肠愈宁调控Rho/ROCK/MLCK信号通路保护溃疡性结肠炎肠黏膜屏障的作用机制研究

Ulcerative colitis (UC) is a refractory disease of digestive system. Although modern clinical drugs have some advantages in controlling symptoms in a short time, the healing rate of ulcer is low in long-term use, and there are many problems such as easy recurrence, many adverse reactions, hormone dependence and so on. Traditional Chinese medicine can significantly improve the repair level of mucosa, improve the permeability of mucosa, and prevent intestinal bacteria and endotoxin translocation. It is expected to become a more effective treatment for UC. Modern studies have shown that intestinal mucosal barrier protection is the core of UC treatment, but the specific mechanism is still unclear. Therefore, we further explore the protective mechanism of traditional Chinese medicine on UC intestinal mucosal barrier. Finding more direct and effective targets is the key to UC control in the future. Previously, the applicant has confirmed that Changyuning has a significant clinical effect on UC under the guidance of "thick intestine" theory of traditional Chinese medicine. Previous animal experiments also found that Changyuning could remodel the tight junction (TJ) structure of colonic epithelial cells in UC rats, protect the intestinal mucosal barrier, and regulate the expression levels of Rho, ROCK and MLCK genes, which are key molecules of TJ formation. However, the specific mechanism remains to be further studied. Therefore, we suggest that Changyuning may play a therapeutic role in UC by regulating Rho/ROCK/MLCK signaling pathway, remodeling TJ structure and protecting intestinal mucosal barrier. In this study, Caco-2 cells were used to establish an in vitro barrier model of intestinal epithelial cells. The best concentration of ethanol was used to destroy the intestinal mucosal barrier, and drug serum intervention was carried out to detect the changes of cell transmembrane resistance, phenol red permeability, intestinal barrier function and TJ structure after intervention. UC rat models were prepared in vivo, and drug intervention was carried out to observe the pathological manifestations and ultrastructure of intestinal mucosa, and to detect the changes of serum inflammatory cytokines and intestinal mucosal TJ structure. In vivo and in vitro experiments showed that Changyuning could treat UC by remodeling TJ structure and protecting intestinal mucosal barrier. We further knocked out Rho gene by CRISPR/Cas9 and transfected it into Caco-2 cells to construct an in vitro barrier model of intestinal epithelial cells. After intervention with drug-containing serum, we observed the expression of ROCK, MLCK, p-MLC, claudin-1, ZO-1, occludin and the structural changes of TJ. We further elucidate the specific mechanism of Changyuning in the treatment of UC by protecting intestinal mucosal barrier. The expected results will provide scientific basis for the development of new medicines for UC.

溃疡性结肠炎(UC)属消化系统难治性疾病，现代临床药物虽在短期控制症状方面具有一定优势，但长期应用溃疡愈合率低，且存在停药易复发、不良反应多、激素依赖等诸多问题。中药可明显提高黏膜的修复水平，改善黏膜通透性，阻止肠道细菌和内毒素易位，有望成为治疗UC更为有效的手段。现代研究表明肠黏膜屏障保护是治疗UC的核心环节，但具体机制尚不明确。故进一步探讨中药对UC肠黏膜屏障保护的相关机制，寻找更为直接有效的靶点，是未来UC防治的关键。申请者前期已证实在中医“厚肠”理论指导下的肠愈宁治疗UC临床疗效显著，前期动物实验研究亦发现肠愈宁可重塑UC大鼠结肠上皮细胞紧密连接(TJ)结构，保护肠黏膜屏障，同时还可调控TJ形成的关键分子Rho、ROCK、MLCK基因表达水平，但具体机制尚待进一步研究。因此我们提出肠愈宁可能通过调控Rho/ROCK/MLCK信号通路，重塑TJ结构，保护肠黏膜屏障而发挥治疗UC作用。本研究利用Caco-2细胞建立肠上皮细胞体外屏障模型，采用最佳浓度乙醇破坏肠黏膜屏障，并进行药物血清干预，对干预后细胞跨膜电阻、酚红透过率、肠屏障功能及TJ结构变化情况进行检测。体内制备UC大鼠模型，同时进行药物干预，观察肠黏膜病理表现及超微结构，对血清炎症细胞因子，肠黏膜TJ结构变化情况进行检测。通过体内外实验明确肠愈宁可能通过重塑TJ结构，保护肠黏膜屏障而治疗UC。进一步采用CRISPR/Cas9方法敲除Rho基因，并转染至Caco-2细胞，构建肠上皮细胞体外屏障模型，含药血清干预后观察Rho下游关键因子ROCK、MLCK、p-MLC，TJ关键蛋白claudin-1、ZO-1、occludin表达及TJ结构变化情况。进一步阐明肠愈宁保护肠黏膜屏障而治疗UC的具体作用机制，预期成果将为开发治疗UC中药新药提供科学依据。

肠黏膜损伤修复是溃疡性结肠炎（UC）转归的关键环节，有研究证实，Rho/ROCK/MLCK信号通路可通过调控肠上皮细胞间紧密连接（TJ）关键蛋白表达发挥肠黏膜屏障保护作用。本研究拟从Rho/ROCK/MLCK-TJ角度，探讨具有较好临床疗效的名老中医经验方肠愈宁保护肠黏膜屏障的相关作用机制。本研究体内、外构建了UC大鼠模型及肠上皮细胞屏障模型，对结肠黏膜病理、超微结构、细胞跨膜电阻及TJ结构变化情况进行观察、检测。进一步采用慢病毒转染敲降Rho，药物干预后观察其下游关键因子ROCK、MLCK，TJ关键蛋白表达及TJ结构变化情况。.通过上述研究我们获得以下研究结果：体内研究发现，肠愈宁可增加UC大鼠结肠黏膜TJ关键蛋白claudin-1、ZO-1、occludin的荧光表达。同时可下调UC大鼠血清促炎因子IL-6、TNF-α表达，上调抗炎因子IL-10表达。通过上述途径，可明显改善UC大鼠结肠黏膜病理表现，修复结肠黏膜上皮细胞间紧密连接断裂。进一步体外研究证实，肠愈宁可通过抑制Rho/ROCK/MLCK信号通路激活，增加Caco-2细胞所形成的的致密单层细胞跨膜电阻，增加细胞间致密性，同时还可进一步上调细胞间TJ关键蛋白claudin-1、ZO-1、occludin的表达，从而进一步发挥肠黏膜屏障保护作用。本研究结果为中药复方治疗UC提供实验依据，同时为UC的中药新药开发提供相关研究基础。