益肾泄浊方对高糖环境下人肾小球内皮细胞的保护作用及机制

Protective Effect and Mechanism of Medicated Serum Yishen Xiezhuo Decoction on Human Glomerular Endothelial Cells in High Glucose Environment

目的:探讨益肾泄浊方含药血清对高糖环境下人肾小球内皮细胞的保护作用及其机制。方法:体外培养的人肾小球内皮细胞分为空白组、模型组、空白血清对照组、益肾泄浊方含药血清低剂量组、益肾泄浊方含药血清高剂量组,每组设置3个复孔。除空白组外,每组给予含30 mmol·L^(-1) D-葡萄糖的ECM培养基培养2 h后,给予相应药物进行干预,分别于干预24 h、48 h后,CCK-8法检测各组细胞的增殖情况;ELISA法检测细胞上清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、纤维蛋白原样蛋白2(fibrinogen-like protein2,fgl2)凝血酶原酶水平;RT-PCR法检测细胞中TNF-α mRNA、fgl2凝血酶原酶mRNA的表达情况;分析细胞上清中TNF-α与fgl2凝血酶原酶水平的相关性及细胞中TNF-α mRNA与fgl2凝血酶原酶mRNA表达的相关性。结果:与空白组比较,模型组肾小球内皮细胞的增殖率显著降低(P<0.05),细胞上清中TNF-α、fgl2凝血酶原酶水平及细胞中TNF-α mRNA、fgl2凝血酶原酶mRNA表达水平显著升高(P<0.05);与模型组比较,益肾泄浊方能显著提高人肾小球内皮细胞增殖率(P<0.05),显著降低细胞上清中TNF-α、fgl2凝血酶原酶水平及细胞中TNF-α mRNA、fgl2凝血酶原酶mRNA表达水平(P<0.05),且细胞上清中TNF-α与fgl2凝血酶原酶水平及两者mRNA表达水平呈正相关关系。结论:益肾泄浊方含药血清能保护高糖环境下肾小球内皮细胞损伤,其可能的作用机制是通过纠正由炎症因子TNF-α介导的免疫凝血功能异常,缓解肾脏微循环障碍,保护肾脏功能。

Objective: To investigate the protective effect and mechanism of medicated serum Yishen Xiezhuo Decoction on human glomerular endothelial cells in high glucose environment.Methods: Human glomerular endothelial cells cultured in vitro were divided into blank control group, high glucose induction group, high glucose serum control group, medicated serum Yishen Xiezhuo Decoction low-dose group and medicated serum Yishen Xiezhuo Decoction high-dose group, with 3 multiple pores in each group.After 24 hours and 48 hours of intervention, the proliferation of cells in each group was detected by CCK-8 method;ELISA method was used to detect the levels of tumor necrosis factor-α(TNF-α) and fibrinogen-like protein 2(fgl2) prothrombinase in the cell supernatant;RT-PCR method was used to detect cell TNF-α and fgl2 prothrombinase mRNA expression;the correlation between TNF-α and fgl2 prothrombinase levels in the cell supernatant and the cell TNF-α and fgl2 prothrombinase mRNA expression were analyzed.Results: Compared with the blank control group, the proliferation rate of glomerular endothelial cells in the high glucose induction group was significantly reduced(P<0.05).The levels of TNF-α and fgl2 prothrombinase in the cell supernatant and the mRNA expression levels of TNF-α and fgl2 prothrombinase were significantly increased(P<0.05).Compared with the high glucose induction group, Yishen Xiezhuo Decoction could significantly increase the proliferation rate of human glomerular endothelial cells(P<0.05),and significantly reduce the levels of TNF-α,fgl2 prothrombinase and cell TNF-α in the cell supernatant, fgl2 prothrombinase mRNA expression level(P<0.05).The cell supernatant TNF-α and fgl2 prothrombinase level and the two mRNA expressions were positively correlated(P<0.05).Conclusion: The medicated serum Yishen Xiezhuo Decoction can protect glomerular endothelial cells from damage under high glucose environment.The possible mechanism of action is to correct the abnormal immune coagulation function mediated by the inflammatory factor TNF-α,alleviate the renal microcirculation disorder and protect the function of the kidney.