MBD2 Regulates Th2-type Response And Its Role In Allergic Inflammation

Objective:Methyl-Cp G-binding protein 2(MBD2),as an important member of the methyl-Cp G-binding proteins(MBDs),plays a key role in epigenetics,It has a very high binding affinity for deoxyribonucleic acid(DNA)methylation(the main regulation mode of epigenetics),and is considered to be a reader of DNA methylation encoding information,and can read and translate DNA methylation.This research takes the main regulation method of epigenetics-DNA methylation as the entry point,focuses on the MBD2 protein(the performer of DNA methylation regulation effects)and helper T cell 2(Th2)immune response,simulates allergic inflammation stimulation with ovalbumin(OVA),and applies to cell experiments to preliminarily study that MBD2regulates Th2 differentiation and function and its role in allergic inflammatory response,to provide preliminary experimental evidence for the in-depth study of subsequent airway inflammation,allergic asthma and its epigenetic mechanisms.Methods:After isolating and sorting out wild-type C57BL/6mouse spleen CD4~+T cells,we transfected part of the CD4~+T cells to silence MBD2 gene,and divided into wild-type group,short hairpin RNA(sh RNA)empty group(normal CD4~+T cells transfected with empty lentivirus),and sh RNA-MBD2 group(CD4~+T cells transfected with MBD2 optimal silencing target virus);in vitro simulation of Th2 cell differentiation microenvironment,while adding DNA methylation inhibitors 5-Aza-2’-deoxycytidine(5-Aza-Cd R)for comparative intervention,we collected each group of cells for flow cytometric detection to count the number of Th2 cell,enzyme linked immunosorbent assay(ELISA)to detect the level of interleukin-4(IL-4)secretion,and DNA methylation detection to measure the content of5-methylcytosine(5-mc).In addition,wild-type C57BL/6 mouse spleen-derived mononuclear cells were isolated,and some mononuclear cells were transfected first to obtain wild-type group,sh RNA empty group(mononuclear cells transfected with empty lentivirus),and sh RNA-MBD2 group(mononuclear cells transfected with MBD2 optimal silencing target virus),then added environmental allergen OVA to stimulate the culture;used magnetic beads to sort out CD4~+T cells,and collected cells in each group to observe the number of Th2 cells(flow cytometry),the secretion level of IL-4(ELISA),and the level of DNA methylation(methylation DNA quantification kit).Results:The experiment found that compared with the Th2 differentiation group,the Th2ratio and IL-4 level in the 5-Aza group were increased(P<0.05);under the Th2differentiation and 5-Aza intervention,the Th2 ratio and the secretion of IL-4 in the sh RNA-MBD2 group were higher than those of the wild-type group and the sh RNA empty group(P<0.05).The Th2 ratio and IL-4 level in the 200μg/m L OVA group were higher than those of the control group(P<0.05);under the control and OVA intervention,compared with the wild-type group and the sh RNA empty group,the Th2 ratio of the sh RNA-MBD2 group increased(P<0.05).Conclusion:In vitro,the reduction of DNA methylation level(addition of 5-Aza)can promote Th2 cell differentiation and IL-4 secretion;silencing MBD2 gene also promotes Th2/IL-4 signaling effect,and silencing MBD2 gene promotes the effects of DNA methylation inhibitors on Th2/IL-4.The allergen OVA can synergistically promote Th2 cell differentiation and IL-4secretion,and silencing MBD2 will enhance the signaling effect of OVA on Th2/IL-4.It is speculated that DNA methylation and MBD2 can regulate the differentiation of Th2 cells and the secretion of IL-4,and MBD2 may participate in allergic inflammation by performing DNA methylation effects and affecting the Th2/IL-4 axis.Combined with our previous studies,it is suggested that this may also be one of the important mechanisms by which MBD2 participates in airway inflammation of allergic asthma.