DNA甲基转移酶及分泌型卷曲相关蛋白1在糖尿病肾脏疾病大鼠肾组织中的表达研究

Expression of DNA methyltransferase and sfrp1 in renal tissue of rats with diabetic kidney disease

目的 探讨DKD大鼠肾组织中DNA甲基转移酶(Dnmts)及分泌型卷曲相关蛋白1(Sfrp1)的表达变化及意义。方法 16只雄性SD大鼠随机分为DKD组与正常对照(NC)组,每组8只。造模成功10周后处死,检测相关生化指标并计算肾脏指数(KI);HE、Masson染色观察肾组织病理学改变;Western blot检测Dnmts、Sfrp1、β-catenin、GSK3β、p-GSK3β、col-Ⅲ、col-Ⅳ蛋白表达变化;q-PCR检测Sfrp1mRNA表达水平;分析Sfrp1与Dnmts相关性。结果 与NC组比较,DKD组血糖[(8.51±1.19)vs(21.00±2.24)mmol/L]、24hUAlb[(12.22±4.91)vs(65.31±18.03)mg]、KI[(6.80±0.53)vs(12.15±1.57)mg/g]均增高(P<0.01);Dnmt1、Dnmt2、Dnmt3a、Dnmt3b、β-catenin、p-GSK3β蛋白表达水平上调(P<0.05);Dnmt3l未见明显变化;Sfrp1 mRNA和蛋白表达水平均下调(P<0.05);Dnmt1、Dnmt2、Dnmt3a、Dnmt3b与Sfrp1的蛋白表达呈负相关(r=-0.811、-0.692、-0.855、-0.858;P<0.01);与Dnmt3l无相关性(r=-0.262,P=0.411)。结论 DKD大鼠肾组织Dnmts蛋白表达增加,可能引起sfrp1表达下调,导致对Wnt信号通路的抑制作用减弱,Wnt信号通路异常活化并促进DKD时肾纤维化的发生发展。

Objective To investigate the changes of DNA methyltransferase（Dnmts）and secreted frizzled-related protein 1（Sfrp1）in renal tissue of rats with diabetic kidney disease. Methods A total of16 male Sprague-Dawley rats were randomly divided into two groups：diabetic kidney disease group（DKD,n=8）and normal control group（NC,n=8）.Ten weeks after building successful models,all the rats were sacrificed.Relative biochemical parameters were tested and renal index（KI）was calculated.The pathological changes of renal tissue were observed by HE and masson staining.The protein expression of Dnmts,Sfrp1,β-catenin,GSK3β,p-GSK3β,col-Ⅲ and col-Ⅳ were detected by Western blot.The expression of Sfrp1 mRNA was detected by q-PCR.The correlation between Sfrp1 and Dnmts was analyzed. Results The levels of blood glucose,24 hurinary albumin（24 hUAlb）and KI were increased in DKD group than in NC group[（8.51±1.19）vs（21.00±2.24）mmol/L,（12.22±4.91）vs（65.31±18.03）mg,（6.80±0.53）vs（12.15±1.57）mg/g,（P〈0.01）].The protein expressions of Dnmt1,Dnmt2,Dnmt3 a,Dnmt3 b,β-catenin and p-GSK3βwere up-regulated in DKD group than in NC group（P〈0.05）.There was no significant change in Dnmt3 lin DKD and NC groups.The mRNA and protein levels of Sfrp1 were down-regulated in DKD group than in NC group（P〈0.05）.The protein expressions of Dnmt1,Dnmt2,Dnmt3 a,Dnmt3 bwere negatively correlated with Sfrp1（r=-0.811,-0.692,-0.855,-0.858;P〈0.01）,and have no correlation with Dnmt3 l（r=-0.262,P=0.411）. Conclusion Dnmts protein expression is increased in DKD rats,which may induce the down-regulation of sfrp1 expression,and caused diminished inhibition in wsnt signaling pathway,resulting in the development of renal fibrosis in DKD.