雷帕霉素对葡萄糖诱导SD大鼠阴茎海绵体平滑细胞自噬与凋亡的影响

Effects of rapamycin on glucose-induced autophagy and apoptosis of corpus cavernosum smooth muscle cells in SD rats

目的:观察雷帕霉素对葡萄糖诱导SD大鼠阴茎海绵体平滑细胞(CCSMC)自噬与凋亡的影响。方法:原代分离纯化鉴定CCSMC后分别用5.6、10、20、30、40 mmol/L葡萄糖处理CCSMC 48 h,mCherry-GFP-LC3B双荧光腺病毒转染细胞检测自噬流情况;Western印迹及PCR检测自噬相关指标、自噬相关基因5(ATG5)、微管相关蛋白1轻链3(LC3)、P62、Beclin-1的蛋白和mRNA水平的变化;流式细胞术检测不同浓度葡萄糖处理后细胞凋亡情况,然后再检测用600 nmol/L雷帕霉素预处理2 h再用40 mmol/L葡萄糖处理CCSMC 48 h后自噬相关指标LC3、Beclin1、P62的蛋白和mRNA水平的变化以及细胞凋亡的变化情况。结果:mCherry-GFP-LC3B双荧光腺病毒转染及PCR、Western印迹均提示在不同浓度葡萄糖处理后均发生了自噬现象,随着葡萄糖浓度升高,在10 mmol/L葡萄糖组自噬现象最强(P<0.05),之后随着葡萄糖浓度升高自噬现象逐渐减弱(P<0.05);流式细胞术检测发现相对于5.6 mmol/L葡萄糖组,10 mmol/L葡萄糖组细胞凋亡率无显著升高(P=0.302),但随着葡萄糖浓度升高,细胞凋亡率逐渐升高(P<0.01)。给予雷帕霉素预处理后,Western印迹及PCR检测均提示LC3的蛋白表达量,Beclin1的蛋白和mRNA表达量增多(P<0.05),而P62的蛋白和mRNA表达量降低(P<0.05),表明自噬增强。细胞凋亡率在雷帕霉素预处理后也显著降低(P<0.01)。结论:当不同浓度葡萄糖体外培养CCSMC,葡萄糖浓度在一定范围内时可能促进自噬,从而对CCSMC起到保护作用,但葡萄糖浓度超过一定浓度后,可能超过了CCSMC承受的范围,出现自噬降低,促进细胞凋亡,从而损害细胞活力和功能。而高浓度葡萄糖作用于CCSMC,雷帕霉素可能通过增强自噬,抑制细胞凋亡水平,从而对CCSMC起到保护作用。

Objective:To observe the effects of rapamycin on glucose-induced autophagy and apoptosis of corpus cavernosum smooth muscle cells(CCSMC)in SD rats.Methods:After primary isolation,purification and identification,CCSMCs were treated with glucose at 5.6,10,20,30 and 40 mmol/L for 48 hours.The autophagy flow was detected in the cells transfected with mCherry-GFP-LC3 B double fluorescent adenovirus,the protein and mRNA expressions of autophagy-related gene 5(ATG5),microtubule-associated protein 1 light chain 3(LC3),P62 and Beclin-1 determined by Western blot and PCR and the apoptosis of the CCSMC measured by flow cytometry,followed by detection of the changes in the protein and mRNA levels of LC3,Beclin-1 and P62 and the apoptosis of the cells after 2-hour pretreatment with 600 nmol/L rapamycin and then 48-hour treatment with 40 mmol/L glucose.Results:Autophagy was observed in the CCSMCs treated with different concentrations of glucose,the strongest in the 10 mmol/L group(P<0.05)and then gradually decreasing with the increase of glucose concentration(P<0.05).The apoptosis rate of the CCSMCs was remarkably increased in a concentration-dependent manner after treated with glucose(P<0.01)though with no statistically significant difference between the 5.6 and 10 mmol/L groups(P=0.302).After pretreatment with rapamycin,the expression of the LC3 protein and those of the Beclin-1 protein and mRNA were markedly up-regulated(P<0.05),while those of the P62 protein and mRNA remarkably down-regulated(P<0.05),indicating the enhancement of autophagy,and the apoptosis rate of the CCSMCs dramatically decreased(P<0.01).Conclusion:In vitro culture of CCSMCs with glucose within a certain range of concentration can promote autophagy,inhibit the apoptosis and thus have a protective effect on the cells,but when the proper concentration exceeded,it may also reduce autophagy,promote the apoptosis and consequently affect the vitality and function of CCSMCs.