Snail在AP-1介导的肾小管上皮细胞转分化中的作用

Role of Snail in the renal tubular epithelial-mesenchymal-transition mediated by activator protein-1

目的:探讨Snail在AP-1介导的肾小管上皮细胞转分化中的作用及对细胞外基质分泌的影响。方法:将体外培养的人肾小管上皮细胞(HK2)分为正常对照组、高糖组、AP-1阻断组。细胞免疫化学法检测E-钙黏蛋白(E-cadherin)和波形蛋白(vimentin)的表达,ELISA法测定培养液上清中纤连蛋白(FN)的浓度变化,用凝胶电泳迁移率改变分析法(EMSA)检测HK2细胞AP-1的改变,RT-PCR法检测vimentin mRNA和Snail mR-NA的表达,Western blotting检测E-cadherin的表达。结果:高糖作用HK2细胞48h后,高糖组FN浓度较正常对照组显著增加(P<0.05),而AP-1阻断组浓度显著低于高糖组(P<0.05);高糖能够刺激AP-1结合活性,AP-1阻断剂可显著抑制AP-1的活化;高糖组Snail mRNA表达水平较正常对照组显著增加(P<0.05),而AP-1阻断组其水平显著低于高糖组(P<0.05);与正常对照组相比,高糖组E-cadherin蛋白表达明显降低(P<0.05),而AP-1阻断组其水平显著高于高糖组(P<0.05);高糖组vimentin mRNA和蛋白水平明显高于正常对照组(P<0.05),而AP-1阻断组显著低于高糖组(P<0.05)。结论:高糖可导致肾小管上皮细胞AP-1活化,诱导Snail表达而下调E-cadherin,同时导致vimentin蛋白的表达、细胞外基质FN的分泌增加,诱导其表型转化,参与糖尿病肾病的纤维化进程。

AIM ： To study the role of Snail in the renal tubular epithelial - myofibroblast transdifferentiation and fibronectin synthesis mediated by activator protein - 1. METHODS： The cultured HK2 cells were divided into three groups ： normal glucose group （ NG）, high glucose group （HG） and activator protein -1 （ AP - 1 ） inhibited group （AG）. Concentration of fibronectin into the culture media was determined by ELISA. The activity of activator protein - 1 was assessed with electrophoretic mobility shift assay （EMSA）. The protein of E -cadherin and vimentin was determined by immunocytochemistry. RT - PCR was used to detect the mRNA expression of vimentin and Snail. Western blotting was used to detect the protein expression of E - cadherin. RESULTS ： Secreted FN level was significantly up - regulated by the stimulation of high glucose （ P 〈 0. 05 ）, but the level significantly decreased in AP - 1 inhibited group than that in high glucose group （ P 〈 0. 05 ）. AP - 1 binding activity was significantly stimulated by high glucose and the inhibitor of AP - 1 inhibited high glucose induced AP - 1 activation in HK - 2. High glucose induced Snail mRNA expression, while the level significantly decreased in AP - 1 inhibited group than that in high glucose group （ P 〈 0. 05 ）. Upon the stimulation with high glucose, the expression of E - cadherin protein decreased markedly （ P 〈 0. 05 ）, while the level was higher in AP - 1 inhibited group than that in high glucose group （P 〈 0.05）. Cultured with high glucose, the expression of vimentin mRNA and protein significantly increased （ P 〈 0.05 ）, but the level significantly decreased in AP - 1 inhibited group than that in high glucose group （ P 〈 0. 05 ）. CONCLUSION： High glucose induces the expression of Snail through the activation of AP - 1. The expression of Snail downregulates E - cadherin expression and induces transdifferentiation of renal tubular ceils characterized by vimentin expression and fibronectin synthesis.