Molecular mechanism of calpain in regulating macrophage polarization mediated by NF-κB/STAT3 signaling pathways / 复旦学报（医学版）

ABSTRACT

Objective To investigate the molecular mechanism of calpain in regulating macrophage polarization.Methods Macrophages (RAW264.7) were transfected with siRNA by Lipofectamine 2000 to konckdown calpain1 and calpain2,respectively.The mRNA levels of markers in M1 (IL-23,TNF-α and iNOS) and M2 (IL-10,TGF-β and Arg-1) were measured by qRT-PCR.The levels of proteins in signaling pathways (NF-κB/STAT3) were measured by Western blot.The ability of macrophage migration was detected by Transwell assay.Results The expression level of calpain1 was lower in M1 than that in M2 (P＜0.05),but the expression level of calpain2 was significantly higher in M 1 than that in M 2 (P＜0.05).In calpain1-siRNA group,the mRNA levels of M1-type macrophages markers and M2-type macrophages markers were decreased by lipopolysaccharide (LPS) stimulation;The mRNA levels of M1-type macrophages markers in calpain2-siRNA group were significantly reduced (P ＜ 0.05),while the mRNA levels of M2-type macrophages markers were significantly increased (P ＜ 0.05).In calpain2-siRNA intervention group,the total phosphorylation inhibitor of nuclear factor kappa-B kinase (p-IKK) protein level of LPS-induced macrophages was decreased;in IL-4-induced macrophages,the protein level of plasmic signal transducers and activators of transcription 3 (STAT3) was also decreased,but there was no significant difference in total level of phosphorylation-p65 (p-p65).It was also found that the ability of migration was reduced by the interventions of calpain1-siRNA and calpain2-siRNA as compared with control-siRNA intervention (P＜0.05).Conclusions Calpain2 may potentially promote M1 polarizations by NF-κB and STAT3 signaling pathways,and inhibit the ability of its migration by the interventions of calpain1-siRNA and calpain2-siRNA.