目的 應用網(wǎng)絡藥理學和分子對接方法預測二至丸治療代謝相關(guān)脂肪性肝病的相關(guān)作用靶點以及潛在的作用機制,并進行實驗驗證。方法 檢索中藥系統(tǒng)藥理學數(shù)據(jù)庫與分析平臺(TCMSP)篩選出二至丸的有效成分以及作用靶點,通過檢索GEO數(shù)據(jù)庫得到代謝相關(guān)脂肪性肝病的相關(guān)差異基因。利用Cytoscape 3.7.2軟件進行調(diào)控網(wǎng)絡以及蛋白相互作用(PPI)網(wǎng)絡的構(gòu)建,利用R包開展基因本體(GO)和京都基因和基因組百科全書(KEGG)分析,并利用AutoDockTools軟件進行分子對接驗證。分別用不同濃度(0.1、0.5、1、5、10、50、100、200 μg/mL)二至丸醇提物培養(yǎng)液作用于肝細胞L02,根據(jù)吸光度(A)值,計算細胞增殖率。將肝細胞L02分為對照組、模型組、二至丸醇提物各劑量(1、2、4 μg/mL)組,采用油紅O染色法,通過顯微鏡進一步觀察各組細胞內(nèi)的脂滴堆積情況。采用酶標儀測定各組細胞內(nèi)總?cè)８视?TG)、總膽固醇(TC)、高密度脂蛋白(HDL-C)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、丙氨酸氨基轉(zhuǎn)移酶(ALT)的含量,并以qPCR法檢測肝細胞L02白細胞介素-6(IL-6)和JUN mRNA表達情況。結(jié)果 共篩選出二至丸治療代謝相關(guān)脂肪性肝病11個主要的靶點基因和12個活性成分,GO功能富集提示治療代謝相關(guān)脂肪性肝病的生物學過程主要有急性期反應、調(diào)節(jié)血管生成、神經(jīng)炎癥反應的調(diào)節(jié)等;KEGG通路主要涉及晚期糖基化終末化產(chǎn)物(AGE)-晚期糖基化終末產(chǎn)物受體(RAGE)信號通路外,還作用于C型凝集素受體信號通路、白細胞介素(IL)-17信號通路、腫瘤壞死因子(TNF)信號通路和Toll樣受體信號通路等;分子對接驗證結(jié)果顯示,木犀草素、槲皮素、β-類固醇、山柰酚與轉(zhuǎn)錄因子AP-1(JUN)、IL-6能穩(wěn)定結(jié)合。實驗表明,二至丸可以顯著改善代謝相關(guān)脂肪性肝病模型的肝細胞L02的脂質(zhì)堆積以及炎癥反應。結(jié)論 二至丸2味藥中的木犀草素、槲皮素、β-類固醇、山柰酚在抗氧化應激、炎癥相關(guān)通路下,作用于前列腺素內(nèi)過氧化物酶2(PTGS2)、透明質(zhì)酸合酶2(HAS2)、JUN、IL-6等靶點發(fā)揮對代謝相關(guān)脂肪性肝病的治療作用。

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

天津中醫(yī)藥大學中西醫(yī)結(jié)合學院2020年度研究生創(chuàng)新基金(ZXYCXLX202017)