基于肌球蛋白轻链激酶MLCK的“益气开秘方”对慢传输型便秘肠道平滑肌细胞生物学机制研究

Our group has been using Yiqi Kaimi Recipe (YQKM) on slow transit constipation (STC) for a decade and have achieved notable curative effect. Early study showed that the therapy on transport constipation chronic intestinal damage power recovery was closely related to cGMP-PKG pathway. Small pilot experiment suggested that the content of intestinal smooth muscle cGMP increased significantly, the expressing quantity of MLCK in the ascending colon increased apparently while downtrend in the descending colon, the intestinal dynamic enhanced significantly. We believed that YQKM affected MLCK on the control of intestinal smooth muscle contraction and relaxation, may be one of the important mechanisms of STC intestinal motility. In order to clarify the mechanism of phosphorylation of the MLCK signal pathway when using YQKM to control constipation, the project intends to study YQKM on the basis of the biological function, from both intracellular and extracellular of the intestine smooth muscle. The study will focus on the regulation of YQKM on PKG-CaMK-MLCK-MLC pathway at different time points, different parts of the intestinal tissue genes, protein expression and phosphorylation of dynamic change, which provides convincing experimental data and new theoretical interpretation for the onset of STC as well as the regulation mechanism of the recipe.

我们课题组十年来采用益气开秘方治疗慢传输型便秘，取得显著临床疗效。前期研究提示该方对慢性传输性便秘肠动力损害恢复与cGMP-PKG作用途径密切相关。小样本预初实验提示肠动力障碍小鼠灌胃给予益气开秘方24h后，肠道平滑肌cGMP含量显著提高，MLCK蛋白表达量在升结肠显著升高而在降结肠部分出现下降趋势，肠道动力显著增强。我们认为益气开秘方影响结肠不同部位MLCK激酶控制肠道平滑肌收缩舒张，可能是慢传输性便秘肠动力调控重要作用机制之一。本项目拟在系统研究益气开秘方体内生物学功能基础上，从结肠平滑肌胞内、胞外两个层面，进一步研究益气开秘方调控PKG-CaMK-MLCK-MLC通路不同时间点和结肠不同部位基因、蛋白表达和磷酸化动态改变，以期明确MLCK通路磷酸化在益气开秘方调控便秘过程中作用机制，为慢传输型便秘发病和益气开秘方调控机制研究提供有说服力的实验数据和新的理论解释。

肠道动力障碍是慢传输型便秘的主要发病机制。肠道平滑肌肉收缩和舒张功能异常可能是发病因素之一。“益气开秘方”治疗慢传输型便秘的经验方，临床疗效好；预初研究表明“益气开秘方”能够显著提高肠道平滑肌cGMP含量；肠动力障碍小鼠MLCK蛋白表达量在升结肠处显著降低，而在降结肠部分显著提高，提示肠动力紊乱和MLCK表达密切相关。本研究拟从慢传输型便秘动物模型的建立和验证，深入探索“益气开秘方”对小鼠便秘模型排便时间、频次、肠容积、小肠推进运动和肠动力等生物学功能影响以及改善慢传输型便秘肠动力障碍的生物学效应和分子机制。探索“益气开秘方”治疗中的量效关系。研究发现益气开秘方调控便秘小鼠结肠平滑肌MLCK磷酸化表达的变化。利用PEX-100磷酸化芯片检测空白组和益气开秘方组结肠平滑肌细胞，进行蛋白磷酸化检测，科学的阐释益气开秘方干预后MLCK磷酸化位点的调变情况及生物学功能变化，为慢传输型便秘肠动力功能障碍提供新的调控靶点和机制。