Effect of Allicin against Ischemia/Hypoxia-Induced H9c2 Myoblast Apoptosis via eNOS/NO Pathway-Mediated Antioxidant Activity
Effect of Allicin against Ischemia/Hypoxia-Induced H9c2 Myoblast Apoptosis via eNOS/NO Pathway-Mediated Antioxidant Activity
Lina Ma Yikui Li
Shangke Chen 0
Hao Li 0
Shaochun Li 1
Lijuan Deng 0 2
0 Xiyuan Hospital, China Academy of Chinese Medical Sciences , Beijing , China
1 Xinjiang Ailexin Pharmaceutical Co., Ltd. , Urumqi, Xinjiang , China
2 School of Basic Medical Sciences, Hebei University , Baoding, Hebei , China
Allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester, diallyl thiosulfinate) is the main biologically active ingredient in garlic. The present study investigated the protective effect of allicin against cardiomyocyte apoptosis that was induced by ischemia in vitro and the potential molecular mechanisms that were involved in this antiapoptotic effect. The results indicated that allicin increased H9c2 cell activity and attenuated the rate of apoptosis that was induced by ischemia/hypoxia. Intracellular calcium concentrations significantly decreased in the allicin-treated groups. Bax expression significantly decreased, and Bcl-2 expression increased in allicin-treated rats. Nitric oxide blockade significantly inhibited these effects. Allicin also increased the activity of SOD and NO release and decreased MDA levels. Allicin significantly increased the expression of eNOS, Nrf2, and HO-1 proteins. Collectively, these findings demonstrate that allicin protects H9c2 cells against apoptosis, and this protective effect appears to occur via eNOS/NO pathway-mediated antioxidant activity.
Ischemic heart disease (IHD) is one of the leading causes of
global mortality, thus resulting in substantial social burdens
]. Several studies have suggested that
apoptosis occurs during the overall pathophysiological process of
ischemia and plays an important role in the progression of
cardiovascular diseases [
]. Apoptosis is a pivotal form
of cell death after acute myocardial infarction, which leads
to abnormal loading conditions and, further, left ventricular
dilatation and consequently symptomatic heart failure [
Therefore, the inhibition of apoptosis may be an effective
way to prevent heart failure and improve cardiac function.
Numerous cardiovascular protective compounds have been
discovered to limit the ischemic damage, but no satisfactory
drug treatments have been developed for clinical practice.
Natural antioxidants from food sources have been used
to prevent and treat IHD. Garlic has attracted particular
attention because of its beneficial cardiovascular effects [
Allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester,
diallyl thiosulfinate) is the main biologically active ingredient
in garlic. Allicin has been shown to exert a wide range of
biological effects, including antioxidant activity, the inhibition
of platelet activation, antitumor activity, and antimicrobial
]. Our previous studies in rats found that allicin
mitigates ischemic injury via inhibiting apoptosis . It
was also shown to prevent against myocardial apoptosis and
fibrosis in a streptozotocin-induced diabetic models in rats
]. Although abundant evidence supports a link between
allicin and cardioprotection, the precise mechanisms by
which allicin prevents IHD remain largely unknown.
Therefore, the present study evaluated the protective effects of
allicin against cardiomyocyte apoptosis that was induced
by ischemia in vitro and investigated the potential
molecular mechanisms that are involved in this antiapoptotic
2. Materials and Methods
2.1. Materials. Allicin injection (5 mg/ml) was obtained from
Xinjiang Ailexin Pharmacy Co., Ltd. (Urumqi, China).
NGnitro-l-arginine methyl ester (l-NAME), a specific
pharmacological blocker of nitric oxide (NO), was purchased
from Sigma (St. Louis, MO, USA). Anti--actin antibody was
purchased from Beijing Zhongshan Golden Bridge
Biotechnology (Beijing, China). Antibodies against Bax and Bcl-2
were purchased from Cell Signaling Technology (Danvers,
MA, USA). Antibodies against endothelial nitric oxide
synthase (eNOS), nuclear factor erythroid 2-related factor 2
(Nrf2), and heme oxygenase-1 (HO-1) were purchased from
Abcam (Cambridge, UK). Horseradish peroxidase- (HRP-)
conjugated anti-mouse and anti-rabbit immunoglobulin G
antibodies were purchased from Beijing Zhongshan Golden
Bridge Biotechnology (Beijing, China).
