Effects Of Pathogen-associated Molecular Patterns(PAMPs) On The Expression Of Inflammatory Cytokines And Steroid Hormone Receptors In Endometrial Stroma Cell Of Rabbit

The female reproductive tract opens directly to the body surface,and its physiological structure makes it vulnerable to various pathogenic microorganisms.Microbial infections can lead to reproductive tract infections,which leads to infertility,miscarriage or even fetal death.As a protective barrier for innate immunity of uterus,endometrium expresses almost all Toll-like receptors（TLRs）.When pathogenic microorganisms invade,endometrium recognizes different pathogen-associated molecular patterns through TLRs,activates the innate immune response,and cleans the pathogen together with the acquired immune response.The aim of this study was to investigate the effect of Pathogen-associated Molecular Patterns（PAMPs）on the expression of inflammatory cytokine and steroid receptor genes in ESCs cultured in vitro.In addition,the effects of P₄ and E₂ on the inflammatory response of ESCs were studied by adding P₄ and E₂ to simulate the levels of hormones in different stages of estrus cycle in vitro.ESCs was isolated from endometrial tissue of healthy New Zealand white rabbits（n=5）and tested for bacteria,fungi and mycoplasma.After 24 hours of serum-free starvation,cultured cells were treated with 5 kinds of PAMPs（Gram-negative bacteria lipopolysaccharide（LPS）,double-stranded virus RNA mimic polyinosine-polycytidine（Poly I:C）,Gram-negative flagellin（Flagellin）,Single-stranded RNA virus mimics R848 and unmethylated oligodeoxynucleotides（Cp G-ODN）widely present in bacterial and viral DNA）for 6 hours.The expression of corresponding TLRs and the expression of downstream inflammatory cytokine IL6,IL8,IL1β,TNFα,IFNγand ovarian steroid receptor were detected respectively.In addition,the effect of Poly I:C on the immune stress of ESCs in vitro was studied after 0,6,12,24h treatment by Poly I:C.Adding different concentrations of P₄ and E₂ in the cell culture system to study the effects of P₄ and E₂ on the induction of inflammatory response by Poly I:C.Select the best concentration of P₄ and E₂.Simulating the E₂-dominated follicular phase and P₄-dominant luteal phase in the uterine environment to study the effect of ovarian steroid hormone on the expression of inflammatory cytokines IL6,IL8,IL1β,TNFαand steroid hormone receptor induced by Poly I:C.The results show that by enzyme digestion primary cells and purifing with the method of differential attachment during passage cultures,the purity of ESCs was 98.3%and without the infection of fungi,bacteria and common mycoplasma,chlamydia.After 6h of treatment,Poly I:C up-regulation expression of TLR3 and inflammatory cytokines IL6,IL8,IL1β,TNFαin ESCs and down-regulate the expression of ER-αand PGRMC1.Poly I:C up-regulated the m RNA and proteins expression level of inflammatory cytokines IL6,IL8,IL1βand TNFα,and down-regulated the proteins expression level of ER-αand PGRMC1 in a time-dependent manner within 24h.P₄ inhibited the upregulation of ESCs proinflammatory cytokines IL6,IL8,IL1βand TNFαinduced by Poly I:C in a dose-independent manner,while E₂ inhibited the up-regulation of IL6,IL8,IL1βand TNFαexpression of ESCs induced by Poly I:C in a dose-independent manner.P₄ and E₂ did not show the additive effect on the inhibition of inflammation after combined treatment of ESCs.After adding P₄ or E₂ to the medium,Poly I:C treatment for 6h did not inhibit the expression of ER-αand PGRMC1 in ESCs,while PGR was easily regulated by Poly I:C in the presence of progesterone.This study further reveals the relationship between the expression of the inflammatory cytokines and the steroid hormone receptor gene in the endometrium,and the role of the ovarian steroid hormone in the defense of the pathogenic microorganism infection in different stages of the estrus cycle.This study provides a theoretical basis for the reproductive dysfunction caused by various bacteria and viruses in the reproductive tract of female animals.