非酒精性脂肪性肝病大鼠肠粘膜紧密连接蛋白ZO-1及肌球蛋白轻链激酶的变化

目的:研究不同阶段非酒精性脂肪性肝病(NAFLD)大鼠肠粘膜紧密连接蛋白ZO-1及肌球蛋白轻链激酶(MLCK)的变化。方法:将60只雄性清洁级SD大鼠随机分为两组:正常饮食组(NDG)和高脂饮食组(FDG)。于4周、8周、12周时观察各组大鼠肝脏病理学改变;应用ELISA方法测定各阶段大鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、胆固醇(CHOL)的变化;应用鲎实验测定各阶段大鼠门静脉血中内毒素(ET)水平;应用免疫组化方法检测肠道粘膜肌球蛋白轻链激酶(MLCK)及紧密连接蛋白(ZO-1)的表达及分布情况。结果:随着高脂饮食时间增长,高脂饮食组大鼠肝脏脂肪变性程度逐渐加重;血清ALT、AST、TG、CHOL水平逐渐升高(4周时,P0.05,8周和12周时P0.05);大鼠血浆内毒素的水平逐渐上升(0.11±0.01比0.11±0.01,P0.05;0.36±0.01比0.11±0.01,P0.05;0.44±0.15比0.18±0.03,P0.05);高质饮食组较正常饮食组大鼠紧密连接蛋白ZO-1表达逐渐下降(3.6±0.7比3.9±0.32,P0.05;2.8±0.63比3.8±0.42,P0.05;1.8±0.79比3.7±0.48,P0.05),MLCK表达逐渐增多(0.5±0.53比0.3±0.48,P0.05;1.3±0.48比0.4±0.52,P0.05;1.9±0.74比0.5±0.53,P0.05)。结论:肠粘膜紧密连接蛋白ZO-1及MLCK可通过影响肠粘膜屏障功能,改变肠粘膜的通透性,促进非酒精性脂肪肝病发生发展。

Changes of Intestinal Mucosal Tight Junction Protein ZO-1 and MLCK in Different Stages of Nonalcoholic Fatty Liver Disease Rats

ABSTRACT Objective: To investigate the expression and distribution of the intestinal mucosal tight junction protein zonula occluden-1(ZO-1) and myosin light chain kinase(MLCK) in rats from the different stages of nonalcoholic fatty liver disease(NAFLD). Methods: Sixty Sprague-Dawley(SD) rats were randomly divided into two groups: normal diet group(NDG) and high fat diet group(FDG). The pathologic changes of hepatic tissue were assessed by Hematoxylin-Eosin(HE) staining at 4 weeks, 8 weeks, 12 weeks. The expression levels of alanine aminotransferase(ALT), aspartate transaminase(AST), triglyceride(TG), cholesterol(CHOL) of two groups in serum were evaluated by enzyme linked immune sorbent assay(ELISA). The expression levels of the plasma Endotoxin(ET) in rats from different stages of NAFLD were quantified by limulus test. The expression and distribution of ZO-1 and MLCK were determined by immunohistochemistry. Results: As time went on, the degree of hepatic steatosis gradually increased in rats feed with high fat diet. The levels of ALT, AST, TG, CHOL in serum and ET in plasma were gradually elevated in high fat diet group (0.11±0.01 vs 0.11±0.01, P>0.05; 0.36±0.01 vs 0.11±0.01, P<0.05; 0.44±0.15 vs 0.18±0.03, P<0.01). The result of immunohistochemistry showed that the expression of tight junction ZO-1 in high fat diet group gradually decreased (3.6±0.7 vs 3.9±0.32, P>0.05; 2.8±0.63 vs 3.8±0.42, P<0.05; 1.8±0.79 vs 3.7±0.48, P<0.01), while the expression of MLCK gradually increased (0.5±0.53 vs 0.3±0.48, P>0.05; 1.3±0.48 vs 0.4±0.52, P<0.05; 1.9±0.74 vs 0.5±0.53, P<0.01). Conclusion: Intestinal mucosal tight junction protein ZO-1 and MLCK play a role in intestinal barrier dysfunction, which may result in the increase of intestinal mucosal permeability and the development and progression of NAFLD.