基于TGF-β/Smads和MAPK通路调控EMT探讨黄蜀葵花总黄酮抑制和/或逆转克罗恩病肠道纤维化

Intestinal fibrosis is a common and serious complication of the Crohn’s disease. Currently, no specific medical therapy exists to treat fibrotic intestinal strictures. It has been proved that epithelial-mesenchymal transition (EMT) affair is one of the critical steps during the development of intestinal fibrosis. TGF-β, activated Smads transcription factors and MAP kinase signaling pathways may be involved in the process of EMT and improved the forming of intestinal fibrosis. The experiments performed by our group have confirmed that total flavone of Abelmoschus manihot (L.) Medicus (TFA) can inhibit intestinal fibrosis in the TNBS-induced model of CD. The mechanism of this is related to activated MAPK/NF-κB signaling pathway, relieving intestinal inflammation by inhibitation TNF-α、INF-γ, and improving intestinal fibrosis by decreasing TGF-β、α-SMA和MMP-2. The aim of this study is to elaborate the molecular mechanisms of EMT in forming intestinal fibrosis in vivo and in vitro, with special signaling pathways related to EMT, such as TGF-β、Smads and MAPK. On this basis, we will explore the effect and molecular mechanisms of TFA on EMA that inhibit or reverse intestinal fibrosis in Crohn’s disease. This study will be not only an endeavor to look for new target and drug for the treatment of intestinal fibrosis of crohn’s disease, but also a new exploration on the effective component of Chinese medicine.

肠道纤维化是克罗恩病（CD）常见的严重并发症，但目前没有能够特异性抑制肠道纤维化的药物。研究证实TGF-β及其下游Smads和MAPK信号通路可能通过介导上皮间质转化（EMT）参与并促进了肠道的纤维化形成。课题组前期研究证实黄蜀葵花的主要成分-黄蜀葵花总黄酮（TFA）通过干预MAPK/NF-κB信号通路，抑制促炎因子TNF-α、INF-γ的过度释放来减轻CD肠道炎症，降低模型动物结肠组织中TGF-β、α-SMA和MMP-2等纤维化标志，减轻CD模型小鼠肠道纤维组织增生。本研究拟通过在体模型、细胞水平研究调控TGF-β及其下游Smads和MAPK信号通路介导EMT进程的机制，阐明EMT在CD肠纤维化过程中的作用及调控机制；并通过分析EMT进程中关键信号通路与分子变化，明确TFA抑制/逆转肠纤维化的作用和可能机制，进一步深化研究黄蜀葵花效用机制，为开发治疗CD肠纤维化的新型中药提供依据。

尽管克罗恩病（Crohn’s disease, CD）在亚洲地区发病率低于西方国家，但值得关注的是，中国CD发病率随着城市化迅速发展而大幅上升。基于我国庞大的人口基数，CD发病率持续上升给个人和国家卫生保健系统造成沉重的经济和社会负担。. 寻找和发现新的干预靶点和治疗药物依然是CD治疗面临的重大挑战。依据中医“异病同治”之理论，基于“内疡与外疡同理，内疡与外疡同治”的论述，项目组临床应用黄蜀葵花治疗炎症性肠病取得较好疗效。本项目研究证实①黄蜀葵花总黄酮（TFA）通过抑制Smad和MAPK信号通路阻止TGF-β1诱导的小肠上皮细胞向间质转化，上皮标记物E-钙黏蛋白上调，间质标记物N-钙黏蛋白和波形蛋白明显下调；②TFA显著降低了TNBS诱导的CD模型动物血清和肠壁组织内过表达的T辅助细胞（Th1和Th17）相关的因子INF-γ、IL-6、IL-17，增加了T调节细胞（Treg）的因子IL-10、TGF-β的表达；③TFA显著降低模型动物肠壁组织内胶原蛋白（Col1a2、Col3a2），上调金属蛋白酶组织抑制剂（TIMP-1、TMP）表达并抑制基质蛋白酶（MMP2和MMP9）的表达。说明TAF能够通过恢复CD模型动物TH17/Treg细胞平衡并抑制肠道纤维化而减轻肠道炎症反应和损伤。④Central carbon metabolism in cancer通路可能参与了黄蜀葵花抑制TNBS小鼠CD模型肠道炎症与纤维化过程。. 到目前为止，凝缩蛋白I复合体非SMC亚基D2（Non-SMC condensin I complex subunit D2, NCAPD2）在肠道相关疾病发生、发展进程中的作用与分子机制研究尚未见报道。项目组基于临床样本发现炎症性肠病患者（UC和CD）肠道粘膜组织中NCAPD2表达水平显著升高，提示NCAPD2在炎症性肠病发生发展过程中可能扮演重要角色。通过体外研究显示，脂多糖刺激结肠NCM460上皮细胞炎症反应后，NCAPD2表达异常升高，伴随炎症因子IL-1β，IL-6和TNF-α升高；siRNA敲减NCM460细胞NCAPD2蛋白表达，LPS刺激诱导后IL-1β，IL-6和TNF-α表达降低，同时伴随IKK/NF-κB信号通路抑制。据此，项目组首次提出NCAPD2激活IKK/NF-κB通路，促进炎症因子IL-1β，