丹芪益肾汤含药血清调控SIRT1/FoxO1通路对高糖诱导的肾系膜细胞氧化应激损伤的影响

Effect of Danqi Yishen Decoction Drug-containing Serum on Regulating SIRT1/FoxO1 Pathway for High Glucose-induced Oxidative Stress Damage in Renal Mesangial Cells

目的:探讨丹芪益肾汤含药血清调控沉默信息调节因子1(SIRT1)/叉头盒蛋白O1(FoxO1)通路对高糖诱导的肾系膜细胞氧化应激损伤的影响。方法:制备丹芪益肾汤含药血清,体外培养人肾系膜细胞,将其分成6组:正常组(加入10%正常血清)、高糖组(加入30mmol/L葡萄糖和10%正常血清)、低、中、高剂量丹芪益肾汤含药血清组(分别加入30mmol/L葡萄糖和10%低、中、高剂量丹芪益肾汤含药血清)、高剂量丹芪益肾汤含药血清+EX527组(分别加入30mmol/L葡萄糖、10%高剂量丹芪益肾汤含药血清和100nmol/L SIRT1抑制剂EX527)。MTT法检测各组细胞活性;流式细胞仪测定细胞凋亡率;ELISA法检测细胞内活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)含量;Western blot检测细胞中促凋亡蛋白caspase-3、caspase-9、Bax和抗凋亡蛋白Bcl-2以及SIRT1/FoxO1通路蛋白表达水平。结果:与正常组比较,高糖组人肾系膜细胞活性、Bcl-2、SIRT1蛋白表达水平、SOD、GSH-Px含量显著降低,凋亡率和caspase-3、caspase-9、Bax、FoxO1表达水平、MDA、ROS含量显著升高(P<0.05)。与高糖组相比,低、中、高剂量丹芪益肾汤含药血清组人肾系膜细胞活性和Bcl-2、SIRT1蛋白表达水平、SOD、GSH-Px含量明显升高,细胞凋亡率和caspase-3、caspase-9、Bax、FoxO1蛋白表达水平、MDA、ROS含量明显降低(P<0.05),且均表现为剂量依赖性。在高剂量丹芪益肾汤含药血清组中加入SIRT1抑制剂EX527后,细胞活性、Bcl-2、SIRT1蛋白表达水平及SOD、GSH-Px含量明显降低,凋亡率、caspase-3、caspase-9、Bax、FoxO1蛋白表达水平以及MDA、ROS含量显著上升(P<0.05)。结论:丹芪益肾汤含药血清可通过激活SIRT1/FoxO1通路,减缓高糖诱导的肾系膜细胞的氧化应激损伤。

Objective:To investigate the effect of Danqi Yishen Decoction drug-containing serum on regulating silent information regulator 1(SIRT1)/forkhead box protein O1(FoxO1)pathway for high glucose-induced oxidative stress damage in renal mesangial cells.Methods:Danqi Yishen Decoction drug-containing serum was prepared,human renal mesangial cells were cultured in vitro,and divided into six groups:a normal group(added 10%normal serum),a high glucose group(added 30 mmol/L glucose and 10%normal serum),a low,medium,and high-dose Danqi Yishen Decoction drug-containing serum groups(added 30 mmol/L glucose and 10%low,medium,and high-dose Danqi Yishen Decoction drug-containing serum),a high-dose Danqi Yishen Decoction-contained serum+EX527 group(added 30 mmol/L glucose,10%high-dose Danqi Yishen Decoction drug-containing serum and 100 nmol/L SIRT1 inhibitor EX527).MTT method was used to detect cell activity in each group;flow cytometry was used to measure the rate of apoptosis;ELISA method was used to detect the contents of intracellular reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione peroxidase(GSH-Px);Western blot was used to detect the expression levels of pro-apoptotic proteins caspase-3,caspase-9,Bax,anti-apoptotic protein Bcl-2 and SIRT1/FoxO1 pathway proteins in the cells.Results:Compared with the normal group,the human renal mesangial cell activity,Bcl-2,SIRT1 protein expression levels,SOD and GSH-Px contents reduced significantly in the high glucose group,the apoptotic rate and the caspase-3,caspase-9,Bax,FoxO1 expression levels,MDA,and ROS contents increased significantly(P<0.05).Compared with the high glucose group,the human renal mesangial cell activity,Bcl-2,SIRT1 protein expression levels,SOD and GSH-Px contents increased significantly in the low,medium,and high-dose Danqi Yishen Decoction drug-containing serum groups,the apoptotic rate and the caspase-3,caspase-9,Bax,FoxO1 expression levels,MDA,and ROS contents reduced significantly(P<0.05),and showed a dose-dependent manner.After adding the SIRT1 inhibitor EX527 to the high-dose Danqi Yishen Decoction drug-containing serum group,the cell viability,Bcl-2,SIRT1 protein expression levels,and SOD and GSH-Px levels reduced significantly,the apoptosis rate,caspase-3,caspase-9,Bax,FoxO1 protein expression levels,MDA and ROS content increased significantly(P<0.05).Conclusion:The serum containing Danqi Yishen Decoction can slow down the oxidative stress damage of renal mesangial cells induced by high glucose by activating the SIRT1/FoxO1 pathway.