2.2. H9c2 Cell Culture and Ischemia/Hypoxia Model. H9c2
cells were cultured in Dulbecco?s modified Eagle?s medium
(DMEM) that contained 10% fetal bovine serum, 2 mM
glutamine, and antibiomycin (10 mM penicillin G and 10 mM
streptomycin) at 37?C in a humidif ied 5% CO2 atmosphere
and subcultured to approximately 80?90% confluence
An I/H model was established to induce apoptosis. The
culture medium was replaced with serum-free DMEM, and
then H9c2 cells were transferred to a hypoxia chamber
that was controlled by a ProOxC system balanced with 5%
CO2/95% N2 at 37?C (Biospherix, Redf ield, NY). T he 2O
concentration was ?1%. The cells were pretreated with allicin
at different concentrations (0.2, 1, and 5 M) for 1 h and
then exposed to I/H for 12 h. The negative control cells
were cultured in DMEM containing 10% fetal bovine serum
2.3. Cell Viability Assay. Cell viability was examined using
bromide (MTT) assay kit (Promega, Madison, WI, USA)
according to the manufacturer?s protocol. H9c2 cells were plated
in 96-well plates at a density of 1 ? 104 cells/well. The cells
were pretreated with various doses of allicin (0.2, 1, and 5 M)
for 1 h and then exposed to I/H for 12 h. Afterward, a 100 l
cell suspension was incubated with 20 l of CellTiter 96
AQueous One Solution Reagent for 1 h with 5% CO2 at 37?.
Absorbance was read at 490 nm. T he mean optical density
(OD) of f ive wells was used to calculate the percentage of
cell viability: % cell viability = ODtreatment group/ODcontrol group ?
2.4. Flow Cytometry Assay. The rate of apoptosis was
determined by f low cytometry af ter staining the cells with Annexin
V-FITC/PI (key GEN Bio TECH, Nanjing, China) according
to the manufacturer?s protocol. The cells were cultured in
six-well plates at a density of 1 ? 105 cells/well, treated
with allicin (0.2, 1, and 5 M) for 1 h, and then exposed
to I/H for 12 h. The cells were harvested, washed twice
with cold 1x phosphate-buffered saline (PBS), centrifuged
at 2000 rotations per minute for 6 min, and resuspended
in 0.5 ml of cold 1x binding buffer. The cell suspension
was stained with 5 l of Annexin V-FITC for 10 min in the
dark, and 5 l of propidium iodide (PI) was then added for
5?15 min in the light at room temperature. Flow cytometry
was performed using a fluorescence-activated cell sorting
instrument. The data were analyzed using WinMDI/PC
2.5. Measurement of Calcium Ion Concentrations. The cells
were pretreated with various doses of allicin (0.2, 1, and 5 M)
for 1 h and then subjected to I/H for 12 h. The cells were
then incubated with Fluo-3/AM (Molecular Probes, USA)
for 30 min at 37?C and then observed under a confocal laser
2.6. Measurement of Oxidative Activity. The content of NO
and malondialdehyde (MDA) and activity of sodium oxide
dismutase (SOD) were assessed using specific kits (Nanjing
Jiancheng Biological Engineering Institute, Nanjing, China).
All of the procedures were performed according to the
2.7. Western Blot. Cell lysates (20 g of protein) were
analyzed by 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and electrotransferred to polyvinylidene
difluoride membranes. The membranes were blocked with
5% bovine serum albumin and then probed with specific
antibodies at 4?C overnight. After three washes in Tris
PBS (TPBS), the membranes were incubated with
HRPconjugated secondary antibodies, followed by
electrochemiluminescent detection. Blot densitometry was then
performed. The bands were analyzed using a Gene Genius Bio
2.8. Statistical Analyses. The data were analyzed using SPSS
19.0 software. The values are expressed as mean ? SEM.
