| Background:Gonorrhea is a sexually transmitted disease caused by the infection of Neisseria gonorrhoeae(hereafter this text will be abbreviated as Ng).It has a high incidence rate and is still a major global public health problem.Most of the men infected with Ng showed purulent discharge from the urethra,accompanied by fever,pain and other discomfort.These patients often took the initiative to see a doctor for timely treatment because of obvious symptoms.However,unlike male gonorrhea,about 50-80%of female infected with gonococcus have no symptoms such as vaginal and cervical pain and local inflammatory cell infiltration,which is manifested as female asymptomatic gonorrhea.Due to the lack of effective molecular diagnostic markers,female asymptomatic gonorrhea often delays the timely diagnosis and treatment of patients.The delay of latent infection may lead to complications such as salpingitis,pelvic inflammatory disease,infertility,and even the hidden transmission of gonorrhea and HIV,endangering public health and safety.However,the specific pathogenesis of female asymptomatic gonorrhea is still unclear.Clarifying the pathogenesis of female asymptomatic gonorrhea is of great significance for screening molecular diagnostic markers of the disease and for the prevention and control of sexually transmitted diseases.High levels of IL-17 can be detected in the serum of gonorrhea patients.IL-17 is the main effector of TH17 cells,suggesting that TH17 cells play a key role in host anti-infection.When infected with Ng,Estradiol(E2)can inhibit the differentiation of Th17 cell and the production of pro-inflammatory factors such as IL-1β,IL-6 and IL-17 on monocyte to down-regulates mucosal immunity and promotes vaginal pathogenic bacteria infection,suggesting that E2 can inhibit the progress of inflammation and promote the onset of asymptomatic gonorrhea during gonococcal infection.Janus kinase/signal transduction and activator of transcription(JAK/STAT)pathway is a signal transduction pathway that is widely expressed in cells and participates in the key biological processes of cell proliferation,differentiation,apoptosis and immune regulation.More and more evidences show that the continuous activation of JAK/STAT signaling pathway is closely related to many immune and inflammatory diseases,such as psoriasis,rheumatoid arthritis,Parkinson’s neuritis,inflammatory bowel disease,multiple sclerosis,etc.Previous studies have shown that E2 can inhibit the release of inflammatory factors in ischemic encephalopathy by inhibiting the phosphorylation of JAK/STAT3 to reduce brain damage.Therefore,we speculate that E2 can inhibit the local inflammatory reaction during gonococcal infection by inhibiting the activation of JAK/STAT signal pathway,leading to the occurrence of female asymptomatic gonorrhea.This topic will explore the effect of E2 on the phosphorylation of JAK/STAT in the gonococcal infection model by constructing a mouse spleen mononuclear cell gonococcal infection model.Objective:1.The model of gonococcal infection of spleen mononuclear cell was established,and the cell viability of each group was measured by CCK8;2.Exploring the effect of E2 on the expression of pro-inflammatory factors IL-17A,IL-17F,IL-36α,IL-36β,IL-36γ and anti-inflammatory factors IL-10 and TGF-β in gonococcal infection model.3.To determine the effect of E2 on JAK/STAT pathway regulation in gonococcal infection model of spleen mononuclear.Method:The spleen of 8-10-week-old SPF grade C57BL/6 female mice was isolated and ground to prepare cell suspension.Mononuclear cell suspension was obtained by density gradient centrifugation and erythrocyte lysis.The living cells were counted by trypan blue staining.Prepare Ng lysate and E2 solution.The experiment was divided into four groups:blank control group,estradiol(E2)group,gonococcus(Ng)group,gonococcus+estradiol(Ng+E2)group.The blank control group was divided into 10%FBS+RPMI 1640 medium and mononuclear cells,E2 group includes 10%FBS+RPMI 1640 medium,mononuclear cells and E2 solution,Ng group includes 10%FBS+RPMI 1640 medium,mononuclear cells,and Ng lysate,Ng+E2 group includes 10%FBS+RPMI 1640 medium,mononuclear cells,Ng lysate and E2 solution.The number of mononuclear cells in each group is 2.0×107,E2 concentration is 10-8 mol/L,the concentration of Ng suspension corresponding to Ng cracking solution is 2.0×107CFU/mL.The CCK8 activity of four groups of cells was measured after 24 hours of culture,and the expression of IL-17A,IL-17F,IL-36α,IL-36β,IL-36γ,IL-10 and TGF-β of cells in each group were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Enzyme-linked immunosorbent assay(ELISA).The phosphorylation of JAK(1-3),Tyk2 and STAT(1-6)in each group was determined by Western blot.Result:1.CCK-8 test results showed that the cell viability of Ng group was the highest,followed by Ng+E2 group(P<0.05 or 0.01),E2 group and blank group were the third(the difference between groups was not statistically significant,P>0.05).2.RT-qPCR and ELISA results showed that compared with the blank group,the pro-inflammatory cytokines IL-17A,IL-17F,IL-36α,IL-36β,IL-36γ in Ng group was significantly up-regulated and the expression of IL-10 and TGF-β was significantly down-regulated(P<0.01 or 0.001);Compared with Ng group,the expression of IL-17A,IL-17F,IL-36α,IL-36β,IL-36γ was significantly decreased and the expression of IL-10 and TGF-β was significantly increased in Ng+E2 group(P<0.05);There was no significant difference in the expression of inflammatory factors between E2 group and blank group(P>0.05).3.Western blot showed that the phosphorylation degree of JAK(1-3),Tyk2 and STAT(1-6)in the Ng group was significantly higher than that in the blank group,and the phosphorylation degree of Tyk2 and STAT2 was the most obvious(P<0.001 or 0.01);Compared with Ng group,the phosphorylation of JAK(1-3),Tyk2 and STAT(1-6)in Ng+E2 group was inhibited to varying degrees,and the phosphorylation of Tyk2 was the most inhibited(P<0.01);There was no statistically significant difference in phosphorylation of JAK(1-3),Tyk2 and STAT(1-6)between E2 group and blank group.Conclusion:E2 can inhibit the phosphorylation of JAK(1-3),Tyk2 and STAT(1-6)in mouse spleen mononuclear model infected with Ng to varying degrees,and inhibit the express of pro-inflammatory factors IL-17A,IL-17A,IL-17F,IL-36α,IL-36β and IL-36y,promote the expressionof anti-inflammatory factors IL-10 and TGF-β.E2 regulates the JAK/STAT signaling pathway,thereby affecting the inflammatory expression of the spleen mononuclear model infected with Ng. |
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