目的 探讨肠道菌群、肠组织中内质网应激蛋白GRP78（glucose-regulated protein 78）、模式识别受体TLR4（toll-like receptors 4）表达水平的改变在大鼠肝硬化发生发展中的作用。 方法 采用复合致病因素法诱导大鼠肝硬化。HE染色观察肝与小肠病理学改变，ARISA（automated ribosomal intergenic-spacer analysis）分析回肠内容物肠道菌群组成，培养法检测各组动物细菌易位情况，ELISA（enzyme-linked immunosorbent assay）测定血浆生化指标，quantitative real-time PCR、Western blotting检测肝、肠组织GRP78、TLR4的表达。 结果 肝硬化模型动物肝、肠组织病理学改变明显。随着肝硬化的进展，模型动物血浆中ALT（alanine aminotransferase）、内毒素和Hcy（homocysteine）的水平逐渐增高。模型组与对照组间肠道菌群的组间相似性明显降低。模型组发生细菌易位的大鼠数量、易位率显著升高。模型组动物肝、肠组织中GRP78的表达水平均随病程进展显著升高；肝组织中TLR4的表达水平在6周之前随病程进展显著升高，8周仍明显高于正常动物，但低于6周时的表达水平；肠组织中TLR4的表达水平在6周之前随病程进展显著升高，8周则明显降低。 结论 肠道微生物群落结构的生态失调、应激水平升高、免疫功能下降，导致肠道细菌易位，加重肝损伤，促进肝硬化形成。
Abstract
Objective To study the effects of gut microbiota, glucose-regulated protein 78 (GRP78) and toll-like receptors 4 (TLR4) on the development of liver cirrhosis in rats. Methods Thirty-six male SD rats were randomly divided into a normal control group (N) and a hepatic cirrhosis model group (M). The liver cirrhosis was induced by compound factors. Each group was sampled at 4, 6 and 8 week time points. Injuries of liver and intestinal mucosa were assessed with H&E stain. The gut microbiota was examined with automated ribosomal intergenic-spacer analysis (ARISA) on fecal DNA, and the bacterial translocation was assessed by standard microbiological techniques on blood, mesenteric lymph nodes (MLN), ascites and liver. The levels of alanine aminotransferase (ALT), endotoxin and homocysteine (Hcy) in the plasma were detected by enzyme-linked immunosorbent assay (ELISA). The expression of GRP78 and TLR4 was analyzed using quantitative real-time PCR and Western blotting. Results Compared to group N, the pathologic change of liver and small intestine was obvious in group M, and the levels of ALT, endotoxin, Hcy in the plasma were significantly and gradually increased. The occurrence of bacterial translocation (BT) was increased in the liver of group M compared to that of group N. BT was not detected in the blood in any group. The expression of GRP78 was significantly and gradually increased in the tissues of liver and ileum in group M. In liver homogenate, the expression of TLR4 in group M was significantly and gradually increased in 6 weeks, and significantly higher than group N at 8 weeks, but lower than group M at 6 weeks. In ileal homogenate, the expression of TLR4 in group M was also significantly and gradually increased in 6 weeks, but was significantly decreased than group N at 8 weeks. Conclusion With the progress of liver cirrhosis, the dysbiosis of gut microbiota becomes more and more serious, the expression of GRP78 increases significantly and the expression level of TLR4 decreases significantly in ileal homogenate, which may result in the gradual increase of the intestinal mucosal barrier damage, and cause significant increase of the occurrence of BT and level of endotoxin, eventually affecting the expression of GRP78 and TLR4 in liver, and worsening liver injury then.
关键词
肝硬化 /
肠道菌群 /
细菌易位 /
GRP78 /
TLR4
Key words
Hepatic cirrhosis /
Gut microbiota /
Bacterial translocation /
GRP78 /
TLR4
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基金
国家自然科学基金(81070339)；山西省自然科学基金(201601D011092)；山西省卫生计生委科研课题(2017156)；山西省高等学校大学生创新创业训练计划项目(2017313)；长治医学院创新团队资助经费项目(CX201508)；长治医学院普及项目(QDZ201631)
京ICP备08002354号-3
