雷公藤红素减轻肝脏炎性损伤网络靶标预测及实验验证

Prediction and experimental verification of network target of celastrol in alleviating hepatic inflammatory injuries

目的运用网络药理学方法探讨雷公藤红素减轻肝脏炎症性损伤的作用靶点及机制,并采用缺血再灌注致肝脏炎症小鼠模型对预测靶点进行实验验证。方法检索SymMap、BATMAN-TCM、TCMSP、HIT 2.0、LigTMap、SEA、SwissTarget、Super-PRED、STITCH数据库获得雷公藤红素靶点,检索GeneCards、DisGeNET数据库获得肝脏炎症性疾病靶点。通过绘制Venn图对药物靶点和疾病靶点取交集,获取雷公藤红素减轻肝脏炎症性损伤的可能靶点。利用STRING数据库构建PPI网络,并利用Cytoscape 3.9.1软件分析关键靶点,通过DAVID数据库进行GO功能和KEGG通路富集分析;基于关键靶点,检索starBase数据库并绘制竞争性内源RNA(ceRNA)网络。对雷公藤红素与核心治疗靶点进行分子对接。将小鼠按体重分为假手术溶剂组,假手术给药组,模型组,雷公藤红素低(0.1 mg/kg)、中(0.3 mg/kg)、高(1 mg/kg)剂量组,每组3~4只。相应药物干预7 d后,除假手术溶剂组、假手术给药组外,其余各组制备缺血再灌注致肝脏炎症小鼠模型。检测小鼠血清转氨酶水平,采用HE染色观察小鼠肝组织病理学形态,采用免疫组化染色检测肝组织IL-6、TNF-α表达。结果雷公藤红素减轻肝脏炎症关键靶点为IL6、TNF等炎症因子,功能富集结果分析显示,雷公藤红素减轻肝脏炎性损伤关键信号通路包含炎症反应、细胞凋亡和增殖、HIF1等通路。雷公藤红素预处理可降低肝缺血再灌注致肝脏炎症小鼠血清转氨酶水平(P<0.01),下调肝组织IL-6、TNF-α炎症因子表达(P<0.05或P<0.01)。结论雷公藤红素可减轻肝缺血再灌注损伤,其作用机制与降低IL-6、TNF-α等炎症因子水平、减轻肝脏炎性损伤密切相关。

Objective To investigate the action targets and mechanism of celastrol in alleviating liver inflammatory injuries using network pharmacology;To verify the key targets through liver ischemia-reperfusion injury inducing inflammation mouse model.Methods The targets of celastrol were integrated by searching SymMap,BATMAN-TCM,TCMSP,HIT 2.0,LigTMap,SEA,SwissTarget,Super-PRED,STITCH databases.The molecular targets of hepatic inflammatory injuries were investigated by GeneCards and DisGeNET databases.The intersection of drug targets and disease targets was obtained by Venn diagram to obtain the possible targets of celastrol in alleviating hepatic inflammatory diseases.Cytoscape 3.9.1 software was used to analyze the key targets of protein interaction(PPI)networks based on STRING database,and enrichment analysis was conducted through DAVID database.Based on the key targets,the ceRNA network was mapped by retrieving the starBase database.Molecular docking was used to evaluated the binding of celastrol with the key target proteins.The mice were divided into sham-operation solvent group,sham-operation medication group,model group,and Tripterygium wilfordii extract low-(0.1 mg/kg),medium-(0.3 mg/kg),and high-(1 mg/kg)dosage groups according to body weight,with 3-4 mice in each group.After 7 days of corresponding drug intervention,except for the sham-operation solvent group and sham-operation medication group,other groups were prepared with ischemia-reperfusion induced liver inflammation mouse models.The serum transaminase levels in mice were detected;the pathological morphology of mouse liver tissue was observed using HE staining;the expressions of IL-6 and TNF-αwere detected in liver tissue using immunohistochemistry staining.Results The key targets of celastrol in alleviating liver inflammation were inflammatory cytokines such as IL6 and TNF.The analysis of functional enrichment results showed that the key signaling pathways of Tripterygium wilfordii extract in reducing liver inflammatory injury included inflammatory response,cell apoptosis and proliferation,HIF1,and other pathways.Triptolide pretreatment could reduce serum aminotransferase level(P<0.01)and liver inflammatory factors expression such as IL-6 and TNF-α(P<0.05,P<0.01)after hepatic ischemia-reperfusion.Conclusion Celastrol can alleviate hepatic ischemia-reperfusion injury,and its mechanism is closely related to the reduction of inflammatory factors such as IL-6 and TNF-αand the alleviation of hepatic inflammatory injury.