Modulatory Effects Of 17β-estradiol On Human β-defensin-2 And Pto-inflammatory Cytokines In Oral Mucosal Epithelial Cells

Objectives:To investigate the modulatory effects of 17β-estradiol（E2）on the expression of humanβ-defensin-2（hBD-2）,tumor necrosis factor（TNF）-α,interleukin（IL）-6,and IL-8 as well as the activation of the estrogen receptors（ERs）in Classical Genomic Mechanism and GPR30 in Non Classical Genomic Mechanism by IL-1β-induced human oral mucosal epithelial cells（hOMECs）in culture.Also to discuss the effects and mechanisms of E2 on innate immume responses in oral epithelium.Method:（1）Oral mucosal tissue was obtained from infant girls who underwent cleft lip surgery.Using the method of enzymatic digestion primary cultures of epithelial cells were obtained.The epithelial cells from sceond passages were pretreated with different concentrations of E2(10^-7M,10^-8M,10^-9M)for 36h,and were stimulated by 100ng/ml IL-1βfor 12hours.Quantitative protein secretion levels of cultured cells were measured for hBD-2,IL-6,IL-8 via enzyme-linked immunosorbent assays（ELISA）,and adherent cells were used to extract total RNA for detecting hBD-2,TNF-α,IL-6 and IL-8mRNA as well as ERα,ERβand GPR30mRNA via real-time quantitative polymerase chain reaction（RT-qPCR）.Adherent cells are also used to extract total protein to quantify the protein levels ERβand GPR30 with Western Blot（WB）.（2）Culture cells were pretreated for 1h with ICI182780,a specific estrogen receptor antagonist,and G1,GPR30 agonist,followed by E2 culturing36h,and then were stimulated by100ng/ml IL-1βfor 12h.Culture media were collected for quantifying the protein secretion levels of hBD-2 via ELISA,and adherent cells collected for detecting hBD-2mRNA via RT-qPCR.Using SPSS 21.0 software,data obtained from three independent experiments were statistically analysed.Data were expressed as means±SD.All data were subjected to analysis of variance（ANOVA）,P-values<0.05 were considered to be statistically significant.Results:（1）The modulatory effects of different concentrations of E2(10^-7M,10^-8M,10^-9M)on the expression of hBD-2,TNF-α,IL-6 and IL-8 in IL-1β-induced hOMECs.（1）Our results showed that IL-1βcould induce the expression of hBD-2,TNF-α,IL-6,IL-8 at mRNA and hBD-2,IL-6,IL-8protein secretions by hOMECs compared to control group（P<0.05）;（2）10^-7M,10^-8M E2 suppressed IL-1β-induced hBD-2mRNA expression compared to IL-1β-induced group（P<0.05）,but only 10^-8M E2 suppressed the secretion of hBD-2.（3）10^-7M,10^-8M,10^-9M E2 significantly suppressed IL-1β-induced mRNA expression of TNF-α,IL-6 as well as the secretion of IL-6 in a dose-dependent manner;（4）10^-7M E2 can inhibit IL-1β-induced mRNA and secretion of IL-8.（2）The expression of ERα,ERβof Classical Genomic Mechanism and GPR30 of Non Classical Genomic Mechanism in hOMECs with treatment of E2 and/or IL-1β.（1）IL-1βby hOMECs resulted in decreased modulation of ERαmRNA（P<0.05）;whereas IL-1βresulted in up-regulation of ERβat mRNA expression,on the contrary,decreased modulation of ERβprotein secretions;IL-1βhad no effect on GPR30 at mRNA expression,but GPR30protein secretions appeared slightly supressed（P>0.05）.（3）Three concentrations of E2 couldn’t attenuate IL-1βinhibition of ERαmRNA;10^-8M E2 could increase ERβmRNA expression（P<0.05）,and partly revers IL-1βinhibition of ERβprotein secretions levels simultaneously（P<0.05）;10^-8M E2upregulated the production of GPR30 at mRNA and 10^-7,10^-88 M E2 protein secretions in IL-1β-stimulated hOMECs.（3）We employed a specific estrogen receptor antagonist,ICI182780,and GPR30 agonist,G1,to detect the changes of hBD-2 in hOMECs.（1）hOMECs preincubated with E2 and ICI abrogated the estradiol inhibitory effect on IL-1β-mediated hBD-2mRNA（P<0.05）,but the difference of hBD-2 secretion was not statistically significant between the two groups of ICI182780+E2+IL-1βand E2+IL-1β.（2）G1 could inhibite the IL-1β-mediated mRNA expression and secretion of hBD-2.Conclusions:（1）E2 inhibits IL-1β-mediated production of hBD-2 and proinflammatory cytokines by hOMECs.（2）E2 supresses IL-1β-mediated expression of hBD-2 via activation of GPR30 and ERβby hOMECs.（3）Non Classical Genomic Mechanism and Classical Genomic Mechanism are involved in E2 regulation of IL-1β-induced immune activation.