中文摘要：

目的 研究肌球蛋白轻链激酶（MLCK）在非酒精性脂肪性肝炎（NASH）小鼠模型肠黏膜屏障变化中的作用,探讨肠黏膜屏障在NASH发病中的作用机制.方法 将C57BL/6小鼠随机分为正常饮食对照组、单纯性脂肪肝（NAFL）组、NAFL加MLCK特异性抑制剂ML-7组（NAFL＋ ML-7组）,NASH组以及NASH＋ ML-7组,每组为10只小鼠.HE染色法观察各组肝组织病理改变,免疫组化法测定小肠上皮中MLCK的表达,透射电镜观察小肠上皮细胞间紧密连接形态并测量细胞间隙宽度,ELISA法检测门静脉中内毒素脂多糖（LPS）浓度,测定外周血中转氨酶水平,ELISA和实时定量PCR法检测肝脏组织中炎性因子的浓度和mRNA表达.结果 NASH组小肠上皮细胞中MLCK表达量明显高于正常饮食对照组（P＜0.01）.NASH组紧密连接结构紊乱,细胞间隙宽度显著大于正常饮食对照组[（26.60±1.20） nm比（14.90±0.33） nm,P＜0.05],给予MLCK抑制剂ML-7后（NASH＋ML-7组）,紧密连接结构恢复,细胞间隙宽度[（14.90 ±0.67）nm]较NASH组明显减小（P＜0.05）.NASH组小鼠门静脉中LPS浓度显著高于正常饮食对照组[（7.260 ±3.184） U/L比（2.962±0.845）U/L,P＜0.05],NASH＋ ML-7组门静脉中LPS浓度[（3.772±1.033） U/L]显著低于NASH组（P＜0.05）.NASH组外周血ALT及AST水平明显高于正常饮食对照组（P均＜0.05）,肝脏中炎性因子TNFα、IL-6浓度及TNFα、IFM、NF-κB的mRNA水平显著高于正常饮食对照组（P均＜0.05）;给予MLCK抑制剂后（NASH＋ ML-7组）,外周血ALT和AST水平、肝脏中TNFα和IL-6浓度、肝脏中TNFα和NF-κB的mRNA水平均显著低于正常饮食对照组（P均＜0.05）.结论 NASH阶段小鼠肠黏膜通透性增加,MLCK可能在其中具有十分重要的调节作用,抑制MLCK后可以有效降低实验动物肠黏膜通透性,从而防治NASH.

英文摘要：

Objective To investigate the role of myosin light chain kinase （MLCK） in intestinal barrier function in a mouse model with nonalcoholic steatohepatitis （NASH）.Methods The C57BI/6 mice were randomly divided into five groups including control group,nonalcoholic fatty liver （NAFL） group,NAFL administrated with MLCK inhibitor ML-7 group,nonalcoholic steatohepatitis （NASH） group,NASH administrated with ML-7 group.Plasma ALT and AST were tested.The degree of liver steatosis was assessed by hematoxylin-eosin staining on liver tissue sections.Intestinal mucosal tight junction was observed by electron microscope.The expression of MLCK on intestinal mucosa was detected by immunohistochemistry staining.The level of lipopolysaccharide （LPS） in portal vein was determined by enzyme linked immune sorbent assay （ELISA）.The protein and mRNA expression of inflammatory cytokines in liver tissue were tested using ELISA and real-time PCR.Results MLCK expression in intestinal mucosa was increased in NASH group compared with control group（P 〈 0.01）.The tight junctions of intestinal barrier were disrupted in NASH group and intercellular space was larger than control group [（26.60 ± 1.20） nm vs （14.90 ± 0.33）nm,P 〈 0.05],which were improved after ML-7 administration [（14.90 ± 0.67） nm].The LPS in portal vein was higher in NASH group than control group [（7.260 ±3.184） U/L vs （2.962 ±0.845） U/L,P 〈 0.05],suggesting that the permeability of intestinal barrier was impaired,however the level of LPS was reduced by ML-7 [（3.772 ± 1.033） U/L,P 〈 0.05].ALT and AST in plasma,TNFα and IL-6 in liver tissue,the mRNA levels of TNFα and NF-κB in liver tissue were all elevated in NASH group compared with control group （all P 〈 0.05）,which were reduced by MLCK inhibitor ML-7.Conclusion Epithelia MLCK probably plays a role in intestinal barrier impairment,which is critical to the pathogenesis of NASH.