| Objective:To research whether the pprI gene of Deinococcus radiodurans can play a protective role in mitomycin C inhibiting the proliferation and inducing apoptosis of H9C2 cardiomyocytes,and preliminarily explore the protective mechanism in cell apoptosis.Methods:1.Lipo8000TM liposome mediated p CMV-C-Flag-pprI eukaryotic expression vector was transfected into H9C2 cardiomyocytes,and the expression of Flag-Ppr I fusion protein was verified by Western blot;2.The CCK8 method was used to detect the effects of different concentrations of mitomycin C（0,6.25,12.5,25 and 50 mg/L）and pprI gene on the survival rate of H9C2 cardiomyocytes;3.Apply 25 mg/L mitomycin C in H9C2 cardiomyocytes for follow-up experiments:DCFH-DA method to detect ROS level,TBA method to detect MDA content,micro-enzyme method to detect GSH content,and WST-8 method to detect SOD activity;Color method to detect LDH release and CK enzyme activity,micro-enzyme standard method to detect ATPase activity,TUNEL method to detect apoptosis,JC-1 method to detect mitochondrial membrane potential;q RT-PCR to detect apoptosis-related genes bax and bcl-2,cycs,caspase-9,caspase-3and parp m RNA expression levels.Results:1.Western blot results showed that Flag-Ppr I fusion protein was successfully expressed in H9C2 cardiomyocytes;2.The results of CCK8 method showed that mitomycin C can promote the apoptosis of H9C2 cardiomyocytes in a dose-dependent manner.The apoptosis rate of H9C2 cardiomyocytes treated with 25mg/L mitomycin C for 24 h was about 50%;pprI gene transfection can improve the survival rate of H9C2 cardiomyocytes;3.After 25 mg/L mitomycin C treated H9C2 cardiomyocytes for 24h,compared with the blank control group,the SOD activity and GSH content in the cells of the untransfected group were significantly reduced（P<0.05）,and the ROS level and MDA content were significantly increased（P<0.05）;compared with the untransfected group,the empty plasmid transfection group had no significant difference in he SOD activity and GSH content,ROS and MDA content in the cells（P>0.05）;compared with the untransfected group,the activity of SOD and GSH in the cells of the pprI gene transfection group was significantly increased（P<0.05）,and the content of ROS and MDA was significantly reduced（P<0.05）;4.After 25 mg/L mitomycin C was applied to H9C2 cardiomyocytes for 24 h,compared with the blank control group,the amount of LDH release,CK enzyme activity,and cell apoptosis in the untransfected group were significantly increased,ATPase activity was significantly reduced,and mitochondrial membrane potential was reduced,and there were statistical differences（P<0.05）;compared with the untransfected group,the amount of LDH released,CK enzyme activity,and the number of apoptosis in the empty plasmid transfection group were not significantly different（P>0.05）;compared with the untransfected group,the intracellular LDH release,CK enzyme activity and apoptosis number in the pprI gene transfection group were significantly reduced,the ATPase activity was significantly increased,and the mitochondrial membrane potential was increased,and there were statistical differences（P<0.05）;5.After 25 mg/L mitomycin C was applied to H9C2 cardiomyocytes for 24 h,compared with the blank control group,the expression of bax,cycs,caspase-9,caspase-3 and parp in the untransfected group were significantly increased,all of which were statistically significant difference（P<0.05）,and the expression of bcl-2 m RNA and the ratio of bax/bcl-2 were significantly decreased,all of which were statistically significant difference（P<0.05）;compared with the untransfected group,the empty plasmid transfection group showed no significant difference in the intracellular bax,bcl-2,cycs,caspase-9,caspase-3,parp m RNA and bax/bcl-2 ratio（P>0.05）;compared with the untransfected group,the expression of bax,cycs,caspase-9,caspase-3 and parp of the gene transfection group was significantly reduced,and there are statistical differences（P<0.05）,and bcl-2 m RNA expression and the ratio of bax/bcl-2 were significantly increased（P<0.05）.Conclusions:1.DR-pprI gene can be successfully expressed in eukaryotic H9C2cardiomyocytes;2.Mitomycin C can promote the apoptosis of H9C2 cardiomyocytes in vitro,and DR-pprI gene can increase the survival rate of H9C2cardiomyocytes;3.Mitomycin C induce oxidative damage and apoptosis of H9C2cardiomyocytes through the mitochondrial pathway;4.DR-pprI gene can play a protective role in mitomycin C-induced H9C2 cardiomyocyte apoptosis. |
