PDF下载 ( 4089 KB)
表没食子儿茶素没食子酸酯治疗实验性代谢相关脂肪性肝炎的效果及机制分析
DOI: 10.12449/JCH250716
伦理学声明：本研究方案于2017年10月20日经由上海中医药大学实验动物伦理委员会审批，批号：SZY201710017，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：徐笑负责设计论文框架，起草论文；张倩负责实验操作，研究过程的实施；辛鑫负责数据收集，统计学分析，绘制图表；冯琴、辛鑫、孙沁梅负责论文修改；冯琴、胡义扬负责拟定写作思路；冯琴、辛鑫指导撰写文章并最后定稿。
Efficacy and mechanism of epigallocatechin-3-gallate in treatment of experimental metabolic dysfunction-associated steatohepatitis
-
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。 -
关键词:
- 代谢相关脂肪性肝炎 /
- 表没食子儿茶素没食子酸酯 /
- 药理作用分子作用机制 /
- 信号传导
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 t-test 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. -
注： a，小鼠大体和肝脏形态肉眼观；b，小鼠30周末体质量和肝湿重；c，肝组织病理染色；d，肝组织TG含量、NAS评分和HYP含量；e，血清ALT和AST水平。
图 1 EGCG对HFHC饮食诱导MASH小鼠肝脂质沉积、炎症及纤维化程度的影响
Figure 1. Effect of EGCG on HFHC diet-induced liver lipid deposition， inflammation， and fibrosis in MASH mice
注： a，L02细胞生存率及L02细胞内TG含量；b，细胞油红O染色；c，各组细胞TNF-α、CCL2和CXCL10 mRNA相对表达量。
图 2 EGCG对FFA诱导L02细胞模型脂质沉积和炎症程度的影响
Figure 2. Effect of EGCG on free fatty acid-induced lipid deposition and degree of inflammation in the L02 cell model
图 3 HFHC组与EGCG组主成分分析图、差异基因聚类热图、表达量差异火山图（n=5）
Figure 3. PCA plot， differentially expressed gene clustering heatmap， and volcano plot of expression differences between HFHC and EGCG groups（n=5）
图 4 代谢相关通路GSEA曲线及部分基因验证
Figure 4. GSEA curves of metabolism-related pathways and partial gene validation
图 5 炎症相关通路GSEA曲线及部分基因验证
Figure 5. GSEA curves of inflammation-related pathways and partial gene validation
-
[1] SCHWÄRZLER J, GRABHERR F, GRANDER C, et al. The pathophysiology of MASLD: An immunometabolic perspective[J]. Expert Rev Clin Immunol, 2024, 20( 4): 375- 386. DOI: 10.1080/1744666X.2023.2294046. [2] WONG VW, EKSTEDT M, WONG GL, et al. Changing epidemiology, global trends and implications for outcomes of NAFLD[J]. J Hepatol, 2023, 79( 3): 842- 852. DOI: 10.1016/j.jhep.2023.04.036. [3] TARGHER G, BYRNE CD, TILG H. MASLD: A systemic metabolic disorder with cardiovascular and malignant complications[J]. Gut, 2024, 73( 4): 691- 702. DOI: 10.1136/gutjnl-2023-330595. [4] LI F, LI MW, WANG YS. Therapeutic paradigms and potential therapies for nonalcoholic steatohepatitis[J]. J Clin Hepatol, 2024, 40( 10): 2082- 2086. DOI: 10.12449/JCH241025.李凤, 李茂微, 王雨杉. 非酒精性脂肪肝病的治疗模式和潜在疗法[J]. 临床肝胆病杂志, 2024, 40( 10): 2082- 2086. DOI: 10.12449/JCH241025. [5] YOUNOSSI ZM, STEPANOVA M, RACILA A, et al. Health-related quality of life(HRQL) assessments in a 52-week, double-blind, randomized, placebo-controlled phase 3 study of resmetirom(MGL-3196) in patients with metabolic dysfunction associated steatohepatitis(MASH) and fibrosis[J]. Hepatology, 2025, 81( 4): 1318- 1327. DOI: 10.1097/HEP. 0000000000001084. [6] SHIMOTOYODOME A, HARAMIZU S, INABA M, et al. Exercise and green tea extract stimulate fat oxidation and prevent obesity in mice[J]. Med Sci Sports Exerc, 2005, 37( 11): 1884- 1892. DOI: 10.1249/01.mss.0000178062.66981.a8. [7] YE Q, LIU Y, CHEN JP, et al. Research progress on chemical constituents and pharmacological activities of green tea[J]. Drug Eval Res, 2021, 44( 12): 2711- 2719. DOI: 10.7501/j.issn.1674-6376.2021.12.026.叶晴, 刘毅, 陈金鹏, 等. 绿茶化学成分及药理作用研究进展[J]. 药物评价研究, 2021, 44( 12): 2711- 2719. DOI: 10.7501/j.issn.1674-6376.2021.12.026. [8] SINGH BN, SHANKAR S, SRIVASTAVA RK. Green tea catechin, epigallocatechin-3-gallate(EGCG): Mechanisms, perspectives and clinical applications[J]. Biochem Pharmacol, 2011, 82( 12): 1807- 1821. DOI: 10.1016/j.bcp.2011.07.093. [9] HE MJ, CHU TH, WANG ZT, et al. Inhibition of macrophages inflammasome activation via autophagic degradation of HMGB1 by EGCG ameliorates HBV-induced liver injury and fibrosis[J]. Front Immunol, 2023, 14: 1147379. DOI: 10.3389/fimmu.2023.1147379. [10] CZECH TY, SEKI E. Kupffer cell TLR2/3 signaling: A pathway for EGCG amelioration of ethanol-induced hepatic injury[J]. Cell Mol Gastroenterol