Effects of estradiol on the JAK/STAT pathway and the expression levels of inflammatory cytokines in a gonococcal infection model
Objective To explore the effects of Estradiol(E2)on the JAK/STAT pathway and the expression levels of inflammatory cytokines in a mouse spleen mononuclear cell model of gonococcal infection.Methods Mononuclear cell suspension was obtained from 8-10-week-old female C57BL/6 mice.The cells were divided into 4 groups:blank control group,E2 group,Neisseria gonorrhoeae(Ng)group,and Neisseria gonorrhoeae+Estradiol(Ng+E2)group.Mononuclear cells in the blank group were incubated with 10％FBS+RPMI1640 medium,while cells in E2 group were incubated with 10％FBS+RPMI1640 medium+E2 solution.Mononuclear cells in the Ng group were cultured with 10％FBS+RPMI1640 medium+Ng lysate,and mononuclear cells in the Ng+E2 group were cultured with 10％FBS+RPMI1640 medium+Ng lysate+E2 solution.The number of mononuclear cells in each group was 2.0× 107,and the final concentration of E2 was 108 mol/L.The Ng lysate was obtained by sonication of a Ng suspen-sion in 1 mL of 2.0 ×107 CFU/mL.Real-time fluorescence quantitative PCR(RT-qPCR)was used to measure the expression levels of mRNA for IL-17A,IL-17F,IL-36α,IL-36β,and IL-36γ.Enzyme-linked immunosorbent assay(ELISA)was used to measure the expression levels of inflammatory cytokines in each group.Western blot was used to determine the levels of non-phos-phorylated and phosphorylated JAK(1-3),Tyk2,and STAT(1-6)in each group.Results RT-qPCR and ELISA results showed that compared with the blank controls,the expression levels of proinflammatory cytokines(IL-17A、IL-17F、IL-36α AIL-36β、IL-36γ)in the Ng group were signifi-cantly up-regulated,while the expression levels of anti-inflammatory cytokines(IL-10、TGF-β)were significantly down-regulated.In contrast,the expression levels of proinflammatory cytokines in the Ng+E2 group were significantly down-regulated,while the expression levels of anti-inflam-matory factors was significantly up-regulated compared with the Ng group.However,the expres-sion levels of inflammatory cytokines did not differ significantly between E2 and blank group(P＞0.05).Western blot results showed that the levels of phosphorylated JAK(1-3),Tyk2,and STAT(1-6)were significantly higher in the Ng group than in the blank control(P＜0.01).Moreover,the levels of phosphorylated JAK(1-3),Tyk2,and STAT(1-6)were lower in the Ng+E2 than in the Ng group.The phosphorylated levels of JAK(1-3),Tyk2,and STAT(1-6)were comparable between the E2 and the blank group(P＞0.05).Conclusions E2 can inhibit the phosphoryla-tion of JAK(1-3),Tyk2,and STAT(1-6)and lower expression levels of proinflammatory cyto-kines,while up-regulating the expression levels of anti-inflammatory factors in a mouse spleen mononuclear cell model of gonococcal infection.E2 regulates the expression of inflammatory cyto-kines by modulating the JAK/STAT signaling pathway in a mouse spleen mononuclear cell model of gonococcal infection.
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