TRPM7 Modulates Macrophage Polarization By STAT1/STAT6 Pathways In RAW264.7 Cells

Macrophages are innate immune cells of human body,which not only participate in inflammation and repair damage,but also influence the development of various immune diseases.In recent years,it has been found that macrophages are a kind of plastic immune cells,and more and more scholars pay attention to their various morphological and functional changes.Macrophages can be divided into inflammatory(M1)macrophages and tissue repair(M2)macrophages in complex and changeable microenvironment.At present,it is believed that M1 macrophages can secrete a variety of proinflammatory factors,Interleukin-1β(IL-1β),Tumor necrosis factor-α(TNF-α),high expression of inducible nitric oxide synthase(i NOS),induce helper T cell(Th1)type immune response and promote inflammatory response;M2 type macrophage can increase the expression of Interleukin-10(IL-10),human Arginase-1(Arg-1),FIZZ1,induce Th2 type immune response and inhibit it can inhibit inflammatory reaction and promote tissue repair.In recent years,it has been found that Transient Receptor Potential Melastatin 7(TRPM7)can play a role in immune diseases through immune cells.As a novel immune regulatory molecule,TRPM7 not only regulates the proliferation and function of macrophages,but also affects the secretion of inflammatory factors.However,the mechanism of TRPM7 on macrophage polarization is not clear,so this study selected mouse macrophage line RAW264.7 for further study.Objective:Investigate the effect and mechanism of TRPM7 on macrophage polarization.In addition,detect the effect of TRPM7 on macrophage secretion factors,and to analyze and explore the mechanism of TRPM7 regulating macrophage polarization.This study can be divided into three parts.Methods:(1)M1 macrophages were induced with LPS(1000 ng/ml)and IFN-γ(20 ng/ ml)for 24 h in vitro to establish M1 macrophage model,and IL-4(20 ng/ml)for 24 h to establish M2 macrophage model.The expression of TRPM7 in M1/M2 macrophage model was detected by q-PCR.(2)Administration of 2-APB(100 μm)and TRPM7 si RNA blocked TRPM7 in RAW264.7 cells.Western blot was used to detect the protein changes of TNF-α,i NOS,Arg-1 and CD206 in RAW264.7 cells.Meanwhile,the changes of M1/M2 biomarkers(IL-6,TNF-α,i NOS,CD86,IL-10,TGF-β,Arg-1,CD206)in RAW264.7 cells were detected by q-PCR.In addition,the changes of IL-6,TNF-α,IL-10 and TGF-β in supernatant were detected by ELISA.(3)After TRPM7 was blocked,the total protein was extracted.The protein changes of p-stat1,STAT1,p-STAT6 and STAT6 were detected by Western blot.Results:(1)The results of q-PCR showed that TRPM7 was highly expressed in M1 macrophages and decreased in M2 macrophages.These results indicate that TRPM7 may be involved in the polarization process of RAW 264.7.(2)Blocking TRPM7 could inhibit the protein expression of TNF-α and i NOS induced by LPS-IFN-γ,and increase the protein expression of Arg-1 and CD206 induced by IL-4.q-PCR showed that TRPM7 blockade reduced the m RNA expression of M1biomarkers(IL-6,TNF-α,i NOS and CD86)induced by LPS-IFN-γ,and increased the m RNA expression of M2 biomarkers(IL-10,TGF-β,Arg-1,CD206)induced by IL-4.These results indicate that TRPM7 blockade can reduce the polarization of macrophages to M1 and increase M2 macrophages.In addition,TRPM7 blocked the secretion of IL-6and TNF-α in the supernatant of M1 macrophages,and increased the secretion of IL-10 and TGF-β in the supernatant of M2 macrophages.(3)Blockade of TRPM7 inhibited the increase of p-STAT1/STAT1 induced by LPS-IFN-γ,and further promoted the increase of p-STAT6 / STAT6 induced by IL-4.Conclusion:In this study,our results suggested that TRPM7 might participated macrophage polarization.Specifically,blockade of TRPM7 suppressed the activation of M1-type macrophages,while it enhanced the activation of M2-type macrophages.The mechanism of TRPM7 regulating macrophage polarization may be that inhibiting STAT1 phosphorylation and promoting STAT6 phosphorylation.