of the bands of fibrotic factors. Myr was effective in reducing T2D-induced
increase in the expressions of Bad (in mitochondria), Cyt C (in cytosol), cleaved caspase 9, cleaved caspase 3, P-IκBα
κ α (in cytosol), and
P-NF-κB (in nucleus),
Keap1 (in cytosol), TGF-β1, P-Smad3,
κ
β and collagen IV in rat kidneys, while it activated the expressions of Bcl-2,
P-Nrf-2 (in nucleus), and Smad7 in the renal cells of T2D rats. The intensity of normal control band was assigned 1. β-actin served as
β normalization. Data are expressed as mean ± SD (n = 6). # Values significantly (p < 0.01) differed from the
the loading control for
− from the diabetic control (T2D+) group. ** Values significantly
normal control (T2D−) group. * Values significantly (p < 0.05) differed
(p < 0.01) differed from the diabetic control (T2D+) group.
2.2.10. Effect on Renal Histology of T2D Rats
Representative histological sections of kidneys of rats received different treatments
were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT) (Figure 13).
The medulla portion of H&E-stained kidney sections of T2D rats showed thickening of
Bowman’s capsules (yellow arrow), glomerular hypercellularity (blue arrow), and cloudy
appearance of tubules (green arrow) when compared with the kidneys of normal control rats (Figure 13a). On the other hand, Myr (300 mg/kg) treatment reciprocated the
histological abnormalities in the kidneys of T2D rats and restored the histo-architecture
to near normal status (Figure 13a). MT-stained kidney sections of T2D rats exhibited
enhanced collagen deposition (red arrows) (Figure 13b). In contrast, Myr (300 mg/kg)
treatment attenuated collagen deposition in the kidneys of T2D rats (Figure 13b). In the morphometric analysis, the thickening of Bowman’s capsules was measured in H&E-stained
kidney sections (100×) taking the arbitrarily selected areas containing one glomerulus. The
⁓
kidney sections of T2D rats showed
a ~2.8-fold (p < 0.01) increase in the capsular space
⁓
(Figure 13c). Histo-quantification of MT-stained kidney sections of T2D rats showed
a ~3.7fold (p < 0.01) increase in collagen deposition (Figure 13d). In contrast, Myr (300 mg/kg)
treatment significantly (p < 0.05–0.01) reciprocated histological abnormalities in the kidneys
of T2D rats (Figure 13c,d). Myr (300 mg/kg) treatment to the non-diabetic rats did not
show any considerable change in the histo-architecture as compared to normal control
rats (Figure 13).
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Figure 13. The effects of Myr on the histological structures of the kidneys of T2D rats. (a) H&E-stained sections of mice kidneys.
(b) The MT-stained sections of mice kidneys. (c) The widening of capsular space is shown as a percentage of the blank staining
of glomerular membrane compared to the whole area of the H&E-stained photomicrograph. (d) Histo-quantification of collagen
deposition in MT-stained kidney sections. T2D rats showed thickening of Bowman’s capsules (yellow arrow), glomerular
hypercellularity (blue arrow), cloudy appearance of tubules (green arrow), and collagen deposition (red arrows). Data are
represented as the mean ± SD, n = 6 × 5. Five randomly selected portions containing a glomerulus from kidney section of
each mouse were chosen for the quantification. # Values significantly (p < 0.01) differed from the normal control (T2D−) group.
* Values significantly (p < 0.05) differed from the diabetic control (T2D+) group. ** Values significantly (p < 0.01) differed from
− group.
the diabetic control (T2D+)
2.3. In Silico Analyses
2.3.1. ADMET and Drug-Likeness Prediction
Chemometric ADMET profiles of Myr were evaluated to explore few pharmacokinetic
and other important physicochemical characteristics. The result of the predicted ADMET
profile of Myr is presented in Table 3. From most of the predicted ADMET values, it
was observed that Myr possesses acceptable drug-like characteristics with no such severe
indication of toxicity issues, and hence, it could be considered as a good bioactive candidate
molecule for exerting essential biological activity.
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Table 3. Computationally predicted absorption distribution metabolism excretion, and toxicity (ADMET) and other
drug-likeness profiles of Myr.
ADMET Properties
Studied
Descriptors/Physiochemical
Properties
Predicated Values
Recommended
Ranges/Indices
Properties under Lipinski’s
rule of five (RO5)
Molecular weight
H-bond donor
H-bond acceptor
QPlogPo/w
464.382
7
12
3.69
130.0 to 725.0
0.0 to 6.0
2.0 to 20.0
−2.0 to 6.5
WPSA
glob
QPlogS
QPlogHERG
QPPCaco
QPlogBB
QPlogKhsa
0
0.84
−2.74
−4.72
2.69
−3.63
−0.74
0.0 to 175.0
0.75 to 0.95
−6.5 to 0.5
Concern below −5
<25 poor; >500 great
−3.0 to 1.2
−1.5 to 1.5
Hepatotoxicity
Mutagenicity
Non-hepatotoxic
Non-mutagenic
Toxic or non-toxic
Mutagenic or non-mutagenic
Other important
physiochemical/ADME
properties
Toxicity profiles
QPlogPo/w: Predicted octanol/water partition coefficient; WPSA: Weakly polar component of the SASA (solvent accessible surface area);
glob: Globularity descriptor; QPlogS: Predicted aqueous solubility; QPlogHERG: Predicted IC50 value for blockage of HERG K+ channels;
QPPCaco: Predicted apparent Caco-2 cell permeability; QPlogBB: Predicted brain/blood partition coefficient; QPlogKhsa: Prediction of
binding to human serum albumin.
