目的:观察高糖刺激下人肾小球系膜细增殖分化及Col IV、MCP-1 mRNA和蛋白活性表达变化,探究益气解毒活络中药有效成分防治糖尿病肾病的分子机制,讨论正交配伍中最优药物组合。方法:以体外培养人肾小球系膜细胞株,传代3~5代后接种于96孔培养板,采用4因素3水平分为正常对照组,高糖模型组、中药1、2、3、4、5、6、7、8、9组,以相应药物对HMCs干预24、48、72 h。MTT法检测24、48、72 h各时间点HMCs增殖分化情况;以qRT-PCR法检测48 h Col IV、MCP-1 mRNA表达;以ELISA法检测48 h Col IV、MCP-1蛋白活性表达。结果:(1)高糖环境刺激作用下HMCs及胞外基质均能够显著增殖,且在48 h达到顶峰;(2)益气解毒活络中药有效成分药防治DN作用机制与通过下调高糖作用下HMCs Col IV、MCP-1 mRNA及蛋白活性表达,进而抑制高糖刺激HMCs增殖相关;(3)益气解毒活络中药有效,最佳配伍为黄芪甲苷低剂量、盐酸小檗碱低剂量、水蛭素低剂量、木犀草苷低剂量。
Objective: To observe the mRNA and protein expressions of Col IV and MCP-1 in HMC with high glucose and fat-induced and discuss the prevention and treatment of diabetic nephorpathy and discuss the best scheme of orthogonal compatibility. Methods: HMCin vitro cultured were divided into 11 groups common control group model control group and 9 orthogonal groups. MTT method was used to quantitatively detect the proliferation and differentiation of HMC. The qRT-PCR method was used for detection of expressions of Col IV,MCP-1 mRNA and ELISA method was used for test the expression activity and content of Col IV,MCP-1. Results:( 1) HMC in vitro was highly proliferation anddifferentiation.( 2) Active components of supplementing qi and detoxification activating can down regulate the protein and mRNA expressions of Col IV and MCP-1 in HMC with high glucose and fat-induced.( 3) The best scheme of orthogonal compatibility is low dose of astragaloside IV,low dose of berberine,low dose of Hirudin and low dose of Cynaroside. Conclusion: