【摘要】 目的 探究表没食子儿茶素没食子酸酯（EGCG）对代谢相关脂肪性肝炎（MASH）体内外模型的影响及作用机制，为临床开发应用提供依据。方法 32只实验用C57BL/6J小鼠随机分为正常饮食组（Con,n=8）和模型组（n=24）。模型组小鼠使用高反式脂肪酸高糖（HFHC）饮食诱导24周，建立MASH模型，第24周末将模型组小鼠随机分为HFHC组、EGCG组(给药剂量为每只小鼠100 mg·kg^-1·d^-1)和奥贝胆酸用药组(给药剂量为每只小鼠10 mg·kg^-1·d^-1)（OCA组），每组8只。EGCG组和OCA组给药6周后收集标本，观察小鼠一般情况；检测肝组织甘油三酯（TG）含量，肝组织羟脯氨酸（HYP）含量，血清ALT、AST水平；使用HE染色、油红O染色、天狼星红染色观察肝组织病理学改变。体外实验采用游离脂肪酸（FFA）诱导L02细胞脂质沉积模型；设对照组（Con组）、FFA组和EGCG处理组（EGCG组）。检测细胞内TG含量、油红O染色以及TNF-α、CCL2、CXCL10 mRNA相对表达量。运用转录组学技术对Con组、HFHC组和EGCG组小鼠肝组织进行差异基因分析和基因集富集分析，挑选富集通路差异程度P-adjust<0.05且与MASH相关的通路进一步分类（代谢相关类和炎症类）分析，对两类通路中的具体信号通路按照富集程度分别从大到小排列，对排名前三信号通路中的关键基因进行体内PCR验证，并挑选NOD样受体信号通路中重要基因通过蛋白质印迹法验证。符合正态分布和方差齐性的计量资料，多组间比较采用单因素方差分析，进一步两两比较使用LSD-t检验。结果 与HFHC组相比，EGCG组小鼠肝组织TG含量显著下降（P<0.05）；血清ALT和AST显著降低（P值均<0.05）；油红O染色显示EGCG组小鼠肝细胞脂肪变性明显改善；HE结果显示EGCG可显著减轻炎症；天狼星红染色结果显示，EGCG给药之后纤维组织的数量明显减少；EGCG干预后肝组织HYP含量明显减少（P<0.01）。细胞实验结果发现，与FFA组相比，EGCG组TG含量显著降低，油红O结果显示EGCG组与FFA组相比脂滴明显消散，炎症因子TNF-α、CCL2和CXCL10 mRNA相对表达显著降低（P值均<0.01）。肝组织转录组学结果显示，HFHC组和EGCG组之间共有230个差异表达基因，其中上调基因108个，下调基因122个。EGCG可以显著降低小鼠肝组织中NOD样受体信号通路上关键蛋白TLR4、NLRP3、IL-1β的表达水平（P值均<0.05）。结论 EGCG可显著改善MASH小鼠模型脂质沉积、炎症和纤维化程度，改善肝细胞L02脂质沉积和炎症损伤，其机制可能与调控NOD样受体信号通路有关。更多还原

【Abstract】 Objective To investigate the effect and mechanism of action of epigallocatechin-3-gallate（EGCG） in the treatment of experimental metabolic dysfunction-associated steatohepatitis（MASH）, and to provide a basis for clinical development and application. Methods A total of 32 experimental C57BL/6J mice were randomly divided into normal diet group（Con group with 8 mice） and model group with 24 mice. The mice in the model group were given a high-trans fatty acid high-carbohydrate（HFHC） diet for 24 weeks to establish a model of MASH, and at the end of week 24, the mice in the model group were further divided into HFHC group, EGCG treatment group(100 mg·kg^-1·d^-1), and obeticholic acid treatment group(10 mg·kg^-1·d^-1), with 8 mice in each group. After 6 weeks of treatment, samples were collected to observe the general conditions of mice; the content of triglycerides（TG） and hydroxyproline in liver tissue and the serum levels of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were measured; HE staining, oil red O staining, and picrosirius red staining were used to observe liver histopathological changes. In the in vitro experiment, L02 cells were induced with free fatty acid（FFA） to establish a model of lipid deposition, and the cells were divided into Con group, FFA group, and EGCG group. The content of TG in cells was measured, as well as the results of oil red O staining and the relative mRNA expression levels of TNF-α, CCL2, and CXCL10. The transcriptomics technique was used to identify differentially expressed genes between the Con group, the HFHC group, and the EGCG group and perform the GSEA analysis, and pathways with a P-adjust value of <0.05 that were associated with MASH were further classified into metabolism-related pathways and inflammation-related pathways. The specific signaling pathways in each category were ranked based on the degree of enrichment, and key genes in the top three pathways were verified by PCR in vivo. Key genes in the NOD-like receptor signaling pathway were verified by Western blotting. A one-way analysis of variance was used for comparison of normally distributed continuous data with homogeneity of variance between multiple groups, and the least significant difference ttest was used for further comparison between two groups. Results Compared with the HFHC group, the EGCG group had significant reductions in the content of TG in liver tissue（P<0.05） and the serum levels of ALT and AST（P<0.05）. Oil red O staining showed significant alleviation of hepatocyte fatty degeneration in the EGCG group, HE staining showed that EGCG effectively alleviated inflammation, and picrosirius red staining showed a significant reduction in the number of fibrous tissue after EGCG treatment. There was a significant reduction in the content of hydroxyproline in liver tissue after EGCG intervention（P<0.01）. Cell experiments showed that compared with the FFA group, the EGCG group had a significant reduction in the content of TG, and oil red O staining showed the disappearance of lipid droplets in the EGCG group compared with the FFA group, with significant reductions in the relative mRNA expression levels of the inflammatory factors TNF-α, CCL2, and CXCL10（all P<0.01）. The transcriptomics analysis identified 230 differentially expressed genes between the HFHC group and the EGCG group, among which there were 108 upregulated genes and 122 downregulated genes. EGCG significantly reduced the levels of the key proteins TLR4, NLRP3, and IL-1β in the NOD-like receptor signaling pathway in liver tissue（all P<0.05）. Conclusion EGCG can significantly alleviate lipid deposition, inflammation, and fibrosis in the mouse model of MASH and improve lipid deposition and inflammatory injury in L02 cells, possibly by regulating the NOD-like receptor signaling pathway.更多还原