| Apoptosis is an active death process caused by the triggering of the cell death signal or the control of its own genetic genes.It plays a very important role in regulating the growth and development of organisms.Many studies have indicated that apoptosis is accompanied by the pathological process of ischemic heart disease.At present,the clinical treatment of ischemic heart disease mainly include angiotensin inhibitors,beta adrenergic receptor blockers,nitrates and calcium channel blockers.The effect of chemical drugs is fast,and the target is clear,but it is often accompanied by some side effects,affecting the efficacy of drugs.In recent years,many researchers working on myocardial ischemia,and accumulated rich experience in the treatment of ischemic heart disease,and confirmed that many Chinese herbs or extracts possess a variety of biological activities.At the same time.they also have the advantages of high safety and less adverse reaction.Allicin(diallyl thiosulfinate),the primary active ingredient in garlic,is produced by tissue damage from alliin in a reaction that is catalyzed by the enzyme alliinase in fresh garlic.It has been shown to exert a wide variety of biological properties,such as the function of lowering blood fat,anti-oxidation,improving myocardial fibrosis etc.,which has great development prospects and research value in the field of cardiovascular diseases.However,potentially beneficial effects of allicin as a cardioprotective agent against myocardial ischemia and mechanisms have not been reported yet.Objective:This study aimed at observing the protective effect of allicin on cardiomyocyte apoptosis induced by ischemia in vitro and in vivo,and investigating the potential molecular mechanisms involved in the anti-apoptosis effect.Methods:The first experiment:1.Myocardial infarction(MI)was induced by left anterior descending coronary artery ligation.The rats were randomly assigned to six groups:sham control group,MI control group,Diltiazem Hydrochloride(DIL)positive control group(1.5mg/kg).MI+allicin group(1.2 mg/kg),MI+allicin group(1.8 mg/kg),and MI+allicin group(3.6 mg/kg).All of the drugs were administered intraperitoneally daily for 21 days.2.HE staining was used to observe the pathological changes.The content of CK?LDH?MDA and the activity of SOD in serum were detected by specific kits.A modified methylene blue method was used to measure H2S levels in plasma.The myocardial apoptotic index was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)staining.The expression of and mRNA content of Bax and Bcl-2 were detected by Western blot and qRT-PCR.The second experiment:1.The H9c2 cells were treated with allicin(0.1?100?M)for 12 hours and the cell viability were examined by the MTT assay.2.The ischemia/hypoxia(I/H)model was established.H9c2 cells were cultured in incubator of 37??5%CO2.Access to 95%N2,and the O2 concentration is less than or equal to 1%.3.H9c2 cells were divided into five groups:control group,I/H group,I/H+allicin group(0.2?M),I/H+allicin group I/H+allicin group(5?M).The morphological characteristics of H9c2 cells were observed by inverted microscope.The cell viability was examined by the MTT assay.The apoptosis rate was determined by flow cytometry assay after stained with Annexin V-FITC/PI.The calcium ions in the cells were labeled with Fluo-3/AM,and observed under confocal laser scanning microscope.Mitochondrial membrane potential was detected by JC-1.The content of MDA and the activity of SOD were detected by specific kits.The expression of Bax?Bcl-2?caspase-3?Nrf2 and HO-1 were detected by Western blot.4.Pharmacological blockade of H2S(PAG)was used to elucidate the mechanism of action of allicin.H9c2 cells were divided into four groups:I/H group,I/H+allicin group(5?M),I/H+allicin(5?M)+PAG group,I/H+PAG group.The expression of Bax and Bcl-2 were detected by Western blot.5.Pharmacological blockade of NO(LNAME)was used to elucidate the mechanism of action of allicin.H9c2 cells were divided into four groups:I/H group,I/H+allicin group(5?M),I/H+allicin(5?M)+LNAME group,I/H+LNAME group.The content of NO was detected by specific kit.The expression of eNOS?Bax and Bcl-2 were detected by Western blot.Results:The first experiment:1.The architecture of myocardium in the sham control group rats were normal,cardiomyocyte arranged closely and regularly,and the nuclei were clearly visible.In the MI group rats,cardiomyocytes presented extensive edema,intercellular space enlarged,and the nuclei were not clear or even fusion.After treatment with DIL and allicin,the changes of the myocardium were markedly relieved,cardiomyocytes presented mild edema,and arranged regularly.2.CK and LDH levels were both elevated in the MI control group compared with the sham control group.After treatment with DIL and allicin,the levels of CK and LDH dose-dependently decreased(P<0.05).In the MI group,MDA level was significantly increased,and the activity of SOD was significantly decreased compared with the sham control group(P<0.05).MDA level were significantly decreased,with SOD activity significantly were increased in the allicin-treated groups and positive control group(P<0.05).3.H2S levels were increased in the allicin-treated groups compared with the untreated groups in a dose dependent manner(P<0.05).4.In the MI group,a significant increase in the number of apoptotic cell nuclei was observed compared with the sham control group(P<0.05).In contrast,the apoptotic index in the allicin-treated groups were lower than in the MI group as well as in the positive control group(P<0.05).5.Bax expression was significantly increased in the MI group,and Bcl-2 was significantly decreased compared with the sham control group(P<0.05).Bax expression was significantly decreased in the 3.6 mg/kg allicin-treated group,and Bcl-2 expression were significantly increased in the 1.8 and 3.6 mg/kg allicin-treated groups and positive control group(P<0.05).Then the mRNA abundance of