Analysis of variance (ANOVA) was conducted, followed by
Bonferroni correction, to test for differences in mean values
between groups. The results were considered significant at
3.1. Allicin Improves H9c2 Cell Morphology and Viability.
Morphological variations of H9c2 cells were observed under
an inverted microscope. In the I/H group, the cells were
severely damaged, with edge blurring, shrunken, shedding,
and floating in the cell culture medium. In the
allicinpretreated groups, the degree of cell injury decreased, and
cellular survival increased, especially in the group that was
pretreated with 5 M allicin (Figure 1(a)).
Cell viability was examined using the MTT assay kit. Cell
viability in the I/H group significantly decreased compared
with the control ( < 0.05). Cell viability significantly
increased in the groups that were treated with 1 and 5 M
allicin compared with the untreated I/H group ( < 0.05). No
significant difference ( > 0.05) was observed between the
Control I/H + AL (0.2 ?M) # I/H
I/H + AL (1 ?M)
group that was treated with 0.2 M allicin and the untreated
I/H group (Figure 1(b)).
3.2. Allicin Decreases Rate of I/H-Induced Apoptosis in H9c2
Cells. After incubation under conditions of I/H for 12 h, the
rate of apoptosis was determined by flow cytometry after
staining the cells with Annexin V-FITC/PI. In the untreated
I/H group, apoptosis significantly increased compared with
the sham control group ( < 0.05). Apoptosis in the
allicintreated groups significantly decreased compared with the
untreated I/H group ( < 0.05; Figure 2).
3.3. Allicin Decreases Intracellular Ca2+ Concentrations. To
detect intracellular Ca2+ concentrations, the cells were
incubated with Fluo-3/AM and then observed under a
confocal laser scanning microscope. Ca2+ concentrations in the
untreated I/H group signif icantly increased compared with
the control group ( < 0.05; Figure 3). In the allicin-treated
groups, a significant decrease in Ca2+ concentrations was
observed compared with the untreated I/H group ( < 0.05).
Control I/H I/H + AL (0.2 ?M) 2000
2 ? 500
Control I/H 0.2
I/H + allicin (?M)
3.4. Allicin Suppresses the Expression of Markers of Apoptosis.
Bax and Bcl-2 are markers of apoptosis. Bax and Bcl-2 protein
expression was detected by Western blot. Bax expression
significantly increased and Bcl-2 expression significantly
decreased in H9c2 cells that were subjected to I/H compared
with the control group. Bax expression significantly decreased
in the group that was pretreated with 5 M allicin, and
Bcl2 expression significantly increased in the groups that were
3.5. Allicin Increases eNOS Expression and NO Levels. Nitric
oxide is a potent gaseous signaling molecule. eNOS-derived
NO may participate in the pathophysiological regulation of
ischemic heart disease. We detected eNOS expression and
NO levels that were induced by I/H in H9c2 cells. eNOS
expression significantly decreased in untreated cells that were
subjected to I/H compared with the control group ( <
0.05). In the allicin-pretreated groups (0.2, 1, and 5 M),
eNOS expression significantly increased compared with the
untreated I/H group ( < 0.05; Figure 5(a)). NO levels
significantly decreased in untreated cells that were subjected
to I/H compared with the control group. In cells that were
treated with allicin (1 and 5 M), NO levels dose-dependently
increased ( < 0.05; Figure 5(b)).
stress, we measured MDA and SOD in H9c2 cells. Our results
showed that MDA levels significantly increased and SOD
activity significantly decreased during I/H, and these effects
were reversed by allicin pretreatment (Figures 7(a) and 7(b)).
3.8. Effect of Allicin on Nrf2 and HO-1 Expression in H9c2
Cells. We further examined whether allicin affects activation
of the Nrf2/HO-1 signaling pathway. I/H induced a trend
toward higher Nrf2 expression in H9c2 cells ( > 0.05).
Treatment with allicin (1 and 5 M) significantly increased
Nrf2 expression ( < 0.05; Figure 8(a)). Moreover, I/H
significantly increased HO-1 expression in H9c2 cells. Treatment
with allicin (1 and 5 M) induced a further dose-dependent
increase in HO-1 expression ( < 0.05; Figure 8(b)).
