长链非编码RNA-ncRuPAR介导云母促非甾体抗炎药肠病肠黏膜机械屏障修复机制的研究

The basic pathological basis of the development of non-steroidal anti-inflammatory drug enteropathy is the dysfunction of the intestinal mucosal mechanical barrier. And PAR-2 blocker can treat NSAIDs enteropathy through promoting the repairing of intestinal mucosal mechanical barrier. However nowadays, the clinical application of PAR-2 blocker is limited. According to the previous research, we found that muscovite had the same effect as PAR-2 blocker. However, its specific mechanism remains to be elucidated. We have confirmed a long non-coding RNA (lncRNA) called ncRuPAR in our previous bioinformatics analysis, and we have confirmed that there is a strong correlation between lncRNA and PAR-2. Therefore, we propose that ncRuPAR can regulate the expression of PAR-2 and reduce the intestinal mechanical barrier dysfunction among NSAIDs enteropathy patients. We propose a hypothesis that muscovite which takes a role as PAR-2 blocker and it can interfere ncRuPAR. Our project is to use the means of serum pharmacology of traditional Chinese medicine and molecular biology to elaborate the mechanism of ncRuPAR to regulate the expression of PAR-2 and the regulation effect of Muscovite to the expression of ncRuPAR, in the levels of whole (animal model and patients), cell (Intestinal epithelial cells cultured in vitro) and molecular biology, which may provide new targets and theoretical basis towards the TCM therapy of NSAIDs enteropathy.

肠黏膜机械屏障功能障碍是非甾体抗炎药(NSAIDs)肠病发生发展的基本病理基础，而PAR-2阻断剂能通过促进肠黏膜机械屏障修复从而起到治疗NSAIDs肠病的目的，但目前PAR-2阻断剂临床应用尚存巨大限制。我们前期研究发现：云母有类PAR-2阻断剂作用，但具体机制有待进一步阐明。有一长链非编码RNA（lncRNA）ncRuPAR在我们前期生物信息学分析证实，该lncRNA与PAR-2间存在很强的调控相关性。据此提出ncRuPAR可通过调节PAR-2的表达，减轻NSAIDs所致肠机械屏障功能障碍；云母可通过靶向干预ncRuPAR从而起到类PAR-2阻断剂作用的假说。本项目拟采用中药血清药理学、分子生物学等手段，在整体（动物模型、患者）、细胞及分子水平阐明ncRuPAR调控PAR-2表达水平的作用机制及云母对ncRuPAR的表达调控作用，为中医药治疗NSAIDs肠病提供新思路。

项目背景：非甾体抗炎药(NSAIDs) 对消化道的副反应日益受到关注，NSAIDs肠病以其较高发生率和致死率严重危害人民的健康，目前有关于NSAIDs肠病的发病机制仍无法明确，治疗方法十分有限，近年来研究发现肠黏膜机械屏障受损在NSAIDs肠病发病中起重要作用，但具体机制不明，而中药云母是中国传统医学中治疗肠炎、下痢的有效药物。.研究内容：本研究首先以正常人和大鼠的小肠组织作为对照组与NSAIDs 肠病患者和NSAIDs肠病大鼠的小肠组织比较，了解NSAIDs肠病小肠黏膜机械屏障的损伤情况及ncRuPAR、PAR-2的表达水平，然后采用RNA基因干扰以及基因激活技术，建立ncRuPAR过表达的Caco-2细胞，并用云母含药血清及PAR-2 阻断剂进行干预，测定细胞通透性，检测细胞连接蛋白（occludin、claudin-1），PAR-2以及ncRuPAR、PAR-2 mRNA的表达水平，从而揭示ncRuPAR对PAR-2的表达调控作用，阐明云母调控ncRuPAR 的作用机制，为中医药治疗NSAIDs肠病提供新靶点和理论基础。.实验结果：NSAIDs肠病患者和大鼠肠黏膜机械屏障功能受损，ncRuPAR及PAR-2表达增加。细胞实验证NSAIDs处理Caco-2细胞后ncRuPAR及PAR-2表达增加，Claudin-1及Occludin表达降低，肠上皮细胞机械屏障完整性破坏，肠通透性增加。而云母和PAR-2阻断剂干预后能拮抗上述过程，起到修复肠黏膜机械屏障的作用。ncRuPAR过表达后，PAR-2明显活化，肠上皮通透性进一步增加，紧密连接蛋白进一步被破坏。加入PAR-2阻断剂或云母干预后能明显下调PAR-2 表达，减少紧密连接蛋白Claudin-1、Occludin损伤，修复肠黏膜机械屏障。.科学意义：NSAIDs肠病的发病机制与ncRuPAR激活后PAR-2活化，Claudin-1及Occludin表达降低，肠上皮细胞机械屏障完整性破坏，肠通透性增加有关。云母可以通过抑制ncRuPAR、下调PAR-2 表达、减少Claudin-1、Occludin损伤，修复肠黏膜机械屏障，改善细胞通透性有效防治NSAIDs肠病。