牛蒡子抗溃疡性结肠炎的效应成分激活ERβ保护黏膜屏障的机制研究

Defective gut barrier is deeply involved in the initiation and development of ulcerative colitis (UC), and mucosal barrier protector has been thought to be an innovative approach in the management of UC. Arctium lappa L. fruit is a traditional Chinese medicine with efficiencies of dispelling wind, expelling heat and removing toxicity, and can be used in the treatment of diseases induced by toxic heat invasion, such as UC. Arctigenin, the major effective constituent of A. lappa, belongs to phytoestrogen. It could attenuate the epithelial damage of the colon, selectively up-regulated the expressions of the target genes of estrogen receptor β (ERβ), and showed moderate anti-inflammatory and immunosuppressive activities. The purpose of this project is to clarify the underlying mechanism of arctigenin for strengthening the tight junctions and mucus barrier in view of enhancement of the expressions of junction-related proteins and mucins via activation of ERβ. In colitis mice and colonic epithelial cells, the effects of arctigenin on epithelial integrity, permeability and mucus secretion should be studied to confirm its barrier protective effect. Next, we will clarify whether arctigenin is an agonist of ERβ, and reveal the key role that ERβ plays in the arctigenin-mediated protection of the barrier using specific antagonists and RNA interference technique. Finally, we investigate the effects and signal pathways of arctigenin on the expressions of tight junction-related proteins and mucins, and elucidate the molecular mechanisms by which it activates ERβ and consequently regulates the key signal pathway. The findings will provide evidence for the development of arctigenin as a mucosal barrier protector to treat UC.

肠道屏障缺损与溃疡性结肠炎（UC）的发生发展紧密相关，黏膜屏障保护剂是治疗UC的最新手段。牛蒡子为散风除热解毒之要药，可用于治疗UC等热毒入侵所致疾患，其抗UC的效应成分牛蒡子苷元属于植物雌激素，可显著改善结肠上皮损伤，选择性上调雌激素受体ERβ的靶基因表达，并兼备一定的抗炎和免疫抑制活性。本项目从激活ERβ受体，促进结肠紧密连接相关蛋白和黏蛋白表达的角度，阐明牛蒡子苷元增强上皮紧密连接和黏液屏障的机制。主要研究内容包括：在UC模型小鼠和结肠上皮细胞，考察苷元对上皮完整性、结肠通透性及黏液分泌的影响，探明其屏障保护作用和特点；明确苷元为ERβ激动剂，并采用特异性拮抗剂和基因沉默等手段，证实ERβ是其保护屏障的关键中介；体内外研究苷元对上皮连接相关蛋白和黏蛋白表达的影响及其信号途径，揭示其活化ERβ调控关键信号通路的分子机制。研究成果为牛蒡子苷元作为黏膜屏障保护剂用于UC的治疗提供理论依据。

本课题组前期研究发现，中药牛蒡子提取物具有显著的抗结肠炎作用，其主要活性成分是牛蒡子苷元，而非牛蒡子苷。鉴于黏膜屏障保护剂在防治炎症性肠病等疾病中的重要价值，本课题探索牛蒡子苷元对上皮屏障和黏液屏障的保护作用和机制。.1）牛蒡子苷元对炎症性肠病所致结肠上皮屏障破坏的保护作用和特点：在DSS和TNBS所致结肠炎小鼠，牛蒡子苷元灌胃给药明显缓解结肠组织病理学改变，下调促炎因子表达，降低结肠上皮通透性升高。体外研究发现，牛蒡子苷元可明显上调结肠上皮细胞紧密连接蛋白ZO-1和occludin表达。在非炎性条件下，牛蒡子苷元仍能促进结肠上皮细胞单层膜结构的形成，且与细胞增殖无关，提示其对结肠上皮屏障的保护作用可能不依赖于抗炎活性。.2）牛蒡子苷元保护结肠上皮屏障的关键机制：证实了牛蒡子苷元通过ERβ而非ERα和GPER呈现上皮屏障保护作用。此外，牛蒡子苷元上调紧密连接蛋白表达的作用与MLCK信号相关，而与MAPK和mTOR途径无关。在ERβ敲减的结肠炎小鼠，牛蒡子苷元上调紧密连接蛋白表达、降低肠通透性的作用明显减弱，表明ERβ-MLCK/MLC通路在牛蒡子苷元保护上皮屏障、抑制结肠炎中起着至关重要的作用。.3）牛蒡子苷元抑制炎症性肠病所致结肠黏液屏障破坏的作用和机制：牛蒡子苷元灌胃给药可促进结肠炎小鼠结肠黏液分泌，此作用与抑制杯状细胞凋亡相关，且该作用主要通过抑制线粒体途径，而非死亡受体或内质网应激途径。牛蒡子苷元可显著增加ERβ与PHB1复合物的形成，阻止PHB1与TRIM21结合，抑制PHB1泛素化降解，进而抑制杯状细胞凋亡，这一结果在ERβ敲减的结肠炎模型小鼠得到了验证。.综上，本课题从激活ERβ的角度诠释了牛蒡子苷元保护肠黏膜屏障的作用和机制，为其用于炎症性肠病的防治提供了新的证据，同时对ERβ激动剂的开发利用具有指导意义。