目的 应用网络药理学和分子对接方法预测二至丸治疗代谢相关脂肪性肝病的相关作用靶点以及潜在的作用机制,并进行实验验证。方法 检索中药系统药理学数据库与分析平台(TCMSP)筛选出二至丸的有效成分以及作用靶点,通过检索GEO数据库得到代谢相关脂肪性肝病的相关差异基因。利用Cytoscape 3.7.2软件进行调控网络以及蛋白相互作用(PPI)网络的构建,利用R包开展基因本体(GO)和京都基因和基因组百科全书(KEGG)分析,并利用AutoDockTools软件进行分子对接验证。分别用不同浓度(0.1、0.5、1、5、10、50、100、200 μg/mL)二至丸醇提物培养液作用于肝细胞L02,根据吸光度(A)值,计算细胞增殖率。将肝细胞L02分为对照组、模型组、二至丸醇提物各剂量(1、2、4 μg/mL)组,采用油红O染色法,通过显微镜进一步观察各组细胞内的脂滴堆积情况。采用酶标仪测定各组细胞内总三酰甘油(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)、天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的含量,并以qPCR法检测肝细胞L02白细胞介素-6(IL-6)和JUN mRNA表达情况。结果 共筛选出二至丸治疗代谢相关脂肪性肝病11个主要的靶点基因和12个活性成分,GO功能富集提示治疗代谢相关脂肪性肝病的生物学过程主要有急性期反应、调节血管生成、神经炎症反应的调节等;KEGG通路主要涉及晚期糖基化终末化产物(AGE)-晚期糖基化终末产物受体(RAGE)信号通路外,还作用于C型凝集素受体信号通路、白细胞介素(IL)-17信号通路、肿瘤坏死因子(TNF)信号通路和Toll样受体信号通路等;分子对接验证结果显示,木犀草素、槲皮素、β-类固醇、山柰酚与转录因子AP-1(JUN)、IL-6能稳定结合。实验表明,二至丸可以显著改善代谢相关脂肪性肝病模型的肝细胞L02的脂质堆积以及炎症反应。结论 二至丸2味药中的木犀草素、槲皮素、β-类固醇、山柰酚在抗氧化应激、炎症相关通路下,作用于前列腺素内过氧化物酶2(PTGS2)、透明质酸合酶2(HAS2)、JUN、IL-6等靶点发挥对代谢相关脂肪性肝病的治疗作用。

Objective To predicted the relevant targets and potential mechanisms of action of Erzhi Pills in treatment of metabolic-dysfunction-associatedfatty liver disease by network pharmacology and molecular docking methods. Methods The active components and targets of Erzhi Pills were screened by searching the TCM Pharmacology Database and Analysis Platform (TCMSP), and the differential genes related to metabolic-dysfunction-associatedfatty liver disease were obtained by searching GEO database. Cytoscape 3.7.2 software was used to construct regulatory networks and protein interaction networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out using R package, and molecular docking verification was performed using AutoDockTools software. Hepatocyte L02 was treated with alcohol extract medium with different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 200 μg/mL) of Erzhi Pills, and cell proliferation rate was calculated according to the value of absorbance (A). Hepatocytes L02 were divided into control group, model group, and Erzhi Pills alcohol extract (1, 2, 4 μg/mL) groups. Oil red O staining method was used to observe the accumulation of lipid droplets in each group. The contents of total triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-C), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in each group were determined by enzyme-labeled instrument. mRNA expressions of L02 interleukin-6 (IL-6) and JUN were detected by qPCR. Results Eleven potential target genes and 12 active components were screened out for Erzhi Pills in treatment of metabolic-dysfunction-associatedfatty liver disease GO enrichment suggested that the biological process of treatment for metabolic-dysfunction-associatedfatty liver disease mainly includes acute phase reaction, regulation of angiogenesis, and regulation of neuroinflammatory response. KEGG pathway mainly involves AGE- RAGE signaling pathway, C-type lectin receptor signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway, etc. Molecular docking verification results showed that luteolin, quercetin, β-steroid, kaempferol could stably bind JUN and IL-6. The experiment showed that Erzhi Pills can significantly improve the lipid accumulation and inflammatory response of liver cells L02 in the model of metabolic-dysfunction-associatedfatty liver disease. Conclusion Luteolin, quercetin, β-steroid, and kaempferol in Erzhi Pills on the targets of peroxidase 2 (PTGS2), hyaluronic acid synthase 2 (HAS2), transcription factor AP-1 (JUN), and IL-6 in the anti-oxidative stress and inflammation-related pathways to exert the therapeutic effect on metabolic-dysfunction-associatedfatty liver disease.

R975

天津中医药大学中西医结合学院2020年度研究生创新基金(ZXYCXLX202017)