基于5-HT4R信号系统及下游Ca2+/CaM/MLCK信号通路探讨枳术方治疗STC的机制研究

Slow transit constipation(STC) is a clinical refractory disease with high incidence. Research shows that the decreased expression of 5-HT4R in colon tissue is clearly associated with the incindence of STC. Colonic smooth muscle cells play a key role in colonic motility. 5-HT4R can activate the downstream Ca2+/CaM/MLCK signal pathway to regulate the contraction of colonic smooth muscle cells. We used “Zhi-Zhu Decoction" as a remedy for STC, which the principle for treatment is to strengthen the spleen. It has been shown efficacy in the early clinical studies of this ancient formula. The corresponding basic research has shown that the clinical effect of Zhi-Zhu Decoction, which can observably up-regulate the expression level of 5-HT4R mRNA and protein in colon tissue of the constipation rat models, is almost equal to Prucalopride. This study intends to use STC rat models of 5-HT4R gene knockout, compared with normal STC rat models, observe the colonic transit function, expression level of related protein affected and so on by the decoction in order to elucidate the mechanism of the treatment of STC and the role of 5-HT4R in the treatment. Besides, the changes of 5-HT4R signal system and Ca2+/CaM/MLCK signal pathway will be observed in the experimental cells treated by Zhi-Zhu Decoction. We are supposed to interpret the internal mechanism of colonic smooth muscle cells contraction, and to provide scientific basis for Chinese medicine treatment of STC.

慢传输型便秘（STC）是临床难治性疾病，且发病率高。研究表明，5-HT4R在结肠组织中表达下降与STC发病明确相关；结肠平滑肌细胞在结肠动力功能中处于关键地位；5-HT4R可以通过激活下游Ca2+/CaM/MLCK信号通路调节结肠平滑肌细胞收缩。我们应用枳术方治疗STC，以运脾为治则，古方今用，临床疗效确切；前期研究显示该方对STC大鼠肠道促动作用与普芦卡必利疗效相当，且能显著上调大鼠结肠组织中5-HT4RmRNA及蛋白表达。本研究拟以5-HT4R基因敲除的STC大鼠模型，与普通STC大鼠对照，观察枳术方对大鼠肠道传输功能及相关蛋白表达等的影响，阐明该方治疗STC的机制及5-HT4R在治疗中的作用；通过细胞实验探讨该方对5-HT4R信号系统及下游Ca2+/CaM/MLCK信号通路的影响，阐明该方促进结肠平滑肌细胞收缩的内在机制，为中医药治疗STC提供科学依据。

慢传输型便秘（slow transit constipation，STC）的发病率约占慢性便秘的45.5%，是临床常见的慢性胃肠病，严重影响人类的身心健康。白术、枳实配伍治疗功能性胃肠病历史悠久，大剂量生白术（70g）配伍枳实（30g）（命名为枳术方）治疗便秘临床疗效显著。前期研究发现枳术方能显著提高STC大鼠肠道5-HT4受体（5-HT4R）表达。Ca2+-CaM-MLCK通路是调节平滑肌细胞收缩的核心通路，其作为5-HT4R的下游信号通路，在结肠平滑肌细胞动力信号传导中起关键作用。因此，本课题以5-HT4R信号系统及下游Ca2+-CaM-MLCK信号通路为焦点，探索枳术方治疗STC的部分作用机制。.本课题以STC大鼠模型、5-HT4R基因敲除（5-HT4R（-/-））的STC小鼠模型、大鼠结肠平滑肌细胞为研究对象，通过观察枳术方对模型动物肠道传输功能、结肠平滑肌细胞收缩等的影响，采用免疫组化、荧光定量PCR、Western blot等方法，检测5-HT4R信号系统及Ca2+-CaM-MLCK通路中相关蛋白表达情况。.结果：①枳术方能促进STC模型动物肠道传输功能和结肠平滑肌收缩，同时促进结肠组织、平滑肌细胞中5-HT4R、CaM、MLCK mRNA和蛋白表达。②枳术方对5-HT4R（-/-）小鼠的5-HT4R表达无促动作用，对其下游CaM、MLCK 表达有促进作用，但较野生型小鼠作用减弱，说明枳术方能通过5-HT4R调控下游Ca2+-CaM-MLCK通路促进肠道动力，但5-HT4R不是该通路唯一的调控系统。