2.3.2. In Silico Molecular Docking Analysis between Myr and Signal Proteins
Molecular docking was executed to analyze the binding interactions between Myr and
various signaling proteins for elucidating possible intermolecular interaction mechanisms
and binding affinity towards each studied protein. Few proteins, such as IRS-1, Akt, Bcl-2,
caspase 3, Cyt C, and Nrf-2 did not show significant docked poses. Such outcomes in
docking might have appeared due to the non-availability of the complete crystallographic
structures of some of these proteins and/or lack of appropriate binding regions in obtained structures. Myr exhibited several types of molecular interactions, such as hydrogen
bond (H-bond), hydrophobic, π-cation, π-stacking, and salt bridge interactions with studied signaling proteins. Overall, molecular docking analyses suggested that majorities of
the signaling proteins, such as PI3K, Bad, caspase 9, IκB, NF-κB, Keap1, TGF-β, Smad3,
Smad7, and collagen IV (Figure 14) exhibited moderate to strong binding interaction affinity towards Myr with Glide dock scores ranging between −2.26 and −14.56 Kcal/mol
(Table 4). In molecular docking, PI3K kinase protein revealed the lowest dock score of
−14.56 kcal/mol and exhibited as the most potential binder of Myr. In particular, docking
analyses revealed that Myr establishes H-bond interactions with Ser614, Tyr670, Ile685, and
Ser687 residues of PI3K (Figure 14a). In addition, few hydrophobic residues (Ile634, Phe684,
Pro689, Leu750, and Ile760) of PI3K protein also mediated to form hydrophobic contacts
with Myr (Figure 14a). Interestingly, the predicted docking-based interactions obtained for
PI3K were also compared with available known ligand (ligand ID: JXM) which revealed
that amino acid residues, such as Phe612, Ly636, Phe684, Ile685 Leu750, Ile760, and Asp761
of PI3K, were involved in several types of intermolecular interactions (Supplementary
Figure S1a). Appearance and involvement of common amino acid residues in molecular
binding interactions were found for Myr and JXM, which undoubtedly supported that
Myr could be a potential and reasonable binder for PI3K. Binding interactions between
Myr and Bad protein revealed five H-bond interactions mediated through Glu311 and
Glu318 residues along with a salt bridge interaction with residue Arg314 (Figure 14b). Myr
interacted with caspase 9 to form several numbers of H-bond interactions through Arg180,
Ser236, His237, Gln285, Ser287, and Arg355 residues along with hydrophobic interactions
through Trp354 and Arg355 residues (Figure 14c). When comparing the predicted binding
interactions of Myr with known ligand (ligand ID: MLT) of caspase 9 that also demonstrated
the participation of H-bond interaction with Arg355 residue and suggested the potentiality
of Myr to be a good binder for caspase 9 (Supplementary Figure S1b). Eight numbers of
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H-bond interactions were identified between Myr and the residues (Thr23, Phe26, Gln48,
Leu173, Gly176, and Ser177) of IκB protein (Figure 14d). Known ligand (ligand ID: KSA)
of IκB protein was also exhibited some common and close proximity residues (Leu21,
Thr23, Val29, Lys44, Tyr98, Cys99, Val152, Ale165, and Asn166) involved in intermolecular interaction with IκB protein as obtained for Myr (Supplementary Figure S1c). Myr
established H-bond interactions with Arg59, His67, Ser243, Asn250, Lys252, and Asp274
κ interaction between Keap1 protein
residues of NF-κB protein (Figure 14e). Intermolecular
and Myr exhibited a number of H-bond interactions through Ser363, Arg380, Gln530,
Ser555, Tyr572, and Ser602 residues and a hydrophobic interaction with Tyr334 residue
of Keap1 (Figure 14f). It was interesting to observe that known ligand (ligand ID: 08A) of
Keap1 also revealed similar types of amino acid residues (Tyr334, Arg380, Asn382, Asn414,
Tyr572, and Phe577) which participated in intermolecular interactions with Myr, and hence
revealing that Myr is a plausible binder for Keap1 (Supplementary Figure S1d). Myr established H-bond interactions with TGF-β through Asp3, Leu83, Glu84, and Ser108βresidues
at the active binding site (Figure 14g). In addition to H-bond interactions, two residues
hydrophobic interactions
(Ala82 and Glu84) of TGF-β protein also participated in the β
with Myr (Figure 14g). In docking analyses, three types of intermolecular interactions,
such as H-bonds (through Tyr363, Arg367, Arg372, Glu396, and His398), hydrophobic
(through Thr370 and Leu403), and π-stacking (through His398)
π were predicted between
Smad3 protein and Myr (Figure 14h). Another Smad family protein, Smad7, exhibited
the involvements of two common residues (Glu203 and Pro207) in establishing both the
H-bond and the hydrophobic interactions with Myr (Figure 14i). Docking analysis of Myr
with collagen IV displayed H-bond interactions through Ser170, Ser190, Trp192, Leu210,
and His218 residues along with the hydrophobic contacts through Leu215 and His218
residues of collagen IV (Figure 14j).
Figure 14. Cont.
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Figure 14. In silico molecular docking analysis revealed several types of possible molecular interactions between Myr and signal
κ (e), Keap1κ (f), TGF-β (g), Smad3β(h), Smad7 (i), and collagen IV (j).
proteins, such as PI3K (a), Bad (b), caspase 9 (c), IκB (d), NF-κB
Table 4. XP-Glide score and interacting residues of different bioactive signaling receptors/proteins in molecular docking
analysis with Myr.
Proteins
Glide Dock Score
(Kcal/mol)
Interacting Residues in
H-bond Interaction
PI3K
−14.56
Ser614, Tyr670, Ile685, Ser687
Bad
Caspase 9
IκB
NF-κB
Keap1
TGF-β1
κ
−
−3.64
−5.11 −
−5.69
−7.06 −
−7.98
−2.99 −
Glu311, Glu318
Arg180, Ser236, His237, Gln285, Ser287, Arg355
Thr23, Phe26, Gln48, Leu173, Gly176, Ser177
Arg59, His67, Ser243, Asn250, Lys252, Asp274
Ser363, Arg380, Gln530, Ser555, Tyr572, Ser602
Asp3, Leu83, Glu84, Ser108
Smad3
−4.71
Tyr363, Arg367, Arg372, Glu396, His398
Collagen IV
−7.99
Glu203, Pro207
Ser170, Ser190, Trp192, Leu210, His218
Smad7κ
β
−2.26 −
−
Involvement of Other Type of
Molecular Interactions
Ile634, Phe684, Pro689, Leu750, Ile760
(Hydrophobic)
Arg314 (Salt bridge)
Trp354, Arg355 (Hydrophobic)
Leu173 (Hydrophobic)
Arg57 (Salt bridge)
Tyr334 (Hydrophobic)
Ala82, Glu84 (Hydrophobic)
Thr370, Leu403 (Hydrophobic)/His398
(π-Stacking)
Glu203, Leu204, Pro207 (Hydrophobic)
Leu215, His218 (Hydrophobic)
−3. Discussion
T2D is among the most prevalent chronic metabolic syndromes affecting a huge num−ber of global populations [14]. The pathological events in T2D include a number of slowly or
π
rapidly developing lethal macro- or microvascular complications [14]. Diabetic nephropa−thy is among the major concerns in T2D patients [15]. Persistent hyperglycemia causes an
increase in ROS production through polyol activation and other mechanisms, which subse−quently endorses redox stress and activation of pathological signaling events [6]. Apoptotic,
inflammatory, and fibrotic signaling events are proposed to be the key pathological signaling in diabetic nephropathy [16]. In this study, we evaluated the protective effect of Myr, a
natural antioxidant in M. esculenta bark, against T2D and associated diabetic nephropathy.
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In this study, a high-fat diet + a low dose of streptozotocin-induced T2D rat model
was used. Persistently high blood glucose levels confirmed that diabetes was induced in
the experimental rats. Significantly high HOMA-IR and low HOMA-β scores in diabetic
rats as compared to normal rats confirmed the establishment of insulin resistance and
partial loss of β cell functions, which signified the induction of T2D in rats [17]. Myr
treatment to T2D rats caused a substantial reduction in fasting blood glucose level and
improved serum insulin level, which might be attributed to a reversal of insulin resistance
and restoration of β cell functions. Myr-treated T2D rats caused a substantial reduction
in HOMA-IR and escalation in HOMA-β scores as compared to T2D control rats, which
proposed that Myr reversed insulin resistance and restored β cell functions. In vitro assay
revealed that Myr could improve glucose uptake by L6 myotubes cultured in HG condition.
