健脾止泻法通过miR-29及靶基因对脾虚泄泻IBS-D肠道敏感性作用机制研究

IBS-D is a bowel disorder with recurring abdominal boating, abdominal pain and diarrhea. Furthermore, it is considered as spleen-deficiency diarrhea in traditional Chinese Medicine. The primary physiological and pathologic basis of IBS-D is visceral hypersensitivity, which presents as increased intestinal permeability. However, there is no definite biomarker in the diagnosis of IBS-D at present. Recent studies have found that miR-29 plays a pivotal role in the regulation of intestinal permeability. It is proved that NFκB repressing factor and CLDN1 are the target genes of miR-29, but the pathway how they regulate the related structural proteins and the intestinal mechanical and immune barriers is unknown. Our previous studies indicate that the representative formula of strengthening spleen and anti-diarrhea has significant curative effect on IBS-D, that is associated with the mechanisms that it could regulate the function of 5-HT, MC and TC and decrease the expression of NFκB and CLDN1. Nevertheless, it remains further interpretation how it improves intestinal hypersensitivity and decreases intestinal permeability. Therefore, we intend to conduct clinical trials to verify the relationship between miR-29 and intestinal sensitivity. Moreover, we use animal and cell models to illuminate how the Chinese compound formula actions on intestinal permeability through regulating miR-29 and target genes. It is of both theoretical and clinical significance that we further study the molecular mechanism of traditional Chinese medicine intervening in IBS-D and explore whether miR-29 can be used as a biological marker in the diagnosis of IBS-D.

IBS-D以反复发作的腹胀痛、腹泻为主要症状，属中医脾虚泄泻范畴。内脏高敏感性是其主要生理病理学基础，肠道高通透性是主要表现，但缺乏确切的生物学诊断标记物。近期研究发现miR-29在肠道通透性调节中具有关键作用，已证实的靶基因有NFκB抑制因子及CLDN1，但其调控相关结构蛋白、影响肠道机械及免疫屏障的途径未明。我们前期研究表明:健脾止泻代表方对IBS-D疗效确切，与调节5-HT、MC及TC功能，下调NFκB及CLDN1表达等机制相关，但其改善肠道敏感性、降低通透性的作用机制有待深入阐明。本项目拟通过临床研究，验证miR-29和肠道敏感性的关系；采用基因敲除、siRNA干扰及共聚焦显微镜等方法，通过动物及细胞模型，验证中药复方调控miR-29及靶基因对肠道敏感性的作用。本项目对深入研究中医药干预IBS-D分子机制，探索miR-29作为IBS-D的一种生物学诊断标记物，具有重要的临床意义。

腹泻型肠易激综合征（Diarrhea-predominant irritable bowel syndrome，IBS-D）是以反复发作的腹胀、腹痛、腹泻为主要症状，缺乏胃肠道结构和生化异常的肠道功能紊乱性疾病，属中医“泄泻、腹痛、郁证”的范畴。其诱发因素包括心理、肠道感染、遗传等方面。肠道屏障功能异常和内脏高敏感性是其重要的病理机制。肠道屏障功能受损可以引起肠粘膜的通透性升高，并以内脏高敏感性为主要表现。本研究通过临床研究、模式动物和细胞实验探讨了miRNA-29a及其靶基因对肠道通透性和内脏敏感性的调控作用，初步阐明肠激安方改善肠道屏障功能异常和内脏高敏感性的作用机制，探索miRNA-29a作为IBS-D的一种潜在的生物学诊断标记物，具有重要临床意义。研究结果发现：（1）IBS-D患者结肠粘膜超微结构屏障异常，结肠粘膜中miRNA-29a上调，CLDN1、NKRF和ZO-1下调；（2）在基因敲除小鼠中，miRNA-29a具有调节肠道通透性和敏感性的作用；（3）在NCM460细胞中，miRNA-29a对CLDN1和NKRF具有调控作用；（4）肠激安方可改善IBS-D模型大鼠肠道通透性及敏感性.结论：（1）IBS-D患者结肠粘膜屏障功能异常可能与miRNA-29a及其靶基因的调控有关；（2）miRNA-29a可通过NKRF和CLDN1调控WAS小鼠肠道通透性和内脏敏感性；（3）在NCM460细胞中，miRNA-29a在NCM60细胞种对CLDN1和NKRF具有反向调控的作用；（4）肠激安方对脾虚泄泻的症状有改善作用，并可调节IBS-D模型大鼠肠道通透性和内脏敏感性；IBS-D模型大鼠肠道通透性升高、内脏高敏感性可能与miRNA-29a及其靶基因NKRF和CLDN1的调控有关；肠激安方能够降低IBS-D模型大鼠结肠组织中miRNA-29a的含量，提高CLDN1、NKRF的表达，提示肠激安方可能通过对miRNA-29a及其靶基因的调控改善IBS-D大鼠肠道通透性和内脏敏感性。.综上所述，本研究结果表明，miRNA-29a及其靶基因对IBS-D肠道通透性和内脏敏感性具有调控作用。肠激安方可能通过对miRNA-29a及其靶基因的调控作用，改善IBS-D肠道通高透性和内脏高敏感性。