抑制a7nAchR相关炎症信号：小檗碱改善2型糖尿病胰岛素抵抗的分子机制？

Based on the facts in our previously study, that Berberin as the inhibiting agent of activity acetylcholinesterase（AchE）regulated not only the expression of inflammatory factors but also ameliorated insulin-resistance (IR) in T2DM, our research group will deeply explore the mechanism of Berberin in the improvement of IR.According to the theory of chronic inflammation of T2DM and the crucial role of "the cholinergic anti-inflammatory pathway" in the mediating IR of T2DM, we pay attention to the "cross-talk" sites between the insulin signal pathway and the cholinergic anti-inflammatory pathway, specially focusing on the study of up-stream molecular events of "the cholinergic anti-inflammatory pathway". Taking the experimental techniques at deferent level of the animal model or cellular and molecular model, in vivo and in vitro, and the integration method of functional and metabolic analysis, we try to certify the hypothesis that "Berberin improves IR in T2DM via regulating the level of Ach through inhibition of activity of AchE, exciting the α7nAChR in insulin-sensitive tissues/cells, and inhibiting the activation of NFκB, finally reducing the effect of inflammatory factors, such as TNFα.IL6... on interference against the insulin signal pathway".Our study is able to find the new target of Berberin as a therapeutic drug of T2DM, furthermore to enrich the theory about multi-way on the regulation of glucose metabolism, also provide the theoretical foundation and a new strategy for the further clinical application/ development of Berberin and its analogues.

课题组前期研究发现作为乙酰胆碱酯酶（AchE）抑制剂的黄连有效成分- - 小檗碱，除了调节胆碱能神经递质释放外，还具有改善2型糖尿病（T2MD）胰岛素抵抗（IR）的作用，因此，本项目拟进一步以T2MD"慢性炎症学说"为基础，以新近提出的"胆碱能抗炎通路"为核心，深入探讨该通路及胰岛素信号通路与T2MD炎症学说之间的联系。本申请首次提出"小檗碱可能通过调节Ach水平、兴奋胰岛素敏感靶组织/细胞中α7 nAChR、抑制NFκB 激活、减少TNFα等炎症因子对胰岛素信号传递的干扰，改善IR的工作假说"，拟在整体与离体实验、以机能和代谢检测相结合的方法对该假说进行系统验证，重点放在胆碱能抗炎通路上游分子事件的调查与确认。该课题的完成，将找到小檗碱治疗T2MD的新靶点从而丰富体内参与血糖调控的多元化途径，深化T2MD中IR的机制和内涵；并为研发以小檗碱为代表的治疗T2MD的小檗碱类新药奠定理论基础。

本研究以链脲佐菌素(STZ)尾静脉注射+高脂高糖诱导建立胰岛素抵抗的大鼠模型，以高糖高胰岛素诱导HepG2、3T3-L1建立两种胰岛素抵抗的细胞模型，采用体外分子模拟、免疫荧光、ELISA、Western blot、SiRNA干扰等现代分子生物学技术，系统探讨了小檗碱基于胆碱能抗炎通路抑制炎症因子改善胰岛素抵抗的分子机制。动物实验表明：小檗碱能显著抑制血清胆碱酯酶活性、激活肝脏、肌肉组织中烟碱样型乙酰胆碱受体-α7nAChR从而抑制炎症通路中关键蛋白NF-κB p65，下调IL-1β、IL-6、TNF-α等炎症因子并通过上调肝脏、肌肉、脂肪组织中胰岛素信号转导相关蛋白PI-3K p85、GLUT2、GLUT4、下调pIRS-1 ser307/ IRS-1比值而增加葡萄糖的摄取改善胰岛素抵抗；离体研究表明：小檗碱能显著改善高胰岛素诱导的HepG2、3T3-L1细胞胰岛素抵抗增加细胞对葡萄糖的摄取，小檗碱能显著抑制细胞上清中胆碱酯酶活性，上调α7nAChR，对NF-κB p65，P-IKKβser181及IL-1β、IL-6、TNF-α等炎症介质及细胞因子有明显的抑制作用，通过上调PI3K、P-AKT473 、GLUT4等蛋白水平而使胰岛素信号转导通路畅通，动物和离体研究均表明当α7nAchR基因沉默后，其乙酰胆碱酯酶活性抑制作用依旧存在，但却不能起到显著降糖和改善胰岛素抵抗的作用。因此，小檗碱改善2型糖尿病胰岛素抵抗的作用可能与α7nAchR介导的胆碱能抗炎通路相关。