In search of mechanism, the signaling event involved in glucose utilization by the skeletal
muscle was studied. Skeletal muscle cells play a key role in glucose metabolism [17].
IRS-1, PI3K, Akt, and GLUT4 are the key signaling molecules, which are involved in the
signal transduction of insulin responsiveness in regulating glucose uptake by the skeletal
muscle for subsequent utilization [2]. The insulin signaling of glucose uptake in muscle
cell initiates with tyrosine phosphorylation of IRS-1, which sequentially endorses PI3K
activation, Akt phosphorylation, and GLUT4 translocation to the membrane where GLUT4
acts as a key transporter of glucose into the cell for metabolism [2]. In this study, HGexposed L6 cells and the soleus muscle of T2D rats exhibited substantial downregulation
in insulin signaling which signified a lack of glucose utilization. On the other hand, Myr
could activate IRS-1/PI3K/Akt/GLUT4 signaling in the muscle cells in both in vitro and
in vivo systems, thereby improving glucose utilization in the diabetic milieu. Thus, it could
be said that Myr exerts an anti-diabetic effect by improving glucose uptake by reversing
insulin resistance, activating insulin signaling, and restoring β cell functions.
Serum and urine biochemical profiles can give a primary indication of pathological
status in the body [18]. In this study, a high level of glycosylated hemoglobin in the sera of
T2D rats is an indication of persistent hyperglycemia [2,15,17]. Persistent hyperglycemia
can endorse several pathological incidences, including dyslipidemia [2]. In this study, T2D
rats exhibited high levels of total cholesterol, triglycerides, and LDL-cholesterol in the
sera along with a reduction in HDL-cholesterol level, which signified the establishment
of dyslipidemia in the diabetic milieu. Dyslipidemia had also been regarded to incite
and promote several diabetic complications through oxidative stress and inflammatory
injuries [2,17]. In contrast, Myr treatment to T2D rats could attenuate dyslipidemia, thus alleviating lipotoxicity in the diabetic milieu. The increased levels of tissue-specific enzymes,
such as LDH and CK in sera, reveal tissue damage. Significant elevation in the levels of
LDH and CK in the sera of T2D rats signified the establishment of cellular damage in the
internal tissues [19]. Myr treatment caused a reduction in serum LDH and CK levels in
T2D rats, which signified the protective role of Myr against T2D-provoked damage to the
internal tissues. In this study, an increase in the level of C-reactive proteins in the sera
of T2D rats justified the induction of systemic inflammation in the diabetic milieu [20].
In contrast, Myr treatment caused a reduction in serum C-reactive proteins level, which
might predict the anti-inflammatory role of the compound. In this study, increased levels
of creatinine, urea, and uric acid in the sera of T2D rats and an escalation in the release
of urinary albumin predicted the glomerular damage and the progression of diabetic
nephropathy [2]. In addition, T2D rats exhibited a lack of creatinine clearance through
urine, which could occur due to the loss of renal function [2]. In contrast, Myr treatment
could rescue T2D-triggered abnormalities in creatinine, urea, and uric acid levels in the sera,
albuminuria, and abnormal creatinine clearance; thus, Myr could be effective in alleviating
diabetic nephropathy.
In vitro studies revealed that HG could impart toxic effects to the renal cells resulting
in the loss of cell viability. In contrast, Myr was able to protect the renal cells against
HG-induced cytotoxicity in vitro evidenced by both cell viability and image assays. The
experimental observations both in vitro and in vivo suggested that HG imparted toxic
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manifestation by inducing oxidative stress, apoptosis, inflammation, and fibrosis to the
renal cells.
In this study, hyperglycemia caused a significant increase in intracellular ROS content,
NO concentration, and NADPH oxidase level in the kidney cells in vitro and in vivo. High
glucose can trigger ROS production via multiple mechanisms. Polyol activation is among
the major pathways of ROS production in the diabetic milieu [15]. In this study, T2D rats
exhibited a significant activation in the polyol pathway in the renal tissue. Aldose reductase
and sorbitol dehydrogenase are two key enzymes in the polyol pathway [6]. Aldose reductase catalyzes the conversion of glucose to sorbitol with the help of NADPH as a cofactor,
while sorbitol dehydrogenase assists in the formation of fructose from sorbitol using its
co-factor NAD+ [6]. Thus, activation of these enzymes causes the reduction in cellular
NADPH resulting in an increment of oxidative stress [6]. In addition, polyol activation
triggers AGEs formation, which endorses ROS production via NADPH activation [6]. In
this study, T2D rats showed elevated levels of aldose reductase and sorbitol dehydrogenase
in the renal tissue resulting in an upregulation of AGEs in the sera. In contrast, Myr treatment significantly reduced aldose reductase and sorbitol dehydrogenase in renal tissue
homogenate and consequently reduced AGE accumulation in sera. Thus, Myr could attenuate ROS accumulation and oxidative stress in renal cells via inhibiting polyol enzymes and
AGEs formation. High glucose-provoked activation of NADPH oxidase can also trigger
ROS production via catalyzing electron transfer from NADPH to molecular oxygen and
thereby produces superoxide which can subsequently be converted into H2 O2 by reacting
with water [21]. H2 O2 can generate hydroxyl radical via Haber–Weiss reaction [21]. This
hydroxyl radical further reacts with NO to generate reactive nitrogen species (RNS) [21].
In this study, both the renal tissue of T2D rats and HG-exposed renal cells exhibited considerably high NADPH oxidase level, which resulted in an enhancement in ROS production
in renal cells. Hyperglycemia also triggered NO level, which could endorse the generation
of RNS, thus further enhancing redox stress. In contrast, Myr treatment lowered ROS
accumulation, NADPH oxidase activation, and NO content in the renal tissue of T2D rats
and HG-exposed renal cells. Endogenous antioxidant molecules play key roles in scavenging/neutralizing the oxidative free radicals. SOD catalyzes scavenging of superoxide
radical to yield H2 O2 and O2 , while CAT and GPx accelerate the dismutation and reduction
of H2 O2 , respectively [18]. GST catalyzes the detoxification of lipid peroxides [18]. The
catalytic properties of GST and GPx depend on GSH concentration [18]. In addition, GSH is
a thiol-based metabolite, which scavenges ROS and consequently converted into GSSG [18].
