| Objective:Arsenic is a metal-like element that exists widely in nature.Long-term exposure to arsenic can cause a variety of systemic diseases and even induce a variety of cancers.As the first line of defense of human immunity,macrophages play an important role in the process of resisting pathogens.Macrophages can change their phenotype under the influence of the surrounding environment,which can be mainly divided into M1 type(classical activated macrophages)and M2 type(vicarious activated macrophages).M1 and M2 type macrophages have pro-inflammatory and anti-inflammatory biological functions,respectively,and participate in the interaction between cells in the tumor microenvironment.Macrophages gathered in the surrounding tissues play an important role in the process of tumor invasion and metastasis,and are called tumor-related macrophages.They are mainly characterized by M2 macrophages,which promote tumor metastasis by enhancing the motility of tumor cells,promoting angiogenesis and degrading the extracellular basal layer.MiRNAs can regulate many pathophysiological processes such as development,metabolism,cell proliferation,growth,differentiation and death,among which miRNA125 family plays an important role in macrophage polarization and tumor development.However,whether miR-125 family regulates arsenic-induced macrophage polarization has not been reported in the literature.To this end,we studied the role of miR-125a-5p in macrophage polarization by establishing the polarization model induced by sodium arsenite.Methods:1.In vivo experiment:A subchronic arsenic exposure model of rats in drinking water was established,and the rats were randomly divided into three groups:control group,10 mg/L arsenic staining group and 50 mg/L arsenic staining group.The rats were killed after 12 weeks of continuous exposure,liver and bladder tissues were taken,paraffin embedded and sliced.The expressions of M1 and M2 macrophages(CD206、Arg1、iNOS、IL-1β and TNF-α)in rat liver and bladder were detected by Western Blot or immunofluorescence staining.2.In vitro experiments:THP-1 cells were stimulated with different concentrations of PMA for 48 h,and the expression of macrophage surface marker CD11b was detected by flow cytometry.CCK8 was used to detect the cell viability of THP1-derived macrophages treated with different concentrations of sodium arsenite(NaAsO2)for 48 h.RT-qPCR and Western Blot were used to detect the expression of signature genes and proteins in M1 and M2 macrophages after 48 h of THP-1 derived macrophages treated with NaAsO2.The cell localization and fluorescence intensity of CD206 and IL-1β were detected by immunofluorescence staining.The mRNA expression level of miR-125a-5p was detected by RT-qPCR.MiR-125a-5p binding sites were screened by GO enrichment analysis of miRDB,Targetscan and miRTarB ase databases and DAVID database.The miR125a-5p overexpression model was constructed,and the expression of M1 type and M2 type signature proteins in THP-1-derived macrophages treated by IRF4 and NaAsO2 after miR-125a-5p overexpression was detected by Western Blot.Results:1.In vivo experiment:after 12 weeks of arsenic exposure in drinking water,M2 type macrophage markers CD206 and Argl in liver tissues were significantly higher than those in control group(P<0.01),M1 type macrophage markers iNOS and IL-1β were significantly lower than those in control group(P<0.05).The expression of M2 type macrophages in the bladder tissues of arsenic exposed rats was increased:CD206 and Arg1 were significantly increased in the bladder tissues compared with the control group(P<0.01),while TNF-α and IL-1β were significantly decreased(P<0.05).2.Morphological characteristics of THP-1 cells after differentiation:compared with monocytes,THP-1 cells after PMA treatment transformed from suspension cells into adherent cells,extended pseudopodia,stopped division and proliferation,and increased cytoplasmic volume;The number of CDl1b cells treated with different concentrations of PMA was significantly higher than that of control group(P<0.001),and there was