Gallic Acid Inhibits Polarization Of M1 Macrophages Through AMPK/STAT1 Signaling Pathway In Vitro

ObjectiveTo investigate the effect of Gallic acid(GA)on the polarization of M1 macrophages and its mechanism by establishing the polarization model of M1 macrophages,MethodsThe peritoneal fluid of 8-week-old C57BL/6 female mice was collected by lavage with 2%FBS-PBS phosphate buffer under aseptic condition.The peritoneal macrophages were extracted and purified and cultured in vitro.M1 polarization model was induced by LPS(100ng/mL)combined with IFNγ(20ng/mL).Cells were randomly divided into M φ group,M1 polarized group,GA treated group(6.25,12.5,25,37.5μg/mL)and AMPK antagonistic group.The morphological manifestations of cells were observed by inverted microscope.Cell viability was detected by trypan blue staining,MTT assay and Annexin V-PE/7-AAD double staining;The expression of F4/80+iNOS+M1 macrophages was detected by flow cytometry;The expression of inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR);Cyclooxygenase-2(COX-2),signal transduction and activator of transcription 1(STAT1),phosphorylated signal transduction and activator of transcription 1(p-STAT1),adenosine 5’-Monophosphate-activated protein kinase(AMPK),phosphorylated adenosine 5’-Monophosphate-activated protein kinase(p-AMPK)and β-actin protein were detected by Western blot.ResultsCompared with M1 polarized group,spindle cells decreased,round cells increased and pseudopodia decreased in GA treated group;The expression level of F4/80+iNOS+M1 cells decreased(P<0.01);The expression of iNOS,TNF-α,IL-1β mRNA,COX-2,p-STAT protein decreased,but the expression of p-AMPK protein increased(P<0.01).After adding Compound C to block AMPK signaling pathway,round cells decreased and spindle cells increased in AMPK antagonistic group;The expression of F4/80+iNOS+M1 cells increased(P<0.01);The expression of iNOS,TNF-α,IL-1β mRNA,COX-2,p-STAT1 protein increased,but the expression of p-AMPK protein decreased(P<0.01).ConclusionsGA(6.25～37.5μg/mL)had no significant effect on macrophage activity,but inhibited M1 macrophage polarization in a concentration-dependent manner through AMPK/STAT1 signaling pathway.