miR-129-3p调控结肠动力在“泻剂结肠”发病中作用及机制研究

“Cathartic colon” caused by long-term use of laxatives anthraquinones is a major type of slow transit constipation (STC). Pathogenesis of “cathartic colon” remains unknown. Recent evidence suggests that enteric neuronal apoptosis in mice induced by abnormal expression of microRNAs (miRs) may lead to pathogenesis of delayed gastrointestinal (GI) transit. Our previous researches indicate that STC patients with long-term use of anthraquinones cathartic have decreased expression of miR-129-3p in the colon tissue and reduction of myenteric neurons and interstitial cells of Cajal (ICC). The use of miR-129-3p inhibitor in fibroblast cells suppresses activity of PI3K signaling pathway and reduces the level of stem cell factor (SCF) expression through directly targeting PIK3IP1 expression. Basing on previous studies and above results, we hypothesis that long-term use of laxatives anthraquinones will induce downregulation of expression of miR-129-3p, to suppress SCF expression by inhibiting PI3K signaling pathway activity in intestinal neuronal via targeting the expression of PIK3IP1, leading to morphological changes of ICCs and colonic motility disorder. It may represent a novel mechanism of “cathartic colon” pathogenesis. In this research, we plan to confirm the above reasoning using molecular and cellular biology technology in both cellular and animal model. Furthermore, we will explore the effects of colonic motility disorder regulated by abnormal miRs expression on pathogenesis of “cathartic colon”. This research may provide a novel reference for diagnosis and treatment of STC in clinic.

长期使用蒽醌类泻剂导致的“泻剂结肠”是慢传输便秘（STC）重要类型且发病机制不明。研究显示微小RNA（miR）表达异常能够诱发肠道肌层神经元细胞凋亡导致小鼠肠道传输减缓。我们前期研究发现长期服用蒽醌类泻剂的STC患者结肠组织miR-129-3p表达下降，肠肌层神经元及Cajal间质细胞（ICC）减少。miR-129-3p抑制物能够通过促进靶基因PIK3IP1表达，抑制成纤维细胞PI3K信号通路活性，降低干细胞因子(SCF)表达水平。由此推测：长期应用蒽醌类泻剂诱发结肠miR-129-3p低表达，通过上调靶基因PIK3IP1表达抑制肠道神经元PI3K信号通路活性下调SCF表达，导致ICC数目形态异常参与结肠动力异常及“泻剂结肠”发病。本研究拟通过分子和细胞生物学技术在细胞和动物模型中对上述推论加以证实，探索miR表达异常调控的结肠动力异常在“泻剂结肠”发病机制中的作用，为临床诊治提供新思路。

探讨miR-129-3p 及其调控的靶基因在长期应用蒽醌类泻剂诱发 “泻剂结肠”发病机制中的作用及其分子机制。 ⑴采用BALB/c小鼠模拟临床慢传输便秘患者发病过程构建慢传输型便秘模型。行钡剂灌胃后再次评估结肠传输情况，确证造模成功后断颈法处死老鼠，取小鼠结肠标本行形态学检查并分段保存结肠肠管大体标本。⑵MicroRNA芯片检测：采用Agilent mouse miRNA（8*60K）V21.0芯片，共计检测吗啡组、吗啡+大黄组（n=5）小鼠模型1881个成熟miRNA。⑶q-PCR法检测动物模型标本miR-129-3p表达量。⑷q-PCR法检测生理 盐水正常组、大黄组、吗啡组、吗啡+大黄组中miR-129-3p 靶基因PIK3IP1、PI3K/Akt 信号通路、肠神经元相关指标的表达量。⑸成功分离，培养与鉴定小鼠原代肠道神经元细胞，通过慢病毒（miR-129-3p过表达以及低表达）感染小鼠原代肠道神经元细胞模型。通过高剂量大黄素处理神经元细胞后，再以 miR-129-3p mimics/inhibitor 慢病毒感染神经元细胞。 ⑴结肠传输实验证实实验组出现显著结肠传输减缓（P＜0.05），吗啡+大黄组较大黄组粪便明显干结、体积减小，HE结果示该组较对照组肠管明显变薄且肌层结构紊乱。⑵MicroRNA芯片检测结果显示：与吗啡对照组相比miR-129-3p等共计42个miRNA明显表达下调。⑶与吗啡组相比，吗啡+大黄组小鼠结肠组织中PIK3IP1 明显上调、NSE及SCF在组织中表达下调。⑷肠道神经元细胞中PIK3IP1 是miR-129-3p 靶基因。miR-129-3p 及其靶基因能够通过影响肠道神经元细胞PI3K/Akt 信号通路活性导致肠道神经元细胞凋亡以及形态学变化，影响SCF表达水平。成功在小鼠中构建STC模型以及小鼠原代肠道神经元细胞模型；在两种模型内均证实蒽醌类泻药能够诱发结肠miR-129-3p 表达异常，通过上调靶基因 PIK3IP1 表达抑制肠道神经元细胞内 PI3K/Akt 信号通路活性诱导神经元细胞凋亡，减少神经元细胞表达 SCF，进而造成结肠 ICCs 数目形态异常，最终导致结肠动力异常参与“泻剂结肠”的发病。本研究初步探索miR表达异常调控的结肠动力异常在“泻剂结肠”发病机制中的作用，为临床诊治提供新思路。