Hepatol, 2020, 9( 1): 187- 188. DOI: 10.1016/j.jcmgh.2019.10.001. [11] LIN YX, HUANG J, GAO TF, et al. Preliminary study on hepatoprotective effect and mechanism of(-)-epigallocatechin-3-gallate against acetaminophen-induced liver injury in rats[J]. Iran J Pharm Res, 2021, 20( 3): 46- 56. DOI: 10.22037/ijpr.2020.112727.13918. [12] TANG GY, XU Y, ZHANG C, et al. Green tea and epigallocatechin gallate(EGCG) for the management of nonalcoholic fatty liver diseases(NAFLD): Insights into the role of oxidative stress and antioxidant mechanism[J]. Antioxidants(Basel), 2021, 10( 7): 1076. DOI: 10.3390/antiox10071076. [13] KLEINER DE, BRUNT EM, van NATTA M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease[J]. Hepatology, 2005, 41( 6): 1313- 1321. DOI: 10.1002/hep.20701. [14] OHISHI T, GOTO S, MONIRA P, et al. Anti-inflammatory action of green tea[J]. Antiinflamm Antiallergy Agents Med Chem, 2016, 15( 2): 74- 90. DOI: 10.2174/1871523015666160915154443. [15] ALAM M, ALI S, ASHRAF GM, et al. Epigallocatechin 3-gallate: From green tea to cancer therapeutics[J]. Food Chem, 2022, 379: 132135. DOI: 10.1016/j.foodchem.2022.132135. [16] MASTERJOHN C, BRUNO RS. Therapeutic potential of green tea in nonalcoholic fatty liver disease[J]. Nutr Rev, 2012, 70( 1): 41- 56. DOI: 10.1111/j.1753-4887.2011.00440.x. [17] DING Y, SUN X, CHEN YN, et al. Epigallocatechin gallate attenuated non-alcoholic steatohepatitis induced by methionine- and choline-deficient diet[J]. Eur J Pharmacol, 2015, 761: 405- 412. DOI: 10.1016/j.ejphar.2015.05.005. [18] LIU JJ, SUN JY, YU JK, et al. Gut microbiome determines therapeutic effects of OCA on NAFLD by modulating bile acid metabolism[J]. NPJ Biofilms Microbiomes, 2023, 9( 1): 29. DOI: 10.1038/s41522-023-00399-z. [19] YOUNOSSI ZM, RATZIU V, LOOMBA R, et al. Obeticholic acid for the treatment of non-alcoholic steatohepatitis: Interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial[J]. Lancet, 2019, 394( 10215): 2184- 2196. DOI: 10.1016/S0140-6736(19)33041-7. [20] HOU HM, YANG WL, BAO SQ, et al. Epigallocatechin gallate suppresses inflammatory responses by inhibiting toll-like receptor 4 signaling and alleviates insulin resistance in the livers of high-fat-diet rats[J]. J Oleo Sci, 2020, 69( 5): 479- 486. DOI: 10.5650/jos.ess19303. [21] XIAO J, HO CT, LIONG EC, et al. Epigallocatechin gallate attenuates fibrosis, oxidative stress, and inflammation in non-alcoholic fatty liver disease rat model through TGF/SMAD, PI3K/Akt/FoxO1, and NF-kappa B pathways[J]. Eur J Nutr, 2014, 53( 1): 187- 199. DOI: 10.1007/s00394-013-0516-8. [22] LIN QW, ZHANG S, LU WQ. Research progress of NOD-like signaling pathways and the relationship between NOD and tumor[J]. China Oncol, 2019, 29( 3): 223- 228. DOI: 10.19401/j.cnki.1007-3639.2019.03.011.林巧卫, 张思, 陆维祺. NOD样受体介导的信号转导通路及其与肿瘤关系的研究进展[J]. 中国癌症杂志, 2019, 29( 3): 223- 228. DOI: 10.19401/j.cnki.1007-3639.2019.03.011. [23] BAUER S, HEZINGER L, REXHEPI F, et al. NOD-like receptors-emerging links to obesity and associated morbidities[J]. Int J Mol Sci, 2023, 24( 10): 8595. DOI: 10.3390/ijms24108595. [24] LI HX, CAO ZQ, WANG LL, et al. Chronic high-fat diet induces galectin-3 and TLR4 to activate NLRP3 inflammasome in NASH[J]. J Nutr Biochem, 2023, 112: 109217. DOI: 10.1016/j.jnutbio.2022.109217. [25] ZHENG D, KERN L, ELINAV E. The NLRP6 inflammasome[J]. Immunology, 2021, 162( 3): 281- 289. DOI: 10.1111/imm.13293. [26] HUANG CY, LIU QH, TANG Q, et al. Hepatocyte-specific deletion of Nlrp6 in mice exacerbates the development of non-alcoholic steatohepatitis[J]. Free Radic Biol Med, 2021, 169: 110- 121. DOI: 10.1016/j.freeradbiomed.2021.04.008. [27] HASEGAWA M, FUJIMOTO Y, LUCAS PC, et al. A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation[J]. EMBO J, 2008, 27( 2): 373- 383. DOI: 10.1038/sj.emboj.7601962. -
下载:
本文二维码
图(6)
计量
- 文章访问数: 390
- HTML全文浏览量: 149
- PDF下载量: 32
- 被引次数: 0