Bax and Bcl-2 were also determined.Compared with the MI group,Bax mRNA levels were significantly decreased,with Bcl-2 levels were significantly increased in the 1.8 and 3.6 mg/kg allicin-treated groups and positive control group(P<0.05).The second experiment:1.Effects of allicin on normal H9c2 cellsCompared with the control group,the H9c2 cells activity treated with allicin(0.1?10?M)had no significant difference(P>0.05),but the H9c2 cells activity were decreased significantly when the concentrations of allicin were up to 25?100?M(P<0.05).2.Effects of allicin on H9c2 cells apoptosis induced by I/H2.1 In the control group,H9c2 cells grew well,the morphology were clear and complete,and spindle.In the I/H group,H9c2 cells were severely damaged,cells with blurred edge,shriveled go round,fell off,and floated in the cell culture medium.In theallicin-treated groups,cells damage degree was significantly reduced and improved,the number of shrinkage loss and floating cells were decreased.2.2 Compared with the control group,H9c2 cells activity was significantly decreased in the I/H group(P<0.05),but the H9c2 cells activity were increased significantly in the allicin-treated group(P<0.05).2.3 Compared with the control group,the apoptotic rate was significantly increased in the I/H group(P<0.05).In contrast,the apoptotic rate in the allicin-treated groups were lower than in I/H group(P<0.05).2.4 Compared with the control group,the intracellular calcium concentration significantly increased in the I/H group(P<0.05),indicating that H9c2 cells appear calcium overload.Compared with the I/H group,intracellular calcium concentration were significantly decreased significantly in the allicin-treated group(P<0.05).2.5 In the control group,H9c2 cells mainly showed red fluorescence.The green fluorescence was very strong in the I/H group,suggesting that mitochondrial membrane potential was decreased significantly.In contrast,the green fluorescent were decreased,and red fluorescence were enhanced in the allicin-treated groups.2.6 I/H increased the expression of pro-apoptosis protein Bax and caspase-3 and decreased the expression of anti-apoptosis protein Bcl-2 compared to control(P<0.05).Bax and caspase-3 expression were significantly decreased in the allicin(5?M)-treated groups,and Bcl-2 expression were significantly increased in the allicin(1?M and 5pM)-treated groups(P<0.05)compared to I/H.3.The relationship of allicin against H9c2 cell apoptosis and H2SThe regulation effects of allicin on apoptosis related protein Bax and Bcl-2 were significantly inhibited by PAG(P<0.05),Compared with the I/H group,the changes of Bax and Bcl-2 expression in H9c2 cells were not obvious in PAG group alone(P>0.05).4.The relationship of allicin against H9c2 cell apoptosis and NOCompared with the control group,NO level and eNOS expression were decreased significantly in the I/H group,while the data were statistically significant(P<0.05).Compared with the I/H group,NO level and eNOS expression were increased in the allicin-treated groups in a dose dependent manner,but the regulation effects of allicin were inhibited by LNAME.Meanwhile,the regulation effects of allicin on apoptosis related protein Bax and Bcl-2 were also significantly inhibited by LNAME(P<0.05).In PAG group alone,the changes was not obvious compared with the I/H group(P>0.05).5.The relationship of allicin against H9c2 cell apoptosis and oxidative stressMDA level was significantly increased in I/H group,and the activity of SOD was significantly decreased compared with the control group(P<0.05).MDA levels were significantly decreased in the allicin(1?M and 5pM)-treated groups(P<0.05),with SOD activity were significantly increased in the allicin(0.2?M and 5?M)-treated groups(P<0.05).The results showed that allicin has antioxidant effect.Compared with the control group,Nrf2 expression was increased in the I/H group,but the data was not statistically significant(P>0.05),and HO-1 expression was significantly increased(P<0.05).Compared with the I/H group,Nrf2 and HO-1 expression were all increased significantly in the allicin(1?M and 5?M)-treated groups(P<0.05).Conclusion:1.Allicin improved myocardial pathological alterations,decreased CK and LDH levels in serum and the myocardial apoptotic index,and the expression of Bax was significantly increased,and the expression of Bcl-2 was significantly decreased.These findings suggested that the protective effect of allicin may be linked to blocking apoptosis induced bymyocardial ischemia.2.I/H could induce H9c2 cells apoptosis.The effects mainly showed by:I/H significantly decreased cell viability,increased cell apoptosis rate and intracellular calcium concentration,decreased mitochondrial membrane potential,and increased the expression of pro-apoptosis protein Bax and caspase-3 and decreased the expression of anti-apoptosis protein Bcl-2.3.Allicin could significantly increase cell viability,inhibit the intracellular calcium overload and translocation of mitochondrial membrane potential,and decrease the expression of pro-apoptosis protein Bax and caspase-3 and increase the expression of anti-apoptosis protein Bcl-2.4.The regulation effects of allicin on apoptosis related protein Bax and Bcl-2 were significantly inhibited by PAG,suggesting that anti-apoptosis of allicin may be related to H2S.5.Allicin could increase NO level and eNOS expression.Meanwhile,the regulation effects of allicin on apoptosis related protein Bax and Bcl-2 were significantly inhibited by LNAME.These results showed that anti-apoptosis of allicin may be related to NO.6.Allicin could significantly decrease MDA level and increase SOD activity.It could also promote Nrf2 synthesis and nuclear translocation,and further increase the expression of HO-1 protein,suggesting that anti-apoptosis of allicin may be associated with the activation of Nrf2/HO-1 signaling pathway of oxidative stress. |
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