小檗碱经由抑炎性胃肠肽抗类风湿关节炎的机制研究

Berberine, orally administered, has marked anti-rheumatoid arthritis effect. However, its plasma/tissue concentrations are far below the minimal effective concentrations of in vitro cell experiments, and the in vivo mechanisms of action remain to be clarified. In accordance with our preliminary studies, this project confirms the intestine-dependent characteristics of the anti-arthritic effect of berberine based on its pharmacokinetic behaviors after multiple oral dosings and results obtained from parenteral administration experiment. The effects of berberine on the expressions of anti-inflammatory gastrointestinal peptides at different sites, such as intestinal dust and synovium, are comparatively studied in an attempt to identify that cortistatin or/and somatostatin are the key players for the anti-arthritic action of berberine. Furthermore, we will investigate the effects of berberine on diversity and richness of the gut microbiota as well as production of short-chain fatty acids (SCFAs) in arthritis rats, and gain insight into the molecular mechanisms for SCFAs enhancing the expressions of cortistatin or/and somatostatin. Finally, specific antagonists and RNA interference technique are used to verify the hypothesis that berberine functions by “regulation of gut microbiota-promotion of SCFAs production-induction of cortistatin or/and somatostatin expression-restore of Th17/Treg balance-amelioration of arthritis”. The findings will provide evidence for the development and utilization of berberine, offer a paradigm for the mechanistic studies of anti-arthritic compounds with similar pharmacokinetic characteristics to berberine, and enhance our understandings of the relationship between intestinal bacterial flora and rheumatoid arthritis.

小檗碱口服给药具有显著的抗类风湿关节炎作用，但血浆和组织浓度远低于体外细胞试验的最低有效浓度，其体内效应机制尚不清楚。本项目基于前期研究，考察小檗碱在胶原关节炎大鼠多次口服给药后的药动学行为，并结合肠道外给药试验证实其抗关节炎作用的肠道依赖性；筛选研究小檗碱对肠道和滑膜等部位抑炎性胃肠肽表达的影响，明确皮质抑素或/和生长抑素是其抗关节炎效应的关键中介；考察小檗碱对关节炎大鼠肠道菌群多样性和丰度的调控及其对短链脂肪酸产生的促进作用，阐明短链脂肪酸诱导上述抑炎性胃肠肽表达的分子机制；采用受体拮抗剂和基因沉默等手段，验证小檗碱“通过调节肠道菌群、促进短链脂肪酸产生，诱导皮质抑素或/和生长抑素表达，恢复Th17/Treg平衡，从而改善关节炎”的科学假说。预期成果为小檗碱的开发利用提供理论依据，亦为具有类似药动学特点化合物的抗类风关机制的研究提供借鉴，并加深对肠道微生态与类风关发生发展相关性的认识。

文献报道，中药活性成分小檗碱口服给药可显著改善大鼠胶原诱导的关节炎（CIA），但小檗碱难以吸收，血浆和组织暴露量极低，呈现“药动学-药效学不相关”现象，其抗关节炎作用机制有待阐明。本课题在CIA模型动物，从调节肠道菌群结构和促进抑炎性胃肠肽表达的角度，探究小檗碱改善类风湿关节炎的机制。. 首先验证小檗碱口服给药对大鼠CIA的抑制作用，比较其在正常和关节炎模型大鼠的药动学特征，结合肠道外给药试验和肠道菌群失调对小檗碱抗关节炎作用的影响，探索其抗关节炎作用的肠道依赖性。其次，通过16s rRNA测序，进一步了解小檗碱对CIA大鼠肠道菌群及其生存环境的影响。然后考察小檗碱对大鼠肠道短链脂肪酸（SCFAs）产生的影响，明确效应SCFAs的类型，并从调节抑炎性胃肠肽分泌的角度，探讨小檗碱调节肠道菌群呈现抗关节炎效应的机制。最后考察肠道菌群和正丁酸在小檗碱促进皮质抑素表达作用中的参与，验证小檗碱缓解大鼠CIA作用的“肠道菌群—正丁酸—皮质抑素”途径，并通过联合使用SSTRs或GHSR1拮抗剂判定皮质抑素经由何种受体亚型介导小檗碱对 CIA 的缓解作用。. 研究证实：小檗碱对CIA大鼠的Th17细胞反应、关节炎症状的抑制作用呈现肠道依赖性，其机制可概括为：上调肠道菌群BUT的表达和活力→促进肠道菌群产生正丁酸→增强抑炎性胃肠肽皮质抑素表达→抑制Th17细胞反应→缓解CIA。上述研究结果表明，调节肠道菌群是防治类风湿关节炎的有效途径之一，肠道皮质抑素促释剂有望用于治疗包括类风关在内的Th17细胞相关性疾病。