In this study, suppression of cellular antioxidant molecules, such as SOD, CAT, GPx, GST,
and GSH in the renal tissue of T2D rats and HG-exposed renal cells hampered the scavenging/neutralizing capacity of these oxidative free radicals resulting in an excess of oxidative
free radicals to impart oxidative damage. In contrast, Myr treatment significantly enhanced
the levels of cellular antioxidant molecules in the renal tissue of T2D rats and HG-exposed
murine kidney cells, thus contributing to redox defense mechanism. A high extent of lipid
peroxidation, protein carbonylation, and DNA oxidation was observed in the renal tissue of
T2D rats and HG-exposed murine kidney cells. Enhanced production of ROS and RNS and
lack of their scavenging in diabetic milieu resulted in these oxidative insults to the cellular
macromolecules. In contrast, Myr treatment could reduce hyperglycemia-provoked redox
insult to the lipids, proteins, and nucleic acid in renal cells in vitro and in vivo. Myr itself
can neutralize oxidative radicals by donating protons from the phenolic groups. Thus, Myr
could attenuate hyperglycemia-provoked renal oxidative stress via multiple targetings,
such as scavenging oxidative radicals, suppressing polyol activation, inhibiting NAHPH
oxidase, reducing intracellular NO level, and endorsing cellular redox defense. In search of
the mechanism by which Myr endorses cellular redox defense, the effect on Nrf-2/Keap1
signaling was studied. Nrf-2 is a key transcription protein, which regulates the cellular
redox defense mechanism [21]. Nrf-2 signaling begins with the cleavage of Nrf-2 from the
Keap1–Nrf-2 complex, followed by its phosphorylation, and translocation to the nucleus
where it triggers transcriptional activation of phase 2 antioxidant enzymes [21]. In this
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study, Myr could significantly reverse Nrf-2 suppression in diabetic milieu evidenced by
upregulation of P-Nrf-2 expression in the nucleus and activation of Keap1 expression in the
cytosol. Activation of Nrf-2 signaling transcriptionally activated endogenous antioxidant
enzymes and GSH level and endorsed redox defense mechanism in diabetic condition. In
silico molecular docking predicted the interaction between Keap1 and Myr, which might
be the cause of dissociation of Keap1–Nrf-2 complex.
In addition to oxidative damages to major cellular components, ROS can endorse
several pathological events, such as apoptosis [21,22]. Oxidative radicals can improve
mitochondrial permeability, thus promoting mitochondrial translocation of Bad, a proapoptotic factor [21]. These can simultaneously suppress Bcl-2 via inhibition of cAMP
response element-binding protein [21]. Activation of pro-apoptotic protein and suppression of anti-apoptotic factor endorse the release of Cyt C to the cytosol, which activates
downstream signaling of apoptosis [21]. Oxidative stress can also trigger caspase activation via cysteine oxidation at their catalytic sites and suppression of GSH-mediated
S-glutathionylation [21]. In this study, Hoechst nuclear staining of HG-exposed renal cells
exhibited signs of apoptosis induction. In addition, hyperglycemia-provoked oxidative
stress endorsed apoptosis to HG-exposed renal cells and the kidneys of T2D rats evidenced
by enhanced mitochondrial translocation of Bad, suppression of Bcl-2, the cytosolic release
of Cyt C, and cleavage of caspases. In contrast, Myr treatment could significantly attenuate
renal apoptosis in the diabetic milieu, which may be achieved through inhibition of oxidative stress and/or direct interaction with pro-apoptotic factor and caspase 9, as predicted
in molecular docking analysis.
Fibrosis is another redox-sensitive pathological event [21,22]. Biochemical analysis of
renal tissue homogenate of T2D rats exhibited high accumulation of TGF-β1, collagen IV,
and hydroxyproline. Histological sections of the kidneys of T2D rats also showed an excess
of collagen deposition. Both these results signified the establishment of renal fibrosis in the
diabetic milieu. In contrast, Myr treatment could attenuate T2D-mediated renal fibrosis as
observed in both biochemical and histological findings. Fibrosis is principally regulated by
TGF-β1/Smad/collagen IV signaling pathway. TGF-β1 and Smad have been reported to
play crucial roles in developing glomerulosclerosis and tubulointerstitial fibrosis [21]. ROS
can directly promote TGF-β1 activation, which can endorse Smad3 phosphorylation and
trigger downstream fibrotic signaling including collagen deposition. Smad7 can inhibit
TGF-β1 activation via serving as a ”negative feedback loop” [21]. In this study, HG-exposed
renal cells and the kidneys of T2D rats exhibited upregulation of TGF-β1, P-Smad3, and
collagen IV expressions with concomitant downregulation of Smad7. In contrast, Myr
treatment could suppress the TGF-β1/Smad/collagen IV signaling in renal cells in the
diabetic milieu, which may be achieved via inhibition of oxidative stress and/or direct
interaction with fibrotic factors as predicted in molecular docking analysis.
Hyperglycemia is known to endorse low-grade inflammation to the renal cells, which
have been proposed to play a significant role in developing diabetic nephropathy [2]. In this
study, significantly high levels of pro-inflammatory mediators, such as IL-1β, IL-6, and TNFα were observed in renal tissue homogenate of T2D rats, which proposed the occurrence of
renal inflammation [2]. These pro-inflammatory mediators are the down-stream targets of
NF-κB. Briefly, NF-κB signaling begins with the phosphorylation-mediated degradation of
NF-κB from its association with IκB which prevents NF-κB activation [23]. Then, P-NF-κB
translocates to the nucleus and triggers the transcription of inflammatory genes [22]. In this
study, HG-exposed renal cells and the kidneys of T2D rats exhibited activation of NF-κB
signaling evidenced by an upregulation P-NF-κB expression in the nucleus. In contrast,
Myr treatment significantly suppressed NF-κB activation in the renal cells in the diabetic
milieu, and thus prevented renal inflammation evidenced by the reduction in IL-1β, IL-6,
and TNF-α levels in the kidneys and C-reactive protein in the sera of T2D rats. An in silico
molecular docking study also predicted the interactions of Myr with NF-κB and IκB.
Histological assessment of the kidneys of T2D rat showed thickening of Bowman’s capsules, glomerular hypercellularity, and cloudy appearance of tubules. In addition, a significant
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increase in collagen deposition was observed in the kidneys of T2D rats. In contrast, Myr
could reverse the histological abnormalities of the kidney sections of T2D rats and restored
the histo-architecture to near-normal status, which supports the protective role of Myr against
diabetic nephropathy.
In silico ADMET profiles predicted that Myr possesses acceptable drug-like characteristics with no such indication of severe toxicity issue, and hence it could be considered as a
good drug candidate.
4. Materials and Methods
4.1. Plant Material and Extraction of Myr
The powdered bark of M. esculenta was macerated with methanol with continuous
stirring. The extract was fractioned successively with hexane and ethyl acetate. The ethyl
acetate fraction was subjected to silica gel column chromatography using mixtures of
n-hexane-ethyl acetate and ethyl acetate-methanol of increasing polarity, to yield seven
major fractions (A–G). Fraction C was chromatographed with hexane-dichloromethane and
dichloromethane-methanol with an increasing gradient to yield Myr (m.p. 359 ◦ C). The
structure (Figure 1) was elucidated employing NMR (1 H and 13 C) and mass spectroscopic
data [24,25].
