抗糖尿病肾病活性灵芝杂萜的定向获取及其调控TGF-β/Smad信号通路机制研究

Up till now, diabetic nephropathy (DN) is still the leading cause of end-stage renal dysfunction.Kidney transplantation is considered as the only or the most effective way to save patients having renal failure. However, the rapid increase of patiens sufferring from renal dysfunction due to the lack of effective drugs against DN makes kidney transplantation a big problem in clinic practice,because the source of kidney is much limited. Altough drug R & D in this aera is hot, actually, little progress has been made in the recent years. New idea is still much required for DN. We hypothesize that DN patient should be deficient due to DN is a long-term disorder which need to be treated with tonic medicine. In our previous study, we have identified a series of structurlly novel meroterpenoids from a famous and typical tonic herbal medicine "Ling-Zhi". These structures were found to inhibit over secretion of proinflammatory cytokines,extracellular matrix and reactive oxygen species in high-glucose induced mesangial cells which are implicated in the progress of DN. These meroterpenoids could also activate Nrf2 and specificly inhibit phosphorylation of Smad3 showing in vivo efficacy in rodent experiments, implying their potential for DN. Previous study also indicated that meroterpenoidal analogues are rich in two Ganoderma species (G.applanatum and G. cochlear). However, Whether the other analogues are also biologically active, their potential, primary mechanism need to be further clarified. In this project, we plan to utilize "personalized" partition technique according to the property of meroterpenoids, TLC chemical screening tracking strategies to purposely obtain a series of meroterpenoid derivatives. And we will test their activity against DN by using multiple indicators, will undertake their primary mechanism of action study focusing on TGF-beta/Smad signaling pathway. We hope to provide valuable data for future drug development against DN.

糖尿病肾病(DN)是导致肾衰竭的主要原因，由于临床缺乏有效的抗DN药物，致使透析患者显著增加，肾移植与肾源供应矛盾突出。抗DN药物研究虽是全球热点但多年来进展缓慢。我们认为"久病入络（微血管）为虚"，因此从扶正固本中药中可能寻找到防治DN的有效分子。前期，我们从临床有效的补药赤芝（仙草）中发现结构新颖的杂萜具有显著抑制肾系膜细胞分泌促炎性因子和细胞外基质作用，也可激活Nrf2和特异性抑制Smad3 磷酸化，动物体内实验有效，提示该类成分的抗DN前景。前期，我们根据UV/DAD等特征也发现该类杂萜在树舌灵芝和反柄紫芝中丰富存在。但其结构、抗DN活性及作用机理等尚不清楚。本项目拟采用薄层化学筛选等方法定向获取二种灵芝中的杂萜，并围绕DN发病机制，从抗氧化应激、抑制细胞外基质和促炎性细胞因子分泌等角度研究其抗DN活性，从TGF-beta/Smad通路探讨其机制，期望为抗DN药物研发提供重要基础。

糖尿病肾病作为糖尿病主要并发症之一，其结局是通过肾脏纤维化导致肾衰竭，因此干预糖尿病肾病，阻断肾脏纤维化意义明显。本项目在前期灵芝杂萜研究基础上立项，旨在以灵芝杂萜结构发现—活性测试—功效验证—机制探索为主线进行研究。项目历经四年努力，开发了以HPLC-UV/DAD-MS分析为代表的灵芝杂萜定向研究策略，以至于高效分离鉴定杂萜530余个，其中271个成分源自树舌灵芝与反柄灵芝。鉴定成分中新化合物达到380余个。围绕与DN相关的体内外模型，共发现约40个活性成分。完成了2个化合物的全合成，并对其中1个化合物进行了简单结构修饰。对天然或全合成富集的3个灵芝杂萜主要围绕TGF-β/Smads通路进行了作用机制研究和动物药效验证，并发现lingzhifuran A还可通过抑制Wnt/β-catenin/RAS 信号通路，在单侧输尿管结扎、单侧肾缺血再灌注及5/6肾切模型上均表现出确切的抗肾纤维化效果。采用分子对接、分子探针、点突变等方法已发现其靶蛋白（axin）及新的结合位点。此外基于化合物的杂泛性特点，并结合灵芝传统功效，还在肿瘤、多种激酶（JAK3、DDR1、HDAC1）、神经、结核等多个模型上发现活性化合物约20个，其中抗胰岛素抵抗、抗肺动脉高压和抗结核成为了项目新的学科增长点。项目迄今共发表论文30篇，其中SCI 24篇（通讯作者24篇，Org Lett（Nature Index收录）论文5篇）；申请中国发明专利并获得授权7项。本项目取得成果的意义在于：（1）在灵芝多糖和三萜研究惯性背景下，开辟了灵芝杂萜研究，该研究丰富了灵芝杂萜的结构多样性和复杂性，将人们对灵芝杂萜的认识推向了一个新的阶段；（2）灵芝杂萜的结构多样性有利于对其构效关系的定性认识；（3）结构多样性利于基于杂泛性的客观特点进行生物功能的开拓（本项目已经在胰岛素抵抗等方面进行了尝试）；（4）活性结构的有趣性必将带动相关学科如合成化学、化学生物学及合成生物学等的发展；（5）本项目研究成果的丰富性体现了研究策略的高效性，将对相关行业可能产生示范作用，灵芝药效物质的深入研究也将对仙草灵芝产业做出贡献。