黄芪甲苷对高糖环境下人肾小球系膜细胞的氧化应激损伤的保护作用及机制

Protective effect of astragaloside Ⅳ on the oxidative stress injury of human glomerular mesangial cells under high glucose conditions and its mechanism

目的研究黄芪甲苷对高糖环境下肾小球系膜细胞氧化应激损伤的保护作用及机制。方法将肾小球系膜细胞分为模型组、对照组和低、中、高剂量实验组。模型组用25 mmol·L^(-1)葡萄糖处理,低、中、高剂量实验组在模型组的基础上分别给予25,50,100μmol·L^(-1)黄芪甲苷,对照组则用等量生理盐水处理,干预后各组继续培养48 h。用四甲基偶氮唑蓝(MTT)法检测各组细胞增殖情况;用荧光探针法检测各组细胞活性氧(ROS)水平及荧光强度;用蛋白免疫印迹法检测细胞Akt/NF-κB信号通路相关蛋白表达情况。结果低、中、高剂量实验组肾小球系膜细胞增殖抑制率分别为(6.79±0.12)%,(17.05±0.15)%,(28.31±0.23)%,且随黄芪甲苷作用浓度增大呈升高趋势(P<0.01)。对照组、模型组和低、中、高剂量实验组肾小球系膜细胞ROS相对表达水平分别为0.02±0.01,0.07±0.01,0.06±0.02,0.04±0.03,0.02±0.02;磷酸化蛋白激酶(p-Akt)蛋白相对表达量分别为0.25±0.03,0.57±0.02,0.46±0.02,0.31±0.04,0.27±0.03;p-IκBα蛋白相对表达量分别为0.44±0.03,0.16±0.01,0.21±0.02,0.26±0.02,0.33±0.02。与对照组比较,模型组肾小球系膜细胞ROS相对表达水平、荧光强度及p-Akt蛋白相对表达量均显著升高(P<0.01),p-IκBα蛋白相对表达量显著降低(P<0.01);与模型组比较,实验组肾小球系膜细胞相对表达ROS水平、荧光强度及p-Akt蛋白相对表达量显著降低,且均随黄芪甲苷作用浓度增大呈逐渐降低趋势(P<0.01);p-IκBα蛋白相对表达量显著升高,且随黄芪甲苷浓度增大呈逐渐升高趋势(P<0.01)。结论黄芪甲苷可显著调节高糖环境下肾小球系膜细胞ROS表达水平,抑制氧化应激反应,其作用机制可能与下调Akt/NF-κB信号通路p-Akt蛋白表达,上调p-IκBα蛋白表达相关,且具有显著的剂量依赖性。

Objective To explore the protective effect of astragalosideⅣ on the oxidative stress injury of human glomerular mesangial cells under high glucose conditions and the mechanism. Methods The glomerular mesangial cells were divided into model group,control group and experimental-L/M/H groups. Model group was treated with 25 mmol·L^-1 glucose,experimental-L/M/H groups were given 25,50,100 μmol · L^-1 astragaloside Ⅳ on the basis of model group,control group was treated with equivalent normal saline. Each group was cultured for 48 h after inervention. The cells proliferation were detected by methyl thiazolyl tetrazolium（ MTT）,the reactive oxygen species（ ROS） leveland fluorescence intensity were detected by fluorescent probe method,the cells Akt/NF-κB signaling pathways related proteins were detected by Western blot. Results The proliferation inhibition rates of glomerular mesangial cells in experimental-L/M/H groups were（ 6. 79 ± 0. 12） %,（ 17. 05 ± 0. 15） %,（ 28. 31 ± 0. 23） %,and increased with the increasing of astragaloside Ⅳ concentration（ P〈0. 01）. The ROS relative expressions of glomerular mesangial cells in control group,model group and experimental-L/M/H groups were 0. 02 ± 0. 01,0. 07 ± 0. 01,0. 06 ± 0. 02,0. 04 ± 0. 03,0. 02 ± 0. 02; the phospho-protein binases（ p-Akt） protein relative expression were 0. 25 ± 0. 03,0. 57 ± 0. 02,0. 46 ± 0. 02,0. 31 ± 0. 04,0. 27 ± 0. 03; p-IκBαprotein relative expression were 0. 44 ± 0. 03,0. 16 ± 0. 01,0. 21 ± 0. 02,0. 26 ± 0. 02,0. 33 ± 0. 02. Compared with control group,the ROS relative expression,fluorescence intensity and p-Akt protein relative expression of model group increased significantly（ P〈0. 01）,the p-IκBα protein relative expression decreased significantly（ P〈0. 01）. Compared with model group,the ROS relative expression,fluorescence intensity and p-Akt protein relative expression of experimental groups decreased significantly with the increasing of astragaloside Ⅳ concentration（ P〈0. 01）; while the p-IκBα relative expression increased with the increasing of astragaloside Ⅳ concentration（ P〈0. 01）. Conclusion Astragaloside Ⅳ can adjust the ROS expression level of glomerular mesangial cells under high glucose conditions effectively,and inhibit the oxidative stress reaction,the action mechanism maybe related to the down-regulation of p-Akt protein and the up-regulation of p-IκBα protein of Akt/NF-κB signaling pathways,which has significant dose-dependent manner.