Effects Of Inorganic Arsenic On The Autophagy-Lysosomal Pathway And Its Upstream Regulator TFEB In J774A.1 Macrophages

Objective:Macrophages are one of the important components in the body’s innate immune response system,and autophagy may affect the normal phagocytosis and cytokine secretion of macrophages.In this study,J774A.1 mouse macrophages were used as the research object.The possible effects of inorganic arsenic on the macrophage autophagy-lysosomal pathway and its upstream regulatory factor TFEB were observed and explored by sodium arsenite exposure in vitro.Methods:1.MTS method was used to determine the proliferation activity of J774A.1macrophages when different concentrations of inorganic arsenic were exposed.2.Western blot analysis of the protein expression levels of LC3,p62,LAMP1,LAMP2,Cathepsin B,Cathepsin D,Cathepsin S,Cathepsin L and TFEB in inorganic arsenic treatment groups.3.J774A.1 macrophages were stained with TFEB immunofluorescence.4.Real-time PCR analysis of Cd80,Cd86,Map1lc3a/b,p62,Lamp1,Lamp2 and Tfeb mRNA expression levels.5.Use SPSS17.0 software for statistical analysis of data.Results:1.Low concentration of 0.25μM As~Ⅲcan slightly promote the proliferation of J774A.1 macrophages,0.5-2μM As~Ⅲhas no toxic effect on cell proliferation activity,2.5-10μM As~Ⅲsignificantly reduces the cell proliferation ability(P<0.05).2.After LPS stimulated macrophages,the cell pseudopods increased significantly.After treatment with 2μM As~Ⅲ,cells became round,pseudopods decreased,and adhesion decreased.Compared with the simple LPS stimulation group,the As~Ⅲ+LPS treatment group performed more obviously.3.After 2μM As~Ⅲtreatment,the expression levels of Cd80and Cd86 mRNA in macrophages decreased.4.Western blot analysis found that different concentrations of inorganic arsenic can increase the expression of LC3(P<0.05).The expression of LC3 protein in 0.5μM As~Ⅲwas significantly increased at different exposure times of 6 h,12 h and 24 h.2μM As~Ⅲcan promote the expression of Map1lc3a/b mRNA.5.Different concentrations of inorganic arsenic can increase the expression of p62 protein(P<0.05).The expression of p62 protein was significantly increased in 0.5μM As~Ⅲtreated cells at 6 h,12 h,and 24 h at different exposure times(P<0.05).2μM As~Ⅲcan promote the expression of p62 mRNA(P<0.05).6.Inorganic arsenic exposure can promote the expression of lysosome-associated membrane protein LAMP1/2 protein and their m RNA to a certain extent in J774A.1 macrophages(P<0.05);treatment with different concentrations of inorganic arsenic can promote CTSB/S/L protein expression(P<0.05).7.As~Ⅲ+LPS co-treatment of macrophages can increase the expression of TFEB whole protein and nuclear protein(P<0.05)and nuclear translocation of TFEB in a dose-dependent manner,while the expression of TFEB cytoplasmic protein did not change significantly.Tfeb mRNA expression increased after2μM As~Ⅲtreatment of macrophages(P<0.05).Conclusion:1.Inorganic arsenic inhibits the expression of macrophages costimulatory molecules CD80 and CD86;2.Inorganic arsenic promotes the expression of autophagy-related molecules LC3 and autophagy substrate protein p62/SQSTM1 of macrophages;3.Inorganic arsenic induces macrophages Protein expression of lysosomal-associated membrane protein LAMP1/2 and cathepsin CTSB/S/L;4.Inorganic arsenic significantly promoted the expression and nuclear translocation of the transcription factor TFEB upstream of the macrophage autophagy-lysosomal pathway.