大黄素对TGF-β1诱导的人肾小管上皮细胞上皮间质转分化及Mfn2表达的影响

Effects of emodin on TGF-β1-induced epithelial-mesenchymal transition and expression ofMfn2 in human renal tubular epithelial cells

目的探讨大黄素(EM)对转化生长因子β1(TGF-β1)诱导的人肾小管上皮细胞(HK-2)上皮间质转分化及线粒体融合蛋白2(Mfn2)的影响.方法采用CCK-8法检测不同浓度EM对HK-2细胞增殖的影响;实验分空白对照组、TGF-β1组、TGF-β1+EM(10、20、40μmol/L)组,EM干预组予以不同浓度的大黄素处理HK-2细胞30 min后,TGF-β1组和EM干预组同时给予浓度为10μg/L的TGF-β1刺激48 h收集细胞,Western blot法检测α-平滑肌肌动蛋白(a-SMA)、E-钙黏蛋白(E-cadherin)、结缔组织生长因子(CTGF)及Mfn2蛋白的表达.结果CCK-8结果提示0、10、20、40、80、160μmol/L浓度的EM处理组的HK-2细胞存活率(%)分别为100.00±0.00、90.89±2.17、88.52±1.67、87.72±2.00、63.46±1.73、52.19±2.60,呈逐渐降低趋势(P<0.01);Western blot结果表明,与空白对照组比较,TGF-β1刺激后,HK-2细胞中α-SMA、CTGF及Mfn2蛋白水平升高,E-cadherin蛋白表达降低(P<0.05),与TGF-β1组比较,给予不同浓度大黄素干预后,α-SMA、CTGF及Mfn2蛋白水平降低,E-cadherin蛋白表达升高(P<0.05),且呈一定剂量依赖性.结论大黄素缓解了TGF-β1诱导的HK-2细胞上皮间质转分化,可能通过减少TGF-β1诱导的Mfn2的表达来减缓肾脏纤维化.

Objective To investigate the effect and mechanism of emodin(EM)on TGF-β1-induced epithelial-mesenchymal transition and the expression of mitofusin 2(Mfn2)in human renal tubular epithelial cells(HK-2).Methods CCK-8 was used to detect the effects of different concentrations of emodin on the proliferation of HK-2 cells.The experiment cells were divided into blank control group,TGF-β1 group and TGF-β1+EM group.The emodin intervention group was treated with different concentrations of emodin(10,20 and 40μmol/L)for 30 min,and the TGF-β1 group and the emodin intervention group were simultaneously stimulated with TGF-β1 at a concentration of 10μg/L for 48 h.The cells were then harvested and the expressions ofα-smooth muscle actin(α-SMA),E-cadherin,connective tissue growth factor(CTGF)and Mfn2 proteins were detected by Western blot assay.Results The CCK-8 result suggested that the survival rate(%)of HK-2 cells were 100.00±0.00,90.89±2.17,88.52±1.67,87.72±2.00,63.46±1.73 and 52.19±2.60 in the EM treatment groups at 0,10,20,40,80,and 160μmol/L concentrations,showing a gradually decreasing trend(P<0.01).Western blot results showed that the protein levels ofα-SMA,CTGF and Mfn2 increased in HK-2 cells,and the expression of E-cadherin protein decreased after stimulation with TGF-β1 compared with those of the blank control group(P<0.05).Compared with the TGF-β1 group,α-SMA,CTGF and Mfn2 protein levels were decreased,and E-cadherin protein expression was increased after treatment with different concentrations of emodin(P<0.05)in a dose-dependent manner.Conclusion Emodin attenuates TGF-β1-induced epithelial-mesenchymal transition in human renal tubular epithelial cells,which may ameliorate renal fibrosis by reducing the expression of TGF-β1-induced Mfn2.