4.2. Reagents
Streptozotocin was procured from Hi-media (Mumbai, India). Myr (96%), Bradford
reagent, cell culture media, fetal bovine serum (FBS), and bovine serum albumin were
procured from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal anti-Akt (SAB4500800), antiBcl-2 (SAB4500003), anti-Cyt C (SAB4502234), anti-P-Nrf-2 (SAB4501984), and anti-Smad7
(SAB4200345) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA); polyclonal anti-PI3K (p85α) (#4292), anti-phospho-Akt (#9271) anti-TGF-β (#3711), anti-P-IRS-1
(#3070), anti-IRS-1 (#2382), and anti-Bad (#9292) antibodies and monoclonal anti-P-NF-κB
(p65) (#3033), anti-P-IκBα (#2859), anti-P-Smad3 (#9520), and β-actin (#4970) antibodies
were obtained from Cell Signaling Technology (Beverly, MA, USA); polyclonal anti-Keap1
(ab139729) antibody was procured from Abcam Inc. (Cambridge, MA, USA). Monoclonal
anti-GLUT4 (NBP2-44298) antibody and polyclonal anti-caspase 9 (NB 100-56366) and
anti-caspase 3 (NB-100-56113) antibodies were procured from Novas Biologicals (Littleton,
CO, USA). HRP-linked antibody (#7074) was procured from Cell Signaling Technology,
(Beverly, MA, USA). Ethylenediaminetetraacetic acid (EDTA), 1-chloro-2,4-dinitrobenzene
(CDNB), dimethyl sulphoxide (DMSO), H2 O2 , N-ethylmaleimide, nicotinamide adenine
dinucleotide reduced disodium salt (NADH), glacial acetic acid, NBT, GSH, GSSG, KH2 PO4 ,
NaN3 , thiobarbituric acid (TBA), and trichloroacetic acid (TCA) were procured from Sisco
research laboratory, India. The kits/reagents for biochemical assays to estimate different
biochemical parameters were purchased from Sigma-Aldrich (St. Louis, MO, USA) and
Span diagnostic Ltd. (Mumbai, India).
4.3. In Vitro Assays
4.3.1. Cell Culture
The L6 myoblast and the NRK epithelial cell lines were gifted by Prof. Parames Sil,
Department of Molecular Medicine, Bose Institute, Kolkata, India. These cells were cultured
in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% FBS and antibiotics.
The cells were maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . The cells were
passaged in every 3 days.
4.3.2. Glucose Uptake Assay
The L6 cells were exposed to high glucose (HG+) and glucose uptake assay was performed in the presence of Myr as per established protocol [26]. Briefly, cells (2 × 104 ) were
pre-incubated with Myr (10, 20, 30, and 50 µM) in a 96-well culture plate for 2 h followed
by HG+ (30 mM) exposure for the following 20 h at 37 ◦ C in a humidified atmosphere of
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5% CO2 . A set of L6 cells cultured with 5.5 mM glucose served as the control (HG−). The
glucose uptake was estimated using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyd-glucose (6-NBDG) following the established protocol [26]. The treated cells were incubated
with a serum-free medium containing 6-NBDG (20 µM). After 30 min of incubation, cells
were washed, lysed, and kept in the dark for 10 min. The cells were homogenized with
30 µL of DMSO and the plate was read immediately using a microplate reader at λexcitation
466 nm/λemission 540 nm.
4.3.3. Immunoblotting of Signal Proteins in L6 Myoblasts
The L6 myoblast cells (2 × 104 ) were pre-incubated with Myr (30 µM) in a 96-well
culture plate for 2 h followed by HG+ (30 mM) exposure for the following 20 h at 37 ◦ C in
a humidified atmosphere of 5% CO2 . The concentration of Myr was calibrated on the basis
of the glucose uptake assay. L6 cells that received different treatments were washed with
cold PBS, lysed in the radio-immunoprecipitation assay (RIPA) buffer supplemented with
protease and phosphatase inhibitors, and the protein samples were separated following
the standard sequential fractionation process as described by Baghirova et al. [27]. Protein
samples were quantified by ELISA (Bio-Rad, CA, USA). The sample proteins (20 µg) were
resolved in 10% SDS-PAGE gel electrophoresis, and immunoblotting was performed as
per the established protocol by our group [22]. The blot was developed by ECL substrate
(Millipore, MA, USA) and the protein expression was detected in a ChemiDoc Touch
imaging system (Bio-Rad, USA). The densitometric analysis was executed using Image
Lab software (Bio-Rad, USA). The membranes were further subjected to mild stripping to
detect the expressions of other proteins in the same membrane [28]. The expressions of
PI3K (p85), P-IRS-1 (Tyr 895), total IRS-1, P-Akt (Ser 473), total Akt, and GLUT4 (in the
membrane fraction) were studied. β-actin was used as a loading control for normalization.
4.3.4. Concentration and Time-Dependent Toxic Effect of D-Glucose to NRK Cells
To establish a model of diabetic nephropathy, the concentration and time-dependent
toxic effect of D-glucose was measured. For measuring the concentration-dependent toxic
effect of D-glucose, NRK cells (2 × 104 ) were seeded in a 96-well culture plate. After
24 h, the cells were treated with D-glucose (5, 10, 20, 30, and 40 mM) and incubated for
72 h. The cell viability was measured using resazurin as per the protocol established by
our group [21]. Briefly, 5 µL of 600 µM resazurin was added to the wells and incubated
for 2 h, and the plate was read using a microplate reader at λexcitation 535 nm/λemission
590 nm. To determine the time-dependent toxic effect of D-glucose, the cells were treated
with D-glucose (30 mM), and the cell viability was measured at 6, 12, 24, 48, and 72 h
using the resazurin-based assay. Based on the assays, D-glucose concentration of 30 mM
and the incubation period of 48 h were optimized as the in vitro condition for diabetic
nephropathy assay.
4.3.5. In vitro Model of Diabetic Nephropathy
The NRK cells (2 × 104 ) were seeded in a 96-well culture plate, and after 24 h, the
cells were treated with Myr (30 µM) and the nephroprotective assay was performed in the
presence of HG+ (30 µM). A set of NRK cells cultured with 5.5 mM glucose served as the
control (HG−). A set of cells treated with HG+ served as the hyperglycemic control, and
another set of cells treated with Myr (30 µM) in the HG−condition was kept to observe the
effect of Myr (30 µM) in the normoglycemic cellular environment. The cells were incubated
for 48 h at 37 ◦ C in a humidified atmosphere of 5% CO2 .
4.3.6. Cell Viability Measurement
The cell viability was measured using the resazurin-based assay [21]. The Hoechst
staining was performed as per the protocol established by our group [21]. Briefly, cells under
different sets were fixed with paraformaldehyde (4%) in phosphate buffer saline (PBS) of
pH 7.4 for 20 min and were strained with Hoechst 33,258 (5 µg/mL in PBS) for 20 min. The
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cells were washed with PBS and counted under fluorescence microscope (Olympus-1 × 70,
Japan, software-Metamorph).