Allicin is one of the critical bioactive organosulfur
com3.6. Ef fect of L-NAME on Antiapoptotic Ef fect of Allicin. pounds in garlic. It has been reported to have a number of
We used l-NAME, a specific pharmacological blocker of bioactivities including antioxidant, cardioprotective activity
NO, to elucidate the mechanism of action of allicin. l- [
]. However, due to its unstable nature, allicin is
NAME significantly inhibited eNOS expression ( < 0.05; rapidly degraded with time depending on environmental
Figure 6(a)) and inhibited the cardioprotective effect of allicin and processing conditions, such as temperature, light, and
against apoptosis, reflected by an increase in Bax expression concentration [
]. Document has also shown that the
and decrease in Bcl-2 expression ( < 0.05; Figures 6(b) and biological half-life of allicin is significantly longer in alcoholic
6(c)). and aqueous extracts than the chemical one . In the
present study, allicin is a water-soluble injection and stored
3.7. Effect of Allicin on Oxidative Activity in H9c2 Cells. Mal- in dark place at 4?C to ensure its stability. Then, we assessed
ondialdehyde and SOD are biomarkers of oxidative stress. To the antiapoptotic effect of allicin in H9c2 cells. Allicin
signifdetermine whether allicin functions at the level of oxidative icantly decreased intracellular calcium concentration and the
rate of apoptosis. Meanwhile, allicin significantly increased
Bax expression and decreased Bcl-2 and eNOS expression.
However, the NO blocker l-NAME partially reversed these
beneficial effects of allicin, indicating an involvement of
eNOS/NO signaling pathway. Allicin also increased SOD
activity, NO release, and Nrf2 and HO-1 expression and
decreased MDA levels. Altogether, these findings suggest
that activation of the eNOS/NO pathway by allicin and its
antioxidant and antiapoptotic effects play an important role
in its cardioprotective effects.
Apoptosis is a key regulator in the pathogenesis of
myocardial ischemia. Bcl-2 family members act upstream
of mitochondrial-mediated apoptosis and play a central role
in cell fate and homeostasis. Bcl-2 (prosurvival) protein
expression determines whether the cell undergoes apoptosis
or reenters the cell cycle. Bax protein expression integrates
important functions that are related to apoptosis and
facilitates the release of cytochrome c from mitochondria [
Therefore, the ratio of Bcl-2/Bax expression is regarded as a
hallmark in cell survival or death upon apoptotic stimulation
]. In the present study, we utilized I/H method
to induce apoptosis in H9c2 cells, and allicin significantly
increased cell activity and decreased the rate of apoptosis
that was induced by I/H, reflected by a decrease in Bax
expression and increase in Bcl-2 expression. Cellular injury
caused I/H is also accompanied by intracellular calcium
overload. Increasing intracellular Ca contributes to the
development and progression of myocardial apoptosis .
Our results showed that intracellular calcium concentrations
significantly decreased in the allicin-pretreated groups.
Nitric oxide is a potent gaseous signaling molecule
that is synthesized by a family of NOS enzymes, including
inducible, neuronal, and endothelial forms [
]. eNOS, also
known as NOS III, is a low-output enzyme that is
constitutively expressed in H9c2 cells. eNOS-derived NO has been
reported to participate in the pathophysiology of ischemic
heart disease, such as myocardial infarction and
myocardial ischemia-reperfusion injury [
]. A previous study
reported that both skin and flesh garlic extracts effectively
prevented norepinephrine-induced cardiomyocyte
hypertrophy and cell death, and these beneficial effects were partially
mediated by NO and H2S . Our previous f indings showed
that plasma H2S levels dose-dependently increased in
allicintreated rats, indicating that allicin may be an H2S donor
]. The present study demonstrated the involvement of NO
in this process. Allicin treatment increased eNOS protein
expression and NO levels. To further explore the mechanism
by which allicin attenuates myocardial cell apoptosis, we
pharmacologically blocked NO using l-NAME. l-NAME
significantly inhibited the cardioprotective effect of allicin
