肌球蛋白轻链磷酸化在腹泻型IBS大鼠肠黏膜屏障功能障碍中的作用

Role of Myosin Light Chain Phosphorylation in Intestinal Mucosal Barrier Dysfunction in Diarrhea-predominant IBS Rats

背景:肌球蛋白轻链(MLC)是调控细胞间紧密连接和肠道通透性的启动因子,磷酸化MLC(pM LC)可引起紧密连接相关蛋白重新分布,破坏紧密连接结构和完整性,最终引起肠黏膜屏障功能障碍。目的:探讨MLC磷酸化在腹泻型IBS(IBS-D)大鼠肠黏膜屏障功能障碍中的作用。方法:将8只Sprague-Dawley孕鼠随机分为模型组和对照组,采用母婴分离法构建IBS-D大鼠模型。记录大鼠造模成功后1 h内粪便性状和颗粒数,采用腹壁回撤反射(AWR)评分评估大鼠内脏敏感性,免疫荧光法观察结肠组织纤维状肌动蛋白(F-actin)、细胞紧密连接相关蛋白ZO-1、claudin-1的分布,蛋白质印迹法检测肠黏膜MLC、pM LC、F-actin、ZO-1、claudin-1蛋白的表达,透射电镜观察细胞间紧密连接。结果:与对照组相比,模型组内脏敏感性显著升高;免疫荧光法显示claudin-1荧光强度减弱,结构紊乱,重新分布,F-actin和ZO-1结构模糊;蛋白质印迹结果显示pM LC/MLC比值和pM LC表达明显升高,claudin-1表达明显降低,MLC、F-actin和ZO-1表达无明显差异;透射电镜显示细胞紧密连接破坏,细胞间隙扩大。结论:MLC磷酸化水平升高介导细胞骨架蛋白F-actin重组,引起细胞紧密连接蛋白结构和功能的改变,细胞间隙增加,导致肠黏膜屏障功能障碍,在IBS-D的发病中发挥重要作用。

Background： Myosin light chain （MLC） is the initiating factor that regulating tight junction and intestinal permeability. Phosphorylated MLC （pMLC） can cause redistribution of tight junction related protein, and break the structure and integrity of tight junction, thus induces the dysfunction of intestinal mucosal barrier. Aims： To explore the role of MLC phosphorylation in intestinal mucosal barrier dysfunction in diarrhea-predominant IBS （IBS-D） rats. Methods： Eight Spague-Dawley pregnant rats were randomly divided into model group and control group, IBS-D rat model was established by maternal separation. The character and particles of feces were record 1 hour after establishing the model. Abdominal withdrawal reflex （AWR） was used to evaluate visceral sensitivity. The distributions of fibrous actin （ F- actin ）, tight junction associated protein ZO-1 and elaudin-1 in colon were assessed by immunofluorescence. The expressions of MLC, pMLC, F-actin, ZO-1 and claudin-1 were measured by Western blotting. Tight junction was observed by transmission electron microscope （TEM）. Results： Compared with control group, visceral sensitivity was significantly increased. Immunofluorescence showed the decrease in fluorescence intensity, structural distortion and redistribution of claudin-1 and the fussy structure of F-actin and ZO-1 in model group. Western blotting showed that pMLC/MLC ratio and expression of pMLC were significantly increased and expression of claudin-1 was significantly decreased in model group, but no significant differences in expressions of MLC, F-actin and ZO-1 were found between the two groups. TEM showed that tight junction was broken and cell space was enlarged. Conclusions： The increased MLC phosphorylation may induce the redistribution of cell cytoskeletal protein F-actin, cause the change of structure and function of intercellular tight junction proteins and enlargement of intercellular space, thus may induce the dysfunction of intestinal mucosal barrier, and play an important role in the pathogenesis of IBS-D.