ATP敏感性钾通道开放剂吡那地尔增高Bcl-2表达而抑制PC12细胞缺血性凋亡

Pinacidil,an ATP-sensitive potassium channel opener,inhibits ischemic apoptosis via up-regulating Bcl-2 expression in PC12 cells

目的探讨ATP敏感性钾通道开放剂吡那地尔对缺血缺氧PC12细胞凋亡及对Bcl-2 mRNA和蛋白表达的影响。方法取传代后3d的PC12细胞,分为正常对照组、缺血对照组、吡那地尔处理组、吡那地尔+格列吡嗪处理组共4组。吡那地尔处理组在PC12细胞缺血缺氧前20min加入浓度为100μmol·L-1的吡那地尔;吡那地尔+格列吡嗪处理组则加入浓度100μmol·L-1的吡那地尔和浓度为500μmol·L-1的KATP通道阻断剂格列吡嗪。采用Annexin-V FITC/PI双染流式细胞分析仪检测凋亡率;应用免疫荧光染色和Western blot检测Bcl-2蛋白表达水平;应用RT-PCR检测Bcl-2 mRNA表达水平。结果缺血缺氧后缺血对照组、吡那地尔处理组、吡那地尔+格列吡嗪处理组细胞凋亡率随时间增加而增加,24h达高峰。吡那地尔组与其余组比较差异均有显著性(P<0.01)。缺血对照组、吡那地尔处理组、吡那地尔+格列吡嗪处理组细胞Bcl-2 mRNA及蛋白表达各时间点均增加,12h达高峰。吡那地尔组与其余组比较差异均有显著性(P<0.05,或P<0.01)。缺血对照组和吡那地尔+格列吡嗪处理组比较差异均无显著性(P>0.05)。结论ATP敏感性钾通道开放剂可能通过提高Bcl-2 mRNA及蛋白表达来减轻缺血缺氧后PC12细胞凋亡,发挥保护作用。

Aim To study the effect of ATP sensitive potassium channel opener-pinacidil on the apoptosis of PCl2 cells induced by ischemia and hypoxia and expression of Bcl-2 mRNA and protein. Methods Cultured PC12 cells were divided into control group, ischemia and hypoxia group, pinacidil and pinacidil ＋ glipizide （ATP sensitive potassium channel blocker） treatment groups. In pinacidil treatment group, pinacidil （100 μmol ·L^-1 ） was added 20 min before PC12 cells induced by ischemia and hypoxia;in pinacidil ＋ glipizide treatment group pinacidil （100 μmol ·L^-1） and glipizide （500 μmol ·L^-1 ） were added. Neuronal apoptosis was detected by Annexin-V FITC/PI doubledyed flow cytometry, the expression of Bcl-2 protein was detected by immunofluorescent staining and Western blot. The expression of Bcl-2 mRNA was detected by RT-PCR. Results The apoptotic ratio in ischemia and hypoxia group, pinacidil and pinacidil ＋ glipizide treatment groups was increased with the time passing by. PC12 cells induced by ischemia and hypoxia for 24 hours yielded the highest apoptotic ratio. The apoptotic ratio of pinacidil treatment group is significant less than that in the other groups（P 〈0. 01 ）. The expression of Bcl-2 mRNA and protein in ischemia and hypoxia group, pinacidil and pinacidil ＋ glipizide treatment groups were higher at all time points. PC12 cells induced by ischemia and hypoxia for 12 hours yielded the highest. The expression of Bcl-2 mRNA and protein in pinacidil treatment group was significant higher than that of the other groups （P 〈 0. 05, or P 〈 0. 01 ）. There were no differences between ischemia and hypoxia group and pinacidil ＋ glipizide treatment group at all time points （P 〉 0. 05 ）. Conclusions KATP opener can increase the expression of Bcl-2 mRNA and protein, decrease neuronal apoptosis and attenuate the PC12 cell injury caused by ischemia and hypoxia. KATP open- er has a protective effect on the apoptosis of PC12 cells induced by ischemia and hypoxia and it is correlated with the up-regulation of the expression of Bcl-2 mRNA and protein.