淋巴增强子结合因子1在肾间质纤维化中的作用及机制

Role and Mechanism of Lymphoid Enhancer Binding Factor 1 in Renal Interstitial

目的探讨淋巴增强子结合因子1(lymphoid enhancer binding factor 1,LEF1)在肾间质纤维化中的作用及机制。方法将18只C57BL/6小鼠,采用完全随机设计分为单侧输尿管梗阻(unilateral ureteral obstruction,UUO)3天组、UUO 7天组和假手术组。苏木精-伊红(hematoxylin-eosin,HE)染色及Masson染色观察肾组织病理变化,Western blot法及免疫组化法检测肾组织中LEF1的表达。Western blot法检测10ng/ml及20ng/ml转化生长因子-β1(transforming growth factor-β1,TGF-β1)刺激24h后LEF1的蛋白表达。体外培养HK-2细胞,并分为4组,即Ctrl siRNA组、LEF1siRNA组、Ctrl siRNA+TGF-β1组和LEF1siRNA+TGF-β1组。Western blot法检测HK-2中LEF1、纤连蛋白(fibronectin,FN)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(collagenⅠ)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、多聚磷酸核糖聚合酶剪切体(cleaved poly ADP-ribose polymerase,cleaved PARP)、半胱氨酸蛋白酶-3剪切体(cleaved caspase-3)等蛋白的相对表达水平,比较4组细胞各蛋白相对表达水平的差异。流式细胞术检测各组细胞的凋亡率。结果UUO组出现小管扩张、炎性细胞浸润及胶原沉积,LEF1表达高于假手术组(P<0.05)。TGF-β1可导致HK-2细胞中LEF1的蛋白表达水平升高(P<0.05)。LEF1siRNA转染HK-2细胞敲低其表达可以减轻细胞的纤维化反应(P<0.05)。敲低LEF1可以延缓上皮间充质转化(epithelial–mesenchymal transition,EMT)进程并减轻HK-2的凋亡(P<0.05)。结论LEF1可以通过促进肾小管上皮细胞发生EMT进而诱导凋亡,最终导致肾间质纤维化。

Objective To investigate the role and mechanism of lymphoid enhancer binding factor 1(LEF1)in renal interstitial fibrosis.Methods Eighteen C57BL/6 mice were randomly divided into unilateral ureteral obstruction(UUO)3day group,UUO 7day group and sham operation group.The pathological changes of renal tissue were observed by hematoxylin-eosin staining and Masson staining,and the expression of LEF1 was detected by Western blot and immunohistochemistry.Human renal tubular epithelial cells(HK-2)were cultured in vitro,LEF1 was detected by Western blot after treatment with 10ng/ml and 20ng/ml transforming growth factor-β1(TGF-β1)for 24h.HK-2 cells were divided into 4 groups:Ctrl siRNA group,LEF1siRNA group,Ctrl siRNA+TGF-β1group and LEF1siRNA+TGF-β1group.The expression levels of LEF1,fibronectin,α-smooth muscle actin(α-SMA),collagenⅠ,E-cadherin,vimentin,cleaved poly ADP-ribose polymerase(cleaved PARP)and cleaved caspase-3 were detected by Western blot,and the relative expression levels of each protein in the 4groups were compared.Flow cytometry was used to detect the apoptosis rate of each group.Results Tubular dilation,inflammatory cell infiltration and collagen deposition were observed in UUO group.The expression level of LEF1 in UUO group was higher than sham operation group(P<0.05).TGF-β1 could increase LEF1 protein expression in HK-2 cells(P<0.05).LEF1siRNA transfection of HK-2 cells decreased the fibrotic response(P<0.05).The knockdown of LEF1 can alleviate the epithelial–mesenchymal transition(EMT),meanwhile reduced the apoptosis induced by TGF-β1 in HK-2 cells(P<0.05).Conclusion LEF1 can induce renal interstitial fibrosis by promoting EMT and apoptosis of renal tubular epithelial cells.