4.3.7. Measurement of Redox Status
The intracellular ROS production was measured employing a 2’,7’-dichlorofluorescein
diacetate (DCFH-DA)-based assay, and DCF fluorescence was measured at λexcitation
485 nm/λemission 525 nm under a fluorescence microscope (Olympus-1 × 70, Japan, softwareMetamorph) [29]. Briefly, the cells that received different treatments were incubated with
10 mM DCFH-DA for 1 h at 37 ◦ C in the dark. Then, the cells were washed and suspended
in PBS, and fluorescence was measured. Firstly, DCFH-DA is deacetylated by viable cells
to non-fluorescent DCFH, which in turn form fluorescent DCF by reacting with ROS [29].
NADPH oxidase level was measured as per the method described elsewhere [30]. Briefly, cells
that received different treatments were detached by acutase and centrifuged at 2500× g for
5 min. the pellet was resuspended in PBS and the cells were incubated with NADPH (250 µM).
NADPH consumption was checked by the decrease in absorbance at λ 340 nm for 10 min. For
NADPH oxidase activity, the rate of NADPH consumption was inhibited by adding 10 µM
diphenyleneiodonium 30 min prior to the assays. The amount of NADPH consumption
was estimated using an absorption extinction coefficient of 6.22 mM−1 cm−1 . The cellular
NO content was measured using a colorimetric assay kit and following the manufacturer’s
protocol (Cayman Chemical Company, Ann Arbor, MI, USA). The lipid peroxidation index
was measured by quantifying the TBARS concentration by following the protocol of Fraga
and co-workers with little modification [31]. Briefly, 50 µL of cell extract was mixed with
50 µL SDS (3%), and the mixture was heated in a water bath after adding 200 µL of 0.1 N HCl,
30 µL of phosphotungstic acid (10%), and 100 µL of 2-TBA (0.7%). The TBRAS was extracted
by n-butanol, and fluorescence was measured at λexcitation 515 nm/λemission 555 nm under
a fluorescence microscope (Olympus-1 × 70, Japan, software-Metamorph). The degree of
protein carbonylation was assayed as per the established protocol [32]. Briefly, the sample
was treated with an equal volume of 2,4-dinitrophenylhydrazine (1%) in 2 N HCl. After an
hour, the mixture was treated with TCA (20%) and centrifuged. The precipitate was extracted
with ethanol/ethyl acetate and dissolved in 8 M guanidine hydrochloride in 133 mM tris
solution containing 13 mM EDTA. The absorbance was recorded at 365 nm. The extent of
protein carbonylation was calculated using a molar extinction coefficient of 22,000 M−1 cm−1 .
The levels of the CAT, SOD, GPx, and GST were assayed following methods described elsewhere [33]. In SOD assay, cell suspension containing 5 µg of protein was mixed with nitroblue
tetrazolium (NBT), phenazine methosulphate, and sodium pyrophosphate, and the reaction
was initiated by adding NADH. After 90 s, the reaction was terminated by adding glacial
acetic acid, and the absorbance was measured at 560 nm. SOD activity was calculated as the
enzyme concentration required inhibiting (µ-moles) of NBT-reduction/min. CAT activity was
estimated spectrophotometrically by measuring the decomposition of 7.5 mM H2 O2 at 240 nm
for 10 min. CAT activity is defined as H2 O2 consumption/min. In GPx estimation, H2 O2
and NADPH were used as substrates, and NADPH to NADP+ conversion was estimated
by measuring the changes in absorption intensity at 340 nm. GPx activity is defined as the
amount of enzyme required to catalyze the conversion of 1 mol NADPH/minute. GST activity
was measured spectrophotometrically at 340 nm based on the conjugation reaction with GSH
in the first step of mercapturic acid synthesis. The reaction mixture comprises supernatant
protein sample, EDTA, CDNB, KH2 PO4 buffer, and GSH. The GST activity was measured as
µmol of CDNB conjugate formed/min/mg protein. GSH and GSSG levels were estimated
following the protocols developed by Hissin and Hilf [34]. In GSH estimation, the assay
mixture comprising diluted cell extract, phosphate-EDTA buffer pH 8.0, and o-phthalaldehyde
solution was incubated for 15 min. Fluorescence at 420 nm was measured with the activation
at 350 nm. The GSH activity was expressed as nmol/mg of protein. In GSSG assay, diluted
cell extract was mixed with 0.04 M N-ethylmaleimide and incubated at 30 min. Then, 0.1 N
NaOH was added to the mixture. GSSG activity was measured following the procedure
outlined above for GSH assay. The redox ratio was calculated as GSH/GSSG.
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4.3.8. Immunoblotting of Signal Proteins in NRK Cells
The cellular/subcellular protein samples (20 µg) obtained from NRK cells that received
different treatments were resolved in 10% SDS-PAGE gel electrophoresis and immunoblotted as described earlier. The expressions of Bcl-2, Bad in the mitochondria, Cyt C in the
cytosol, cleaved caspase 9, cleaved caspase 3, P-IκBα (Ser 32) in the cytosol, P-NF-κB p65
(Ser 536) in the nucleus, Keap1 in the cytosol, P-Nrf-2 (Ser40) in the nucleus, TGF-β1,
P-Smad3 (Ser423/Ser425), Smad7, and collagen-IV were studied.
4.4. In Vivo Assay
4.4.1. Animals
Male Wistar rats (3–4 months old, 150 ± 20 g) were used in this study. The rats were
housed in separate polypropylene cages in our departmental animal house and maintained
at the temperature of 22 ± 2 ◦ C, relative humidity of 45 ± 5%, light–dark schedule of
12 h, standard rat diet (Agro Corporation Private, Ltd., Bangalore, India), and water
ad libitum [35]. The experiment was performed at the animal house of the Department
of Pharmaceutical Technology, Jadavpur University, India. The animal experiment was
approved (Reference no. AEC/PHARM/1701/08/2017, dated 30.7.2017) by the animal
ethical committee of our institute (Registration no.: 0367/01/C/CPCSEA, UGC, India),
and the principles of laboratory animal care were followed during the experiment [36]. The
animals were allowed to be acclimatized for 15 days before performing the experiment.
Myr was freshly dissolved in 2% Tween 80 before each dosing.
4.4.2. Induction of Diabetes and Experimental Scheme
A high fat-fed and a single low-dose streptozotocin (STZ) model for type 2 diabetes
has been standardized by our group, which was used in this study [2,15,17]. Briefly, the
Wistar rats were fed a high-fat diet [15] and water ad libitum for 2 weeks. After 2 weeks, the
rats were injected with a single dose of STZ (35 mg/kg body weight, i.p.). One week after
STZ treatment, the blood samples were taken from each rat for HOMA-IR and HOMA-β
analysis to screen type 2 diabetic animals [17]. The diabetic rats were continued with
high-fat diet throughout the course of the study.
