清化颗粒启动胃肠胰岛轴治疗糖尿病的内在机制及其主要成份研究

Taste receptors exist wide in human body (tongue, pancreas, brain, gut) and show endocrine functions in addition to taste chemical signal. They are involved in glucagon-like peptide-1 (GLP-1) secreting in intestinal L cells, which become a new target for anti-diabetic drugs regarding ameliorating beta cell function. Our preliminary studies confirmed that Qing-Hua Granule (QHG) could up-regulate GLP-1 expression in L cell, improve insulin secretion and regulate hyperglycaemia via type 2 taste receptors (TAS2Rs) and its signaling pathway (α-gustducin, PLCβ2, TRPM5). Accompanying TAS2R, type 1 taste receptors (TAS1R) also exist wide in human body. They can also induce GlP-1 secretion in intestinal L cell and share same signaling pathway with TAS2R. Maybe, QHG could be kind of agonist for TAS1R, activate same signaling pathway (α-gustducin, PLCβ2, TRPM5). This study is to testify this hypothesis. Furthermore, 17 compounds have been accessed in QHG by using UPLC-MS technology. In this study, we also try to find the active compounds as agonist for TAS1Rs in vivo and in vitro models. We will analyze TAS2Rs (TAS1R1, TAS1R2, TAS1R3), α-gustducin, Gβ, PLCβ2, TRPM5, IP3, GLP-1, PDX-1, cAMP and Ca2+, in db/db mice and NCI-H716 cells. Our study is to explore QHG main active compounds and its molecular mechanism of augmenting GLP-1 secretion via TAS1Rs signaling pathway. Its results will support proofs for optimizing QHG.

味觉受体(TAS1Rs和TAS2Rs)存在舌、消化道、胰腺和脑，既形成味觉，又具内分泌效应。它可调控胰高血糖素样肽-1(GLP-1)，后者作为胃肠胰岛轴的代表是糖尿病药物研发新靶点。前期研究(国自然30801472，81403359)发现：验方清化颗粒促进肠L细胞分泌GLP-1 ，改善胰岛β细胞功能，调节糖代谢；通过调控TAS2Rs及下游通路，上调回肠L细胞GLP-1表达 ，影响回肠形态，促进GLP-1分泌。TAS1Rs和TAS2Rs分享相同下游信号通路，促进肠L细胞分泌 GLP-1。清化颗粒是否也调控TAS1Rs启动胃肠胰岛轴？主要成分是哪些？这是本课题拟解决的问题。本研究以清化颗粒及其成分(UPLC-MS分析)为研究对象，采用体内动物和体外细胞两种模型，观察TAS1Rs及下游信号分子、GLP-1、Ca2+的变化,探索主要成分，揭示作用机制，为优化复方提供依据。

味觉受体(TAS1Rs/TAS2Rs)的内分泌效应之一是调控胰高血糖素样肽-1(GLP-1)，后者也是糖尿病治疗主要靶点。在前期研究(国自然30801472/81403359)发现验方清化颗粒可通过TAS2Rs促进肠L细胞分泌GLP-1的基础上，通过本项目，课题组发现验方也可以通过调控TAS1R1和TAS1R2及其下游信号因子，促进肠L细胞合成、分泌GLP-1，影响转运因子和肠道环境，改善糖尿病小鼠糖代谢和体重。通过以NCI-H716内分泌细胞株为L细胞模型载体，经多次筛选，发现黄芩素和表小檗碱可能是复方调控GLP-1表达的主要成分。黄芩素和表小檗碱的高剂量干预在糖尿病小鼠上重复了GLP-1的表达增强和糖代谢的改善。然而，黄芩素和表小檗碱分别影响TAS1R1/TAS1R2及其下游信号因子表达的实验结果并不一致，结合近年研究报道，提示上述成分仍有可能通过其它机制调控GLP-1表达，需要进一步探索。本项目拓展了验方的作用机制，为优化复方提供了重要参考依据，也丰富了中医四气五味理论的科学内涵。