ANO1钙激活氯通道在FD肝郁脾虚证大鼠胃窦ICC中的作用及疏肝健脾方的干预机制

Functional dyspepsia (FD) is a disease characterized by gastrointestinal motility disorders and often accompanied by mental disorders. There are also multiple symptoms that overlap, vary, or interconvert, ,and repeated attacks, more difficult to cure and other characteristics.Gastrointestinal motility disorder is the main pathophysiological basis of FD，however, TCM believes that liver depression and spleen deficiency are the crux of the gastrointestinal motility disorder of FD. Using the advantages of Chinese medicine overall regulation has the unique curative effect to its treatment. Research findings: As pacemaker cells of gastrointestinal tract, Cajal interstitial cells (ICC) play an important role in gastrointestinal dynamic regulation. Calcium activated chloride channel protein ANO1 in ICC cell membrane regulates pacemaker current, meanwhile, functional expression of ANO1 regulates ICC proliferation. Hypothesis was proposed based on previous research: ANO1 electrophysiological characteristics and proliferation function were taken as the entry point, the animal models of FD liver depression and spleen deficiency and ICC cells model , as subjects, were established by complex etiology. To investigate the electrophysiological characteristics of ANO1 and the role of PI3K/Akt signaling pathway in the regulation of ANO1 expression in the ICC of the gastric antrum of FD rats with liver depression and spleen deficiency syndrome. Then to explore the mechanism of Shugan Jianpi Recipe (Shuwei Decoction) in the intervention of gastrointestinal motility and electrogastric rhythm in FD syndrome of liver depression and spleen deficiency. To provide new ideas and theoretical basis for prevention and treatment of FD.

功能性消化不良（FD）是以胃肠动力障碍为主并多伴有心理精神障碍的一种疾病，有多种症状重叠、多变或相互转换以及反复发作、较难治愈等特点。胃肠动力障碍是FD主要的病理生理学基础，而中医认为肝郁脾虚是FD胃肠动力障碍的症结所在，运用中医药整体调节的优势对其治疗有独特的疗效。研究发现：Cajal间质细胞（ICC）作为胃肠道的起搏细胞，对胃肠动力调控具有重要作用，而ICC细胞膜中钙激活氯离子通道蛋白ANO1可调节ICC起搏电流，且ANO1功能性表达调控ICC的增殖。基于前期研究基础提出假说：以ANO1电生理特性与增殖功能为切入点，采用复合病因建立FD肝郁脾虚证动物及ICC细胞模型为受试对象，研究ANO1电生理特性及PI3K/Akt信号通路调控ANO1表达在FD肝郁脾虚证大鼠胃窦ICC中的作用；继而探讨疏肝健脾方（舒胃汤）干预FD肝郁脾虚证胃肠动力、胃电节律的作用机制，为防治FD提供新思路和理论。

功能性消化不良是最常见的功能性胃肠道疾病之一，影响超过20%的人口。其定义为存在以下一种或多种症状：上腹部疼痛或烧灼感、早饱和餐后饱腹感，但影像学检查或内镜检查显示无结构性疾病。胃肠Cajal间质细胞(ICCs)一般认为是胃和小肠内的一种主要起搏细胞，它可通过平滑肌细胞传播慢波活动，是胃肠运动的基础。功能性消化不良的患者相对于一般人群生活质量明显下降，PI3K/Akt信号通路调节多种细胞ANO1表达介导细胞增殖，该信号通路也可调控STZ诱导的糖尿病小鼠胃窦ICCs的含量。本项目组通过研究舒胃汤通过调控PI3K/Akt信号通路介导ANO1表达，调控胃窦ICCs含量，进而影响胃肠动力，由此揭示FD肝郁脾虚证的本质。与对照组比较，FD模型组大鼠中WB法显示ANO1、PI3K、p-Akt、AKT蛋白表达水平下降，RT-qPCR法显示PI3K、AKT相关mRNA表达水平下降，CCK8法显示ICCs增殖水平下降，流式细胞仪检测显示凋亡水平上升；与模型组组比较，舒胃汤组WB法显示ANO1、PI3K、p-Akt、AKT蛋白表达上升，RT-qPCR法显示PI3K、AKT相关mRNA表达水平上升，CCK8法显示ICCs增殖水平上升，流式细胞仪检测显示凋亡水平下降。给予ANO1抑制剂T16Ainh-A01干预后PI3K、AKT相关mRNA表达水平较干预前下降更为明显，ICCs增殖水平较干预前下降更为明显，而凋亡水平较干预前上升更为明显。模型组的CACCs的电流大小及密度分别有了显著下降。与对照组相比（P<0.05），舒胃汤组和莫沙必利组的电流大小及密度均有显著增加。FD胃肠动力障碍与PI3K/Akt信号通路调控ANO1表达介导ICC增殖相关。FD肝郁脾虚证胃肠动力障碍与ANO1调控ICCs的增殖有关，而ICCs的增殖受PI3K/Akt信号通路介导ANO1表达调控；舒胃汤通过调控PI3K/Akt信号通路促进ANO1表达，增加胃窦ICCs含量；舒胃汤促进胃窦 ICC跨膜蛋白 ANO1表达增加影响 ICC形成慢波形成，促进胃肠动力，从而有效的治疗FD肝郁脾虚证。