Network pharmacology analysis of chlorogenic acid combined with forsythoside in regulating cytokine storm and synergistic anti-inflammatory effect based on TLR4/TRAF6/PI3KC3 pathway
Objective To explore the mechanisms by which chlorogenic acid combined with forsytho-side regulates cytokine storms through network pharmacology and in vitro experiments.Methods The targets of chlorogenic acid and forsythoside were predicted using Swiss Target Prediction,PharmMapper,SEA,and TCMSP databases.Targets related to cytokine storms were retrieved from GeneCards,OMIM,DRUGBANK,and DisGeNET databases.Intersection targets were identified and depicted in a Venn diagram.A protein inter-action network was constructed using the STRING database and Cytoscape software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed using the DAVID database.A cytokine storm model was established in RAW264.7 macrophages induced by lipopolysaccharide(LPS).Cell viability was assessed using the Cell Counting Kit-8(CCK-8)method.Nitric oxide(NO)levels in the supernatant were measured by the Griess method.Levels of inflammatory cytokines,tumor necrosis fac-tor-alpha(TNF-α),interleukin-6(IL-6),inducible nitric oxide synthase(iNOS),and cyclooxygenase-2(COX-2)were determined by enzyme-linked immunosorbent assay(ELISA).The expression of TNF receptor-associated factor 4(TRAF4),TNF receptor-associated factor 6(TRAF6),and phosphatidylinositol 3-kinase 3(PIK3C3)proteins were analyzed by Western blot.Results Network pharmacology analysis identified eight core net-work targets for the treatment of cytokine storms with chlorogenic acid and forsythoside,including serine/threonine-protein kinase(AKT1),albumin(ALB),hypoxia-inducible factor 1-alpha(HIF1A),IL-6,matrix metalloproteinase-9(MMP-9),peroxisome proliferator-activated receptor gamma(PPARG),tyrosine kinase(SRC),and Toll-like receptor 4(TLR4).Key pathways included the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.In vitro experiments showed that compared to the control group,levels of NO,TNF-α,IL-6,iNOS,and COX-2 were significantly higher in the model group(P＜0.001).After treat-ment,levels of NO,TNF-α,IL-6,iNOS,and COX-2 significantly decreased compared to the model group(P＜0.001).The combined treatment of chlorogenic acid and forsythoside(1:1)more effectively reduced inflam-matory indicators compared to single treatments(P＜0.001).Protein expression of TLR4,TRAF6,PIK3C3,and phosphorylation levels of PIK3C3 significantly decreased in the treatment group compared to the model group.Conclusion Chlorogenic acid combined with forsythoside synergistically suppresses the expression of pro-inflammatory cytokines and regulates cytokine storms,potentially through the TLR4/TRAF6/PIK3C3 signaling pathway.
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维普
