Mechanistic Study of Xuefu Zhuyu Capsules in the Treatment of Acetaminophen-induced Liver injury

FAN Zhenli; FANG Wei

doi:10.3969/j.issn.2096-305X.2025.06.002

Abstract：Objective To explore the mechanism of Xuefu Zhuyu capsules(XFZYC)in the treatment of acetaminophen(APAP)-induced liver injury through network pharmacology,molecular docking and experimental verification.Methods The active components of Xuefu Zhuyu Capsule were screened through the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)and the herbal medicine database(HERB).Their corresponding targets were predicted and selected through the SwissADME and Swiss Target Prediction databases.The relevant targets of drug-induced liver injury were obtained from the Human Genome Database(GeneCards),the Online Mendelian Inheritance in Man(OMIM)database,and the Database of Gene-Disease Associations(DisGeNET).The intersection targets of drugs and diseases were imported into the STRING database to construct a protein-protein interaction(PPI)network model.Topological parameter analysis was conducted in Cytoscape 3.9.1.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of the intersection targets were carried out using the database for annotation,visualization and integrated discovery(DAVID).Molecular docking verification was performed using the CB-Dock2 online molecular docking server.Animal experiments verified the prediction results of network pharmacology.A mouse model of drug-induced liver injury was established using APAP,and different doses of Xuefu Zhuyu Capsules were administered for intervention.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue.Serum biochemical indicators such as alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),and total bilirubin(TBIL)were measured by the microplate method.The levels of oxidative factors such as superoxide dismutase(SOD),glutathione(GSH),catalase(CAT),malondialdehyde(MDA),and inflammatory factors such as tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1β),and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA).The expression of key proteins in the phosphatidylinositol 3-kinase-protein kinase B(PI3K-AKT)and mitogen-activated protein kinase(MAPK)pathways was detected by Western Blot.Results A total of 158 active components,914 potential targets,1657 drug-induced liver injury disease targets,348 intersecting targets,and 67 core targets(including protein kinase B1(AKT1),interleukin 6(IL6),tumor necrosis factor(TNF),tumor protein p53(TP53),interleukin 1β(IL1β),epidermal growth factor receptor(EGFR),signal transducer and activator of transcription 3(STAT3),caspase 3(CASP3),B lymphocyte tumor-2(BCL2),and proto-oncogene tyrosine kinase(SRC))were identified through PPI network analysis.The KEGG enrichment analysis indicates that the effect of Xuefu Zhuyu Capsule is mainly achieved through multiple biological pathways,including cancer-related pathways,metabolic pathways,PI3K-AKT signaling pathway,and MAPK signaling pathway,etc.The molecular docking results indicate that the main active components in Xuefu Zhuyu Capsule,namely quercetin,kaempferol,baicalein,β-sitosterol,stigmasterol,isoquercitrin,naringenin,and luteolin,have good binding with the target proteins AKT1,IL6,TNF,TP53,IL1β,and EGFR.Animal experiments show that Xuefu Zhuyu Capsule reduces the levels of serum ALT,AST,ALP,TBIL and inflammatory factors TNF-α,IL-1β,and IL-6 in liver tissue,increases the levels of oxidative factors SOD,CAT,and GSH in liver tissue,and reduces the level of MDA.Western blot results show that Xuefu Zhuyu Capsule reduces the expression levels of phosphorylated phosphatidylinositol 3-kinase(p-PI3K),phosphorylated protein kinase B(p-AKT),phosphorylated c-Jun N-terminal kinase(p-JNK),phosphorylated extracellular signal-regulated kinase(p-ERK),and phosphorylated p38 mitogen-activated protein kinase(p-P38)in the PI3K-AKT and MAPK signaling pathways.Conclusion This study,by combining network pharmacology,molecular docking and animal experiments,confirmed that the Xuefu Zhuyu Capsule exerts an anti-liver injury effect induced by acetaminophen through multiple components,multiple targets and multiple pathways.The mechanism may be to inhibit the expression of proteins related to the PI3K-AKT and MAPK signaling pathways to regulate the oxidative stress and inflammatory response caused by acetaminophen stimulation.