补阳还五汤类方提取物对PC12细胞氧化应激损伤模型凋亡与自噬的调控

Regulation of Buyang Huanwu Decoction extracts on apoptosis and autophagy of PC12 cells model with oxidative stress injury

目的从凋亡与自噬的调控探讨补阳还五汤类方提取物对PC12细胞氧化应激模型的保护机制。方法以不同浓度H2O2刺激PC12细胞制备不同损伤程度的氧化应激模型,采用MTT法确定补阳还五汤类方提取物对氧化应激损伤初期和加剧期发挥保护作用的有效浓度,流式细胞术和TUNNEL法检测细胞凋亡情况,透射电镜和mRFP-GFP-LC3腺病毒双标法检测细胞自噬情况,蛋白质印迹法检测凋亡相关蛋白(Bcl-2和Bax)与自噬相关蛋白(Beclin1、LC3A和LC3B)的表达。结果分别以0.5、2.0 mmol/L H2O2处理PC12细胞制备氧化应激损伤的初期模型和加剧期模型;与对照组比较,模型组细胞凋亡率增加,自噬程度加剧,Bax/Bcl-2比例增加,Beclin1和LC3B表达上调,LC3A表达下调(P<0.05);与氧化应激损伤初期的模型1组比较,补阳还五汤类方提取物均能下调Bax/Bcl-2,抑制凋亡率,并一定程度上调Beclin1和LC3B/LC3A的表达,激活自噬(P<0.05);当氧化应激损伤程度加剧时,模型细胞中出现较高水平的凋亡与自噬,此时,两方则通过抑制凋亡和降低自噬,共同发挥保护作用。结论补阳还五汤类方提取物可通过对凋亡与自噬的交互动态调控对不同程度损伤的氧化应激模型细胞挥发保护作用。

Objective To explore the underlying mechanism of Buyang Huanwu Decoction extracts on apoptosis and autophagy in PC12 cells model with oxidative stress injury.Methods Different level oxidative stress injury models with H2O2 at various concentrations were established.The effective concentrations of Buyang Huanwu Decoction extracts were determined by MTT method in the initial stage and the intensifying period after oxidative stress injury.The apoptosis of PC12 cells were evaluated by FCM and TUNNEL,the autophagy situations were observed by TEM and mRFP-GFP-LC3.Furthermore,the proteins of Bax,Bcl-2,Beclin1,LC3A,and LC3B related to apoptosis were determined by Western blotting.Results The initial stage and the intensifying period of oxidative stress injury cell models were established by H2O2 at the concentration of 1.5 and 2.0 mmol/L,respectively.Compared with the control group,model group appeared increasing apoptosis and autophagy levels,and model group had higher expressions of Bax/Bcl-2,Beclin1,LC3B and lower expression of LC3A(P<0.05).Compared with the initial stage of oxidative stress injury cell models,Buyang Huanwu Decoction extracts could reduce the Bax/Bcl-2 and restrain apoptosis rates,while the autophagy was activated by up-regulation Beclin1 and LC3B/LC3A(P<0.05).When the serious apoptosis and excessive autophagy were observed in the intensifying period of oxidative stress injury cells,the extracts could play the protective effect by apoptosis restraining and autophagy alleviating.Conclusion Buyang Huanwu Decoction extracts can play the protective effects on oxidative stress injury cell models in different period by regulating apoptosis and autophagy.