CHOP Regulates The Molecular Mechanism Of Macrophage Polarization Through The STAT6/KLF4/IL-13 Signaling Pathway

Background:Schistosomiasis is a zoonotic parasitic disease with a long history,which has brought huge health and social and economic burden to human beings.M2 macrophages are mainly involved in the inflammatory response of late schistosoma infection,promoting the regression of inflammation and tissue repair.In our previous animal experiments,we found that the expression of CHOP in the liver of schistosoma infected mice increased in a time-dependent manner,but how CHOP regulates schistosoma induced liver fibrosis through the polarization of M2 macrophages remains to be further studied.Objective:At the cellular level,the molecular mechanism of CHOP regulating macrophage polarization was investigated by RAW264.7 murine mononuclear macrophage line.It provides a possible therapeutic target for the clinical treatment of schistosoma.Methods:(1)RAW264.7 mouse mononuclear macrophage line was routinely cultured,and cells were stimulated by il-4 at different concentrations(10,20,50 and 100 ng/ml).Cells were collected at different time points(6,12,24 and 48 h)after stimulation.①Western blot was used to observe the expression of proteins related to M2 polarization pathway and CHOP protein.②fluorescence quantitative PCR was used to observe the expression of genes related to M2 polarization pathway and chop gene.(2)STAT6 inhibitor was used to act on RAW264.7 macrophages to inhibit the phosphorylation of STAT6.After il-4 stimulation,M2 polarization,protein expression and gene expression of pathway related factors KLF4 and CHOP were observed.(3)After transfection of CHOP overexpressed plasmid into RAW264.7 cells,STAT6 inhibitor and il-4 were used to stimulate the cells to observe the M2 polarization of macrophages,as well as the protein expression and gene expression of p-stat6,KLF4 and CHOP in the polarization pathway.(4)RAW264.7 macrophages were stimulated by SEA,and the phosphorylation of STAT6 and the expression of KLF4 protein were observed by WB.Results:(1)I1-4 induced polarization of RAW264.7 cells into m2-type macrophages(P<0.05,P<0.01).(2)After il-4 stimulated RAW264.7 macrophages,the expressions of p-stat6,KLF4 and CHOP increased(p<0.01).(3)After the inhibition of p-stat6,the m2-type polarization of macrophages was reduced and the expression of KLF4 and CHOP was reduced under the induction of il-4 in the non-inhibitor group.(4)After transfection with CHOP overexpressed plasmids,compared with the normal cell group,il-4-induced macrophage expression increased to m2-type,while KLF4 and CHOP expression increased.(5)After SEA stimulation of RAW264.7 cells,p-stat6 and KLF4 expression increased.Conclusion:(1)Il-4-induced m2-type polarization of RAW264.7 macrophages is partially dependent on STAT6 phosphorylation.(2)CHOP may promote m2-type polarization transformation of RAW264.7 macrophages by regulating KLF4 expression.