Protective Effects Of Triptolide On H9c2 Myocardial Cells Injury Induced By Hypoxia-reperfusion

Objective:To observe the effects of hypoxia-reperfusion induced injure on H9c2 myocardial cells,via detecting the levels of creatine kinase(CK),lactic dehydrogenase(LDH),malondialdehyde(MDA)and superoxide dismutase(SOD),and the level of intracellular reactive oxygen species(ROS)was detected by fluorescence microscope,in addition,cell proliferation,cell viability and morphological changes were also investigated.And than to investigate the effects of the levels of CK,LDH,ROS,MDA and SOD on the H9c2 myocardial cells injury induced by hypoxic-reperfusion;and to investigate the effects of triptolide(TP)on H9c2 myocardial cells injury induced by hypoxic-reperfusion.To explore its function and mechanism on myocardial cells,which can provide new experimental evidence for reducing myocardial hypoxia-reperfusion injury.Methods: 1.Specimen selection: Select the H9c2 myocardial cells which in the exponential growth phase,and according to the different indexes set the 96-well plate or 24-well plate.2.Model preparation: The hypoxia-reperfusion injury model of H9c2 myocardial cells was established via giving hypoxia treatment for 4 hours,and then reperfusion for 4 hours,give the TP with 1nmol/L,5nmol/L,10nmol/L,20nmol/L,50nmol/L,100 nmol/L at the same time,3.test indicators: After the hypoxia-reperfusion injury,using CCK-8 method to measure the cell proliferation and cell viability,SiMultaneously,the cell morphological changes was detected using inverted microscope.The levels of enzymes in H9c2 myocardial cells and oxidative/antioxidant in the culture supernatant of H9c2 myocardial cells were measured by the corresponding detection kit.The activity of creatine kinase(CK),lactate dehydrogenation(LDH)was detected by microplate reader method,The level of reactive oxygen species(ROS)in cells was detected by chemiluminescence method,the level of malondialdehyde(MDA)was detected by TBA method,and the superoxide dismutase(SOD)level was detected by hydroxylamine method.4.Data analysis: All data measured in the experiment,measurement data are expressed as mean ± standard deviation((?)± s),and after the homogeneity of variance test and normal distribution test,using t test for statistical analysis,using GraphPad Prism 6.0 Mapping Software drawing the Statistical graph,P <0.01 or 0.05 was considered the difference between different groups have statistically significant,with all experiments were repeated 3 times.Results:(1).In this experiment,H9c2 myocardial cells were treated with hypoxia for 4 hours and then reperfusion for 4 hours.The hypoxic-reperfusion injury model of myocardial cells was successfully established.Hypoxia-reperfusion could significantly decrease the proliferation rate of H9c2 myocardial cells(P<0.05),suggesting that hypoxia-reperfusion injury can cause myocardial cells death and morphological changes;(2).The viability rate of H9c2 myocardial cells in each concentration of TP group was significantly higher than that in the control group,and the cell morphological changes were also lighter.The protective effect of TP100nmol/L,50nmol/L,20nmol/L and 10 nmol/L group was the most significant(P<0.01).(3).The levels of CK,LDH and MDA in the supernatants of the groups with TP were significantly lower than those in the control group,and the activity of SOD was significantly higher than that in the control group(P<0.01).Fluorescence microscopy showed that TP could decrease the level of ROS in H9c2 myocardial cells in a dose-dependent manner in the concentration range which was set in our experiment.The most significant reduction was observed in TP 50nmol/L and 20nmol/L groups.Conclusion:(1).Giving 4 hours of hypoxia treatment and then 4 hours of reperfusion treatment can cause H9c2 myocardial cells injury,which lead to cell death and morphological changes.(2).TP have a protective effect against H9c2 myocardial cells injury induced by hypoxia-reperfusion,which can reduce cell death and maintain cell membrane completely.The mechanism may be through reducing the lipid peroxidation level of myocardial cells and increasing the antioxidant capacity of myocardial cells,and there is a concentration dependence between this protection function with TP concentration.It is suggested that the application of TP may be a new strategy to reduce myocardial hypoxia-reperfusion injury.