The type 2 diabetic rats were divided into 4 groups (n = 6) and treated as follows:
Group I: Non-diabetic rats were treated with vehicle daily for 28 days;
Group II: Non-diabetic rats were treated with Myr (300 mg/kg body weight, p.o.) daily for
28 days;
Group III: T2D rats were treated with vehicle daily for 28 days;
Group IV: T2D rats were treated with Myr (300 mg/kg body weight, p.o.) daily for 28 days.
The animals were scrutinized at 12 h intervals to observe any symptom/sign of
irregularity. The fasting blood glucose levels were measured on days 0, 1, 3, 7, 14, 21, and
28 using a single touch glucometer (Ascensia Entrust, Bayer Health Care, Maharashtra,
India) [14]. The body weights, food intake, and water intake were also recorded in the
specific intervals. After 28 days, animals were fasted overnight, and the blood samples were
collected from retro-orbital venous plexus after applying tetracaine ophthalmic drop (0.5%)
to the eyes of rats [37]. The animals were euthanatized, and the soleus muscle (skeletal
muscle) and kidneys were excised, and cleaned immediately with cold PBS (pH 7.4) [37].
For albumin and creatinine measurements, urine samples were collected from the bladder
and immediately stored at −80 ◦ C.
4.4.3. Estimation of Serum and Urine Parameters
Serum insulin level was assessed by ELISA using a commercially available kit (SigmaAldrich, St. Louis, MO, USA) following the manufacturer protocol. Homeostatic model
assessments were performed by estimating HOMA-IR and HOMA-β following established
formulae [17]: HOMA-IR = (Fasting serum insulin in U/L × Fasting blood glucose inmmol/L)/22.5; HOMA- β = (Fasting serum insulin in U/L × 20/Fasting blood glucose
in mmol/L) −3.5. Total cholesterol, HDL-cholesterol, and triglyceride levels in the sera
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were measured using commercially available kits (SPAN Diagnostic, Ltd., Mumbai, India)
following manufacturer protocols. LDL-cholesterol level was estimated following Friedewald’s equation, LDL-cholesterol = Total cholesterol −Triglycerides/5−HDL cholesterol.
Glycosylated hemoglobin concentration in sera was estimated according to the protocol
described by Nayak and Pattabiraman [38]. Briefly, the blood sample was hemolyzed by
toluene then the hemolysate was hydrolyzed by 1 M oxalic acid in 2 M HCl. TCA (40%)
was added to the hydrolysate and then centrifuged. The supernatant was subjected to
phenol-sulphuric acid assay. The developed color was measured at 480 nm. The levels of
albumin, C-reactive protein, creatinine, creatine kinase (CK), lactate dehydrogenase (LDH),
urea, and uric acid were estimated using commercially available kits (Span Diagnostic
Limited, India) following the manufacturer’s protocols. Serum AGEs level was estimated
by ELISA (Abcam, Cambridge, UK) following the manufacturer’s instructions.
4.4.4. Immunoblotting of Signal Proteins in Skeletal Muscle
The soleus muscles of experimental rats were homogenized with ice-cold lysis buffer. The
cellular/subcellular protein samples (20 mg) were dissolved in 10% SDS-polyacrylamide gel
electrophoresis and immunoblotted. The expressions of PI3K (p85), phospho-IRS-1(Tyr895),
total IRS-1, Phospho-Akt (Ser473), total Akt, and GLUT4 (in the membrane) were studied.
4.4.5. Estimation of Renal Parameters
The kidneys were homogenized in 0.1 M Tris-HCl-0.001 M EDTA buffer of pH 7.4 and
centrifuged at 12,000× g for 30 min at 4 ◦ C. The supernatants were used for the biochemical
analyses. Aldose reductase activity was assessed in accordance with the established protocol [39]. Briefly, tissue homogenate was added to an assay mixture consisting of 100 mM
sodium phosphate buffer (pH 7.0) containing 10 mM DL-glyceraldehyde and 66 mM
NADPH. Activity was measured by monitoring the decrease in absorbance at 340 nm.
The value was expressed as consumption of NADPH/min/mg of protein. The sorbitol
dehydrogenase activity was estimated as per the method described elsewhere [40]. Briefly,
the renal tissue homogenate was treated with 12 mM NADH and 0.2 M triethanolamine
buffer (pH 7.4). After 30 min, the reaction was initiated by adding 4 M D (−)fructose. Absorbance was determined at 1 min intervals for 5–8 min at 365 nm. Glyoxalase-I activity was
estimated following the protocol developed by McLellan and Thornalley [41]. Briefly, glyoxalase I activity was assayed by measuring the rate of formation of S-D-lactoylglutathione
from hemi-thioacetal in the presence of tissue homogenate, followed by the increase in
absorbance at 240 nm using a molar extinction coefficient of 2.86 mM−1 cm−1 at pH 6.6. The
levels of ROS, NO, NADPH oxidase, SOD, CAT, GPx, GST, GSH, and GSSG in renal tissue
homogenates were estimated following the established protocol mentioned earlier. DNA
oxidation was estimated as per the established protocol [28]. Briefly, DNA was isolated
from renal tissue by the pronase-ethanol method followed by enzymatic digestion. DNA
oxidation assay was performed by RP-HPLC analysis in a Dionex UltiMate 3000 HPLC system (Dionex, Germany), using a C-18 column and equipped with electrochemical detector
and was represented as 7,8-hydroxy-2′ -deoxyguaosine/2′ -deoxyguaosine (8-OHdG/2-dG)
ratio. An isocratic mobile phase (pH 3.0) comprising 0.1 M formic acid, 1 mM citric acid,
7.7 mM NaN3 , 0.5 mM EDTA, 24 mM diethylamine, and acetonitrile (4%) was used to
estimate 8-OHdG, while acetonitrile (4%) in 50 mM NaH2 PO4 (pH 4.5) was used to estimate 2-dG. The applied potential for 8-OHdG and 2-dG was 0.0/+0.5 and +0.4/+0.8 volts,
respectively. Hydroxyproline, TNF-α, IL-1β, and IL-6 levels in the renal tissue homogenate
were measured by ELISA using commercially available kits (Fisher Thermo Scientific Co.,
Waltham, MA, USA). TGF-β1 and collagen IV levels in the renal tissue homogenates were
determined using ELISA kits (R&D Systems, Inc. Minneapolis, USA) according to the
manufacturer’s guidelines.
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4.4.6. Immunoblotting of Signal Proteins in Renal Tissue
The kidneys of experimental rats were homogenized with ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. The cellular/subcellular protein
samples (20 mg) were dissolved in 10% SDS-polyacrylamide gel electrophoresis and immunoblotted. The expressions Bcl-2, Bad in the mitochondria, cytochrome Cyt C in the
cytosol, cleaved caspase 9, cleaved caspase 3, P-IκBα (Ser 32) in the cytosol, P-NF-κB p65
(Ser 536) in the nucleus, Keap1 in the cytosol, P-Nrf-2 (Ser40) in the nucleus, TGF-β1,
P-Smad3 (Ser423/Ser425), Smad7, and collagen-IV were studied.