against apoptosis, evidenced by an increase in Bax expression
and decrease in Bcl-2 expression. These data strongly suggest
that the eNOS/NO pathway is involved in the effects of allicin
Oxidative stress promotes cell death in response to
various pathophysiological conditions. Reaction oxygen species
(ROS) are free radicals, the accumulation of which can
cause oxidative stress that damages the heart in
myocardial ischemia [
]. Previous studies reported that allicin
protected cells against oxidative stress by inhibiting the
generation of intracellular ROS [
]. Nrf2 is a
redoxsensitive transcription factor that plays a key role in cellular
antioxidant defense . In the presence of oxidative stress,
Nrf2 is rapidly degraded by the proteasome system, enters
the nucleus, binds to antioxidant response element, and
upregulates multiple antioxidant and detoxifying genes, such
as HO-1 [
]. Allicin was previously reported to prevent
the development of cardiac remodeling and progression of
cardiac hypertrophy to cardiac dysfunction by enhancing
Nrf2 antioxidant signaling pathways [
]. A reasonable
speculation is that the antiapoptotic effect of allicin may be
associated with activation of the Nrf2 signaling pathway
associated with oxidative stress. Therefore, we tested the
effects of allicin on oxidative stress. Allicin significantly
decreased MDA levels and increased SOD activity. It also
promoted Nrf2 synthesis and nuclear translocation and
further increased the expression of HO-1 protein,
indicating the antioxidative activity of allicin in our I/H model.
Numerous studies have shown that NO can scavenge ROS
and attenuate the detrimental effects of ROS [
the present study, allicin increased eNOS protein expression
and NO levels in H9c2 cells, and the NO blocker l-NAME
significantly inhibited the cardioprotective effect of allicin
against apoptosis. Therefore, we suggest that the antioxidative
activity of allicin may be linked to activation of the eNOS/NO
Overall, our data indicate that allicin has powerful protective
effects against I/H-induced cell apoptosis. The mechanism
appears to involve an antioxidative effect that is mediated by
the eNOS/NO pathway. Allicin treatment may be a promising
clinical approach for IHD.
Conflicts of Interest
The authors declare no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
This study was supported by the Autonomous Region Major
Science & Technology Specific Projects: Innovation and
Industrialization Demonstration of National Medicine (no.
Hindawi Publishing Corporation
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Mathematical in Medicine
 X. Zhu and L. Zuo , ? Characterization of oxygen radical formation mechanism at early cardiac ischemia , ? Cell Death & Disease , vol. 4 , no. 9 , article e787 , 2013 .
 L. Ma , C.- C. Chuang , W. Weng et al., ? Paeonol protects rat heart by improving regional blood perfusion during no-reflow,? Frontiers in Physiology , vol. 7 , article no. 298 , 2016 .
 V. Sivaraman and D. M. Yellon , ? Pharmacologic therapy that simulates conditioning for cardiac ischemic/reperfusion injury , ? Journal of Cardiovascular Pharmacology and Therapeutics , vol. 19 , no. 1 , pp. 83 - 96 , 2014 .
 E. Teringova and P. Tousek , ? Apoptosis in ischemic heart disease , ? Journal of Translational Medicine , vol. 15 , no. 1 , article no. 87 , 2017 .
 V. Boshra and A. Atwa , ? Effect of cerebrolysin on oxidative stress-induced apoptosis in an experimental rat model of myocardial ischemia , ? Acta Physiologica Hungarica , vol. 103 , no. 3 , pp. 310 - 320 , 2016 .
 J. Narula , N. Haider , R. Virmani et al., ?Apoptosis in myocytes in end-stage heart failure,? The New England Journal of Medicine , vol. 335 , no. 16 , pp. 1182 - 1189 , 1996 .
 G. Olivetti , F. Quaini , R. Sala et al., ? Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart , ? Journal of Molecular and Cellular Cardiology , vol. 28 , no. 9 , pp. 2005 - 2016 , 1996 .
 O. Dayoub , R. Andriantsitohaina , and N. Clere , ? Pleiotropic beneficial effects of epigallocatechin gallate, quercetin and delphinidin on cardiovascular diseases associated with endothelial dysfunction,? Cardiovascular & Hematological Agents in Medicinal Chemistry , vol. 11 , no. 4 , pp. 249 - 264 , 2013 .
