黄芪甲苷抑制内质网应激及CHOP信号通道改善衣霉素诱导的系膜细胞凋亡

Influence of AstragalosideⅣon Inhibiting Endoplasmic Reticulum Stress and CHOP Signaling Pathways and Improving Tunicamycin Induced Mesangial Cells Apoptosis

目的观察黄芪甲苷对衣霉素诱导的系膜细胞凋亡、内质网应激标志蛋白和CHOP信号通路表达的影响,从系膜细胞凋亡角度探讨黄芪甲苷治疗早期糖尿病肾病的可能机制。方法采用衣霉素诱导大鼠系膜细胞产生内质网应激,分为模型对照组、黄芪甲苷小剂量组(AS-IVL,50μg·mL^(-1))、黄芪甲苷大剂量组(AS-IVH,100μg·mL^(-1))、4-苯基丁酸组(PBA,10 mmol·L^(-1)),并设置正常对照组。流式细胞术检测各组细胞凋亡水平,Western blotting检测细胞eIF2α、PERK和IRE1α磷酸化水平,测定GRP78、CHOP、BAX、Bcl-2和Cleaved caspase 3蛋白的表达变化。结果与正常对照组比较,模型对照组系膜细胞凋亡水平升高,内质网应激相关蛋白eIF2α、PERK及IRE1α磷酸化水平显著上调,而调控细胞凋亡的CHOP、BAX和Cleaved caspase 3表达明显升高(P<0.05)。与模型对照组细胞比较,2种浓度黄芪甲苷组系膜细胞凋亡水平明显下降(P<0.05),包括eIF2α、PERK和IREα等内质网应激相关蛋白磷酸化水平明显降低(P<0.05),CHOP、BAX和Cleaved caspase 3表达明显下调(P<0.05)。结论黄芪甲苷能抑制细胞内质网应激、缓解CHOP介导的细胞过度凋亡,显著改善衣霉素诱导的大鼠系膜细胞凋亡。

Objective To explore the mechanism of astragalosideⅣ(AS-Ⅳ)in treatment of diabetic nephropathy by effects of apoptosis on mesangial cells induced with tunicamycin,expression of protein of endoplasmic reticulum stress and CHOP pathway.Methods The mesangial cells were respectively cultured in DMEM medium of inactivated fetal bovine serum(normal control group),plus 5μg·mL^(-1) tunicamycin(model control group),5μg·mL^(-1) tunicamycin+AS-Ⅳ50μg·mL^(-1)(AS-IVL group),5μg·mL^(-1) tunicamycin+AS-Ⅳ100μg·mL^(-1)(AS-IVH group),or 5μg·mL^(-1)tunicamycin+PBA10 mmol·L^(-1)(PBA group).Apoptosis of mesangial cells were measured with flow cytometric analysis.The signaling pathways in UPR of phosphorylation of eIF2α,PERK and IRE1α,the apoptotic related factors including GRP78,CHOP,BAX,Bcl-2 and cleaved caspase-3 were determined by Western blotting.Results Compared to the normal control group,the mesangial cells in model control group showed significant increase of apoptosis,and the phosphorylations of eIF2α,IRE1αand PERK were upregulated,and the expressions of CHOP,Bax and cleaved caspase-3 were significantly increased(all P<0.05).Both concentrations of AS-Ⅳtreatment significantly inhibited the apoptosis of mesangial cells induced with tunicamycin,inhibited the phosphorylation of IRE1α,PERK and eIF2α,decreased the expression of CHOP,and inhibited the expression of Bax and cleaved caspase-3(all P<0.05).Conclusion AS-Ⅳsignificantly down-regulated the CHOP pathway,and reduced mesangial cells apoptosis induced by endoplasmic reticulum stress.