基于网络药理学及动物实验探讨苗药地锦草抗肝纤维化的作用机制OA

Objective To explore the mechanism of the Miao medicine Euphorbia humifusa against liver fibrosis based on network pharmacology and animal experiments.Methods(1)Active components of Euphorbia humifusa and their corresponding targets were screened and obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Disease-related targets for liver fibrosis were retrieved from the GeneCard,PharmGkb,OMIM,and TTD databases.The intersection of active component targets and disease targets yielded potential therapeutic targets of Euphorbia humifusa for liver fibrosis.The active components and potential targets were imported into Cytoscape 3.7.2 software to construct an"active component-target"network.A protein-protein interaction(PPI)network of potential targets was constructed using the STRING database.Gene Ontology(GO)functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were performed on the potential therapeutic targets.(2)A liver fibrosis rat model was established by intraperitoneal injection of a carbon tetrachloride-olive oil mixture(volume ratio 2∶3)at a dose of 3 mL·kg-1,twice weekly for 8 consecutive weeks.SD rats were randomly divided into a blank group(n=7),model group(n=7),silibinin group(n=6),and low-,medium-,and high-dose Euphorbia humifusa groups(n=6 each).The doses for the low-,medium-,and high-dose Euphorbia humifusa groups were 0.675,1.35,and 2.70 g·kg-1,respectively,and the silibinin dose was 18.9 mg·kg-1.All drugs were administered intragastrically at a volume of 10 mL·kg-1 once daily for 30 days.Liver tissue pathological changes were observed by HE and Masson staining.The expression levels of α-SMA and TGF-β1 in liver tissue were detected by immunohistochemistry.Serum levels of procollagen type III(PC III),laminin(LN),hyaluronic acid(HA),and collagen type Ⅳ(Col Ⅳ)were measured by ELISA.The protein and mRNA expression levels of HIF-1α and VEGF in liver tissue were detected by Western Blot and qRT-PCR,respectively.Results(1)A total of 147 potential therapeutic targets(intersection targets)for Euphorbia humifusa against liver fibrosis were obtained.Potential targets such as PTGS2,HSP90AA1,AR,GSTP1,TNF,HIF1A,and PGR were closely associated with active components including ellagic acid,sitosterol,quercetin,ensaculin,kaempferol,and 5,4'-dihydroxyflavone.Targets such as TP53,HSP90AA1,AKT1,TNF,IL6,RELA,and HIF1A may be the core targets for Euphorbia humifusa in treating liver fibrosis.The potential therapeutic targets were mainly enriched in pathways including the AGE-RAGE signaling pathway,IL-17 signaling pathway,TNF signaling pathway,and HIF-1 signaling pathway.(2)Compared with the blank group,the model group showed disordered arrangement of hepatocytes,hepatocyte edema,extensive connective tissue proliferation in the portal area,and inflammatory cell infiltration in the interstitium;significant blue collagen fiber deposition and fibrous septa were visible,and the fibrous area ratio was significantly increased(P＜0.01);the expression levels of α-SMA and TGF-β1 in liver tissue were significantly elevated(P＜0.05,P＜0.01);serum levels of PC Ⅲ,LN,HA,and Col Ⅳ were all significantly increased(P＜0.01);and the protein and mRNA expression of HIF-1α and VEGFA in liver tissue were significantly upregulated(P＜0.01).Compared with the model group,all treatment groups showed varying degrees of improvement in inflammatory cell infiltration,connective tissue proliferation,and hepatocyte edema in liver tissue.The fibrous area ratio was significantly reduced in the silibinin group and the high-dose Euphorbia humifusa group(P＜0.05,P＜0.01);the expression levels of α-SMA and TGF-β1 in liver tissue were significantly decreased in the silibinin group and the medium-and high-dose Euphorbia humifusa groups(P＜0.05,P＜0.01);serum levels of PC Ⅲ,LN,HA,and Col Ⅳ were significantly reduced in all treatment groups(P＜0.01);and the protein and mRNA expression of HIF-1α and VEGFA in liver tissue were significantly downregulated(P＜0.05,P＜0.01).Conclusion The Miao medicine Euphorbia humifusa can ameliorate pathological damage and reduce collagen fiber deposition in the liver tissue of rats with liver fibrosis,which may be related to its regulatory effect on hypoxia and angiogenesis via the HIF-1α/VEGF pathway.