黄芪对糖尿病肾病大鼠肾间质Wnt/β-catenin及TGF-β1信号通路表达的影响

摘要

目的:观察黄芪(AM)对糖尿病肾病(DN)大鼠肾间质中Wnt/β - catenin信号通路及转化生长因子-β1( TGF -β1)表达情况的影响,探讨其抗糖尿病肾病肾间质纤维化的作用机制.方法:采用单次空腹腹腔注射60 mg/kg链脲佐菌素(STZ)制备糖尿病大鼠模型.4周后测定24 h尿蛋白排泄量,以24 h尿蛋白定量≥30 mg确定DN大鼠模型成功(n=48).48只DN大鼠随机分为4组:DN模型组(DN组)、黄芪治疗组(DA组)、DN模型组+氯沙坦治疗组(DL组)、DN模型组+黄芪联合氯沙坦治疗组(AL组),每组12只.选取12只健康雄性wistar大鼠作为正常对照组(N组).连续给药12周后,记录大鼠体重,并检测血清尿素氮(BUN)、肌酐(Scr)及24 h尿蛋白定量；处死大鼠,留取肾组织行HE及Masson染色观察肾间质病理改变,采用免疫组织化学技术及实时荧光定量PCR( FQ - PCR)检测肾间质中Wnt4、β - catenin及TGF -β1蛋白及mRNA的表达.结果:与N组比较,12周末DN组大鼠24 h尿蛋白定量、血清BUN、Scr水平明显增高,差异有统计学意义(P＞0.05).与N组比较,12周末DN组、DA组、DL组和AL组肾间质Wnt4、β- catenin及TGF -β1蛋白表达量明显增加(P＜0.05).DA组、DL组和AL组作两两比较,AL组24 h尿蛋白定量、血清BUN、Scr、Wnt4、β - catenin及TGF -β1下降更为明显,差异有统计学意义(P＜0.05).DA组与DL组相比各指标差异无统计学意义(P＞0.05).FQ - PCR结果显示wnt4、β - catenin和TGF -β1 mRNA在各组大鼠肾组织中的表达与Wnt4、β- catenin及TGF-β1蛋白表达趋势一致.结论:黄芪可以降低24 h尿蛋白定量,减轻肾间质病理损伤,下调wnt4、β- catenin及TGF-β1在肾间质中的表达,提示黄芪可能通过下调Wnt4、β- catenin及TGF-β1在肾间质中的表达,延缓DN大鼠肾间质纤维化的进程,而发挥肾脏保护作用.%Objective: To investigate the possible mechanism for atragalus mongholicus to protect renal function, the expression of Wnt/β - catenin signaling pathway and TGF - β1 were analysed in STZ - induced of diabetic model. Methods:The diabetes group rats were induced by injection intra - peritoneally with 60 mg/kg body weight STZ in 0. 1 mol/L sodium citrate solution (pH 4.3). The rats in N group were injected with 0. 1 mol/L sodium citrate solution. Diabetic model was considered to be successful when 24 -hour urinary protein was higher than 30 mg after 4 weeks of the injection(n =48). After model establishment, these rats were randomly divided into 4 groups ;DN group(n = 12) ,DA group(n = 12) ,DL group(n = 12) and ALgroup(n = 12). Meanwhile, 12 rats were selected to normal group. 12 weeks later ,body weight, blood glucose, 24 - hours urine protein quantity and the level of serum creatinine,urea nitrogen were measured, and then all the rats were sacrificed respectively. The methods of immunohistochemistry and real - time fluorescence quantitative PCR were used to detect the expression of Wnt4, β - catenin and TGF - β1 in renal interstitial. Results ;24 - hour urinary protein, serum creatine and blood urea nitrogen were significantly higher than those of the normal control groups(P <0. 05). At 12th weeks, in DN group, the expression of Wnt4、β- -catenin and TGF - β1 was relatively increased(P < 0. 05 ) . In three treatment group, the expression of Wnt4 、β- catenin and TGF - β, were reduced( P < 0. 05 ) . AL group was lower than the other two, while DA group and DL group have no significantly difference. The tendency of Wnt4 、β - catenin and TGF - β1 mRNA expression is tha same with protein expression. Conclusion: AM can decrease 24 - hour urinary protein, serum creatine and blood urea nitrogen, alleviating the degree of renal interstitial fibrosis, reducing the expression of Wnt4、β - catenin and TGF - β1 played a role in renal protection.