基于网络药理学探索环黄芪醇治疗酒精性肝损伤的作用机制研究

Exploring the model of action of Cycloastragenol in treating alcoholic liver injury based on network pharmacology

目的基于网络药理学及细胞实验验证的方法探讨环黄芪醇治疗酒精性肝损伤的作用靶点及机制。方法运用SwissTargetPrediction数据库预测环黄芪醇的潜在作用靶点。于各疾病数据库中检索酒精性肝损伤疾病的相关靶点,提取共同靶点,利用蛋白相互作用数据库(String)以及Cytoscape 3.9.1软件建立“环黄芪醇-核心靶基因”网络,对其进行GO及KEGG富集分析以探究环黄芪醇治疗酒精性肝损伤可能的分子机制。两步灌流法分离C57BL/6小鼠原代肝细胞并采用“三明治”方法体外培养。通过CCK8、MTT和ROS法确定诱导小鼠肝原代细胞氧化应激的最佳乙醇浓度并探索环黄芪醇的最佳治疗浓度,并用试剂盒检测原代肝细胞中ALT、AST的含量。real-time PCR、Western Blot检测NF-κB p65、p-NF-κB p65、AMPK、Nrf2和HO-1等基因和蛋白的表达。结果网络药理学方法筛选除出环黄芪醇治疗酒精性肝损伤关键基因10个,包括热休克蛋白90AA1(HSP90AA1)、糖原合成酶激酶3(GSK3A、GSK3B)、丝裂原活化蛋白激酶1(MAPK1)等。KEGG富集分析预测环黄芪醇主要是通过IL-17信号通路(IL-17 signaling pathway)、趋化因子信号通路(Chemokine signaling pathway)、Rap1信号通路(Rap1 signaling pathway)、癌症信号通路(Prostate cancer、Bladder cancer)等发挥干预作用。体外实验结果显示,环黄芪醇可明显改善小鼠原代肝细胞状态,降低细胞中ALT、AST水平,下调p-NF-κB p65蛋白表达水平,上调AMPK、Nrf2、HO-1基因和蛋白表达水平,差异具有统计学意义(P<0.05)。结论环黄芪醇具有一定的保肝作用,其可能通过NF-κB和AMPK/Nrf2/HO-1信号通路改善酒精性肝损伤。

Objective To investigate the targets and the mechanism of cycloastragenol in the treatment of alcoholic liver injury based on network pharmacology and cell experiment validation.Methods The potential targets of cycloastragenol were predicted with the SwissTargetPrediction databases.The relevant targets of alcoholic liver injury were searched in various disease databases,and the common targets of drugs and diseases were extracted.The“cycloastragenol-core target gene”network was established by using protein-protein interaction(String database)and Cytoscape 3.9.1 software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed on the core targets to explore their possible molecular mechanisms.The primary hepatocytes of C57BL/6 mice were isolated by the two-step perfusion method and cultured in vitro by the“sandwich”method.The optimal ethanol concentration for inducing oxidative stress in mouse primary hepatocytes was determined by CCK8,MTT,and ROS methods,the optimal therapeutic concentration of cycloastragenol was explored,and the content of ALT and AST in primary hepatocytes was detected with a kit.real-time PCR and Western Blot detected the expression of genes and proteins such as NF-κB p65,p-NF-κB p65,AMPK,Nrf2,and HO-1.Results The network pharmacology approach identified 10 key targets for the treatment of alcoholic liver injury about cycloastragenol,including heat shock protein 90AA1(HSP90AA1),GSK3A,GSK3B,MAPK1,and so on.KEGG enrichment analysis predicted that the drug exerted intervention action through IL-17 signaling pathway,chemokine signaling pathway,Rap1 signaling pathway,cancer signaling pathway(prostate cancer,bladder cancer),etc.The results of in vitro primary hepatocytes experiments showed that cycloastragenol could significantly improve cell status,reduce ALT and AST levels in cells,down-regulate p-NF-κB p65 protein expression levels,and up-regulate AMPK,Nrf2,HO-1 genes and protein expression levels,with a statistically significant difference(P<0.05).Conclusions Cycloastragenol has certain hepatocyte protection effect,which may improve alcoholic liver injury in mice through the NF-κB and AMPK/Nrf2/HO-1 signaling pathway.