虫草益肾颗粒对高糖条件下肾小球系膜细胞氧化应激及p22phox表达的影响

Effect of Chongcao Yishen Particle on Oxidative Stress of HMCs and Expression of P22phox

[目的]研究虫草益肾颗粒对高糖环境下人肾小球系膜细胞(human mesangial cells,HMCs)氧化应激及p22phox表达水平的影响,探讨其防治糖尿病肾病(diabetic kidney disease,DKD)的作用机制。[方法]将50只无特定病原体(specific pathogen free,SPF)级Wistar大鼠随机分为正常组,福辛普利组,虫草益肾颗粒低、中、高剂量组,每组10只。以细胞增殖检测(cell counting kit-8,CCK-8)法检测HMCs在24、48、72 h的细胞增殖情况;分别于48、72 h时,以流式细胞术检测各组HMCs的活性氧簇(reactive oxygen species,ROS)水平;以免疫印迹和实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)法检测各组p22phox的表达情况。[结果] HMCs经高糖培养后增殖明显,48 h时的增殖率最高,随后便进入增殖平台期,增殖速度逐渐趋于平缓。各给药组对高糖培养下的HMCs的增殖均有抑制作用(P<0.01),且虫草益肾颗粒对HMCs增殖的抑制作用呈时间-剂量依赖性关系。与正常组比较,48、72 h时各组细胞ROS水平均增高(P<0.05),p22phox的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、高剂量组细胞的ROS水平、p22phox的m RNA及蛋白表达量均明显下降(P<0.05或P<0.01)。[结论]虫草益肾颗粒可能是通过抑制p22phox基因转录,下调p22phox蛋白表达来减少细胞内ROS的生成,从而减轻氧化应激反应,延缓DKD发生发展。

[Objective] To observe the effect of Chongcao Yishen Particle on oxidative stress of human mesangial cells(HMCs) and expression of p22 phox, explore the mechanism of Chongcao Yishen Particle in the treatment of diabetic kidney disease(DKD). [Methods] Fifty specific pathogen free(SPF) Wistar rats were randomly divided into normal group, Chongcao Yishen Particle low, medium and high dosage groups and Fosinopril group, with 10 rats in each group. Cells were collected after respectively given the appropriate treatment. The HMCs proliferative degrees at 24, 48 and 72 hours were detected by cell counting kit-8(CCK-8);in 48 and 72 hours, flow cytometry was used to detect the reactive oxygen species(ROS) level in each group, and Western blot and Real-time quantitative polymerase chain reaction(Realtime q PCR) were used to detected the expressions of p22 phox in each group. [Results] HMCs proliferated significantly under the stimulation of high glucose. The proliferation rate was the highest at 48 h, and then it entered the proliferation platform stage, and the proliferation rate gradually became flat. Each administration group had inhibitory effect on the proliferation of HMCs under high glucose culture(P<0.01),and the inhibitory effect of Chongcao Yishen Particle on the proliferation of HMCs was time-dose-dependent. Compared with normal group, ROS levels of other groups were significantly increased(P <0.05), the expression levels of p22 phox mRNA and protein were significantly upregulated at 48 and 72 hours(P<0.01). Compared with high glucose group, the levels of ROS, p22 phox mRNA and protein in Fosinopril group and Chongcao Yishen Particle low, medium and high dosage groups obviously tend to ascent(P <0.05 or P <0.01).[Conclusion] Chongcao Yishen Particle may reduce the production of ROS in cells by inhibiting p22 phox gene transcription and downregulating p22 phox protein expression, so as to reduce oxidative stress and delay the occurrence and development of DKD.