4.4.7. Histological Assessment
Formalin (10%)-fixed kidneys of mice under different treatments were embedded
within the paraffin blocks. The paraffin-mounted tissue samples were processed for microtome sectioning. Sections (~5 µm) were subjected to H&E and MT staining as per
the established protocol, and the sections were coated with resinous mounting medium
before taking microscopic images [2,17]. For MT staining, the sections were de-paraffinized
and stained with hematoxylin. Sections were washed with warm distilled water and
stained with Biebrich scarlet-acid fuchsin solution. Sections were then differentiated in
phosphomolybdic-phosphotungstic acid followed by staining with aniline blue and differentiation with 1% acetic acid. The sections were washed again and dehydrated with
absolute ethyl alcohol before mounting. Histo-quantification was performed using NIH
IMAGE (Image-J, 1.37v) software.
4.5. Statistical Analysis
The data are presented as the mean ± SD. The statistical analysis was executed using a
one-way analysis of variance (ANOVA) followed by Dunnett’s t-test in the GraphPad InStat
software (version 3.05), San Diego, CA, USA. p value < 0.05 was considered significant.
4.6. In Silico Assays
4.6.1. ADMET properties of Myr
The energy minimized state of Myr was subjected to chemometric ADMET and
other physicochemical parameter prediction. Important ADME and other properties
under Lipinski’s Rule of Five (RO5) [42], such as molecular weight, hydrogen bond donor,
hydrogen bond acceptor, and octanol/water partition coefficient (QPlogPo/w), were
predicted for Myr using Qikprop [43], a module of the Schrödinger software suite. Further
toxicity profiles, such as hepatotoxicity, developmental toxicity, and mutagenicity were
predicted using ADMET descriptors protocol of Discovery Studio 2.5.
4.6.2. Molecular Docking
All the signaling target proteins were retrieved from the protein data bank (PDB)
accessible at www.rcsb.org [44]. High resolution three-dimensional X-ray crystallographic
structures of PI3K (PDB: 4UWH), Akt (PDB: 3D0E), IRS1 (PDB: 5U1M), Bad (PDB: 1G5J),
Bcl-2 (PDB: 4LXD), Cyt C (PDB: 3ZCF), caspase 9 (PDB: 2AR9), caspase 3 (PDB: 5I9B),
IκB (PDB: 4KIK), NF-κB (PDB: 1SVC), Keap1 (PDB: 6TYM), Nrf-2 (PDB: 2FLU), TGF-β
(PDB: 3KFD), Smad7 (PDB: 2DJY), Smad3 (PDB: 1MJS), and collagen IV (PDB: 5NAX)
were selected for the molecular docking. All selected crystal structures were processed
using the ”Protein Preparation Wizard” module of Schrödinger suite [45], and protein
preparation was performed as per the established protocol [21]. The ligand molecule, Myr,
was prepared using the ”LigPrep” [46] module integrated into Schrödinger’s Maestro
interface, which produced the low-energy stereoisomers of the ligand with correct chirality
of the processed structure. To analyze the protein–ligand interactions, the prepared Myr
was docked to the active site of respective proteins employing ”Ligand docking” in Glide
module using the Extra Precision docking method following default settings [47]. However,
only the input parameter was changed in terms of allowing or generating a maximum
of 6 docking poses per ligand. After successful execution of docking, a protein–ligand
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interaction profiler tool [48] was used to detect the interaction patterns and binding contacts
between each signaling protein and Myr. Moreover, docking-based interactions obtained
for Myr were also compared with few signaling target proteins, such as PI3K, Caspase 9,
IκB, and Keap1, available with known ligands obtained from their respective PDBs.
5. Conclusions
In this study, Myr exhibited a protective effect against diabetic nephropathy via
antihyperglycemic, antioxidant, anti-inflammatory, and antifibrotic effects. The antihyperglycemic effect was manifested via endorsing insulin sensitization to improve glucose
uptake by the skeletal muscle evidenced by the activation of IRS-1/PI3K/Akt/GLUT4
in muscle cells in the diabetic milieu. Myr exhibited an antioxidant effect on the renal
cells via multiple mechanisms including neutralization of free radicals, suppression of
NADPH oxidase, inhibition of polyol pathways, and activation of cellular redox defense
system through Nrf-2 activation. Myr inhibited renal inflammation in the diabetic milieu
through NF-κB suppression. In addition, Myr could inhibit renal fibrosis by inhibiting
TGF-1/Smad/collagen IV signaling. The protective mechanism of Myr is proposed in
Figure 15. Molecular docking analysis predicted the interactions between Myr and the
signal proteins. ADMET prediction revealed that Myr supports the drug-likeness character.
Thus, Myr would serve as a potential therapeutic agent for T2D and diabetic nephropathy
in the future.
Figure 15. The possible mechanism of Myr in the management in glucose utilization and diabetic nephropathy. Red arrows
indicate downstream pathway, red lines indicate inhibition, and green lines indicate activation.
κ
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Supplementary Materials: The following are available online, Figure S1: Intermolecular interaction
analyses of known ligands of four signal proteins: PI3K (a), caspase 9 (b), IκB (c), and Keap1 (d).
Predicted molecular docking-based interactions of Myr were compared with these available target
proteins of known ligands, which revealed similar types of interacting amino acid residues involved
in several intermolecular interactions as obtained for Myr.
Author Contributions: Conceptualization, S.D., A.S., and V.D.F.; methodology, S.D., S.J., T.K.D.,
A.S., and V.D.F.; software, S.J. and S.B.; validation, T.K.D. and A.S.; formal analysis, S.J. and S.B.;
investigation, S.J., T.K.D., P.C., and S.B.; resources, S.D., and A.S.; data curation, S.J., T.K.D., and S.B.;
writing—original draft preparation, S.D., S.J., T.K.D., and S.B.; writing—review and editing, S.D.,
A.S., and V.D.F.; visualization, S.D.; supervision, S.D. and A.S.; project administration, S.D.; funding
acquisition, S.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Council of Scientific and Industrial Research, New Delhi,
India, grant number 02(0275)/16/EMR-II to S.D.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Public Health Service Policy on Humane Care and Use of Laboratory Animal; National Institute of Health: Washington, DC, USA, 2015, and approved by the Institutional Animal Ethical Committee (Reg. no.: 0367/01/C/CPCSEA, UGC, India) of Jadavpur University (Reference
no. AEC/PHARM/1701/08/2017, dated 30.7.2017).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data are presented in this study are available in the article and the
supplementary material.
Acknowledgments: Authors sincerely acknowledge Jadavpur University, Kolkata, India and University of Salerno, Salerno, Italy.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are commercially available. Authors will not
provide any sample to others.
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