Abstract：
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 factor β1(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-β1was 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, sh RNA 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 m RNA levels of EMT-related indicators including p-Smad2/3, α-SMA,Snail, and vimentin, while it up-regulated the expression of E-cadherin. After sh RNA silencing of the Snail gene, the protein and m RNA 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.
摘要：
本研究探讨加味防己黄芪汤对转化生长因子β1(TGF-β1)诱导的单侧输尿管梗阻(UUO)小鼠模型和大鼠肾小管上皮细胞(NRK-52E)纤维化模型上皮间质转化(EMT)相关蛋白表达的影响。目的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小鼠进行UUO。手术后,小鼠用0.5倍和2倍浓度的加味防己黄芪汤和福辛普利钠(阳性对照),连续7天。通过Masson染色评估肾脏中的间质胶原沉积。Westernblot和RT-qPCR检测TGF-β1、磷酸化Smad2/3(p-Smad2/3)的表达水平,Smad2/3蜗牛,上皮钙黏着蛋白（E-钙黏着蛋白），α-平滑肌肌动蛋白（α-SMA），还有波形蛋白.采用不同浓度的加味防己黄芪汤处理SD大鼠血清标本,建立TGF-β1诱导的NRK-52E细胞模型。使用CCK-8测定来检查血清样品对NRK-52E细胞增殖的影响。在显微镜下观察不同组的细胞形态。此外，用含1倍汤的血清处理模型细胞。然后采用Westernblot和RT-qPCR来测量p-Smad2/3，Smad2/3，Snail的表达水平，E-cadherin,α-SMA,细胞中的波形蛋白。在同样的条件下,shRNA被用来沉默蜗牛基因，用含1倍汤的血清治疗前后重复测量。结果表明，加味防己黄芪汤减轻了UUO小鼠模型的纤维化损伤和NRK-52E细胞模型的纤维化。汤治疗下调EMT相关指标的蛋白和mRNA水平，包括p-Smad2/3，α-SMA，蜗牛,还有波形蛋白,同时上调E-cadherin的表达。Snail基因的shRNA沉默后，E-cadherin的蛋白质和mRNA水平，α-SMA,含药血清治疗前后波形蛋白无明显差异。提示加味防己黄芪汤可能通过抑制TGF-β1/Smad/Snail信号通路,调节EMT过程减轻肾间质纤维化。
