The Effects Of Heat Shock Pretreatment On Rat Myocardial Apoptosis And Expression Of Metallothionein

Objective:To explore the effects of heat shock pretreatment on rat myocardial apoptosis induced by hydrogen peroxide (H2O2) and the mitochondrial metallothionein expression.Method:The cultured H9C2 rat cardiomyocytes were randomly divided into three groups:Control group,H2O2 injury group and Heat shock pretreatmen group.①Control group (group Ctrl,n=3):Myocardial cells were incubated with serum containing DMEM medium, placed in 37℃,5% CO2 incubator 3 h;②H2O2 injury group (group H2O2, n=3): Myocardial cells were incubated with 0.5 mmol/L H2O2 DMEM serum-no medium, placed in 37℃,5% CO2 incubator for 3 h;③Heat shock pretreatmen group (group HSR,n=3):Myocardial cells were incubated with serum containing DMEM medium, wrapped with a sterile powder-free gloves, myocardial cell culture flask placed in 42℃constant temperature water bath and incubated for 1 h, then recover to 37℃,5% CO2 cell incubator for 6 h, suction of containing serum DMEM medium and clean the culture flasks, then followed by the same to group H2O2.Myocardial cell apoptosis is observed by flow cytometry; and the structure changes of mitochondrial is observed by electron microscopy; Mitochondrial cytochrome C and metallothionein expression are determined by western-blotting technique. Results:1:Flow cytometry scatter shows:group H2O2 apoptotic cells was significantly more than group Ctrl and group HSR, while the apoptotic cells of group HSR is less than group H2O2; the fluorescence figure shows:the apoptosis and necrosis Cells of group H2O2 are more than group Ctrl and group HSR, the apoptosis and necrosis Cells of group HSR are more than group Ctrl.2. Electron microscopy:The complete mitochondrial structure can be seen in the group Ctrl. Although, there is a bit vague, but the outer membrane and ridge are visible in group HSR. While the vacuoles mitochondrial, swelling and irregular outer Membrane, cristae lost in the group H2O2.3. The expression of myocardial cells mitochondrial cytochrome C in each group determined by Western-blotting:the expression of mitochondrial cytochrome C in group heat shock pretreatment is more than group H2O2 and group Ctrl.4. The expression of mitochondrial metallothionein in each group myocardial cells determined by Western-blotting:The expression of mitochondrial metallothionein in group HSR is significantly higher than that of H2O2 injury group and normal group.Conclusion:1.Heat shock pretreatment markedly inhibited the myocardial apoptosis induced by hydrogen peroxide; 2. Metallothionein is correlated with the effect of heat shock pretreatment on myocardial cells injuryed by hydrogen peroxide.