The Study Of The Effect And Mechanism Of Trivalent Inorganic Arsenic Exposure On Inactivation Of Macrophage Polarization

Objective:Inorganic arsenic（iAs）widely exists in the natural environment,and human activities will aggravate the accumulation of arsenic in the natural environment.Arsenic pollution has become a serious public health problem.Studies have shown that the body can be exposed to arsenic through various ways.Exposure to inorganic arsenic has been proved to cause serious damage to multiple organs and multiple systems of the body,while the immune system,as the most important protective system of the body,has more serious consequences.Macrophages,as an important component of the immune system,play a role in phagocytosis,antigen presentation,chemotactic migration,tumor killing,and secretion of various cytokines and active substances during the immune process,participating in and regulating the body’s immune response.After macrophages reach different organs of the body through blood,they polarize into two main phenotypes under the action of specific environment and cytokines:M1 type macrophages and M2 type macrophages,which play different functions respectively.Previous studies have mostly treated mature macrophages with inorganic arsenic to explore the effects of inorganic arsenic on macrophages,while research results have shown opposite effects of promotion and inhibition.Based on the above contradictions,this study not only exposed mature macrophages to inorganic arsenic,but also treated monocytes with inorganic arsenic during the process of inducing differentiation into macrophages,exploring the impact of inorganic arsenic exposure on macrophage function.As a classical signal transduction and transcription activator,STAT plays a role in the polarization of macrophages.At present,studies have shown that STAT1phosphorylation is mainly related to M1 polarization of macrophages,STAT6phosphorylation is mainly related to M2 polarization of macrophages,while the relationship between STAT3 phosphorylation and M1 and M2 polarization is not clear.Therefore,this study uses primary mouse bone marrow mononuclear macrophages,peritoneal macrophages and RAW264.7 mouse macrophage lines as research models to study the effects of inorganic arsenic exposure on the polarization function of macrophages,and to explore the role of STAT in the dysfunction of macrophages caused by arsenic exposure,it provides theoretical basis and experimental basis for the in-depth study of macrophage dysfunction and immune function damage caused by arsenic exposure.Methods:In this study,the primary bone marrow derived macrophages and primary peritoneal macrophages of wild-type C57BL/6 mice,and RAW264.7 mouse macrophage lines were used as models.After low-dose inorganic arsenic treatment,the effects of inorganic arsenic treatment on the polarization of macrophages and the expression of related factors were studied.The specific research methods are as follows:1.Effect of inorganic arsenic treatment on monocyte-macrophage differentiationDuring the induction of differentiation,the primary bone marrow derived macrophages were treated with 1μM iAs³⁺for 5 days,the primary peritoneal macrophages were treated with 1μM iAs³⁺for 24 hours,and the macrophages of RAW264.7 mice were treated with 1μM iAs³⁺for 48 hours.The expression levels of macrophage marker genes CD14,Adgre1,and CD86 were detected by RT-qPCR.2.Effect of inorganic arsenic treatment on the migration ability of macrophagesDuring the induction of differentiation,1μM iAs³⁺was given to treat the primary bone marrow derived macrophages for 5 days,and the migration number of primary bone marrow macrophages was measured by transwell.During the induction of differentiation,the primary bone marrow mononuclear macrophages of wild type mice were treated with 1μM iAs³⁺for 5 days,the primary peritoneal macrophages were treated with 1μM iAs³⁺for 24 hours,and the macrophages of RAW264.7 mice were treated with 1μM iAs³⁺for 48 hours.The expression levels of macrophage chemokine receptors Ccr2,Ccr3,and Ccr5 were detected by RT-qPCR.3.Effect of inorganic arsenic treatment on macrophage response to polarization inductionDuring the induction of differentiation,the primary bone marrow derived macrophages were treated with 1μM iAs³⁺for 5 days,and treated with LPS and IL-4+IL-13 for 6 hours respectively.The expression levels of M1 marker protein inducible Nitric oxide synthase（iNOS）and M2 marker protein CD206 were detected by Western bolt.After 18 hours of treatment with 1μM iAs³⁺of primary peritoneal macrophages,IL-4+IL-13 was given for 6 hours,and the expression level of M2 marker genes Fizz1,Arg1,Mrc1 was detected by RT-qPCR.4.Effect of chronic inorganic arsenic treatment on STAT phosphorylation level of macrophagesDuring the induction of differentiation,the primary bone marrow derived macrophages were treated with 1μM iAs³⁺for 5 days,LPS and IL-4+IL-13 for 6 hours respectively,and the phosphorylation level of STAT1,STAT3,STAT6of primary bone marrow macrophages was detected by Western blot.After treating RAW264.7 with 1μM iAs³⁺for 18 hours and LPS was given for another 6 hours,the phosphorylation level of STAT1 in primary bone marrow macrophages was detected by Western blot.Results:1.Inorganic arsenic treatment during differentiation significantly inhibited the expression level of macrophage differentiation marker genes CD14,Adgre1 and CD86,while mature macrophages did not change the differentiation phenotype after chronic inorganic arsenic exposure,the difference was statistically significant（P<0.05）.2.Inorganic arsenic treatment significantly inhibited the migration function of macrophages and the expression level of chemokine receptors Ccr2,Ccr3,and Ccr5,and the difference was statistically significant（P<0.05）.3.Inorganic arsenic exposure inactivates the polarization function of macrophages.First,the expression level of M1polarization marker protein iNOS were significantly inhibited（P<0.05）.Secondly,the expression level of M2 polarization marker protein CD206 were significantly inhibited,and the expression level of M2 polarization marker genes Fizz1,Arg1,Mrc1 was significantly decreased,with a statistically significant difference（P<0.05）.4.Inorganic arsenic treatment inhibits the phosphorylation level of STAT in macrophages,suggesting that STAT may participate in the polarization dysfunction of macrophages caused by arsenic exposure.Conclusion:1.Arsenic exposure inhibits the differentiation of bone marrow-derived monocytes into macrophages.2.Arsenic damages the chemotactic migration function of macrophages.3.Arsenic inactivates the response of macrophages to LPS and M2.4.Arsenic exposure may inactivate the polarization function of macrophages by inhibiting the phosphorylation level of STAT,thus causing other functional abnormalities of macrophages.