Dexamethasone Suppresses The Beta Defensins And Its Related Toll Like Receptor Expression In Human Oral Mucosal Epithelial Cells

Objective: 1.In this study,to established an inflammatory model in vitro,recombinant human inflammatory factor was used to stimulate human oral mucosal epithelial cells(hOMEC).We observed the effect of inflammatory cytokine on expression of human beta defensin(human Beta-defensins,hBDs)and Toll like receptor(Toll-like receptors,TLR)in h OMECs.2.Adding Toll like receptor inhibitor on hOMEC after stimulation of inflammatory factors,we detected the change of hBD-2 and hBD-3 expression,and explored the molecular mechanism of hBD-2 and h BD-3 secretion stimulated by inflammatory factors in hOMEC.3.To explore the effect and mechanism of dexamethasone(Dex)on oral epithelium,we detect the influence of Dex on the expression of toll like receptors and hBD-2 and hBD-3 hOMEC induced by ininflammatory factor.Methods: 1.A modified tissue block method was used to cultivate the primary oral mucosa epithelial cells from the superfluous lip mucous membrane of cleft lip children in clinic.The second generation hOMEC was stimulated with recombinant human TNF-??IFN-??IL-17 either alone or a combination of these reagents at the final concentration of 50ng/ml.After 24 hours,the relative expression of TLR2,TLR4,h BD-2 and hBD-3 mRNA in the cells were detected by real-time fluorescence quantitative PCR(RT-PCR).Enzyme linked immunosorbent assay(ELISA)was used to detect the protein levels of h BD-2 and hBD-3 in the supernatant.2.The second generation of hOMEC induced by 50ng/ml(TNF-?+ IFN-?)for 24 h was taken as inflammatory model group,the TLR2 inhibitor anti-mTLR2-IgG was incubated in the experimental group for 2h before stimulating cells by inflammatory factors.RT-PCR was used to detect the mRNA level of hBD-2 and hBD-3,and the level of the protein in the supernatant was detected by ELISA.3.After pretreated with different concentrations of Dex(0.1,1,10?M)for 12 h,the second generation of hOMEC was induced by 50ng/ml(TNF-alpha + IFN-gamma)for 24 h.RT-PCR was used to detect the relative expression of TLR2,hBD-2 and h BD-3 mRNA in cells.The total protein was extracted from anchorage-dependent cells and detect the expression of TLR2 protein by Western Blot(WB).ELISA was used to detect the level of hBD-2 protein in the cell supernatant.Results: 1.Under the different inflammatory factors,the relative expression level and protein level of hOMEC hBD-2 mRNA and hBD-3 mRNA expression increased to varying degrees,the difference was statistically significant(P<0.05).Among them,the combinations of TNF-??IFN-??IL-17 had synergistic or superimposed effects on the induction of hBD-2 and hBD-3.The mRNA level of TLR2 increased significantly in the TNF-?group and the TNF-?+IFN-?group(P<0.05),the difference between the other treatment groups was not statistically significant.The mRNA level of TLR4 was increased between the treatment groups,but the difference was not statistically significant(P>0.05).2.After hOMEC pretreated with TLR2 inhibitor for 2h,the mRNA expression of hBD-2 and hBD-3 induced by inflammatory factors was significantly inhibited(P<0.05),and the protein expression level of hBD-2 was consistent with the trend of mRNA level.3.Different concentrations of Dex could decrease the relative expression of hBD-2 mRNA induced by inflammatory factors in hOMEC.And as the concentration of Dex increased,the more obvious hBD-2 mRNA decreased.The difference was statistically significant when the concentration was 10?M(P<0.05).At the level of hBD-2 protein,all concentrations of Dex could decrease the expression of hBD-2(P<0.05),and there was no significant difference between different concentrations of Dex in the expression of hBD-2(P>0.05).However,At any concentration of Dex,the relative expression of hBD-3 mRNA was not statistically significant compared with that of the model group(P>0.05).TNF-?+IFN-? could increase the relative expression of mRNA of TLR2,and the mRNA level of TLR2 increased slightly at concentration of 0.1,1 ?M Dex,but the difference was not statistically significant(P>0.05).The Dex of 0.1,1,10?M could decrease the protein expression of TLR2,and the low concentration of Dex down regulated TLR2 protein expression was the most obvious.Conclusion: TNF-? and IFN-? may induce hOMEC to express hBD-2 and hBD-3 through activating TLR2 signaling pathway.Among them,the expression of hBD-2 can be inhibited by Dex through TLR2 signaling pathway.