基于网络药理学、分子对接和实验验证探讨肉苁蓉治疗肝纤维化的作用机制

Study on mechanism of Cistanches Herba in the treatment of liver fibrosis based on network pharmacology,molecular docking and experimental verification

从药物、疾病、靶点角度研究肉苁蓉中活性成分治疗肝纤维化的作用机制。通过中药数据库(TCMSP)、中医药百科全书数据库(ETCM)、中医药研究综合数据库(TCMID)、CNKI数据库、PubMed等数据库收集肉苁蓉的有效成分,利用Uniprot数据库获取肉苁蓉活性成分对应的靶标基因,通过GeneCards数据库和OMIM数据库收集肝纤维化疾病基因做映射,将有效成分靶点与肝纤维化靶点取交集,并绘制中药-有效成分-疾病网络;通过String数据库及Cytoscape 3.7.1软件构建蛋白相互作用网络(PPI);通过DAVID数据库对交集靶点进行GO功能富集与KEGG通路富集;建立动物肝纤维化模型,采用ELISA试剂盒检测小鼠肝组织中LN、HA、PCIII、Col IV含量变化,并进行HE和Masson染色观察肝病理组织学变化,通过实时荧光定量PCR法检测PI3K、AKT mRNA的转录情况。收集并筛选肉苁蓉有效成分8个,靶点157个,肝纤维化疾病靶点1 005个,二者交集靶点92个,涉及5条主要信号通路,包括癌症信号通路、乙肝病毒信号通路、PI3K-AKT信号通路、肿瘤坏死因子信号通路、蛋白多糖与癌症。动物实验表明:与模型组相比,肉苁蓉苯乙醇总苷高、中、低剂量组(700、350、175 mg/kg)能显著降低肝纤维化小鼠肝组织LN、HA、PCIII、Col IV含量(P<0.01),肝组织病理切片观察发现肉苁蓉苯乙醇总苷能减少肝细胞损伤及胶原纤维沉积,通过实时荧光定量PCR检测表明:与正常组相比,模型组小鼠肝组织中PI3K、AKT的mRNA表达量显著上调;与模型组相比,肉苁蓉苯乙醇总苷高、中、低剂量组中PI3K、AKT的mRNA表达量显著减少,其机制可能是通过抑制PI3K-AKT通路的激活,延缓肝纤维化的进展。肉苁蓉苯乙醇总苷具有抗纤维化作用,本实验为进一步深入探讨肉苁蓉的中药药理作用奠定基础。

To study the mechanism of active components of Cistanches Herba in the treatment of liver fibrosis from the point of view of drugs,diseases and targets.The effective components of Cistanches Herba were collected through traditional Chinese medicine system pharmacology database and analysis platform(TCMSP),traditional Chinese medicine encyclopedia database(ETCM),traditional Chinese medicine research comprehensive database(TCMID),CNKI database,PubMed and other databases.The target genes of active components of Cistanches Herba were obtained by Uniprot database,and the disease genes of liver fibrosis were collected by GeneCards database and OMIM database,the active component targets were intersected with liver fibrosis targets,and the traditional Chinese medicine-active components-disease network was drawn.Protein interaction network(PPI) was constructed by String database and Cytoscape 3.7.1 software.GO function and KEGG pathway were enriched by DAVID database.Animal model of liver fibrosis was established,the contents of LN,HA,PCIII and Col IV in mouse liver tissue were detected by ELISA kit,liver histopathological changes were observed by HE and Masson staining,and the transcription of PI3 K and AKT mRNA was detected by real-time fluorescence quantitative PCR.Eight active components of Cistanches Herba,157 targets and 1 005 targets of liver fibrosis were collected and screened,and 92 targets intersected with each other,involving 5 main signal pathways,including cancer signal pathway,hepatitis B virus signal pathway,PI3 K-AKT signal pathway,tumor necrosis factor signal pathway,proteoglycan and cancer.The animal experiment showed that compared with the model group,the high,middle and low dose groups of total phenylethanol glycosides of Cistanches Herba(700,350,175 mg/kg) could significantly reduce the contents of LN,HA,PCIII and Col IV in liver tissue of mice with liver fibrosis.The pathological observation of liver tissue showed that total phenylethanol glycosides of Cistanches Herba could reduce hepatocyte injury and collagen fiber deposition.The real-time fluorescence quantitative PCR detection showed that the mRNA expression of PI3 K and AKT in the liver tissue of model group was significantly up-regulated compared with normal group.Compared with model group,the mRNA expression of PI3 K and AKT in the high,middle and low dose groups of total phenylethanol glycosides of Cistanches Herba significantly decreased,and its mechanism may delay the progression of liver fibrosis by inhibiting the activation of PI3 K-AKT pathway.The total phenylethanol glycosides of Cistanches Herba have the effect of anti-fibrosis,which lays a foundation for further study on the pharmacological effects of Cistanches Herba.