 X. U. Ying and Y. Zong-Ning , ?Study on antioxidant activity of alliin, alliinase and their mixture in vitro,? West China Journal of Pharmaceutical Sciences , vol. no. 04 , pp. 328 - 330 , 2011 .
 S. J. Thomson , P. Rippon , C. Butts et al., ?Inhibition of platelet activation by lachrymatory factor synthase (LFS)- silenced (tearless) onion juice , ? Journal of Agricultural and Food Chemistry , vol. 61 , no. 44 , pp. 10574 - 10581 , 2013 .
 X. Zou , J. Liang , J. Sun et al., ? Allicin sensitizes hepatocellular cancer cells to anti-tumor activity of 5-fluorouracil through ROS-mediated mitochondrial pathway , ? Journal of Pharmacological Sciences , vol. 131 , no. 4 , pp. 233 - 240 , 2016 .
 D. Wallock-Richards , C. J. Doherty , L. Doherty et al., ? Garlic revisited: antimicrobial activity of allicin-containing garlic extracts against Burkholderia cepacia complex ,? PLoS ONE , vol. 9 , no. 12 , Article ID e112726, 2014 .
 L.-N. Ma , L. -D. Li , S.-C. Li et al., ? Allicin improves cardiac function by protecting against apoptosis in rat model of myocardial infarction,? Chinese Journal of Integrative Medicine , vol. 23 , no. 8 , pp. 589 - 597 , 2017 .
 Y. Liu , H. Qi , Y. Wang et al., ? Allicin protects against myocardial apoptosis and fibrosis in streptozotocin-induced diabetic rats , ? Phytomedicine , vol. 19 , no. 8-9 , pp. 693 - 698 , 2012 .
 J. Y.-Y. Chan , A. C.-Y. Yuen , R. Y. -K. Chan , and S.-W. Chan, ? A review of the cardiovascular benefits and antioxidant properties of allicin ,? Phytotherapy Research , vol. 27 , no. 5 , pp. 637 - 646 , 2013 .
 Y. J. Lee , D. Lee , S. M. Shin et al., ?Potential protective effects of fermented garlic extract on myocardial ischemiareperfusion injury utilizing in vitro and ex vivo models , ? Journal of Functional Foods , vol. 33 , pp. 278 - 285 , 2017 .
 D. Ilic? , V. Nikolic?, M. Stankovic ? et al., ?Transformation of synthetic allicin: the influence of ultrasound, microwaves, different solvents and temperatures, and the products isolation,? The Scientific World Journal , vol. 2012 , Article ID 561823, 7 pages, 2012 .
 H. Wang , X. Li , X. Liu et al., ? Inf luence of pH, concentration and light on stability of allicin in garlic (Allium sativum L.) aqueous extract as measured by UPLC , ? Journal of the Science of Food and Agriculture , vol. 95 , no. 9 , pp. 1838 - 1844 , 2015 .
 M. S. Rahman , ? Allicin and other functional active components in garlic: Health benefits and bioavailability ,? International Journal of Food Properties , vol. 10 , no. 2 , pp. 245 - 268 , 2007 .
 H. Fujisawa , K. Suma , K. Origuchi , H. Kumagai , T. Seki , and T. Ariga, ? Biological and chemical stability of garlic-derived allicin , ? Journal of Agricultural and Food Chemistry , vol. 56 , no. 11 , pp. 4229 - 4235 , 2008 .
 G. Del Poeta , A. Venditti , M. I. Del Principe et al., ?Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML ), ? Blood , vol. 101 , no. 6 , pp. 2125 - 2131 , 2003 .
 J. Martinou and R. J. Youle , ? Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics, ? Developmental Cell , vol. 21 , no. 1 , pp. 92 - 101 , 2011 .
 S. M. Mosaad , S. A. Zaitone , A. Ibrahim , A. A. El-Baz , D. M. Abo-Elmatty , and Y. M. Moustafa , ? Celecoxib aggravates cardiac apoptosis in L-NAME-induced pressure overload model in rats: Immunohistochemical determination of cardiac caspase-3, Mcl-1, Bax and Bcl- 2 , ? Chemico-Biological Interactions , vol. 272 , pp. 92 - 106 , 2017 .
