痛泻要方靶向miR-144调控肠道黏膜通透性治疗肝郁脾虚型肠易激综合征分子机制研究

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common functional bowel disease. Western medicine still lacks exact curative therapeutic methods. As the dominant disease of traditional Chinese medicine, treatment of Tongxie Yaofang to IBS-D with liver depression and spleen deficiency has been widely recognized clinically. Previous studies have found that miR-144 can target tight junction proteins OCLN, ZO1 regulate intestinal mucosal permeability of IBS-D rats. Based on this, we proposed the hypothesis that "Tongxie Yaofang can down-regulate the expression of miR-144, produce the regulation and activation mechanism of intestinal mucosal permeability, release the inhibitory effect on OCLN, ZO1, and thus strengthen the intestinal mucosal barrier function of IBS-D.” This study is intended to explore the drug-target-disease relationship between Tongxie Yaofang, miR-144 and IBS-D from the clinical, cells and animal level, to determine the relationship between miR-144 and the intestinal mucosa in IBS-D patients with liver depression and spleen deficiency before and after treatment. Different expression of OCLN and ZO1 were confirmed by positive and negative validation of plasmid and lentivirus transfection techniques. The direct evidence of Tongxie Yaofang targeted miR-144 regulate of intestinal mucosal permeability in the treatment of IBS-D with liver depression and spleen deficiency was obtained. This will provide the basis for seeking drug treatment targets of Chinese medicine.

腹泻型肠易激综合征（IBS-D）是常见的功能性肠病，目前西医尚缺乏确切疗效的治疗方法，而该病作为中医的优势病种，采用经方痛泻要方治疗肝郁脾虚型IBS-D已获得广泛临床认可，前期研究发现miR-144可靶向紧密连接蛋白OCLN、ZO1调节IBS-D大鼠肠道黏膜通透性，基于此，我们提出“痛泻要方可下调miR-144的表达，产生肠道黏膜通透性的调控激活机制，解除对OCLN、ZO1的抑制效应，从而加强IBS-D肠道黏膜屏障功能”的假说，本研究拟从临床水平、细胞水平、动物水平探索痛泻要方、miR-144与IBS-D的药物-靶点-疾病治疗关系，明确痛泻要方对肝郁脾虚型IBS-D治疗前后肠道黏膜中miR-144、OCLN和ZO1的表达差异，通过质粒、慢病毒转染技术正反验证，获得痛泻要方靶向miR-144调控肠道黏膜通透性治疗肝郁脾虚型IBS-D的直接证据，为寻找中药药物治疗靶点提供依据。

本项目总体研究发现，痛泻要方可以明显改善肝郁脾虚证IBS-D患者的腹痛以及腹泻症状，经长期随访，患者生活质量得到明显改善；全转录组学研究发现，痛泻要方参与调控IBS-D治疗中的CircRNA-miRNA-mRNA-LncRNA调控网络，证实miR-144在IBS-D发病机制中起关键作用，是痛泻要方的作用治疗靶点。痛泻要方可以改善IBS-D大鼠的肠道粘膜炎症反应，上调结肠组织中OCLN和ZO-1的表达，并促进肠粘膜屏障功能恢复，可能通过抑制NF-κB和Notch信号通路起作用。通过紧密连接蛋白调节肠上皮通透性是增强粘膜防御功能和维持肠上皮细胞的重要机制。痛泻要方可以抑制肠上皮细胞中miR-144的表达，并靶向OCLN和ZO1的表达以维持肠上皮细胞稳态。然而，需要体内研究来进一步探索痛泻要方对肠道黏膜屏障功能的确切保护机制。.综上所述，痛泻要方调节肠上皮通透性可能成为治疗肠上皮屏障功能障碍相关疾病的新靶点，miR-144极有可能使其作用靶标，它不仅能为临床个体化中药治疗IBS-D提供新的有价值的分子标志物，并能为加强肠道黏膜屏障功能、找到中药分子治疗靶点提供依据。