基于肠L细胞CaSR-TAS2R受体探讨苦药-苦参刺激GLP-1分泌的作用机制

The five flavors are dialectical cognition of Chinese Medicine's taste perception “feeling” and efficacy characteristics “medicinal taste”. The bitterness of bitter medicine can treat Xiao-Ke and modern clinical use for the treatment of diabetes Taste cells identify bitter "taste" by bitter taste receptors; they also expressed in the intestinal L cells and the calcium-sensing receptors on the intestinal L cells both can activate intracellular inositol trisphosphate signaling pathway, then Increase secretion of insulinotropic hormone GLP-1, promote insulin secretion, and regulate blood glucose. We found that the bitter herbs-Sophora flavescens can reduce the blood glucose of diabetic rats, and the active component of the Matrine can activate the inositol trisphosphate signaling pathway to improve the level of GLP-1. However, it is not clear whether Sophora flavescens stimulates the intestinal bitter taste receptor through its “feeling” or stimulates calcium-sensitive receptors through its “medicinal taste” to its efficacy. This study intends to use self-induced and inducible diabetic model rats, through immunofluorescence and immunoblotting to study the regulation effect of Sophora flavescens on intestinal bitter taste receptor and calcium sensitive receptor; by using siRNA and fluorescence labeling technique in the intestinal L cells, defining the relationship between the molecular sites of GLP-1 secretion and its "bitterness" medicinal properties of Sophora flavescens. The purpose of this study is to reveal the intrinsic relationship between taste feeling and medicinal taste, and also to provide new ideas for the study of the mechanism of the effect of traditional Chinese Medicine.

五味是中药集味觉感受“滋味”与功效特性“药味”于一身的辩证认知。苦药之“苦”可倾泻消渴的肥甘之壅郁，现代临床用于糖尿病治疗。味觉细胞通过苦味受体辨识苦的“滋味”，而苦味受体在肠L细胞亦有表达，并与肠L细胞上钙敏感受体共同通过激活胞内三磷酸肌醇信号途径，提高促胰岛素分泌激素GLP-1的分泌，促进胰岛素分泌，调节血糖。我们发现苦味药-苦参可降低糖尿病大鼠血糖，其活性成分苦参碱可激活三磷酸肌醇信号途径，提高GLP-1水平。但苦参是通过其“滋味”刺激肠苦味受体，还是通过“药味”刺激钙敏感受体发挥其“苦味”药效尚不清楚。本课题拟采用自发型及诱导型糖尿病模型鼠，通过免疫荧光及免疫印迹手段，研究苦参对肠苦味受体及钙敏感受体的调控作用；采用肠L细胞模型，借助siRNA及荧光标记技术，明确苦参促进GLP-1分泌的分子位点与其“苦味”药性的关系，揭示滋味与药味的内在关联，并为中药药味的作用机制研究提供新思路。

二型糖尿病临床高发，糖尿病归于中医消渴范畴，患者多饮食不节，长期过食肥甘脾胃运化失司。苦药之“苦”可倾泻消渴的肥甘之壅郁，现代临床用于糖尿病治疗。味觉细胞通过苦味受体辨识苦的“滋味”，而苦味受体在肠L细胞亦有表达，并与肠L细胞上钙敏感受体共同通过激活胞内三磷酸肌醇信号途径，提高促胰岛素分泌激素GLP-1的分泌，促进胰岛素分泌，降低血糖。本研究旨在通过研究苦参是通过其“滋味”刺激肠苦味受体，还是通过“药味”刺激钙敏感受体发挥其“苦味”药效作用，明确苦参碱促进GLP-1分泌，降低血糖的作用机制。通过研究我们发现苦味药-苦参可降低糖尿病大鼠血糖，其活性成分苦参碱可激活三磷酸肌醇信号途径，提高GLP-1水平。其中通过整体动物实验，细胞实验以及分子对接研究发现，苦参中的苦参碱可明显改善高脂饮食联合链脲左菌素诱导的二型糖尿病模型小鼠的葡萄糖耐受及全身胰岛素抵抗作用，并且具有明显的肠促胰岛素的作用。苦参碱可通过影响小鼠肠分泌细胞（STC-1）PLCβ2的表达，促进胞内Ca2+水平的升高，从而促进GLP-1的分泌。通过计算机辅助分子对接实验发现，苦参碱与钙离子敏感受体（CaSR）具有明显的分子间作用，苦参碱能够有效的嵌入CaSR结合口袋，形成稳定的蛋白—配体复合物。通过采用siRNA敲除STC-1细胞中苦味受体mTas2r108、mTas2r137和mTas2r138后，发现苦参碱并不能影响GLP-1的调节作用。通过以上实验研究初步阐明，苦味药-苦参促进GLP-1分泌的分子位点与其“苦味”药性的关系，揭示了滋味与药味的内在关联，并为中药药味的作用机制研究提供新思路。同时我们研究发现脑-肠循环在代谢综合症的发生中扮演重要作用，而糖尿病引起的认知障碍在临床上严重危害着人们的健康及生活质量，GLP-1在肠道中分泌发挥着降低血糖的作用，其与糖尿病脑病的关系有待进一步研究，也为我们下一步研究提供了新的方向。