 O. Bar-Am , O. Weinreb , T. Amit , and M. B. H. Youdim , ? Regulation of Bcl-2 family proteins, neutrophic factors, and APP processing in the neurorescue activity of propargylamine,? The FASEB Journal , vol. 19 , no. 13 , pp. 1899 - 1901 , 2005 .
 Y. Wang , S. Wei , Y.-L. Wang et al., ? Protective ef fects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/ reperfusion injury , ? Oncotarget , vol. 8 , no. 33 , pp. 54572 - 54582 , 2017 .
 J. Lee , E. H. Bae , S. K. Ma , and S. W. Kim , ? Altered Nitric Oxide System in Cardiovascular and Renal Diseases,? Chonnam Medical Journal , vol. 52 , no. 2 , p. 81 , 2016 .
 M. C. De Waard , J. Van Der Velden , N. M. Boontje et al., ? Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction ,? American Journal of Physiology-Heart and Circulatory Physiology , vol. 296 , no. 5 , pp. H1513 - H1523 , 2009 .
 S. P. Jones , J. J. Greer , A. K. Kakkar et al., ? Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury ,? American Journal of Physiology-Heart and Circulatory Physiology , vol. 286 , no. 1 , pp. H276 - H282 , 2004 .
 X. L. Louis , R. Murphy , S. J. T handapilly , L. Yu, and T. Netticadan, ? Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: A role for nitric oxide and hydrogen sulfide,? BMC Complementary and Alternative Medicine , vol. 12 , article no. 140 , 2012 .
 N. A. Kelsey , H. M. Wilkins , and D. A. Linseman , ? Nutraceutical antioxidants as novel neuroprotective agents , ? Molecules , vol. 15 , no. 11 , pp. 7792 - 7814 , 2010 .
 X. Chen , S. Pang , J. Lin , J. Xia , and Y. Wang , ? Allicin prevents oxidized low-density lipoprotein-induced endothelial cell injury by inhibiting apoptosis and oxidative stress pathway,? BMC Complementary and Alternative Medicine , vol. 16 , no. 1 , article no. 133 , 2016 .
 M. Zhang , H. Pan, Y. Xu , X. Wang , Z. Qiu , and L. Jiang, ? Allicin decreases lipopolysaccharide-induced oxidative stress and inflammation in human umbilical vein endothelial cells through suppression of mitochondrial dysfunction and activation of Nrf2,? Cellular Physiology and Biochemistry , vol. 41 , no. 6 , pp. 2255 - 2267 , 2017 .
 L. Ma , X. Liu, Y. Zhao , B. Chen , X. Li , and R. Qi , ? Ginkgolide B reduces LOX-1 expression by inhibiting Akt phosphorylation and increasing Sirt1 expression in oxidized LDL-stimulated human umbilical vein endothelial cells ,? PLoS ONE , vol. 8 , no. 9 , Article ID e74769, 2013 .
 K. Itoh , N. Wakabayashi , Y. Katoh et al., ?Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain,? Genes & Development , vol. 13 , no. 1 , pp. 76 - 86 , 1999 .
 J. Y. Chan , H. Tsui , I. Y. Chung , R. Y. Chan , Y. Kwan , and S. Chan , ? Allicin protects rat cardiomyoblasts (H9c2 cells) from hydrogen peroxide-induced oxidative injury through inhibiting the generation of intracellular reactive oxygen species ,? International Journal of Food Sciences and Nutrition , vol. 65 , no. 7 , pp. 868 - 873 , 2014 .
 J. Li , T. R. Billiar , R. V. Talanian , and Y. M. Kim , ? Nitric oxide reversibly inhibits seven members of the caspase family via Snitrosylation,? Biochemical and Biophysical Research Communications , vol. 240 , no. 2 , pp. 419 - 424 , 1997 .
 D. A. Wink , I. Hanbauer, M. C. Krishna , W. DeGraf f, J. Gamson , and J. B. Mitchell , ? Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species , ? Proceedings of the National Acadamy of Sciences of the United States of America , vol. 90 , no. 21 , pp. 9813 - 9817 , 1993 .
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