基于调控Wnt/β-catenin/snail1信号通路阻抑EMT研究生地水提物抗肾纤维化机制

Renal fibrosis is the final common pathway of all kinds of chronic kidney disease progressing to end-stage renal failure. Epithelial-to-mesenchymal transition (EMT) is a crucial step in the pathogenesis of renal fibrosis. The key transcription factor responsible for EMT, Snail1, is one of target genes of Wnt/β-catenin signaling. Our previous studies showed that collagen deposition and interstitial fibrosis were decreased in unilateral ureteral occlusion (UUO) rats administrated by the aqueous extract of Radix Rehmanniae, which was accompanied with the decreasing expression of Wnt5a, β-catenin and α-SMA and increasing expression E-cadherin. Thus, it is supposed that the aqueous extract of Radix Rehmanniae may ameliorate renal fibrosis through inhibiting the Wnt/β-catenin/snail1 signal pathway, which may be associated with the prevention of EMT. For that reason, we plan to choose HK-2 cells and UUO rats as the objects of this study for discussing the important role of the Wnt/β-catenin/snail1 signal pathway in the EMT process of the renal fibrosis, and analysis the effect of the aqueous extract of Radix Rehmanniae on Wnt/β-catenin/snail1 signal pathway, which can help us to make clear if EMT inhibited by Wnt/β-catenin/snail1 signal pathway is the key mechanism on ameliorating renal fibrosis induced by the aqueous extract of Radix Rehmanniae. The purpose of this study is benefit to provide experimental evidence for explaining the theory of ameliorating renal fibrosis under Traditional Chinese Medicine mechanism and exploit new drug to control the development of renal fibrosis.

肾纤维化是慢性肾脏病发展到终末期肾衰竭的共同通路，上皮-间质转分化(EMT)是肾纤维化发生的中心环节之一，EMT的关键转录因子Snail1是Wnt通路的下游靶基因。课题组研究发现，生地水提物能减少UUO大鼠肾脏胶原沉积，缓解肾纤维化，并下调Wnt5a、β-catenin、α-SMA表达，上调E-cadherin表达。推测生地水提物可能通过Wnt/β-catenin信号通路缓解肾纤维化。本项目以HK-2细胞和UUO模型为研究对象，探讨Wnt/β-catenin/snail1信号通路在生地水提物抑制EMT，缓解肾纤维化中的重要作用，分析生地水提物对Wnt/β-catenin/snail1信号通路关键蛋白的调控作用，阐明生地水提物抑制Wnt/β-catenin/snail1信号通路，阻抑EMT，缓解肾纤维化的机制。本项目为阐释中医药理论防治肾纤维化提供实验依据，对发掘防治肾纤维化药物有重要意义。

慢性肾脏病(CKD)是全球主要医疗负担之一。肾纤维化是各种病因引起的CKD发展到终末期肾衰竭的共同通路。目前，西医治疗的疗效不甚理想，而中医药或中西医结合治疗有望提高疗效，改善预后。课题组基于前期初步研究基础，提出假说：生地水提液通过抑制Wnt/β-catenin通路活化，下调靶基因Snail1表达，阻抑EMT进程，从而缓解肾纤维化。.在该项目支持下，课题组制备UUO模型研究生地水提液对肾纤维化和Wnt/β-catenin信号通路活化、Snail1表达、EMT的影响，明确了生地水提液缓解肾纤维化与抑制Wnt/β-catenin信号通路、下调Snail1表达有关；通过TGFβ1刺激HK-2细胞构建的体外转分化（EMT）模型，明确了生地水提液阻抑EMT，其机制与抑制Wnt/β-catenin信号通路活化，下调Snail1表达有关，同时运用Wnt/β-catenin信号通路抑制剂DKK1、激动剂LiCl和Snail1 siRNA，进一步证实生地水提液抑制EMT与通过Wnt/β-catenin信号通路，下调Snail1表达有关。.为深入探讨生地水提液缓解肾纤维化的物质基础，课题组还通过体内外实验研究梓醇抑制EMT，缓解肾纤维化的作用机制。因此，本研究一方面明确了生地通过抑制Wnt/β-catenin通路活化，下调Snail1表达，阻抑EMT进程，从而缓解肾纤维化的作用机制，完成了项目任务书研究内容；另一方面，还探明了梓醇是生地通过Wnt/β-catenin通路阻抑EMT进程，从而缓解肾纤维化的物质基础。.在此基础上，课题组开展了以生地为重要组成的国医大师临床经验方-参芪延肾方治疗CKD的作用机制、物质基础、制剂研发等相关研究，围绕真实世界探讨临床有效方药、单味药、单体的组学基础及现代生物学内涵，积极开发医院制剂。先后获批重庆英才﹒创新创业团队（CQYC201903172）、重庆市留学人员回国创业创新支持计划（cx2019060）以及重庆市科技局绩效激励引导专项（csts2019jxjl130024）等支持，建成了集临床研究、基础研究、制剂研发为一体的转化研究团队，基于临床大数据提炼中医药防治慢性肾病的理法方药，运用现代药理学研究方法阐释中医药理论的生物学内涵，探索其药效学物质基础，开展医院制剂研发和新药开发，为中医药传承发展探索新路径。