沉默信息调节因子1调控Zeste同源物2增强子对高糖诱导的大鼠肾小管上皮细胞-间充质转化影响的研究  

Effect of SIRT1 regulating EZH2 expression on EMT induced by high glucose in rat renal tubular epithelial cells

作　　者：李清璇 安小敏 杨鹏[3] 龙天华 王彤 冉瑶 卢雨微 郭兵[1] 丁菁 石明隽 LI Qingruan;AN Xiaomin;YANG Peng(Department of Pathophysiology Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学病理学与病理生理学教研室,贵阳550025 [2]贵州省常见慢性疾病发病机制及药物研究重点实验室 [3]地方病与少数民族疾病教育部重点实验室 [4]省部共建药用植物功效与利用国家重点实验室 [5]贵州省常见重大慢性疾病发病机制及药物开发应用创新基地

出　　处：《中国糖尿病杂志》2023年第6期433-442,共10页Chinese Journal of Diabetes

基　　金：国家自然科学基金(81860135、82060142);贵州省科技计划项目(黔科合基础-ZK[2022]一般378);贵州省研究生科研基金立项课题建设任务合同书(KYJJ2017013)。

摘　　要：目的探讨沉默信息调节因子1(SIRT1)通过调控Zeste同源物2增强子(EZH2)表达影响肾小管上皮细胞-间充质转化(EMT)过程及DKD肾纤维化发病机制。方法12只清洁级雄性SD大鼠,随机分为正常对照(NC)组、DKD组,各6只,检测各组BG、BUN和24 h尿白蛋白(24 hUAlb)。培养大鼠肾小管上皮细胞(NRK-52E),分为正常对照组(Con)、高糖组(HG)和高渗组(HM)。在无血清条件下用脂质体2000(Lip2000)转染法,将空载质粒(Vector)、敲低及过表达SIRT1和EZH2质粒分别转染细胞,将细胞分为正常空载组(Con+Vector)、正常敲低EZH2转染组(Con+sh-EZH2)、正常敲低SIRT1转染组(Con+sh-SIRT1)、正常过表达EZH2转染组(Con+oe-EZH2)、正常过表达SIRT1转染组(Con+oe-SIRT1)、高糖空载组(HG+Vector)、高糖敲低EZH2转染组(HG+sh-EZH2)、高糖敲低SIRT1转染组(HG+sh-SIRT1)、高糖过表达EZH2转染组(HG+oe-EZH2)、高糖过表达SIRT1转染组(HG+oe-SIRT1)、高糖双转过表达组(HG+oe-EZH2+oe-SIRT1)。HE、Masson染色观察肾组织病理形态改变,免疫荧光化学染色观察NRK-52E细胞中SIRT1和EZH2表达,Western blot和qRT-PCR检测肾组织和NRK-52E细胞SIRT1、EZH2、E-cadherin、α-SMA和CollegenⅢ蛋白和mRNA表达。结果DKD组BG、BUN、24 h UAlb高于NC组(P<0.05),HE、Masson染色观察到DKD组肾小管管腔扩张,肾小球和肾间质有蓝紫色胶原纤维沉积;DKD组较NC组α-SMA、CollagenⅢ、EZH2蛋白表达升高(P<0.05),E-cadherin、SIRT1蛋白表达降低(P<0.05)。培养的细胞中,HG组较Con组α-SMA、CollagenⅢ、EZH2蛋白表达升高(P<0.05),E-cadherin、SIRT1蛋白表达降低(P<0.05)。与HG+Vector组比较,HG+sh-EZH2组E-cadherin蛋白表达升高(P<0.05),EZH2、α-SMA、CollagenⅢ蛋白表达降低(P<0.05),HG+oe-EZH2组EZH2、α-SMA、CollagenⅢ蛋白表达升高(P<0.05),E-cadherin蛋白表达降低(P<0.05);HG+sh-SIRT1组较HG+Vector组EZH2、α-SMA、CollagenⅢ蛋白表达升高(P<0.05),SIRT1、E-cadherin蛋白表达降低(P<0.05),HG+oe-SIRT1组SIRT1、Objective To explore the effect of SIRT1 on EMT process of renal tubular epithelial cells by regulating the expression of Zeste homolog-2 enhancer(EZH2),and the pathogenesis of DKD renal fibrosis.Methods Twelve clean grade male SD rats were randomly divided into normal control(NC,n=6)group and DKD group(n=6).Blood glucose,blood urea nitrogen(BUN),and 24-hour urine albumin(24-hour UAlb)were measured in each group.Rat renal tubular epithelial cells(NRK-52E)were cultured and divided into normal control group(Con),high glucose group(HG),and hypertonic group(HM).Empty plasmids(Vector),knockdown and overexpression plasmids SIRT1 and EZH2 were transfected into cells under serum-free conditions using liposome 2000(Lip2000)transfection method.Cells were divided into normal empty group(Con+Vector),normal knockdown EZH2 group(Con+sh-EZH2),normal knockdown SIRT1 group(Con+sh-SIRT1),normal overexpression EZH2 group(Con+oe-EZH2),and normal overexpression SIRT1 group(Con+oe-SIRT1).High glucose empty load group(HG+Vector),high glucose knockdown EZH2 group(HG+sh-EZH2),high glucose knockdown SIRT1 group(HG+sh-SIRT1),high glucose overexpression EZH2 group(HG+oe-EZH2),high glucose overexpression SIRT1 group(HG+oe-SIRT1),and high glucose double transfection expression group(HG+oe-EZH2+oe-SIRT1).Results The levels of BG,BUN,and 24-hour UAlb were higher in DKD group than in NC group(P<0.05).HE and Masson staining showed dilation of the renal tubular lumen and deposition of blue purple collagen fibers in the glomerulus and renal interstitium in DKD group.Compared with NC group,the expression of E-Cadherin and SIRT1 proteins decreased(P<0.05),while the expression ofα-SMA,CollagenⅢ,and EZH2 proteins increased in DKD group(P<0.05).Compared with Con group,the expression of E-cadherin and SIRT1 proteins decreased(P<0.05),while the expression ofα-SMA,CollagenⅢ,and EZH2 proteins increased in HG group(P<0.05).Compared with HG+Vector group,the expression of EZH2,α-SMA,and CollagenⅢproteins decreased(P<0.05),while the expression of E-cad

关 键 词：大鼠 糖尿病肾脏疾病 上皮细胞-间充质转化 沉默信息调节因子1 Zeste同源物2增强子 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]