Research On TLR2/4/NF-?B Signaling Pathway And Downstream Cytokine In The Pathogenesis Of Aerobic Vaginitis

Aerobic vaginitis(AV)is a new type of vaginal infection proposed in 2002,characterized by decreased lactobacillus and increased aerobic bacteria.Common pathogens include both Gram-negative and Gram-positive bacteria,such as Escherichia coli and Staphylococcus aureus.Toll-like receptors(TLR)2 and TLR4 can recognize peptidoglycan(PGN)and lipopolysaccharide(LPS),the main component of the wall of Gram-positive and Gram-negative bacteria,and regulate the release of downstream cytokines.Our previous work revealed excesssive IL-1?,IL-6 and IL-8 presented in AV patients' vagina.However,pathogenesis of AV remained unclear.Therefore,this study was divided into 3 parts: stimulate immortalized human vaginal epithelial cells VK2/E6E7 with Escherichia coli,Staphylococcus aureus,LPS and PGN,and establish SD rats AV model,aiming to investigate the role of TLR2/4/NF-?B signaling pathway and downstream cytokines in AV pathogenesis.Objectives Part 1: To investigate the effects of AV common pathogens on TLR2/4/NF-?B signaling pathway activation and downstream cytokines release in VK2/E6E7 cel s.Part 2: To observe the effects of LPS,PGN and neutralization of TLR2 and TLR4 on TLR2/4/NF-?B signaling pathway activation and downstream cytokines release in VK2/E6E7 cel s.Part 3: To explore the role of TLR2/4/NF-?B signaling pathway in AV pathogenesis by in vivo experiments via establishing SD rat AV model.Methods Part 1: Stimulate VK2/E6E7 cells with Gram-negative Escherichia coli(ATCC 25922)and Gram-positive Staphylococcus aureus(ATCC 29213).Observe TLR2/4 signaling pathway activation and cytokines level changes by Real Time RT-PCR,Western Blot,immunofluorescence,Luminex liquid phase chip and ELISA.Part 2: Stimulate VK2/E6E7 cells with LPS,PGN and TLR2 and TLR4 neutralize antibodies.Observe TLR2/4 signaling pathway activation and cytokines levels changes by Real Time RT-PCR,Western Blot,immunofluorescence,Luminex liquid phase chip and ELISA.Part 3: Establishing SD rats AV model with 25922 and 29213 strains.Evalue vaginal pH and observe TLR2/4 signaling pathway activation via HE staining,paraffin section and frozen section immunohistochemistry.Results Part 1:(1)25922 and 29213 strains stimulation upregulated the expression of TLR2,TLR4 and NF-?B in VK2/E6E7 cells;(2)after 2 hours cocluture with 25922 strain,pro-inflammatory cytokines IL-6 and IFN-? increased significantly,4 hours later,pro-inflammatory cytokines IL-1?,IL-6,IL-8,IFN-? and TNF-? increased significantly;(3)after 2 hours cocluture with 29213 strain,pro-inflammatory cytokines IL-32 and IFN-? increased significantly;4 hours later,IL-2,IL-8,IL-17 A,IL-18,IL-32 and IFN-? increased significantly.Part 2:(1)LPS and PGN stimulation upregulated the expression of TLR2,TLR4 and NF-?B in VK2/E6E7 cells;after neutralization of TLR2 and TLR4,activation of NF-?B p65 by LPS and PGN were attenuated;(2)after LPS treatment,pro-inflammatory cytokines IL-1?,IL-6,IL-8 and IL-23 in VK2/E6E7 cells significantly increased in a time-dependent manner;(3)after PGN treatment,pro-inflammatory cytokine IL-8 increased significantly.Part 3:(1)After modeling,vaginal pH of rats increased;(2)On the 3rd and 7th day after bacteria inoculation,vaginal inflammation were observed in each experimental group;(3)Immunohistochemistry staining indicated higher expression of NF-?B p65 in the experimental group than that in the control group.Conclusions Part 1: AV common pathogens can activate TLR2/4/NF-?B signaling pathway in VK2/E6E7 cel s and promote downstream cytokines release.Part 2: LPS and PGN can activate TLR2/4/NF-?B signaling pathway via TLR2 and TLR4 in VK2/E6E7 cel s and promote downstream cytokines release.Part 3: 25922 and or 29213 strains infection in SD rats with TLR2 and TLR4 expressed in vaginal epithelium can induce TLR2/4/NF-?B signaling pathway activation and resulted in vaginal epithlial inflammatory cell infiltration.