基于miRNA-200c调控肠上皮紧密连接研究血必净注射液保护脓毒症大鼠肠黏膜屏障功能的作用机制

The occurrence and development of sepsis is closely related to intestinal mucosal barrier dysfunction. Intestinal epithelial tight junction is considered to be the structural basis of maintaining intestinal mucosal barrier function. Xuebijing has been widely used for clinical treatment of sepsis. However, the molecular mechanisms of intestinal mucosal barrier remain unclear. Our previous study demonstrated that Xuebijing effectively inhibits the increase of intestinal mucosal barrier permeability, and relieves intestinal epithelial tight junction structure damage in septic rats. Recently, our miRNA-depth sequencing studies showed that Xuebijing significantly downregulated the expression of miR-200c in vitro, suggesting miR-200c as a key target of Xuebijing in rebuilding the intestinal epithelial tight junction. In this study, the model of CLP septic rat was established. The effects of Xuebijing on relieving sepsis-induced intestinal mucosal barrier dysfunction were clarified by detecting its role in intestinal mucosal barrier function, tight junction protein and miR-200c expression. Furthermore, we employed lentiviral transfection, transcriptome sequencing and luciferase reporter system technology to study the mechanism of Xuebijing on "miR-200c→Tight junctions" in vitro and verified the revealed mechanisms in rat CLP sepsis model. The aim of this study was to reveal the molecular mechanism of Xuebijing in improving intestinal mucosal barrier dysfunction by regulating the "miR-200c→Tight junctions" in septic rats, and to provide scientific basis for its clinical application.

脓毒症发生发展与肠黏膜屏障功能障碍密切相关，肠上皮紧密连接是维持肠黏膜屏障功能的结构基础。血必净是防治脓毒症的一线用药，但其对肠黏膜屏障的作用机制尚不清楚。前期研究发现，血必净能有效抑制脓毒症大鼠肠黏膜屏障通透性增加，降低肠上皮紧密连接结构损伤，深度测序发现血必净能显著下调miR-200c表达，提示其可能通过调控miR-200c保护肠上皮紧密连接。本课题通过复制大鼠CLP脓毒症模型，评价血必净对肠黏膜屏障功能、紧密连接蛋白及miR-200c表达的影响，明确血必净改善脓毒症肠黏膜屏障功能障碍的作用。进一步在体外模型中通过慢病毒转染、转录组测序和荧光素酶报告系统等技术，研究血必净对“miR-200c→紧密连接”的干预机制，并将其机制在大鼠CLP脓毒症模型中验证。本研究旨在阐明血必净通过调控“miR-200c→紧密连接”，改善脓毒症大鼠肠黏膜屏障功能障碍的作用机制，为其临床应用提供科学依据。

脓毒症发病率高，病死率高，是危重症研究领域面临的重大难题和挑战。肠黏膜屏障功能障碍常伴随着脓毒症的发生和发展，与脓毒症患者病情转归及预后密切相关。加强对脓毒症肠功能障碍的预防和治疗已成为脓毒症患者诊疗的关键策略。肠上皮紧密连接是维持肠黏膜屏障功能的结构基础。血必净注射液为活血化瘀法治疗脓毒症的代表方，临床疗效确切，在新冠肺炎疫情的防控中也发挥了重要的作用。阿魏酸是血必净注射液主要活性成分。然而血必净注射液和阿魏酸对肠黏膜屏障功能的调控作用及分子机制尚未完全明确。本研究成功建立了盲肠结扎穿孔手术（CLP）大、小鼠脓毒症，以及LPS诱导的肠上皮细胞（Caco-2/IEC-6）屏障功能损伤模型，研究发现：（1）血必净注射液及其主要成分阿魏酸可抑制LPS诱导的Caco-2/IEC-6细胞屏障通透性增加，上调紧密连接蛋白表达，减轻紧密连接结构损伤，保护肠黏膜屏障功能。（2）构建miR-200c过表达/抑制慢病毒载体转染至Caco-2细胞发现，miR-200c是血必净注射液及阿魏酸调控肠黏膜屏障的关键靶点，血必净注射液及阿魏酸能通过调控miR-200c介导的PTEN/PI3K/AKT通路改善LPS诱导的Caco-2细胞屏障功能障碍。（3）血必净注射液和阿魏酸能够提高脓毒症小鼠存活率，抑制肠道细菌移位，增加紧密连接蛋白表达，抑制炎性因子释放，改善肠黏膜屏障功能，其机制与调控miR-200c介导的PI3K/AKT/mTOR通路相关。通过以上研究阐明了“血必净注射液通过调控肠上皮细胞miR-200c，调节紧密连接蛋白表达，保护肠黏膜屏障功能”的科学假说。该研究进一步丰富了血必净注射液应用于临床治疗脓毒症的科学内涵，也为其进行药物二次研发提供科学依据和潜在靶点。