非酒精性脂肪肝大鼠小肠黏膜机械屏障的变化

The alterations of intestinal mechanical barrier in rat models of non-alcoholic fatty liver disease

目的 探讨在非酒精性脂肪肝（NAFLD）的进展过程中小肠黏膜机械屏障的变化.方法 雄性SD大鼠90只均分为3组,即为正常饮食组、高糖饮食组和高脂饮食组,建立NAFLD动物模型,并于4、8、12周各组分别处死10只.另取SD大鼠20只,随机均分为四氯化碳（CCl4）组和对照组,CCl4组予以CCl4建立肝损伤模型,4周时处死两组大鼠.肝脏石蜡切片HE染色观察脂肪变性程度.鲎试验终点显色法检测门静脉血中脂多糖（LPS）水平.免疫荧光法检测小肠黏膜紧密连接蛋白occludin,并对荧光强度进行评分.电镜下观察小肠黏膜紧密连接形态的变化.结果 肝脏组织病理表现提示NAFLD和肝损伤模型成功建立.高糖组LPS含量在各个时间点与正常饮食组差异均无统计学意义（P值均＞0.05）.高脂组4周及12周时LPS含量与正常饮食组差异均无统计学意义（P值均＞0.05）,而8周时显著高于正常饮食组（P＜0.05）.CCl4组与对照组LPS含量差异无统计学意义（P＞0.05）.在8周时正常饮食组、高糖组和高脂组occludin蛋白表量分别为1.80±0.42、1.50±0.53和1.30±0.67,高糖组和高脂组较正常饮食组略有减弱,差异均有统计学意义（P值均＜0.05）;而12周时高糖组和高脂组较正常饮食组明显减弱（P值均＜0.05）.CCl4组occludin的表达明显较对照组减弱（0.60±0.16比1.80±0.42,P＜0.05）.高糖组、高脂组及CCl4组在各个时间点紧密连接形态均无明显变化.结论 NAFLD进程中紧密连接蛋白occludin的表达随肝脏脂肪变的进展逐渐减弱.因此在NAFLD的治疗中除改善胰岛素抵抗、保肝治疗外,尽早保护肠黏膜屏障可能有助于减慢肝损伤的进程.

Objective To explore the alterations of intestinal mechanical barrier during the nonalcoholic fatty liver disease （NAFLD） progression. Methods To establish NAFLD rats＇ model,ninety male SD rats were divided into three groups equally： normal-diet group, high-sucrose diet group and high-fat diet group. At the 4th, 8th and 12th week each time point, ten rats were sacrificed in each group. Another twenty SD rats were randomly divided into carbon tetrachloride （CCl4） group and control group. The liver injured model of CCl4 group was induced by CCl4 ; all the rats of these two groups were killed at the 4th week. The degree of liver steatosis was observed through HE staining of liver paraffin sections. The lipopolysaccharide （LPS） level of portal vein blood was measured by limulus test. The occludin expression in intestinal mucosal was detected by immunofluorescent assay,and the fluorescence intensity was scored. The morphology change of intestinal mucosa tight junction was observed through electron microscopy. Results The liver histopathology suggested that NAFLD and liver injured rats＇ model was successfully established. There was no statistical significance of LPS level between high-sucrose diet group and normal diet group at all the time point （P〉0.05）. At the 4th and 12th week, there was no statistical significance of LPS level between high-fat diet group and normal diet group （P〉0.05）, while it was significantly higher in high-fat diet group than normal diet group at the 8th week （P〈0. 05）. There was no statistical significance of LPS level between CCl4 group and control group （P〉0. 05）. At the 8th week, the expression of occludin in normal-diet group, high-sucrose diet group and high-fat diet group was 1.80±0. 42, 1. 50 ± 0. 53 and 1.30±0.67, respectively, the expression was a little bit lower in high-sucrose diet group and high-fat diet group than in normal diet group, the difference was statistical significance （P〈0. 05）. At the 12th week, the expression of occludin was significantly lower in high-sucrose diet group and high-fat diet group than normal diet group. The expression of occludin was significantly lower in CCl4 group （0.60±0.16） than in control group （1.80±0. 42） （P〈0. 05）. No obvious morphology changes of tight junction was found in high-sucrose diet group, high-fat diet group and CCl4 group at each time point.Conclusion The expression of occludin decreased gradually during the non-alcoholic fatty liver disease progression. So in the NAFLD treatment, besides improving insulin resistance and protecting liver function,the protection of intestinal mucosa barrier as early as possible may slow down the progression of liver injury.