Effects For Receptor-interacting Protein 3 Gene And Ketoleucine On Myocardial Ischemia Reperfusion Injury In Mouse

Objective:To investigate the effects of Rip3 gene and ketoleucine on myocardial ischemia-reperfusion injury in mouse.(1)To evaluate the effects of Rip3 knockout on the H2O2-inducing MEF cell necrosis;(2)To establish mouse model of myocardial ischemia-reperfusion according to langendorf,and determine the effects of Rip3 knockout on myocardial ischemia-reperfusion injury under in vitro condition;(3)To evaluate the effects of BCKAs on the H2O2-inducing NRVM cell necrosis;(4)To establish mouse model of myocardial ischemia-reperfusion according to langendorf,and determine the effects of ketoleucine on myocardial ischemia-reperfusion injury under in vitro condition.Methods:Part 1:(1)To observe the effects of Rip3 knockout on H2O2 inducing cell necrosis:MEF cells were subjected to 30min of H2O2 treatment.Then the cell viability was measured by MTT.(2)To determine the effects of Rip3 knockout on myocardial ischemia-reperfusion injury:To establish myocardial ischemia-reperfusion model in Rip3 knockout mouse according to langendorf,then compare the infarction size at the end of reperfusion,LVDP,±dp/dt and RPP recovery,CFR and LVEDP at different time points of reperfusion between Wild Type(WT)group and Rip3 knockout group.Part 2:(1)To observe the effects of BCKAs on H2O2 inducing cell necrosis:NRVM cells were respectively pretreated by 500uM ketoleucine,ketovaline,ketoisoleucine and BCKAs for 30min,followed by 30min of 20uM H2O2 treatment.Then the cell viability was measured by MTT and by Calcein-AM.(2)To determine the minimal effective concentration of BCKAs:NRVM cells were respectively pretreated by ketoleucine of 0,0.05,0.2,0.5,1,2mM,followed by 30min of 20uM H2O2 treatment.Then the cell viability was measured by MTT.(3)To establish myocardial ischemia-reperfusion model according to langendorf,then compare the infarction size at the end of reperfusion,LVDP,±dp/dt and RPP recovery,CFR at different time points of reperfusion among control group,ketoleucine group and postconditioning group.Results:Part1:(1)There were no statistically significant differences between WT group and Rip3 knockout group in H2O2 inducing MEF cell necrosis(P>0.05).(2)Compared with WT group,Rip3 knockout had no significant effects on myocardial function under physiological condition.(3)Compared with WT group,Rip3 knockout group did not statistically decrease the infarction size,(32.76±9.44%VS 47.12±18.68%,P>0.05),and had no statistically significant differences in LVDP,±dp/dt,RPP coronary during reperfusion phrase(P>0.05).There were no statistically significant differences in CFR and LVEDP during the whole I/R process between the two groups(P>0.05).Part 2:(1)There were statistically significant differences between control group and BCKAs group in H2O2 inducing NRVM cell necrosis(P<0.05).(2)BCKAs at the dosage of 0.5μM can significantly decrease NRVM cell necrosis induced by H2o2.(3)BCKAs had no significant effect on myocardial function compared with control group under physiological condition.(4)Compared with control group,ketoleucine group and postconditioning group could significantly reduce the infarction size at the end of reperfusion(48.31±11.47%VS 33.21±14.88%,P=0.007)and(48.31±11.47%VS 35.25±8.96%,P=0.025).However,there were no statistical differences between these two interventional groups(33.21±14.88%%VS 35.25±8.96%%,P>0.05).Compared with control group,ketoleucine group and postconditioning group could significantly improve LVDP,±dp/dt,RPP recovery during reperfusion phrase(P<0.05),and there were no statistically significant differences between these two interventional groups.There were no differences in CFR during the entire I/R process among control group,ketoleucine group and postconditioning group(P>0.05).Conclusion:Part 1:(1)Rip3 knockout could not decrease H2O2 inducing MEF cell necrosis;(2)Rip3 knockout had no significant effects on myocardial function under physiological condition;(3)Rip3 knockout also could not reduce the infarction size and improve myocardial functional recovery during myocardial I/R process.Part 2:(1)BCKAs pretreatment could decrease H2O2 inducing NRVM cell necrosis;(2)Ketoleucine had no significant effects on myocardial function under physiological condition;(3)Ketoleucine could significantly reduce the infarction size and improve myocardial functional recovery during myocardial I/R process;(4)Ketoleucine had both pharmacological preconditioning and postconditioning effects,and postconditioning determined the cardioprotective effects of ketoleucine.In summary,Rip3 knockout can not attenuate myocardial I/R injury under in vitro condition,however this conclusion needed to be verified by further studies.Ketoleucine can obviously prevent heart from myocardial I/R injury,and may be a promising chemical drug in cardiovasular diseases.