基于网络药理学研究连翘叶提取物通过AMPK抗恩诺沙星诱导肝损伤的作用

doi:10.16431/j.cnki.1671-7236.2022.06.035

摘要/Abstract

摘要： 【目的】运用网络药理学并通过体内试验研究连翘叶提取物（Forsythia suspense leaves extract，FSLE）治疗恩诺沙星（enrofloxacin，ENR）诱导的肝损伤（enrofloxacin induced liver injury，EILI）的作用机制。【方法】采用乙醇-酶法提取连翘叶有效成分，通过液相-质谱法（LC-MS）鉴定FSLE主要的化学成分。利用PubChem、Swiss ADME和Swiss Target Prediction数据库分析预测FSLE有效活性成分及作用靶点，通过GeneCards疾病数据库搜索EILI的作用靶点。运用STRING在线网站及Cytoscape 3.8.0软件构建FSLE治疗EILI的核心靶点，借助Metascape网站进行GO功能及KEGG通路富集分析，并用Cytoscape 3.8.0软件绘制"成分-靶点-信号通路"可视化网络。通过体内试验验证FSLE抗EILI的药理作用，采用实时荧光定量PCR和Western blotting检测不同处理组腺苷酸激活蛋白激酶（AMP-activated protein kinase，AMPK）的表达，通过商业化试剂盒检测还原型谷胱甘肽（glutathione，GSH）、过氧化氢（hydrogen peroxide，H₂O₂）和丙二醛（malondialdehyde，MDA）含量及总超氧化物歧化酶（superoxide dismutase，SOD）的活性。【结果】结合LC-MS与各数据库分析得出FSLE活性成分有20种，对应靶点338个；EILI疾病靶点913个；药物-疾病交集靶点124个，包含FSLE治疗EILI核心靶点12个，KEGG通路20条，其中AMPK通路与FSLE治疗EILI密切相关。动物试验结果显示，FSLE可显著降低EILI小鼠血清中谷丙转氨酶（alanine transferase，ALT）和谷草转氨酶（aspartate transferase，AST）活力（P<0.05）。FSLE可显著升高小鼠肝脏AMPK的基因和蛋白表达量水平（P<0.05）。与正常对照组相比，FSLE可显著降低小鼠肝脏组织中MDA和H₂O₂含量（P<0.05），显著升高GSH含量和SOD活力（P<0.05），抑制EILI诱导的肝脏氧化应激。【结论】 FSLE可通过上调AMPK的表达抑制肝脏氧化应激发挥抗EILI的作用，对肝损伤药物的开发具有重要的意义。

关键词: 连翘叶提取物; 恩诺沙星; 肝损伤; 网络药理学; AMPK

Abstract: 【Objective】 This study was aimed to explore the mechanism of Forsythia suspense leaves extract (FSLE) against enrofloxacin (ENR)-induced liver injury (EILI) by network pharmacology and in vivo experiments.【Method】 The Forsythia suspense leaves were extracted by ethanol-enzymatic method, whose main chemical components were identified by liquid chromatography mass spectrometry (LC-MS).The effective active ingredients and targets of FSLE were analyzed and selected by PubChem, Swiss ADME and Swiss Target Prediction database, while the EILI targets were screened by GeneCards disease database.STRING online analysis and Cytoscape 3.8.0 software were applied to analyze and construct the core targets of FSLE treats EILI.Metascape website was used to perform GO function and KEGG pathway enrichment analysis of the targets, and Cytoscape 3.8.0 software was hired to construct the "component-target-signal pathway" visual network.The protective effects of FSLE were verified in EILI mice in vivo.The expression of AMP-activated protein kinase (AMPK) was tested by Real-time PCR and Western blotting, and the contents of glutathione (GSH), hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and the activity of total superoxide dismutase (SOD) were detected by commercial kits.【Result】 About 20 oral ingredients in FSLE and their related 338 drug targets were analyzed and screened by LC-MS and Network pharmacology, while 913 protein targeting EILI, 124 protein targets which contained 12 core targets, were screened from drug-disease targets intersection.20 signing pathways were obtained by KEGG pathway enrichment analysis.KEGG pathway showed that AMPK signal pathway had a good affinity to EILI.The animal model experiments showed that FSLE could significantly reduce the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum of mice (P<0.05), and could significantly increase the expression of gene and protein of AMPK (P<0.05).Compared with the normal control group, FSLE could significantly reduce the contents of MDA and H₂O₂ in liver of EILI mice (P<0.05), and significantly increase the GSH content and SOD activity (P<0.05), which reduced the EILI-induced oxidative stress significantly.【Conclusion】 FSLE could protect the liver against ENR induced injury by inducing the expression of AMPK to reduce the oxidative stress, which was helpful for the development of new drugs targeting liver injury.

Key words: Forsythia suspense leaves extract; enrofloxacin; liver injury; network pharmacology; AMPK

中图分类号:

S853.74

安志霞, 潘阳阳, 范碧玥, 马小燕, 叶得河, 张治杰, 邱山桐, 姚亚乐, 王萌. 基于网络药理学研究连翘叶提取物通过AMPK抗恩诺沙星诱导肝损伤的作用[J]. 中国畜牧兽医, 2022, 49(6): 2347-2361.

AN Zhixia, PAN Yangyang, FAN Biyue, MA Xiaoyan, YE Dehe, ZHANG Zhijie, QIU Shantong, YAO Yale, WANG Meng. Study on the Effect of Forsythia suspensa Leaf Extract on Enrofloxacin-induced Liver Injury Through AMPK Based on Network Pharmacology[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2347-2361.

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