组蛋白去乙酰化酶6在肾小管上皮细胞⁃间充质转化及成纤维细胞活化中的作用  

Role of histone deacetylase 6 in epithelial⁃mesenchymal transition of renal tubular epithelial cells and activation of renal interstitial fibroblasts

作　　者：施映枫 周璕 马晓燕[1] 陈慧[1] 陶敏 胡艳 陈思 刘娜[1] Shi Yingfeng;Zhou Xun;Ma Xiaoyan;Chen Hui;Tao Min;Hu Yan;Chen Si;Liu Na(Department of Nephrology,Shanghai East Hospital,Tongji University School of Medicine,Shanghai 200120,China)

机构地区：[1]同济大学附属东方医院肾内科,上海200120

出　　处：《中华肾脏病杂志》2021年第11期911-917,共7页Chinese Journal of Nephrology

基　　金：国家自然科学基金(82070791、81670690、81470991、81200492);上海市浦东新区卫生系统领先人才培养计划(PWRl2021-02);上海市科委基金(20ZR1445800、13PJ1406900);上海市浦东新区卫健委重点学科主攻项目(PWZxk2017-05)。

摘　　要：目的探讨组蛋白去乙酰化酶6(histone deacetylase 6,HDAC6)在肾小管上皮细胞⁃间充质转化及肾间质成纤维细胞活化中的作用及其机制。方法体外培养人肾小管上皮细胞(HK⁃2)和大鼠肾间质成纤维细胞(NRK⁃49F),细胞被分为4组:(1)对照组;(2)HDAC6抑制剂Tubastatin A(TA)组:10μmol/L TA干预36 h;(3)转化生长因子(TGF)⁃β1组:10 ng/ml TGF⁃β1刺激36 h;(4)TGF⁃β1+TA组:10 ng/ml TGF⁃β1+10μmol/L TA刺激36 h。Western印迹法检测HK⁃2和NRK⁃49F细胞中纤连蛋白、α平滑肌肌动蛋白(α⁃SMA)、I型胶原、E钙黏蛋白、HDAC6、乙酰化组蛋白H3、组蛋白H3、乙酰化α微管蛋白、α微管蛋白、TGF⁃β受体(TGF⁃βR)1、p⁃Smad3、Smad3、结缔组织生长因子(CTGF)、表皮生长因子受体(EGFR)及p⁃EGFR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。结果(1)与TGF⁃β1组比较,TGF⁃β1+TA组HK⁃2细胞肌成纤维细胞标志物α⁃SMA、细胞外基质蛋白I型胶原和纤连蛋白表达水平显著下调;上皮细胞标志物E钙黏蛋白的表达水平显著上调;HDAC6表达水平下调,乙酰化组蛋白H3和乙酰化α微管蛋白的表达水平上调(均P<0.05)。(2)与TGF⁃β1组比较,TGF⁃β1+TA组HK⁃2细胞TGF⁃βR1、p⁃Smad3、CTGF及p⁃EGFR的表达水平下调(均P<0.05),Smad3和EGFR总蛋白表达水平的差异无统计学意义(均P>0.05)。(3)与TGF⁃β1组比较,TGF⁃β1+TA组NRK⁃49F细胞纤连蛋白、α⁃SMA、I型胶原、TGF⁃βR1和p⁃Smad3的表达水平下调(均P<0.05)。结论HDAC6抑制剂TA可能通过阻断TGF⁃β1/Smad3、CTGF和EGFR多条信号通路,抑制肾小管上皮细胞⁃间充质转化及肾间质成纤维细胞活化。Objective To investigate the role and mechanism of(histone deacetylase 6,HDAC6)in the epithelial⁃mesenchymal transition(EMT)of renal tubular epithelial cells and the activation of renal interstitial fibroblasts.Methods Human renal tubular epithelial cells(HK⁃2)and rat renal interstitial fibroblast(NRK⁃49F)were cultured in vitro,and divided into 4 groups:control group,Tubastatin A(TA)group(treated with 10μmol/L HDAC6 inhibitor TA for 36 h),transforming growth factor⁃β1(TGF⁃β1)group(10 ng/ml TGF⁃β1 for 36 h),and TGF⁃β1+TA group(treated with 10 ng/ml TGF⁃β1 and 10μmol/L TA for 36 h).The expression levels of fibronectin,α⁃smooth muscle actin(α⁃SMA),collagen I,E⁃cadherin,HDAC6,acetyl histone H3,histone H3,acetylα⁃tubulin,α⁃tubulin,TGF⁃βreceptor(TGF⁃βR)1,p⁃Smad3,Smad3,connective tissue growth factor(CTGF),epidermal growth factor receptor(EGFR)and p⁃EGFR in HK⁃2 and NRK⁃49F cell samples were detected by Western blotting,and quantitative analysis was performed according to gray level.Results(1)In HK⁃2 cells stimulated by TGF⁃β1,TA decreased the expression of fibronectin,α⁃SMA,collagen I,and increased the expression of epithelial cell marker E⁃cadherin.Meanwhile,TA decreased the expression of HDAC6 and increased the expression levels of acetyl histone H3 and acetylα⁃tubulin(all P<0.05).(2)Compared with the TGF⁃β1 group,the expressions of TGF⁃βR1,p⁃Smad3,CTGF and p⁃EGFR in TGF⁃β1+TA group were decreased(all P<0.05),while the total protein levels of Smad3 and EGFR were not significantly different(both P>0.05).(3)In NRK⁃49F cells stimulated by TGF⁃β1,TA decreased the expressions of fibronectin,α⁃SMA,collagen I,TGF⁃βR1 and p⁃Smad3(all P<0.05).Conclusions Blockade of HDAC6 by TA may inhibit the EMT of renal tubular epithelial cells and the activation of renal interstitial fibroblasts via regulating multiple signaling pathways including TGF⁃β/Smad3,CTGF and EGFR.

关 键 词：细胞转分化 转化生长因子β1 组蛋白脱乙酰基酶类 上皮细胞⁃间充质转分化 肾间质成纤维细胞 

分 类 号：R692.5[医药卫生—泌尿科学]