The Role Of Foxp3 Gene Promoter Methylation In Immunoregulation Process Of Human Lymphocytes Exposed To Arsenic And The Intervention Effect Of 5-Aaz-CdR

Objective:Investigating the role of Foxp3 gene promoter methylation in immunoregulation process of human lymphocytes exposed to arsenic,and taking targeted intervention,in order to provide scientific basis for the improvement and clarification of arsenic poisoning mechanism and reliable interventions.Methods:?1?Exposure tests:Peripheral blood lymphocytes from healthy adults were isolated and cultured by density gradient method and exposed to0.00,5.00,10.00,20.00,30.00,40.00?mol/L?NaAsO₂?for 24h.Relative survival rates of cells were detected by MTT colorimetric method,HE staining was used to identify the exposed model.Real-time quantitative?RT-qPCR?was taken to detect mRNA transcription levels of Foxp3 and DNMT1.Relative protein expression levels of Foxp3 and DNMT1 were detected by Western blot.Methylation status of Foxp3gene promoter region was detected by bisulfite?BSP?sequencing.Tegulatory T cells?Treg?accounted for CD4⁺T cell ratio was measured by flow cytometry.Enzyme-linked immunosorbent assay?ELISA?was used to detect the levels of IL-10and IL-35 in the supernatant.?2?Intervention tests:control group?without any treatment?,NaAsO₂ group?20?mol/L NaAsO₂?,NaAsO₂+5-Aza-CdR group?20?mol/L NaAsO₂+5.00?mol/L 5-Aza-CdR?,5-Aza-CdR group?5.00?mol/L 5-Aza-CdR?were set up.Relative protein expression levels of Foxp3 and DNMT1 were detected by Western blot.The methylation status of Foxp3 gene promoter was detected by BSP sequencing.Treg was detected by flow cytometry.IL-10 and IL-35 levels were detected by ELISA.Results:1.Exposure tests:?1?The effect of NaAsO₂ exposure on lymphocyte survival rates:With the increase of NaAsO₂ concentration,the survival rates of lymphocytes in each exposed group showed a downward trend(r_{rate of lymphocytes}=-0.96,P<0.05).Compared with control group,the survival rates of each NaAsO₂ group decreased,and the differences were statistically significant?P<0.05?.?2?The expression of Foxp3 and DNMT1 genes:Compared with control group,mRNA and protein expression levels of Foxp3 gene in lymphocytes of all exposed groups significantly decreased?P<0.05?,while mRNA and protein expression levels of DNMT1 significantly increased?P<0.05?.?3?The methylation rates of Foxp3 gene promoter region:The methylation rates of Foxp3 gene promoter region in 0.00,5.00,10.00,20.00,30.00?mol/L NaAsO₂ group were 1.3%?1/80?,5.0%?4/80?,12.5%?10/80?,77.5%?62/80?,87.5%?70/80?respectively.Except for 5.00?mol/L NaAsO₂ exposure group,the methylation rates of Foxp3 gene promoter region in other exposure groups were significantly higher than that in control group?P<0.05?.?4?The proportion of Treg cells:Compared with control group,the proportion of Treg cells in CD4⁺T cells decreased during 10.00?mol/L and above exposure groups,and the differences were statistically significant?P<0.05?.?5?The secretion of cytokines by Treg cells:Compared with control group,the levels of IL-10 in the lymphocytes decreased in 10.00?mol/L and above groups significantly?P<0.05?.IL-35 level decreased obviously in 20.00?mol/L and above exposure groups?P<0.05?.2.Intervention tests:?1?Intervention effect of 5-Aza-CdR on protein expression of Foxp3 gene:Compared with control group,the protein expression of Foxp3 decreased obviously in NaAsO₂ group and NaAsO₂+5-Aza-CdR group?P<0.05?.Compared with NaAsO₂ group,the protein expression level of Foxp3 gene increased in NaAsO₂+5-Aza-CdR group,and the difference was statistically significant?P<0.05?.?2?Intervention Effect of 5-Aza-CdR on protein expression of DNMT1 gene:Compared with control group,DNMT1 protein expression increased in NaAsO₂group,while DNMT1 protein expression decreased in NaAsO₂+5-Aza-CdR group?P<0.05?.Compared with NaAsO₂ group,the expression level of DNMT1protein in NaAsO₂+5-Aza-CdR group decreased,and the difference was statistically significant?P<0.05?.?3?Effects of 5-Aza-CdR intervention on methylation of Foxp3gene promoter region:The methylation rates of Foxp3 gene promoter region were2.5%?2/80?,76.3%?61/80?,57.5%?46/80?,and3.8%?3/77?in control group,NaAsO₂group,NaAsO₂+5-Aza-CdR group,5-Aza-CdR group,respectively.Compared with the control group,the methylation rates of Foxp3 gene promoter region increased in NaAsO2 group and NaAsO2+5-Aza-CdR group significantly??2control group and NaAsO2 group=91.141,?2control group and NaAsO2+5-Aza-CdR group=57.619,P<0.018?.Compared with NaAsO₂ group,the methylation rate of the promoter region of Foxp3 gene decreased in NaAsO₂+5-Aza-CdR group,and the difference was statistically significant??2=6.348,P<0.018?.?4?Intervention effect of 5-Aza-CdR on the proportion of Treg cells:compared with control group,the proportion of Treg cells decreased in NaAsO₂group and NaAsO₂+5-Aza-CdR group in CD4⁺T cells.Compared with NaAsO₂group,the proportion of Treg cells in CD4⁺T cells increased in NaAsO₂+5-Aza-CdR group,and the difference was statistically significant?P<0.05?.?5?Intervention effect of 5-Aza-CdR on the secretion of cytokines by Treg cells:Compared with control group,the expression levels of IL-10 and IL-35 decreased in NaAsO₂ group and NaAsO₂+5-Aza-CdR group.Compared with NaAsO₂ group,the expression levels of IL-10 and IL-35 in NaAsO₂+5-Aza-CdR group increased obviously?P<0.05?.Conclusions:1.In vitro,NaAsO₂ can induce high expression of DNMT1,up-regulate the methylation level of Foxp3 gene promoter region in human lymphocytes,then inhibit the transcription and expression of Foxp3 gene,and at last induce the reduction secreting of Treg cells and the related anti-inflammatory factors.As a result,anti-inflammatory function is weakened.This might be the basis of immune dysfunction of arsenic poisoning.2.5-Aza-CdR can effectively reactive the hypermethylation of Foxp3 gene promoter region of human lymphocyte through inhibiting the expression of DNMT1,thereby reactive the protein expression level of Foxp3 gene.3.5-Aza-CdR intervention can stimulate the activation of Treg cells,promote the secretion of anti-inflammatory factors,and may restore anti-inflammatory and immune regulation functions during arsenic poisoning to a certain extent.