no significant difference between the two dose groups in inducing macrophages between 80 nM and 160 nM PMA.3.Effects of NaAsO2 on the activity of THP-1-derived macrophages:The activity of macrophages was significantly decreased after treatment with 4 μM NaAsO2 or higher dose(P<0.05),the cell activity was lower than 50%after 12 μM NaAsO2 treatment.4.NaAsO2 induced the expression of THP-1-derived macrophage related genes:The mRNA levels of CD206,Arg1 and IL-10 in THP-1-derived macrophages treated with different doses of NaAsO2 for 48 h were significantly higher than those in the control group(P<0.05).The mRNA levels of Arg1 and IL-10 were not significantly different from those of IL-4 treatment group.The mRNA levels of IL-1β and TNF-α were significantly lower than those of control group(P<0.05),compared with IL-4 group(P<0.01).The mRNA level of VEGF after 4 μM NaAsO2 treatment was significantly lower than that of control group,and there was no statistical difference between other treatment groups and control group.5.NaAsO2 induced the expression of THP-1-derived macrophage-related proteins:compared with the control group,the fluorescence intensity of CD206 in NaAsO2 and IL-4 treatment groups was significantly stronger,and the fluorescence intensity of IL-1β was significantly weaker;The expression levels of CD206,Argl and IL-10 in 8 μM NaAsO2 and IL-4 groups were significantly higher than those in control group(P<0.05).The expression levels of IL-1β,TNF-α and iNOS were significantly lower than those of control group(P<0.05).The expression level of VEGF protein was not significantly different from that of control group.6.NaAsO2 induced the expression of miR-125a-5p in THP-1-derived macrophages:The mRNA level of miR-125a-5p in 8 μM NaAsO2 and IL-4 treatment groups was significantly lower than that in control group(P<0.05).7.MiR-125a-5p target gene prediction:GO enrichment analysis of miRDB,Targetscan,miRTarB ase and DAVID databases found the target of IRF4;miR-125a-5p was found to bind to IRF4 3’-UTR 592-599 in Targetscan database;miR-125a-5p overexpression model was constructed,the mRNA levels of miR125a-5p were significantly increased in the control group or NaAsO2 or IL-4-miR-mimic group compared with the control group or NaAsO2 or IL-4 treated group(P<0.001),and there was no significant difference between control or NaAsO2 or IL-4-miR-NC group and control or NaAsO2 or IL-4 treatment group.The expression level of IRF4 protein in 8 μM NaAsO2 and IL-4 treatment groups was significantly higher than that in control group(P<0.05).After miR-125a-5p overexpression,IRF4 protein levels in NaAsO2-miR-NC and IL-4-miR-NC groups were significantly increased compared with the control-miR-NC group(P<0.01),and there was no significant difference between control-miR-mimic group and control-miR-NC group.NaAsO2 or IL-4-miR-mimic group significantly reduced IRF4 protein levels compared with NaAsO2 or IL-4-miR-NC group(P<0.01).8.NaAsO2 induced the expression of macrophage-related proteins after miR-125a-5p overexpression:After miR-125a-5p overexpression,CD206,Argl and IL-10 protein levels in NaAsO2-miR-NC and IL-4-miR-NC were significantly increased compared with the control miR-NC group(P<0.01),iNOS,IL-1β and TNF-α protein levels were significantly decreased(P<0.05),the control-miR-mimic showed no significant difference in M2 markers compared with the control-miR-NC group,while the M1 marker IL-1β protein level was significantly increased(P<0.01),iNOS and TNF-α protein showed no significant difference.CD206,Arg1,and IL-10 protein levels of NaAsO2-miR-mimic and IL-4-miR-mimic groups were significantly reduced compared with those of NaAsO2-miR-NC and IL-4-miR-NC groups(P<0.05),while iNOS,IL-1β and TNF-α protein levels were significantly increased(P<0.01).Conclusion:1.Sodium arsenite can induce M2 type polarization of macrophages.2.Sodium arsenite induces M2 type polarization of THP-1-derived macrophages through reducing the content of miR-125a-5p and promoting its arget protein IRF4. |
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