[肌球蛋白轻链激酶参与非酒精性脂肪性肝炎小鼠模型肠道黏膜屏障功能的改变]

[Myosin light chain kinase involved in change of intestinal mucosal barrier function in nonalcoholic steatohepatitis mice model].

作者信息

Zhang Yuanyuan, Li Jing, Chi Yujing, Li Mei, Pan Xiuying, Zhang Qi, He Xiangjun, Liu Yulan

机构信息

Department of Gastroenterology, Peking University People's Hospital, Beijing 100044, China; Email:

出版信息

Zhonghua Nei Ke Za Zhi. 2015 May;54(5):434-8.

PMID:26080824

Abstract

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 C57BL/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 [(14.9 0 ± 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.

摘要

目的

在非酒精性脂肪性肝炎（NASH）小鼠模型中研究肌球蛋白轻链激酶（MLCK）在肠道屏障功能中的作用。

方法

将C57BL/6小鼠随机分为五组，包括对照组、非酒精性脂肪肝（NAFL）组、给予MLCK抑制剂ML-7的NAFL组、非酒精性脂肪性肝炎（NASH）组、给予ML-7的NASH组。检测血浆谷丙转氨酶（ALT）和谷草转氨酶（AST）。通过对肝组织切片进行苏木精-伊红染色评估肝脏脂肪变性程度。用电子显微镜观察肠道黏膜紧密连接。通过免疫组织化学染色检测肠道黏膜上MLCK的表达。采用酶联免疫吸附测定（ELISA）法测定门静脉中脂多糖（LPS）水平。用ELISA和实时荧光定量聚合酶链反应（PCR）检测肝组织中炎性细胞因子的蛋白和mRNA表达。

结果

与对照组相比，NASH组肠道黏膜中MLCK表达增加（P<0.01）。NASH组肠道屏障紧密连接被破坏，细胞间隙大于对照组[（26.60±1.20）nm对（14.90±0.33）nm，P<0.05]，给予ML-7后有所改善[（14.90±0.67）nm]。NASH组门静脉中LPS高于对照组[（7.260±3.184）U/L对（2.962±0.845）U/L，P<0.05]，提示肠道屏障通透性受损，而ML-7使LPS水平降低[（3.772±1.033）U/L，P<0.05]。与对照组相比，NASH组血浆中ALT和AST、肝组织中肿瘤坏死因子α（TNFα）和白细胞介素6（IL-6）、肝组织中TNFα和核因子κB（NF-κB）的mRNA水平均升高（均P<0.05），MLCK抑制剂ML-7使其降低。