白花丹提取物对高糖诱导的肾小球系膜细胞分泌炎症因子及细胞外基质的影响

Effects of Plumbago zeylanica L extract on the secretion of inflammatory factors and extracellular matrix by high glucose-induced glomerular mesangial cells

目的探讨白花丹提取物对高糖诱导的肾小球系膜细胞分泌炎症因子及细胞外基质的影响。方法体外培养大鼠肾小球系膜细胞,随机分为对照组,模型组,白花丹提取物低剂量(1 mg/L)、中剂量(2 mg/L)、高剂量(4 mg/L)组,甘露醇(24.5 mmol/L)组,收集各组细胞及细胞培养基,以酶联免疫吸附法检测各组细胞上清液中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin-6,IL-6)、转化生长因子β1(transforming growth factor-β1,TGF-β1)水平;检测各组细胞超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)水平;以实时荧光定量法检测各组细胞miR-155、SOCS1 mRNA水平;采用蛋白免疫印迹法检测各组细胞纤维连结蛋白(fibronectin,FN)、Ⅳ型胶原蛋白(collagen IV,Col IV)、细胞因子信号传导抑制蛋白1(cytokine signal transduction inhibitor 1,SOCS1)蛋白表达。结果与对照组相比,模型组、甘露醇组细胞上清液中TNF-α、IL-6及TGF-β1水平升高,细胞MDA、miR-155、FN及Col IV表达升高(P<0.05),细胞SOD水平、SOCS1 mRNA及蛋白表达明显降低(P<0.05)。与模型组相比,白花丹提取物低、中、高剂量组细胞上清液中TNF-α、IL-6及TGF-β1水平降低,细胞MDA、miR-155、FN及Col IV表达降低(P<0.05),细胞SOD水平、SOCS1 mRNA及蛋白表达升高(P<0.05),且各项变化均对白花丹提取物呈剂量依赖性(P<0.05)。结论白花丹提取物可抑制高糖诱导的肾小球系膜细胞分泌炎症因子及细胞外基质,下调miR-155表达、上调SOCS1表达可能是其作用机制。

Objective To investigate the effects of Plumbago zeylanica L extract on the secretion of inflammatory factors and extracellular matrix by high glucose-induced glomerular mesangial cells.Methods Glomerular mesangial mesangial cells from rats were cultured in vitro and randomly divided into control group,model group,low-dose(1 mg/L),medium dose(2 mg/L)and high dose(4 mg/L)Plumbago zeylanica L extract groups,and mannitol(24.5 mmol/L)group.Cells and cell culture media in each group were collected.The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and transforming growth factor-β1(TGF-β1)in cell supernatants were detected by enzyme-linked immunosorbent assay.Superoxide dismutase(SOD)test kit and malondialdehyde(MDA)test kit were used to detect the levels of SOD and MDA in each group.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the levels of miR-155 and SOCS1 mRNA in the cultured cells in each group.The expressions of fibronectin(FN),human type IV collagen(Col IV)and cytokine signal transduction inhibitor 1(SOCS1)were detected by western blotting.Results Compared with the control group,the levels of TNF-α,IL-6 and TGF-β1 in the supernatant,MDA,miR-155,FN and Col IV protein in the cells of the model group and mannitol group were significantly increased(P<0.05),and SOD level,SOCS1 mRNA level and protein expression in the cells were significantly decreased(P<0.05).Compared with the model group,the levels of TNF-α,IL-6 and TGF-β1 in the supernatant,MDA,miR-155,FN and Col IV protein in the cells of the low,medium and high dose groups were decreased(P<0.05);SOD level,SOCS1 mRNA level and protein expression in the cells were increased(P<0.05),and all the above changes exhibited dependent on doses of Plumbago zeylanica L extract(P<0.05).Conclusions Plumbago zeylanica L extract can inhibit the secretion of inflammatory factors and extracellular matrix of glomerular mesangial cells induced by high glucose,and down-regulation of the expression of miR-155 and up-regulation of the expression of SOCS1 may be its mechanism.