加味防己黄芪汤通过抑制上皮间质转化过程中TGF-β1/Smad/Snail信号通路减轻肾间质纤维化

Modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis by inhibiting TGF-β1/Smad/Snail signaling pathway during epithelial-mesenchymal transition

研究加味防己黄芪汤在小鼠单侧输尿管结扎(UUO)模型中,以及转化生长因子β1(TGF-β1)诱导大鼠肾小管导管上皮细胞(NRK-52E)纤维化模型中对上皮间质转化(EMT)相关蛋白表达的影响,探讨加味防己黄芪汤减轻肾间质纤维化的分子机制。对C57/BL小鼠予UUO手术,0.5、2倍加味防己黄芪汤以及蒙诺阳性对照药给药7 d后取材,Masson染色观察小鼠肾间质组织胶原沉积情况,Western blot、RT-qPCR检测肾组织中TGF-β1、磷酸化Smad2/3(p-Smad2/3)、Smad2/3、Snail、E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(vimentin)表达水平。利用SD大鼠制备不同浓度加味防己黄芪汤含药血清,CCK-8检测不同浓度含药血清对NRK-52E增殖活力的影响。TGF-β1诱导NRK-52E纤维化模型,显微镜观察不同浓度含药血清处理后细胞形态。TGF-β1诱导NRK-52E纤维化模型,1倍加味防己黄芪汤含药血清处理,Western blot、RTq PCR检测p-Smad2/3、Smad2/3、Snail、E-cadherin、α-SMA、vimentin表达水平,以及同等模型下sh RNA沉默Snail基因后,1倍加味防己黄芪汤含药血清处理,测量指标同上。结果显示,加味防己黄芪汤能改善UUO模型小鼠纤维化损伤,减轻TGF-β1诱导NRK-52E纤维化模型中的纤维化,使EMT相关指标p-Smad2/3、α-SMA、Snail、vimentin蛋白及m RNA表达降低,E-cadherin蛋白及m RNA表达升高;当sh RNA沉默Snail基因后,加味防己黄芪汤含药血清处理前后EMT相关指标E-cadherin、α-SMA及vimentin蛋白表达及m RNA水平差异没有统计学意义。结果表明,加味防己黄芪汤可能通过抑制TGF-β1/Smad/Snail信号通路减轻肾间质纤维化,参与EMT进程。

This study investigated the effects of modified Fangji Huangqi Decoction on the expression of proteins related to epithelial-mesenchymal transition(EMT)in a mouse model of unilateral ureteral obstruction(UUO)and in a rat renal tubular epithelial cell(NRK-52E)model of fibrosis induced by transforming growth factorβ1(TGF-β1).It aims to decipher the molecular mechanism by which modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis.C57/BL mice were subjected to UUO.After the surgery,the mice were treated with 0.5-fold and 2-fold concentrations of modified Fangji Huangqi Decoction and fosinopril sodium(positive control)for 7 days.The interstitial collagen deposition in the kidney was assessed by Masson staining.Western blot and RT-qPCR were employed to determine the expression levels of TGF-β1,phosphorylated Smad2/3(p-Smad2/3),Smad2/3,Snail,epithelial cadherin(E-cadherin),alpha smooth muscle actin(α-SMA),and vimentin.The NRK-52E cell model induced by TGF-β1 was treated with the serum samples collected from SD rats treated with different concentrations of modified Fangji Huangqi Decoction.The CCK-8 assay was employed to examine the effects of the serum samples on NRK-52E cell proliferation.The cell morphology in different groups was observed under a microscope.Furthermore,the modeled cells were treated with the serum containing 1-fold decoction.Western blot and RT-qPCR were then employed to measure the expression levels of p-Smad2/3,Smad2/3,Snail,E-cadherin,α-SMA,and vimentin in the cells.Under the same conditions,shRNA was used to silence the Snail gene,and measurements were repeated before and after treatment with the serum containing 1-fold decoction.The results indicated that modified Fangji Huangqi Decoction alleviated the fibrotic injury in the mouse model of UUO and the fibrosis in the NRK-52E cell model.The treatment with the decoction down-regulated the protein and mRNA levels of EMT-related indicators including p-Smad2/3,α-SMA,Snail,and vimentin,while it up-regulated the expression of E-cadherin.After shRNA silencing of the Snail gene,the protein and mRNA levels of E-cadherin,α-SMA,and vimentin showed no significant differences before and after treatment with the serum containing the decoction.The results suggest that modified Fangji Huangqi Decoction may alleviate renal interstitial fibrosis by inhibiting the TGF-β1/Smad/Snail signaling pathway and regulating the EMT process.