Protective Effect Of Taurine On Hypoxia/reoxygenation Injury Of H9C2 Cardiomyocytes

Studies have shown that the mortality rate of cardiovascular disease ranks the first in various diseases,and the number of deaths from ischemic heart disease is the main reason.To restore blood supply as soon as possible is the most effective measures to prevent and treat cardiac ischemic death.At present,ischemia/reperfusion(I/R)injury is still a challenge in the clinical treatment of acute myocardial infarction.Studying the mechanism of I/R injury will help us provide theoretical and experimental evidence for the prevention and treatment of myocardial I/R injury.Studies have shown that Tau(taurine)has various functions such as anti-oxidation and regulation of intracellular calcium homeostasis,and has a good preventive and therapeutic effect on myocardial I/R injury,myocardial infarction,heart failure and other ischemic heart diseases.PUMA(p53 up-regulated modulator of apoptosis),as the most pro-apoptotic BH3-only protein family,is considered a new target for the prevention and treatment of ischemic heart disease.Ischemia and hypoxia and other stimulating on the endoplasmic reticulum(ER)result unfolded protein or misfolding protein increased in the ER,induced ERS(endoplasmic reticulum stress).CHOP(CCAAT/enhancer-binding protein homologous protein)is a classical marker of ERS and can induce apoptosis.In this experiment,rat myocardial cells H9C2 was used as a subject,and H/R(hypoxia/reoxygenation)was used to simulate I/R injury to elucidate the effect of PUMA and CHOP over-expression on H/R apoptosis in cardiomyocytes.To explore the way and the molecular mechanism of Tau protecting I/R cardiomyocyte apoptosis,and to provide a new experimental basis for widening the application of Tau in the prevention and treatment of I/R injury.Objective:1.To observe the effect of PUMA and CHOP on the apoptosis of cardiomyocytes under H/R,and explored the effects of PUMA and CHOP on the protective effects of Tau on I/R injury of cardiomyocytes.2.To investigate the protective effect of Tau on myocardial I/R injury and the relationship between ERS and its mechanism.Method:1.Hypoxia for 3 h,and reoxygenated for 2 h,4 h,6 h and 12 h respectively were treated for rat H9C2 cardiomyocytes cell lines.To investigate the effects of H/R on the apoptosis of cardiomyocyte injury and the expression of PUMA,AP-1,CHOP,GRP78,Bcl-2,Bax and Caspase-3.2.H9C2 were treated with different concentrations of Tau(0 mM,10 mM,20 mM,40 m M,80 mM,120 mM,160 mM)for 1 h,and to investigate the protective effect of different concentrations of Tau on cardiomyocyte H/R injury.3.To investigate the effects of Tau on the H/R of H9C2 in cardiomyocytes during overexpression of PUMA and CHOP.4.When the specific si RNA silences the expression of PUMA and CHOP,the molecular mechanism of the protective effect of Tau on myocardial H/R injury is observed.Result:1.Western Blot datas showed that the levels of the expression of PUMA,CHOP,Caspase-3,AP-1 and Bax in H9C2 cells were the highest at hypoxia for 3h and after reoxygenation for 6h,while GRP78 increased with time.In addition,FCM(flow cytometry)data also showed that the rate of apoptosis is time-dependent.2.Western Blot shows that in H9C2 cells,Tau 80 mM group can significantly reduce the expression of PUMA,CHOP,Caspase-3 and Bax compared with other concentrations(P<0.01).RT-qPCR showed that the expression of PUMA,CHOP,Bax,GRP78 and Caspase-3 were significantly decreased(P<0.01)and the expression of Bcl-2 was increased at 3 h after hypoxia and 6 h after reoxygenation.Analysis of the results of Caspase-3 activity assay showed that the cell activity was the strongest when Tau concentration was 80 mM(P<0.05).3.Transfected plasmid p-EGFP-PUMA and established a model,found that Caspase-3 and Bax protein expression increased(P<0.05),while GRP78,AP-1,CHOP expression did not change significantly(P>0.05).Combined treatment with Tau(80 mM)can reduce this effect.RT-qPCR results showed that the mRNA expression of Caspase-3 and Bax in the p-EGFP-PUMA group was significantly increased(P<0.01),and the combined treatment of taurine(80 mM)blocked the high expression of PUMA.The results of Caspase-3 activity test showed that the p-EGFP-PUMA group had statistically significant differences compared with the control group(P<0.05).4.Transfected with PUMA-siRNA and established a model,found that the protein expression of Caspase-3 and Bax decreased(P<0.05),Tau(80 mM)combined treatment makes this effect more significant.The expression of Caspase-3 and Bax mRNA in PUMA-siRNA group was significantly decreased(P<0.01).The results of Caspase-3 activity test showed that the PUMA-siRNA group was statistically different from the control group(P<0.05).5.After in vitro plasmid p-EGFP-CHOP,the protein expression of Caspase-3,PUMA,AP-1,Bax was increased(P<0.05),but the expression of GRP78 did not change significantly(P>0.05).Combined treatment with Tau(80 mM)can reduce this effect.RT-qPCR results showed that the mRNA expression of Caspase-3,PUMA,AP-1 and Bax in p-EGFP-CHOP group was significantly increased(P<0.01),and Tau(80 mM)combined treatment prevented the high expression of CHOP.The results of Caspase-3 activity test showed that the p-EGFP-CHOP group was significantly different from the control group(P<0.05).6.After transfected with CHOP-siRNA,the protein expression of Caspase-3,PUMA,AP-1 and Bax decreased(P<0.05),and the combined treatment of Tau(80 mM)made this effect more significant(P<0.05).RT-qPCR results showed that the mRNA expression of Caspase-3,PUMA,AP-1,and Bax in the PUMA-si RNA group was significantly decreased(P<0.01).The results of Caspase-3 activity test showed that there was a statistically significant difference between the CHOP-siRNA group and the control group(P<0.05).In conclusion:1.Rat cardiomyocyte cell line H9C2 undergoes H/R and induces apoptosis.After Tau pretreatment,the apoptotic rate was significantly decreased,and played an anti-apoptotic effect.2.Tau protects rat cardiomyocyte cell line H9C2 from apoptosis induced by H/R.The mechanism of action is to reduce the damage caused by ERS by decreasing the protein expression of CHOP,which in turn reduces the expression of PUMA and Caspase-3 activity,inhibition of mitochondrial apoptosis pathway.Therefore,CHOP is expected to become a new target for the prevention and treatment of myocardial I/R injury.