补元汤对香烟烟雾提取物诱导的L6大鼠成肌细胞凋亡、增殖及自噬的干预效应与机制研究

Intervention effect and mechanism of Buyuan Decoction on apoptosis,proliferation and autophagy of L6 rat myoblasts induced by cigarette smoke extract

目的:探讨补元汤对体外香烟烟雾提取物(CSE)诱导的L6大鼠成肌细胞(L6细胞)凋亡、增殖及自噬的干预效应及机制。方法:构建CSE诱导L6细胞损伤的体外模型,实验分为对照组、模型组、补元汤组。对照组常规培养,模型组加入20%CSE诱导,补元汤组加入20%CSE及1/500补元汤。流式细胞术检测细胞凋亡及细胞周期分布;CCK8法检测细胞活力;Western blot检测微管相关蛋白1轻链3(LC3)、BCL2/腺病毒E1B 19kDa结合蛋白1(Bnip-1)、自噬相关基因7(Atg7)、p62、磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)蛋白表达水平;透射电镜观察自噬体形成情况。结果:与对照组比较,模型组细胞凋亡率、细胞周期中S期细胞比例升高,进入G_(2)/M期细胞比例降低,细胞活力降低,LC3-Ⅱ/Ⅰ、Atg7、Bnip-1、p-PI3K蛋白表达水平升高,p62蛋白表达水平降低,差异均具有统计学意义(P<0.01),电镜观察到自噬体数量增多;与模型组比较,补元汤组细胞凋亡率、细胞周期中S期细胞比例降低,进入G_(2)/M期细胞比例升高,细胞活力升高,LC3-Ⅱ/Ⅰ、Atg7、Bnip-1、p-PI3K蛋白表达水平降低,p62蛋白表达水平升高,差异均具有统计学意义(P<0.01),电镜观察到自噬体数量减少。结论:补元汤可以抑制CSE诱导的L6细胞过度自噬作用,减少细胞凋亡、促进细胞增殖,对CSE诱导的L6细胞损伤具有保护作用,其机制可能与调节Ⅲ型PI3K信号通路有关。

Objective:To investigate the intervention effect and mechanism of Buyuan Decoction on apoptosis,proliferation and autophagy of L6 rat myoblasts(L6 cells)induced by cigarette smoke extract(CSE)in vitro.Methods:The L6 cell injury model induced by CSE was established in vitro.The experiment was divided into control group,model group and Buyuan Decoction group.According to the concentration screening results,the control group was cultured routinely,the model group was induced by 20%CSE,and the Buyuan Decoction group was induced by 20%CSE and 1/500 Buyuan Decoction.Apoptosis and cell cycle distribution were detected by flow cytometry.Cell viability was detected by CCK8 method.The expression levels of microtubule associated protein 1 light chain 3(LC3),BCL2/adenovirus E1 B 19 kDa binding protein 1(Bnip-1),autophagy related gene 7(Atg7),p62,phosphatidylinositol 3-kinase(PI3K)and phosphorylated phosphatidylinositol 3-kinase(p-PI3K)were detected by Western blot.The formation of autophagosome was observed by transmission electron microscope.Results:In the model group,the apoptosis rate and the proportion of S-phase cells in the cell cycle increased,the proportion of cells entering G_(2)/M phase decreased,the cell viability decreased,the expression levels of LC3-Ⅱ/Ⅰ,Atg7,Bnip-1 and p-PI3K protein increased,and the expression level of p62 protein decreased.Compared with the control group,the difference was statistically significant(P<0.01).The number of autophagosomes increased under electron microscope.Compared with the model group,the apoptosis rate and the proportion of cells in S-phase of cell cycle in Buyuan Decoction group decreased,the proportion of cells entering G_(2)/M phase increased,the cell viability increased,the expression levels of LC3-Ⅱ/Ⅰ,Atg7,Bnip-1 and p-PI3K protein decreased,and the expression level of p62 protein increased.The differences were statistically significant(P<0.01).The number of autophagosomes decreased under electron microscope.Conclusion:Buyuan Decoction can inhibit the excessive autophagy of L6 cells induced by CSE,reduce apoptosis,promote cell proliferation,and protect L6 cells from CSE induced injury.Its mechanism may be related to the regulation of typeⅢPI3K signal pathway.