基于AMPK信号通路探讨血府逐瘀汤治疗代谢相关脂肪性肝病的效果及机制OA

Objective:To investigate the therapeutic mechanism of Xuefu Zhuyutang(XFZYT)for metabolic-associated fatty liver disease(MAFLD)through integrated network pharmacology and animal experiments.Methods:Network pharmacology was utilized to predict the core components,key therapeutic targets,and signaling pathways of XFZYT in the treatment of MAFLD.For animal experiments,a rat model of MAFLD was established by feeding a high-cholesterol diet for 4 weeks.Intervention was then administered with low-dose(2 g·kg-1)and high-dose(4 g·kg-1)XFZYT for 2 weeks.Biochemical assays were performed to measure the serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC),triglycerides(TG),high-density lipoprotein(HDL),and low-density lipoprotein(LDL).In addition,the activities of superoxide dismutase(SOD)and catalase(CAT)and levels of malondialdehyde(MDA)and glutathione(GSH)in the serum were measured.The same way was adopted to measure the levels of TC and TG in the liver tissue.Enzyme-linked immunosorbent assay(ELISA)was employed to quantify the serum levels of interleukin(IL)-6,IL-1β,and tumor necrosis factor-alpha(TNF-α).Histopathological evaluations included hematoxylin and eosin(HE)staining for liver tissue morphology,Oil Red O staining for lipid deposition,and dihydroethidium(DHE)probe staining for reactive oxygen species(ROS)levels.Western blot analysis was conducted to assess the protein levels of AMP-activated protein kinase(AMPK),phosphorylated(p)-AMPK,nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),nuclear factor-kappa B(NF-κB),and p-NF-κB in the liver tissue.Untargeted metabolomics analysis of the serum was performed by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results:Network pharmacology analysis predicted 155 potential targets of XFZYT for MAFLD treatment,with core targets including signal transducer and activator of transcription 3(STAT3),protein kinase B1(Akt1),TNF,and IL-6.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment primarily implicated the AMPK signaling pathway.Animal experiments demonstrated that compared with the normal group,the model group exhibited dyslipidemia,hepatic function impairment,pronounced hepatic lipid deposition,and inflammatory manifestations,with elevated serum levels of AST,ALT,TC,TG,LDL,and MDA(P＜0.05),reduced HDL and GSH levels plus decreased SOD and CAT activities(P＜0.05),downregulated protein levels of Nrf2,HO-1,and p-AMPK(P＜0.05),and upregulated protein level of p-NF-κB(P＜0.05)in the liver tissue.Compared with the model group,XFZYT intervention groups showed significant amelioration of dyslipidemia and hepatic function impairment,markedly reduced hepatic lipid deposition and inflammatory cell infiltration,decreased serum levels of AST,ALT,TC,TG,LDL,and MDA(P＜0.05),increased HDL and GSH levels plus enhanced SOD and CAT activities(P＜0.05),upregulated protein levels of Nrf2,HO-1,and p-AMPK(P＜0.05),and downregulated protein level of p-NF-κB(P＜0.05).Serum metabolomics revealed 511 differentially expressed metabolites(231 upregulated and 280 downregulated)between normal and model groups,while XFZYT groups versus model group showed 94 differential metabolites(51 upregulated and 43 downregulated).Among them,11 metabolites displayed the most significant alterations,with enriched pathways including glycerolipid metabolism,cholesterol metabolism,and insulin resistance,multiple of which demonstrated AMPK association.Conclusion:XFZYT alleviates MAFLD by regulating the AMPK signaling pathway and associated metabolic networks.