All-trans Retinoic Acid Promotes M2 Macrophage Polarization Through P38MAPK/STAT6 Signaling Pathway In Vitro

Objective M2 macrophages are inflammation-suppressing cells that are differentiated after induction by cytokines such as Interleukin-4(IL-4)or Interleukin-13(IL-13),which play an important regulatory role in inflammation and influence the regression of inflammation-related diseases.All-trans retinoic acid(ATRA)has an important role in suppressing immunemediated inflammatory responses but the effect and underlying mechanism of ATRA on the polarization of M2 macrophages remains unclear.IL-4-induced M2 macrophage polarization model was established in vitro to investigate the effects of ATRA on polarization of M2 macrophages and its related mechanisms.Methods Peritoneal macrophages were extracted from eight-week-old C57BL/6female mice via intraperitoneal lavage,and treated by IL-4(20 ng/m L)to establish an M2 macrophage polarization model.The experiment was randomly divided into M group,M2 polarization group,DMSO control group,ATRA treatment group(15,30,45 μg/m L)and SB202190(p38MAPKα antagonist)group.Cell morphology was observed under an inverted microscope;and cell viability was determined by MTT,Taipan blue staining and Annexin V-PE/7-AAD double staining.F4/80~+CD206~+ M2 macrophages were screened by flow cytometry.Real-time quantitative polymerase chain reaction(q RT-PCR)was used to detect mRNA expression of M2 macrophage-associated factor Arginase-1(Arg-1),Interleukin-10(IL-10)and Transforming growth factor-β1(TGF-β1).Protein expression of Arg-1,IL-10,TGF-β1,p38 mitogen-activated protein kinase(p38MAPK),phosphorylated p38 mitogen-activated protein kinase(p-p38MAPK),signal transducer and activator of transcription 6(STAT6),phosphorylated signal transducer and activator of transcription(p-STAT6)and β-actin were detected by Western blot.Results Compared with the M2 polarization group,disc-shaped cells in the ATRA treatment group became larger and the number increased;there was no significant difference in macrophage activity in the ATRA treatment group(P>0.05);the expression of F4/80~+CD206~+M2 macrophage expression showed concentration-dependent increase(P<0.01);the mRNA and protein expression of Arg-1,IL-10,TGF-β1 were increased,and their effects were more significant with the increase of ATRA(P<0.01);the protein expression of p-p38 MAPK and p-STAT6 were also increased,and the expression levels increased with the increase of ATRA concentration(P<0.01).After adding SB202190 to block the p38 MAPK signaling pathway,disc-shaped cells in the SB202190 antagonist group became smaller and the cell number decreased;the expression of F4/80~+CD206~+ M2 macrophage was significantly reduced(P<0.01);the mRNA and protein expression of Arg-1,IL-10,TGF-β1 were significantly reduced(P<0.01);the protein expression of p-p38 MAPK and p-STAT6 were significantly reduced(P<0.01).Conclusions ATRA(15-45 μg/m L)had no significant effect on apoptosis of macrophage but can promote concentration-dependent polarization of macrophages to M2 via p38MAPK/STAT6 signaling pathway.