基于网络药理学探讨消癥活络方通过ERK5通路抗大鼠肝纤维化的机制

Mechanism of Xiaozheng Huoluo prescription in preventing liver fibrosis in rats via the ERK5 pathway:An analysis based on network pharmacology

目的基于网络药理学方法探索消癥活络方(XZHLF)防治肝纤维化的作用机制。方法通过TCMSP数据库、化学专业数据库、ETCM数据库、化源网数据库、PubChem数据库以及文献查阅,收集XZHLF各中药化学成分,利用Swiss ADME数据库筛选出XZHLF各中药活性成分,利用Swiss Target Prediction数据库预测XZHLF各中药活性成分靶点;通过GeneCards、OMIN数据库收集肝纤维化疾病靶点,利用韦恩图获得XZHLF防治肝纤维化的潜在作用靶点。使用Cytoscape 3.7.1软件建立XZHLF“药物-活性成分”网络和XZHLF防治肝纤维化的“活性成分-潜在作用靶点”网络。在Metascape数据库对潜在作用靶点进行GO和KEGG富集分析,选取富集基因数最多的前20条绘制气泡图。对前20条KEGG通路中的MAPK信号通路进行分析,绘制“活性成分-潜在作用靶点-通路”网络图。将健康的SD大鼠随机分为6组,分别为空白对照组(K组)、模型组(M组)、秋水仙碱阳性对照组(Y组)、XZHLF高(G组)、中(Z组)、低(D组)剂量组,利用CCl4诱导大鼠肝纤维化进行造模,造模同时给药,共8周。通过Western Blot法检测各组大鼠肝组织内ERK5、p-ERK5、MEK5、MEKK3蛋白的相对含量和表达情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果从XZHLF中筛选出110个活性成分,作用于923个活性成分靶点,与6823个肝纤维化疾病靶点相映射得到潜在作用靶点647个。XZHLF可能通过MAPK信号通路等通路,通过MAPK级联反应的调控等生物过程,作用于EGFR、AKT1、IKBKB、MAPK8、PDGFRB等多个蛋白靶点,从而发挥防治肝纤维化的作用。M组的大鼠肝组织内ERK5、p-ERK5、MEK5和MEKK3蛋白水平较K、Y、G、Z、D组明显增加,差异均有统计学意(P值均<0.05);K组的大鼠肝组织内ERK5、p-ERK5、MEK5和MEKK3蛋白水平较Y、G、Z、D组明显减少,差异均有统计学意义(P值均<0.05)。结论基于网络药理学的方法预测出XZHLF可能通过MAPK信号通路防治肝纤维化,通过实验验证XZHLF是通过MAPK信号通路家族中ERK5通路抗肝纤维化。XZHLF高剂量组的抗肝纤维化作用效果最为明显。

Objective To investigate the mechanism of Xiaozheng Huoluo prescription(XZHLF)in the prevention and treatment of liver fibrosis based on network pharmacology.Methods TCMSP database,Chemistry Database,ETCM database,chemical source network database,ChemSrc database,and PubChem database were searched and a literature review was performed to collect the chemical components of each traditional Chinese medicine of XZHLF,Swiss ADME database was used to screen out the active components of each traditional Chinese medicine of XZHLF,and Swiss Target Prediction database was used to predict the targets of these active components;GeneCards and OMIN databases were used to collect the disease targets of liver fibrosis,and Venn diagrams were used to obtain the potential targets of XZHLF in the prevention and treatment of liver fibrosis.Cytoscape 3.7.1 software was used to establish a“drug-active component”network for XZHLF and an“active component-potential target”network for XZHLF in the prevention and treatment of liver fibrosis.Metascape database was used to perform GO and KEGG enrichment analysis of potential targets,and bubble charts were plotted for the top 20 pathways with the highest number of enriched genes.The MAPK signaling pathway among the top 20 KEGG pathways was analyzed to plot an“active component-potential target-pathway”network.Healthy Sprague-Dawley rats were randomly divided into blank control group(K group),model group(M group),colchicine-positive control group(Y group),high-dose XZHLF group(G group),middle-dose XZHLF group(Z Group),and low-dose XZHLF group(D group),and a rat model of liver fibrosis was established by CCl4;the drug was administered simultaneously for 8 weeks.Western blot was used to measure the protein expression of ERK5,p-ERK5,MEK5,and MEKK3 in liver tissue.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results A total of 110 active components were screened out from XZHLF and they acted on 923 targets,which were mapped to 6823 disease targets of liver fibrosis to obtain 647 potential targets.XZHLF might act on multiple protein targets such as EGFR,AKT1,IKBKB,MAPK8,and PDGFRB through the pathways including the MAPK signaling pathway and the biological processes including the regulation of MAPK cascade,thereby playing a role in the prevention and treatment of liver fibrosis.The M group had significant increases in the protein levels of ERK5,p-ERK5,MEK5,and MEKK3 in liver tissue compared with the K,Y,G,Z,and D groups(all P<0.05),and the K group had significant reductions in the protein levels of ERK5,p-ERK5,MEK5,and MEKK3 in liver tissue compared with the Y,G,Z,and D groups(all P<0.05).Conclusion Based on the method of network pharmacology,it is predicted that XZHLF may prevent and treat liver fibrosis via the MAPK signaling pathway,and it is verified through experiments that XZHLF prevents and treats liver fibrosis via the ERK5 pathway in the MAPK signaling pathway family.The high-dose XZHLF group shows the most obvious anti-liver fibrosis effect.