补阳还五汤合参芪地黄汤化裁对糖尿病肾病小鼠肾小管间质纤维化的作用及机制研究

Effect and mechanism of revised Buyang Huanwu Decoction combining with Shenqi Dihuang Decoction on renal tubulointerstitial fibrosis in diabetic nephropathy mice

目的研究补阳还五汤合参芪地黄汤化裁对糖尿病肾病小鼠肾小管间质纤维化的作用及机制。方法取8只db/m小鼠作为对照组,16只db/db小鼠随机分为模型组和中药组,给予补阳还五汤合参芪地黄汤化裁干预12周,留尿,检测各组小鼠尿白蛋白/肌酐比值(albumin/creatinineratio,ACR);取肾组织,进行苏木素-伊红(HE)、高碘酸希夫(PAS)和天狼猩红(Pmicrosirius-Red)染色,检测肾组织病理损伤和纤维化程度;采用免疫组化法检测肾组织α-平滑肌肌动蛋白(α-smoothmuscle actin,α-SMA)和E-钙黏蛋白(E-cadherin)表达;采用qRT-PCR法检测肾组织Ⅲ型胶原(collagen-Ⅲ,Col-Ⅲ)和纤黏蛋白(fibronectin,FN) mRNA表达;采用Westemblotting法检测肾组织Col-Ⅲ、FN、α-SMA、E-cadherin及Wnt/β-catenin信号通路相关蛋白表达。结果与模型组比较,中药组小鼠ACR水平明显降低(P<0.05);肾脏病理损伤和纤维化减轻;α-SMA蛋白在肾间质的表达减少,E-cadherin蛋白在肾小管上皮细胞的表达增加;肾组织Col-Ⅲ和FNmRNA表达水平明显降低(P<0.05);肾组织Col-Ⅲ、FN、α-SMA、Wnt4、磷酸化糖原合酶激酶3β(phosphorylated glycogen synthase kinase 3β,p-GSK3β)、β-catenin和转化生长因子-β1 (thansforminggrowthfactor-β1,TGF-β1)蛋白表达水平显著降低(P<0.05),E-cadherin蛋白表达水平显著升高(P<0.05)。结论补阳还五汤合参芪地黄汤化裁可以减轻糖尿病肾病小鼠的肾组织病理损伤,减轻肾小管间质纤维化程度,其机制可能与抑制Wnt/β-catenin信号通路及其介导的肾小管上皮细胞向间充质转化有关。

Objective To study the effect and mechanism of revised Buyang Huanwu Decoction and Shenqi Dihuang Decoction(补阳还五汤合参芪地黄汤化裁)on renal tubulointerstitial fibrosis in diabetic nephropathy(DN)mice.Methods Eight db/m mice were used as control group,16 db/db mice were randomly divided into model group and traditional Chinese medicines(TCM)group,revised Buyang Huanwu Decoction combining with Shenqi Dihuang Decoction was given for intervention for 12 weeks,urine was retained,and albumin/creatinine ratio(ACR)of mice in each group was detected.The renal tissue was taken,degree of pathological injury and fibrosis were detected by HE,PAS and Picrosirius-Red stained method.Expressions ofα-smooth muscle actin(α-SMA)and E-cadherin in renal tissue were detected by immunohistochemistry.Expressions of collagen-Ⅲ(Col-Ⅲ)and fibronectin(FN)m RNA in renal tissue were detected by q RT-PCR.Expression of Col-Ⅲ,FN,α-SMA,E-cadherin and Wnt/β-catenin pathway related proteins were detected by Western blotting.Results Compared with model group,ACR level of mice in TCM group was significantly decreased(P<0.05),renal pathological injury and fibrosis were alleviated;Expression ofα-SMA protein in renal interstitium was decreased,expression of E-cadherin protein in renal tubular epithelial cells was increased;Expression of Col-Ⅲand FN m RNA were significantly decreased(P<0.05),expressions of Col-Ⅲ,FN,α-SMA,Wnt4,phosphorylated glycogen synthase kinase 3β(p-GSK3β),β-catenin and thansforming growth factor-β1(TGF-β1)protein were significantly decreased(P<0.05),expression of E-cadherin protein was significantly increased(P<0.05).Conclusion Revised Buyang Huanwu Decoction combining with Shenqi Dihuang Decoction can reduce the renal pathological damage and degree of renal tubulointerstitial fibrosis in DN mice.The mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway and its mediating renal tubular epithelial cell to mesenchymal transition.