基于网络药理学研究一贯煎治疗肝纤维化的有效成分及作用机制

Investigation of effective components and action mechanism of Yiguanjian in treatment of liver fibrosis based on network pharmacology

背景中药复方具有多成分综合调节的特点,在肝纤维化防治中显示出独特的优势.一贯煎始载于魏之琇所著《续名医类案》,是滋阴疏肝的著名方剂,能够改善肝纤维化症状,研究其抗肝纤维化机制有助于药物开发和推广.目的通过网络药理学探究一贯煎治疗肝纤维化的机制并实验验证.方法利用文献和TCMSP数据库收集一贯煎成分及靶点,GeneCard、OMIM数据库收集肝纤维化疾病靶点.通过STRING数据库构建一贯煎-肝纤维化蛋白互作网络(protein-protein interaction,PPI)子网络,应用重启随机游走算法获得关键基因,通过DAVID数据库进行基因本体(gene ontology,GO)富集分析及京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析.动物实验验证,18只SD大鼠随机分为正常组、模型组、一贯煎组.模型组、一贯煎组大鼠采用腹腔注射50%四氯化碳(carbon tetrachloride,CCl_(4))橄榄油溶液造模6周,正常组注射等量橄榄油.造模结束后一贯煎组每天灌胃一贯煎溶液(6.67 g/kg)共4 wk,其他组灌胃等量蒸馏水,取血和肝脏.全自动生化仪检测血清丙氨酸转氨酶(alanine aminotransferase,ALT)和天冬氨酸转氨酶(aspartate aminotransferase,AST).HE、Masoon染色观察肝脏病理学变化.Western blot法和qRT-PCR法检测肝脏关键蛋白表达水平和mRNA转录水平.结果筛选得到一贯煎成分52个,潜在作用靶点186个,肝纤维化疾病靶点1080个,得到一贯煎治疗肝纤维化的关键基因,其中亲和度前10的基因为STAT6、SRC、MAPK3、STX1A、EP300、STAT3、PLG、CTNNB1、CDKN1B、CANX,亲和度前50的基因KEGG富集得到PI3K-Akt信号通路、FoxO信号通路等134条.动物实验结果发现,一贯煎能够改善CCL4肝纤维化大鼠肝功能和减轻纤维化,降低α-肌动蛋白表达,促进信号转导和转录激活因子6(signal transducer and activator of transcription 6,STAT6)磷酸化,提高过氧化物酶体增殖物激活受体-γ(peroxisome proliferator activated receptor-γ,PPAR-γ)、白细胞分化抗原163(cluster of differentiation 163,CD163)蛋白表达和精氨酸酶1、白细胞分化抗原206、CD163mRNA转录水平,降低白细胞介素6的mRNA转录水平.结论一贯煎治疗肝纤维化作用涉及多个成分、多条信号通路,其中包括STAT6/PPAR-γ通路.

BACKGROUND Traditional Chinese medicine compounds are characterized by the comprehensive adjustment of multiple components and show unique advantages in the prevention and treatment of liver fibrosis.Yiguanjian(YGJ)is a famous prescription for nourishing Yin to soothe the liver,which can improve the symptoms of liver fibrosis,and understanding its anti-liver fibrosis mechanism can promote its development and use.AIM To explore the mechanism of YGJ in the treatment of liver fibrosis through network pharmacology and to experi-mentally validate the initial results obtained.METHODS Components of YGJ and potentially targeted proteins were downloaded from the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database.The targets of liver fibrosis were accessed from GeneCard and OMIM databases.STRING database was utilized to construct a protein-protein interaction(PPI)network based on the components of YGJ and the targets of liver fibrosis.The PPI network was subjected to random walk with restart(RWR)to obtain key genes,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed based on the DAVID database.For animal experimental validation,eighteen SD rats were randomly assigned to a normal group,a model group,and a YGJ group.The rats in the model group and YGJ group were intraperitoneally injected with 50%CCl_(4) olive oil solution for 6 wk to induce liver fibrosis,and rats in the normal group were intraperitoneally injected with the same amount of olive oil solution.Then,the rats of the YGJ group were given YGJ decoction(6.67 g/kg)daily for 4 weeks.Meanwhile,rats in the other groups were given distilled water.Blood and liver samples were collected,and the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum of rats were detected with an automated analyzer.Pathological changes in liver tissue were observed by hematoxylin-eosin(HE)and Masson staining.Western blot and qRT-PCR were used to detect the expression of key proteins and genes in the liver.RESULTS A total of 52 components and 186 potential targets of YGJ were obtained,and 1080 targets of liver fibrosis were screened.The top 10 genes with the high-affinity scores to the drug targets were STAT6,SRC,MAPK3,STX1A,EP300,STAT3,PLG,CTNNB1,CDKN1B,and CANX.The top 50 genes were mainly enriched in response to PI3K-Akt signaling pathway and FoxO signaling pathway,etc.In CCl_(4)-induced liver fibrosis rats,YGJ decoction could significantly improve liver lesions and reduce fibrosis.YGJ decoction could reduceα-SMA expression,promote the expression of phosphorylated STAT6,increase the protein expression of PPAR-γand CD163 and the mRNA expression of Arg-1,CD206,and CD163,and inhibit the gene expression of IL-6.CONCLUSION The therapeutic effect of YGJ decoction for liver fibrosis involves multiple components and multiple pathways,including the STAT6/PPAR-γpathway.