基于网络药理学及实验验证探讨温经汤治疗肝纤维化的作用机制

Exploration on the mechanism of Wenjing Decoction in the treatment of liver fibrosis based on network pharmacology and experimental verification

目的通过网络药理学方法探讨温经汤治疗肝纤维化的作用机制,并对核心靶点进行动物实验验证。方法检索TCMSP筛选温经汤组方药物活性成分及相关靶点,利用UniProt数据库获取温经汤活性成分对应靶点,采用Cytoscape 3.7.2构建“中药-成分-靶点”网络,检索GeneCards数据库筛选肝纤维化相关靶点,使用STRING数据库构建PPI网络,筛选肝纤维化核心成分和关键靶点,并进行GO功能和KEGG通路富集分析。对结果进行动物实验验证,选取10只作为空白组,其余45只采用四氯化碳诱导肝纤维化大鼠模型。造模结束后,将40只造模成功大鼠按随机数字表法分为模型组和温经汤高、中、低剂量组,每组10只。温经汤高、中、低剂量组灌胃1.5×3.18、3.18、0.5×3.18 g/kg温经汤,空白组灌胃等体积蒸馏水,1次/d,连续灌胃8周。采用HE染色观察大鼠肝组织病理形态,全自动生化仪检测大鼠血清GPT、GOT水平,Western blot检测大鼠肝组织TNF、Akt、IL-6蛋白表达。结果获得温经汤有效成分188个,度值较高的有效成分为β-谷甾醇、槲皮素、柚皮素等,收集肝纤维化基因靶点799个,PPI网络核心靶点为TNF、AKT、IL6等,通过GO功能和KEGG通路富集得到抗肝纤维化相关通路包括癌症通路、TNF及PI3K-Akt等信号通路。动物实验结果显示,模型组大鼠肝组织有明显炎症浸润、胶原纤维及假小叶生成,温经汤高、中、低剂量组大鼠肝组织炎症与纤维化水平较模型组均不同程度改善;与模型组比较,温经汤高、中、低剂量组大鼠血清GPT、GOT水平降低(P<0.05),TNF、Akt、IL-6蛋白表达下降(P<0.05)。结论温经汤可能通过干预TNF、AKT、IL6靶点,调节癌症通路及TNF、PI3K-Akt等信号通路,发挥抗肝纤维化作用。

Objective To investigate the mechanism of Wenjing Decoction in the treatment of hepatic fibrosis through network pharmacological methods,and conducting animal experiments to verify the core targets.Methods The TCMSP database platform was used to screen the active components and related targets of Wenjing Decoction,and the Uniprot database was used to obtain the target genes corresponding to the active components of Wenjing Decoction.The network diagram of"Chinese materia medica-compound-target"was constructed in Cytoscape 3.7.2,and the GeneCards database was used to search liver fibrosis related targets.String database was used to construct a protein interaction network(PPI)to screen the core components and key targets of liver fibrosis,and GO analysis and KEGG pathway enrichment were performed.Animal experiments were conducted to verify the results of the analysis.10 mice were selected as the blank group,and the remaining 45 rats were induced with carbon tetrachloride induced liver fibrosis model.After modeling,40 successfully modeled rats were divided into model group and Wenjing Decoction high,medium-,and low-dosage groups using a random number table method,with 10 rats in each group.Wenjing Decoction high,medium-,and low-dosage groups were orally administered with 1.5×3.18,3.18,and 0.5×3.18 g/kg Wenjing Decoction,respectively.The blank group was orally administered with equal volume distilled water once a day for 8 consecutive weeks.HE staining was used to observe the histopathological and morphological changes in the liver of rats.The serum GPT and GOT levels of rats were detected using a fully automated biochemical analyzer,and the expressions of TNF,AKT,and IL-6 proteins in rat liver tissue was detected using Western Blot.Results A total of 188 active components of Wenjing Decoction were obtained,and the active components with higher degree values wereβ-sitosterol,quercetin,naringenin,etc.799 liver fibrosis gene targets were collected,and the core target genes of the PPI network were TNF,AKT,IL6,etc.The key anti-hepatic fibrosis related pathways were obtained by GO function and KEGG analysis,including pathway in cancer,TNF,PI3K-Akt and other signalling pathways.Results of animal experiments showed that there were obvious inflammatory infiltration,collagen fibre and pseudo lobe generation in the liver tissue of rats in the model group,and the levels of inflammation and fibrosis in the liver tissue of rats in the Wenjing Decoction high,medium-,and low-dosage groups were improved to different degrees compared with that of the model group;compared with the model group,the levels of serum GPT and GOT decreased(P<0.05);the protein expressions of TNF,AKT and IL6 in the Wenjing Decoction high,medium-,and low-dosage groups decreased(P<0.05).Conclusion Wenjing Decoction may exert anti-liver fibrosis effects by intervening in TNF,AKT,IL6 targets,regulating cancer pathways,TNF,PI3K Akt and other signaling pathways.