虫草益肾颗粒对高糖诱导人肾小球系膜细胞氧化应激及p47phox表达的影响

Effects of Chongcao Yishen Granule(虫草益肾颗粒)on Oxidative Stress of HMCs Inducd by High Glucose and Expression of p47phox

目的:研究虫草益肾颗粒对高糖培养下人肾小球系膜细胞(HMCs)的氧化应激及p47phox表达水平的影响,探讨其防治糖尿病肾病(DKD)的作用机制。方法:将HMCs随机分为6组:正常组、高糖组、福辛普利组和虫草益肾颗粒低、中、高剂量组,分别给予相应的动物含药血清处理后,收集48 h和72 h细胞上清液,用流式细胞术检测各组细胞的活性氧簇(ROS)水平,用比色法分别检测各组超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量。用Western Blot和Real-time PCR法分别检测p47phox蛋白和mRNA表达。结果:48 h、72 h时,与正常组比较,各组SOD活性均明显下降(P<0.01),各组ROS及MDA含量均明显上升(P<0.01),HMCs p47phox mRNA及蛋白表达量明显升高(P<0.01);与高糖组比较,福辛普利组、虫草益肾颗粒低、中、高剂量组SOD活性明显上升(P<0.05或P<0.01),ROS、MDA含量明显下降(P<0.01),p47phox mRNA及蛋白表达量均明显下降(P<0.05或P<0.01)。结论:虫草益肾颗粒可通过增强SOD活性,降低ROS、MDA含量,改善HMCs氧化应激状态,延缓DKD进程。

Objective:To observe the effects of Chongcao Yishen Granule on oxidative stress of HMCs cultured with high glucose and expression of p47phox and to explore the mechanism of Chongcao Yishen Granule in the treatment of DKD(Diabetic kidney disease).Methods:The human glomerular mesangial cells cultivated with high glucose in vitro were divided into six groups:the normal group,the high glucose group,the Chongcao Yishen Granule low,medium and high dosage groups and Fosinopril positive control group.HMCs in each group were cultured with rats’serum containing medicine,cells supernatant was collected at 48 and 72 hours,the level of ROS in cells were tested by flow cytometry,the levels of SOD and MDA were tested by colorimetric method(DNS method),and the expressions of p47phox protein and mRNA were determined by Western Blot and fluorescence quantitative PCR.Results:At 48 and 72 hours,compared with normal group,SOD activity of other groups were significantly decreased(P<0.01),ROS and MDA levels of other groups were significantly increased(P<0.01),the expression levels of p47phox mRNA and protein were significantly upregulated(P<0.01);Compared with the high glucose group,SOD activity in Fosinopril group and Chongcao Yishen Granule low,medium and high dosage groups were obviously raised(P<0.05 or P<0.01),the levels of ROS and MDA were lowered significantly(P<0.01),and the expressions of p47phox mRNA and protein were obviously decreased(P<0.05 or P<0.01).Conclusion:The possible mechanism of Chongcao Yishen Granule for DKD is related to raising SOD activities,reducing the levels of ROS and MDA,and improving oxidative stress.