异黄芪皂苷Ⅳ对高糖诱导的肾小球系膜细胞活性的影响

Effect of Isoastragaloside Ⅳ on Activity of Mesangial Cells Induced by High Glucose

目的探讨异黄芪皂苷Ⅳ对高糖诱导的人肾小球系膜细胞HRMC活性的影响及相关机制.方法将体外培养的HRMC细胞分为正常对照组、高糖模型组、Tempol阳性对照组、Ly364947阳性对照组、异黄芪皂苷Ⅳ低、中、高浓度组,培养48 h.CCK-8法测定细胞增殖能力;酶联免疫吸附法测定上清液中Ⅳ型胶原含量;DCFH-DA测定活性氧含量;Western blot检测转化生长因子β1(TGF-β1)、p-Smad2/3、NADPH氧化酶4、瞬时受体电位阳离子通道蛋白6(TRPC6)表达.结果与高糖模型组比较,随着异黄芪皂苷Ⅳ浓度的增加,给药组的HRMC增殖水平依次降低,差异具有统计学意义(P<0.05),Tempol阳性对照组、Ly364947阳性对照组的HRMC增殖水平明显低于高糖模型组,差异具有统计学意义(P<0.05);与高糖模型组比较,Tempol阳性对照组、Ly364947阳性对照组、各浓度异黄芪皂苷Ⅳ干预组的Ⅳ型胶原含量均明显下降,差异具有统计学意义(P<0.05),其中,异黄芪皂苷Ⅳ呈剂量依赖性降低HRMC上清液中Ⅳ型胶原表达;与高糖模型组比较,Tempol阳性对照组、Ly364947阳性对照组、各浓度异黄芪皂苷Ⅳ干预组的细胞活性氧含量均明显下降,差异具有统计学意义(P<0.05),其中,异黄芪皂苷Ⅳ呈剂量依赖性降低细胞活性氧含量;与高糖模型组比较,Tempol阳性对照组、Ly364947阳性对照组、各浓度异黄芪皂苷Ⅳ干预组的TGF-β1、p-Smad2/3、NADPH氧化酶4蛋白表达水平均明显下降,TRPC6蛋白表达水平明显上升,差异具有统计学意义(P<0.05).结论异黄芪皂苷Ⅳ对高糖诱导的HRMC损伤具有保护作用,其机制与抑制细胞增殖、降低活性氧生成、下调TGF-β1、p-Smad2/3、NADPH氧化酶4蛋白表达、上调TRPC6表达有关.

Objective To investigate the effect of isoastragaloside Ⅳ on the activity of HRMC induced by high glucose and its mechanism. Method HRMC was cultured in vitro and divided into normal control group,high glucose model group,Tempol positive control group,Ly364947 positive control group and low,medium and high concentration group of isoastragaloside Ⅳ. After 48 hours culture,CCK-8 method was used to measure cell proliferation,enzyme-linked immunosorbent assay(ELISA) was used to measure the content of type Ⅳ collagen,DCFH-DA was used to measure the content of reactive oxygen species,Western blot was used to detect the expression of transforming growth factor β1(TGF-β1),p-Smad2/3,NADPH Oxidase 4,transient receptor potential cation channel protein 6(TRPC6). Results Compared with the high glucose model group,the proliferation level of HRMC in the administration group decreased with the increase of Ⅳ concentration of isoastragaloside(P<0.05);the proliferation level of HRMC in the Tempol positive control group and Ly364947 positive control group was significantly lower than that in the high glucose model group(P<0.05).Compared with the high glucose model group,the content of type Ⅳ collagen in Tempol positive control group,Ly364947 positive control group and each concentration of astragaloside Ⅳ intervention group decreased significantly(P < 0.05),and the expression of type Ⅳ collagen in the supernatant of HRMC decreased in a dose-dependent manner.Compared with the high glucose model group,the content of cellular ROS in the Tempol positive control group,Ly364947 positive control group and the intervention group with different concentrations of astragaloside Ⅳdecreased significantly(P<0.05),among which astragaloside Ⅳ decreased the content of cellular ROS in a dosedependent manner. Compared with the high glucose model group,the expression of TGF-β1,p-Smad2/3 and NADPH Oxidase 4 in the Tempol positive control group,Ly364947 positive control group and all concentrations of astragaloside Ⅳ intervention group decreased significantly,and the expression of TRPC6 protein increased significantly(P<0.05).Conclusion Isoastragaloside Ⅳ has protective effect on HRMC injury induced by high glucose.Its mechanism is related to the inhibition of cell proliferation,the reduction of ROS production,the down regulation of TGF-β1,p-Smad2/3,NADPH Oxidase 4 expression and the up-regulation of TRPC6 expression.