HMGB1 Promotes Th17 Cell Differentiation By Regulating The Expression Of MBD2

Objective:To explore whether High mobilit y group box 1(HMGB1)participates in the differentiation of Th17 cells by regulating the expression of Methyl-Cp G binding domain protein 2(MBD2).Methods:The 6-week-old female C57BL/6 mice were killed by breaking their necks,and the 5min was soaked in 75%alcohol.Put the spleen tissue into an iron filter,grind evenly until the spleen cells are completely separated,then wash the spleen cells attached to the Internet with PBS,and collect the washed spleen cells.After magnetic beads sorting,the immature CD4~+T cells adsorbed on LS column were collected and MBD2 and HMGB1 were silenced by activation and si-RNA cell transfection.(1)Cell grouping:(1)blank group:the cells were not treated;(2)si-NC group:After transfection with si-NC,anti-IL-4(10μg/ml),IL-6(20ng/ml),TGF-β1(5ng/ml),IL-23(50ng/ml),IFN-γ(10μg/ml)were added;(3)Si-HMGB1 group:after transfection of si-HMGB1,IL-23(50ng/ml),IL-6(20ng/ml),IFN-γ(10μg/ml),TGF-β1(5ng/ml)and anti-IL-4(10μg/ml)were added.(4)Si-MBD2 group:after transfection of si-MBD2,anti-IL-4(10μg/ml),IL-6(20ng/ml),IFN-γ(10μg/ml),TGF-β1(5ng/ml)and IL-23(50ng/ml)were added.(2)Experimental methods:(1)RT-q PCR and Western blot were used to detect the expression of RORγt,MBD2,HMGB1 and IL-17A in spleen naive CD4~+T cells of the above 4 groups of mice.(2)Western blot was used to detect the protein expression levels of RORγt,MBD2 and HMGB1 in spleen immature CD4~+T cells of the above four groups of mice.(3)Flow cytometry was used to detect the proportion of IL-17A and IL-17 in spleen immature CD4~+T cells of the above four groups of mice.(4)ELISA method was used to detect the content of IL-17 in the supernatant of spleen immature CD4~+T cells of the above four groups.Results:The results of RT-q PCR showed that after HMGB1 silencing,the gene expression level of MBD2 in si-HMGB1 group was significantly lower than that in blank group and blank group(P<0.05);Compared with the blank group and the blank group,there was no significant difference in the expression of HMGB1 gene in the MBD2 silencing group(P>0.05);When HMGB1 and MBD2 were silenced respectively,the gene expression of IL-17A and RORγt was lower than that of blank group and blank group,and the difference was statistically significant(P<0.05).(2)The results of Western blot showed that there was no significant difference in the expression of HMGB1 protein in si-MBD2 group when MBD2 was silenced compared with blank group and no load group(P>0.05);The results of Western blot showed that when HMGB1 was silenced,the expression of MBD2 protein in si-HMGB1 group was lower than that in blank group and unloaded group,and the difference was statistically signifi cant(P<0.05);The results of Western blot showed that when HMGB1 and MBD2 were silenced respectively,the expression of RORγt protein in si-HMGB1 group and si-MBD2 group was significantly lower than that in blank group and blank group(P<0.05).(3)The results of flow cytometry showed that Th17cells were tested in mouse spleen CD4~+T cells,and then intracellular IL-17A antibody staining was performed by flow cytometry.Compared with blank group and empty load group,the proportion of IL-17A decreased in silent HMGB1 and silent MBD2,and the difference was statistically significant(P<0.05).(4)The results of ELISA showed that the secretion of IL-17 in spleen CD4~+T cells in si-MBD2 group and si-HMGB1 group was significantly lower than that in blank group and emp ty group,the difference was statistically significant(P<0.05).Conclusion:HMGB1 promotes the differentiation of mouse spleen immature CD4~+T cells into Th17 cells by regulating the secretion and expression of IL-17 and RORγt mediated by MBD2.