基于网络药理学和细胞实验探究健脾活血方干预AMPK和Hippo糖脂代谢信号通路治疗肝纤维化研究  

Mechanism of Jianpi Huoxue Decoction in the treatment of liver fibrosis by AMPK and Hippo glycolipid metabolism signaling pathways based on network pharmacology and cellular experiments

作　　者：黄迪 彭辉 李子义 胡高斌 黄龙[1] 张琦[1] 于庆生[1,2,3] HUANG Di;PENG Hui;LI Ziyi;HU Gaobin;HUANG Long;ZHANG Qi;YU Qingsheng(The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,Hefei 230031,China;Institute of Traditional Chinese Medicine Surgery,Anhui Academy of Traditional Chinese Medicine,Hefei 230031,China;Anhui Integrated Traditional Chinese and Western Medicine Hospital,Heifei 230031,China)

机构地区：[1]安徽中医药大学第一附属医院,安徽合肥230031 [2]安徽中医药研究院中医外科研究所,安徽合肥230031 [3]安徽省中西医结合医院,安徽合肥230031

出　　处：《海南医科大学学报》2025年第7期531-540,共10页Journal of Hainan Medical University

基　　金：十二五国家临床重点专科建设项目[脾脏与门静脉高压症专项,财社(2013)239号];国家中医药管理局全国名老中医药专家传承工作室[国中医药办人教函(2021)270号];安徽省“十四五”临床重点专科建设推荐计划[皖卫医秘(2022)105号]。

摘　　要：目的:网络药理学和实验探究健脾活血方干预肝纤维化的作用靶点及机制。方法:检索TCMSP获得健脾活血方各药物的活性成分;检索GeneCards、OMIM、TTD和DrugBank数据库预测肝纤维化疾病靶点;活性成分与靶点交集预测健脾活血方治疗肝纤维化靶点。运用Cytoscape 3.8.0构建“药物-成分-靶点-疾病”网络;运用STRING数据库以0.700为标准构建蛋白质-蛋白质互作网络;运用R软件进行GO富集和KEGG通路富集分析。实验选取人星状细胞LX-2培养,分为空白对照组、模型组[使用5ng转化生长因子-β1(transforming growth factor-beta 1,TGF-β1)诱导]和观察组(5 ng TGF-β1诱导+健脾活血方干预),CCK-8法检测中药干预细胞最佳浓度,免疫印迹法(Western Blot,WB)检测各组肝纤维化指标α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、金属基质蛋白酶组织抑制因子-1(tissue inhibitor of metalloproteinase-1,Timp-1)、结蛋白(Desmin)、胶原蛋白I(Collagen I)表达,WB检测各组AMPK和Hippo信号通路蛋白表达。结果:筛选出114个主要活性成分,涉及146个基因靶点,其中主要活性成分包括木樨草素、槲皮素、β-谷甾醇、当归素、白桦酸等;核心靶点基因包括TNF、IL-6、PTGS2、TP53、MAPK1、JUN、ESR1等;富集到GO生物过程661个、细胞组分467个、分子功能115个,包括AMPK、TNF、Hippo等KEGG信号通路164条。CCK-8结果表明0.625 mg/mL和1.25 mg/mL健脾活血方干预LX-2细胞增殖最佳;WB结果表明相比空白对照组,模型组α-SMA、Collagen I、Desmin、Timp-1表达增高,相比模型组,观察组α-SMA、Collagen I、Desmin、Timp-1表达降低(P<0.05);WB结果表明相比空白对照组,模型组p-YAP、p-TAZ和p-AMPK表达降低,YAP、TAZ、AMPK表达升高,相比模型组,观察组p-YAP、p-TAZ和p-AMPK表达增高,YAP、TAZ、AMPK表达降低(P<0.05)。结论:健脾活血方治疗肝纤维化的机制与健脾活血方多成分多靶点干预AMPK和Hippo信号通路等多通路相关Objective:To explore and verify the target and mechanism of Jianpi Huoxue Decoction in the intervention of liver fibrosis.Methods:The active components of Jianpi Huoxue Decoction were obtained by searching TCMSP,and the disease targets of liver fibrosis were obtained by searching GeneCards,OMIM,TTD and DrugBank databases,and then the intersection of active components and targets was used to obtain the predictive targets of Jianpi Huoxue Decoction in the treatment of liver fibrosis.Cytoscape 3.8.0 was used to construct the'drug-component-target-disease'network,the protein-protein interaction network was constructed by STRING database,and the core network was simplified;R software was used for GO enrichment and KEGG pathway enrichment analysis,and the components with higher degree values in the network were molecularly docked with the core targets.Human stellate cells LX-2 were cultured and divided into the blank control group,model group[induced by 5 ng transforming growth factor-beta 1(TGF-β1)]and observation group(5 ng TGF-β1 induction+Jianpi Huoxue Decoction intervention).CCK-8 method was used to detect the optimal concentration of Chinese medicine intervention cells,Western blot(WB)was used to detect the expression ofα-smooth muscle actin(α-SMA),tissue inhibitor of metalloproteinase-1(Timp-1),desmin,collagen I and AMPK and Hippo signaling pathway proteins in each group.Results:The results showed that 114 main active ingredients were screened out,involving 146 gene targets,including the main active ingredients such as luteolin,quercetin,β-sitosterol,angelica,betulinic acid,ect,the core target genes including TNF,IL⁃6,PTGS2,TP53,MAPK1,JUN,ESR1,etc.A total of 661 GO biological processes,467 cell components,and 115 molecular functions were enriched,including 164 KEGG signaling pathways such as fluid shear stress and atherosclerosis,AMPK,TNF,and Hippo.The results of CCK-8 showed that the proliferation of LX-2 cells was the best at 0.625 mg/mL and 1.25 mg/mL.The results of WB showed that compared with the con

关 键 词：健脾活血方 肝纤维化 网络药理学 AMPK/Hippo信号通路 

分 类 号：R285[医药卫生—中药学]