| ObjectiveThis study aims to clarify the regulatory role of MyD88 DNA methylation in LPS-induced inflammation of human bronchial epithelial cells.Methods1.Constructing a inflammation model of BEAS-2B cells induced by LPS1.1 BEAS-2B cells were stimulated with different concentrations of LPS(0,0.1,1,10,100 ug/mL)for 24 h,and the cell viability was calculated by the CCK-8 array.1.2 After stimulating BEAS-2B cells with 1 ug/mL LPS for 24 h,the relative expression level of IL-6 and IL-8 at mRNA were measured by the real-time fluorescent quantitative PCR.2.The change of inflammatory cytokines after demethylation of BEAS-2B cells induced by 5-AZA-2′-deoxycytidine(5-Aza-CdR)2.1 BEAS-2B cells were stimulated with different concentrations of 5-Aza-CdR(0,1,3,6,9,12 umol/L)for 72 h,and the cell viability was calculated by the CCK-8 array.2.2 After BEAS-2B cells were stimulated with 1 umol/L 5-Aza-CdR for 72 h,cells were stimulated with 1 ug/ml LPS for 24 h.The expression level of IL-6 and IL-8 at mRNA were measured by the real-time fluorescent quantitative PCR,and the expression level of IL-6 and IL-8 at protein were measured by ELISA.3.Analyzing the probable molecular mechanism that DNA methylation affects BEAS-2B cells inflammatory response induced by LPSAfter 5-Aza-CdR pretreatment,BEAS-2B cells were stimulated with LPS for 24 h.Then the protein level of MyD88、IκBα、p-IκBα、p65、p-p65 was detected by Western Blot.4.The change of the methylation level in the promoter region and mRNA expression of MyD88 gene4.1 After 5-Aza-CdR pretreatment,BEAS-2B cells were stimulated with LPS for 24 h.The methylation level of the promoter region of MyD88 gene was detected by Mass ARRAY.4.2 The expression level of MyD88 at mRNA were measured by the real-time fluorescent quantitative PCR.Results1.Compared with the control group,the cell viability of BEAS-2B cells was significantly inhibited by LPS above 1 ug/mL,and the expression levels of inflammatory cytokines IL-6 and IL-8 were significantly increased.2.Compared with the control group,the cell viability of BEAS-2B cells was significantly inhibited by 5-Aza-CdR above 1 umol/L;Compared with the LPS group,after 5-Aza-CdR pretreatment,the mRNA levels and protein levels of the inflammatory cytokines IL-6 and IL-8 were significantly increased.3.Compared with the control group,the protein expressions of MyD88,p-IκBα,and p-p65 in the LPS group were significantly increased;while the protein expressions of MyD88,p-IκBα,and p-p65 after 5-Aza-CdR pretreatment were also significantly increased than the LPS group.4.In LPS-induced BEAS-2B cells,the methylation level of the MyD88 gene promoter region was significantly reduced after 5-Aza-CdR pretreatment,while the expression of MyD88 at mRNA was significantly increased.ConclusionIn conclusion,5-Aza-CdR may increase the expression of MyD88 gene by reducing the methylation level of the MyD88 promoter region in BEAS-2B cells,thereby enhances the activity of NF-κB signaling pathway and releases of LPS-induced inflammatory cytokines.These findings indicate that DNA methylation may play an important role in the inflammatory response of BEAS-2B cells induced by LPS,and provide new ideas for the research of bronchial epithelial inflammation. |
