Anti-Myocardial Ischemia-Reperfusion Injury And Its Mechanism Of Onion Extract Monomer Sulfide S1

Objectives:To establish a model of hypoxia-reoxygenation injury in rat cardiomyocyte H9C2 cells at cellular level to simulate ischemia-reperfusion injury.By prepari-ng sulfide dipropyl disulfide(S1)from onion white extract and pretreating Hypoxic-reoxygenation model cells,the changes of endoplasmic reticulum stress and apoptotic related indicators were observed,and the mechanism of sulfide S1 from onion white extract in endoplasmic reticulum stress-induced apoptotic pathway of H9C2 cells was explored.Methods:Rat H9C2 myocardial cells were cultured and randomly divided into 7 groups.The activity of H9C2 cells in the normal control group(con group),solvent control group(0.1%DMSO)and S1 treatment group(1?g/ml,10?g/ml,50?g/ml,100?g/ml,1000?g/ml)was detected by MTT method at 24 h.Model preparation: H9C2 cells were randomly divided into four groups: control group(con group),hypoxia 4H reoxygenation 0h(4h/0h),hypoxia 4H reoxygenation 4h(4h/4h),hypoxia 4H reoxygenation 16h(4h/16h).The expression of endoplasmic reticulum stress marker protein GRP78 was detected by Western blot,and the time of endoplasmic reticulum stress in H9C2 cells induced by hypoxia and reoxygenation was determined,and the hypoxia and reoxygenation injury model of H9C2 cells was constructed.H9C2 cardiomyocytes were randomly divided into 5 groups according to the early results,control group(con group),hypoxia-reoxygenation group(H/R group),and the hypoxia-reoxygenation group with different concentrations(10?g/ml,50?g/ml,100?g/ml)after S1 pretreatment for 30 min ?hypoxia-reoxygenation group(H/R group).Annexin v-fitc /PI double staining and flow cytometry were used to detect the apoptosis of H9C2 cells in each group.Fluorescence quantitative PCR and Western blot were used to detect the expressions of intracellular related proteins GRP78,CHOP,caspase-3 and Bcl-2.Result:1.MTT results showed that the concentration of sulfide S1 in onion white extract was 10 ?g/ml,50 ?g/ml and 100 ?g/ml,which could promote cell proliferation(P < 0.01).When the concentration of S1 was 1000 ?g/ml,it was toxic to cells(P <0.01).The concentration of low concentration solvent(0.1% DMSO)and S1 was 1?g/ml had no significant effect on cells(P > 0.05).2.Conditions of hypoxia-reoxygenation H9C2 cell injury model: Compared with con group,there was no significant difference in GRP78 expression after hypoxia for 4 hours,reoxygenation for 0 hours and reoxygenation for 16 hours.(P >0.05).GRP78 was up-regulated significantly at 4 h hypoxia and 4 h reoxygenation(P< 0.05).Thus,a hypoxic reoxygenation model was established to induce endoplasmic reticulum stress in H9C2 cells.3.Annexin V-FITC/PI double staining and flow cytometry detection: After S1 pretreatment,the cell survival rate increased significantly and the apoptosis rate decreased significantly in the range of 10-1000 ?g/ml.4.Fluorescence quantitative PCR and Western blot detection showed that the expression of GRP78,CHOP and caspase-3 related proteins in endoplasmic reticulum stress apoptotic pathway was significantly lower than that in H/R group,while the expression of Bcl-2 was significantly increased(P < 0.05).Conclusion:1.The monomer sulfide S1 of onion white extract can promote the proliferation of H9C2 cells in a concentration-dependent manner in the range of 10-100 ?g/ml.2.Hypoxia-reoxygenation injury can significantly reduce the survival rate of myocardial cells and increase the apoptotic rate.Pretreatment with sulfide S1 of onion extract monomer can reduce the apoptotic rate.3.The inhibition of endoplasmic reticulum stress-induced apoptosis by sulfide S1 may be related to the up-regulation of GRP78,CHOP and caspase-3 proteins and the down-regulation of Bcl-2 proteins.