基于TNF-ɑ/Elk-1/MLCK信号通路探讨清热化湿法修复溃疡性结肠炎肠黏膜屏障损伤的作用机制

The damage of intestinal mucosal barrier is strongly associated with the development of ulcerative colitis (UC). The abnormal activation of TNF-ɑ/Elk-1/MLCK signal pathway has been implicated as a key mediator for the disturbance in the intestinal the disturbance in the intestinal TJ barrier, which contributed to the impaired intestinal mucosal barrier. Qing Hua Chang Yin (QHCY) is a traditional Chinese formula, which has been demonstrated to be clinically effective in the treatment of UC. However, the precise mechanisms behind its anti‑inflammatory effects remain largely unknown. Our previous studies showed that QHCY can ameliorate histologcal damage of intertstinal mucosa in DSS-induced UC mouse model and reduce the secretion of TNF-ɑ in LPS-stimulated differciated Caco-2 cells. Our findings suggest that QHCY treat UC could through reverse the damage of Intestinal mucosal barrier, which involving in TNF-ɑ/Elk-1/MLCK signal pathway. In the present project we will study the in vivo and in vitro effects of QHCY on the intestinal permeability by measurement of transepithelial electric resistance (TEER) and detection of sugar molecular probes. the Effect of QHCY on the activation of TNF-ɑ/Elk-1/MLCK pathway and the expression of cell tight junction protein will be explored by immunohistochemical staining, Western blot and Real-time quantitative RT-PCR. Our findings in this study will provide new insight into the molecular mechanism of the anti-inflammatory activity of QHCY in UC. It will provide further experimental evidence for clinically treatment of UC.

溃疡性结肠炎（UC）的发生发展与肠黏膜屏障破坏密切相关。TNF-ɑ/Elk-1/MLCK通路异常激活介导的细胞紧密连接破坏是肠黏膜屏障受损的重要调节机制之一。大肠湿热证为UC的主要证型，清热化湿法为重要治则。清化肠饮是名老中医杨春波经验方，主治大肠湿热型UC，治疗效果显著。课题组前一个国家自然科学基金项目研究表明清化肠饮可明显改善UC小鼠结肠黏膜的组织学损伤并显著降低LPS介导的肠上皮细胞TNF-ɑ的分泌，提示通过调控TNF-ɑ/Elk-1/MLCK通路、继而促进肠黏膜屏障修复可能是清化肠饮治疗UC的重要机制。在此基础上本课题拟通过体内外实验，采用检测跨膜电阻、糖分子探针等方法研究清化肠饮对UC结肠黏膜通透性的影响，并采用Q-PCR、Western-blot等方法研究清化肠饮对该通路活化和细胞紧密连接蛋白表达的影响。研究将助于进一步阐明清化肠饮治疗UC的内在机制，为其临床应用提供实验依据。

溃疡性结肠炎（UC）的发生发展与肠黏膜屏障破坏密切相关。TNF-ɑ/Elk-1/MLCK通路的异常激活是介导UC肠黏膜屏障受损的重要调节机制。清化肠饮（QHCY）治疗UC效果显著。课题组前期研究表明QHCY可改善UC小鼠结肠黏膜的组织学损伤并降低LPS介导的肠上皮细胞TNF-ɑ的分泌，提示通过调控TNF-ɑ/Elk-1/MLCK通路来促进肠黏膜屏障修复可能是QHCY治疗UC的重要机制。在此基础上，本课题通过体内外实验，采用检测跨膜电阻(TEER)、糖分子探针（FD4）、Q-PCR、Western-blot等方法研究了QHCY对UC结肠黏膜屏障的保护作用和对TNF-α/Elk-1/MLCK信号通路的影响。体内实验结果显示：1）QHCY明显改善了小鼠UC的临床表现，包括疾病活动指数（DAI）增加、结肠缩短和结肠黏膜的组织学损伤；2）QHCY显著抑制了UC结肠组织和血清中TNF-α水平的升高、血清中SAA水平的升高以及结肠组织中MPO表达的增加；3）QHCY显著逆转了UC结肠组织中occludin、zo-1 和Claudin-1的mRNA 和蛋白表达水平下降，以及MLCK的mRNA 和蛋白表达水平上调，并减少了Elk-1的磷酸化。体外实验结果显示：1）QHCY逆转了LPS刺激引起的Caco-2细胞TEER的降低和FD4的增加；2）QHCY显著抑制了LPS诱导的细胞分泌TNF-α水平增加；3）QHCY显著逆转了LPS刺激引起的细胞中occludin、zo-1 和Claudin-1的mRNA 和蛋白表达水平下降，以及MLCK的mRNA 和蛋白水平上调，并减少了Elk-1的磷酸化。由此可见，QHCY可通过抑制TNF-ɑ/Elk-1/MLCK 通路的异常活化来调控粘膜紧密连接蛋白的表达进而改善肠屏障功能以达到治疗UC的作用。本研究成果进一步阐明了QHCY治疗UC 的机制，为其临床应用提供了新的实验依据。