Iron Ion Polarizes Macrophages To M1 Subtype Through ROS-acetyl-P53

Background:Macrophages are an important part of the body's immune system,and they also play a very important role in the regulation of iron metabolism.Macrophages limit their growth and proliferation by maximizing uptake and storage of iron,reducing iron excretion,and reducing iron ions that can be used by pathogens.Meanwhile,macrophages use free iron in the cytoplasm and pass through Fenton.The reaction produces a large amount of reactive oxygen species to kill the phagocytosed pathogen.On the other hand,iron overload in macrophages also alters the polarization state of macrophages,but how iron overload promotes changes in the polarization state of macrophages is still lacking.The purpose of this study was to investigate the mechanism by which iron overload promotes macrophage polarization.Methods:DCFH-DA probe was used to detect ROS production in iron-treated macrophages;Western Blot,q RT-PCR,and flow cytometry were used to detect expression levels of macrophage polarization markers?IL-1?,TNF-?,TGF-?,IL-10,CD206,and CD86?;Western Blot and qRT-PCR were use to detect of p53 expression and acetylation in macrophages after treatment with iron;Western Blot and qRT-PCR were use to test the effect of GSH and C646 pretreatment on p53 expression and acetylation and macrophage polarization markers?TNF-?,CD206,Arg-1,and iNOS?;Xenograft model,,were prepared by subcutaneous injection of H22 hepatoma cells and RAW264.7 cells at a ratio of 4:1 in BALB/c nude mice to observe the polarization of macrophages after iron treatment.Result:The DCFH-DA probe showed that compared with the control group,macrophages in the iron-treated group produced a large amount of ROS,whereas no significant ROS production was observed in the macrophages of the GSH+iron-treated group.At the same time,Western Blot,qRT-PCR,and FCM showed that the expression of IL-1?,TNF-?,iNOS,and CD86 in the macrophages in iron group was significantly higher than control group and GSH+iron group?P<0.01?,there was no significant differences in the expression of TGF-?,IL-10 and CD206?P>0.05?.In addition,results of Western Blot and q RT-PCR showed that the expression of p53 in iron group was significantly higher than that in the control group and GSH+iron group?P<0.05?.Subsequently,Western Blot and qRT-PCR showed that the expression of p53 increased with the treatment time increase in the macrophage cells,treated with different time?0-8 hours?at a specific concentration?P<0.05?.Western Blot confirmed that the level of p53 acetylation also increased with the iron treatment time increase.Then,C646 was used to inhibit the acetylation of p53 and detected the inhibitory effect of p53acetylation by Western Blot,it confirmed that the C646 treatment can significantly inhibit p53 acetylation?P<0.01,compared with the iron-treated group?.At the same time,it was also found that the p53acetylation of macrophages in the GSH+iron treatment group was significantly lower than that in iron group?p<0.01?,but higher than that of the control group?P<0.05?.Western Blot and qRT-PCR showed that the expression of IL-1?,TNF-?and iNOS in the C646+iron group was lower than that in the iron group?P<0.01?,and there was no significant difference between the control group and the control group?P>0.05?.H22 murine hepatoma cells and RAW264.7 murine macrophages were subcutaneously injected in BABL/c nude mice at a ratio of 4:1 to prepare animal model.All mice were tail intravenous injected with ferric citrate solution?iron group?or saline?control group?before subcutaneous injection.The tumor volume was measured on the third day after the subcutaneous cell line injection.The tumor was completely harvested and weighed on the 21st day?four tumors in each group?.After statistical calculation,it was found that the subcutaneous tumor growth in iron group was slower than that in control group,and the quality was lower than that in the control group?P<0.01?.Subcutaneous tumor tissue expressed high level of CD86 and low level of CD206 in high iron treatment group,and a higher level of ROS was observed in the high iron treatment group than control group.Conclusion:Iron overload in macrophages can promote the production of ROS,which might result from the Fenton reaction:Fe²⁺+H₂O₂?Fe³⁺+OH^-+OH·.Large amounts of ROS are produced accompanied by macrophage polarization to M1,and macrophage M1 polarization was inhibited by repression of ROS which was induced by GSH treatment,suggesting that iron overload promotes ROS generation and induces macrophage M1polarization.In the subsequent experiments,Western blot and qRT-PCR showed that the expression of p53 was increased with the iron treatment time increase.After ROS repression,iron treatment no longer promoted the increase of p53.In the study,we also found that acetylated p53 also increased after iron treatment.C646 inhibited acetylation of p53 and macrophage M1 polarization.These results indicate that acetylated p53 is also involved in M1 polarization of iron overload-promoting macrophages M1 polarization.All date showed that iron overload in macrophages promoted the production of ROS,increase p53 acetyltransferase activity,induce p53 expression and acetylation,and finally lead to macrophage M1polarization.