The Effects Of17beta-estradiol And Progesterone On Toll-like Receptors In Bovine Endometrial Epithelial Cells (BEnEpC)

Endometritis, one of the major diseases that constraining economic benefits of beef cattlebreeding industry, the main reason for infertility.The endometrium is a crucial barrier and a sitewhere the host's defenses interact with pathogens. Toll-like receptor (TLRs) are associated withthe mechanisms by which endometrial epithelial cells identify pathogenic microbes. In addition,cytokines and inflammatory chemokines, produced in response to endometrial TLRs, arenecessary for both immunity and reproductive function.The incidence of endometritis closelyrelated to the cyclical physiological changes of reproductive hormones, especially estradiol (E2)and progesterone (P). But the molecular mechanism of reproductive hormones associated withthe pathogenesis is unclear.This study was designed in vitro experiments, by different stimuli changes, verify changesof TLR2,4expression and Cytokines and inflammatory chemokines by induced from theperspective of TLR2and TLR4. Analysis of hormone regulation of uterine immune system. Tofind the targets of endometrial immune regulation from the molecular level. Provide atheoretical basis and in vitro experimental data for treatment and prevention of endometritis atthe follow-up tests.In this study, bovine endometrial epithelial cells were cultured and subsequently analyzedfor the expression of TLR2/4mRNA and protein using RT-PCR and Western blot,Immunofluorescence and other test methods. To study the role of hormones, cells were treatedwith10-8M E2and/or10-7M P. Before and after the hormone treatment, the TLR2/4ligandLTA and/or LPS was used to stimulate the cells. Secretion of cytokines and chemokines wasevaluated via ELISA. We successful detected TLR2/4Real-time PCR via preparation ofTaqMan probe. This study investigates the roles of E2and P in the expression and function ofTLR2/4in bovine endometrial epithelial cells in an attempt to elucidate the modulating effect ofhormones on endometrial immune mechanisms involving TLRs.First, the bovine uterine epithelial cells were cultured. To study the role of hormones, cellswere treated with E2/P. Before and after the hormone treatment, the TLR2/4ligand LTA and/orLPS was used to stimulate the cells, determine the optimal working concertration of various additives, build the cell model for the follow-up test to lay a stability and good foundation.Before and after the hormone treatment, the TLR2/4ligand LTA/LPS was used to stimulatethe cells,study the role of E2around the inflammatory substances stimulate the cells. The resultshowed that: Add to LTA/LPS, the expression levels of TLR2/4mRNA and protein weresignificantly increased. Secretion levels of cytokines and inflammatory chemokines that inducedby TLR2/4were significantly increased. Description LTA and LPS as a PAMP, TLR2and TLR4could proinflammatory effect, increasing the biological activity of TLRs, and cytokineproduction and other products in the inflammatory response plays an important role. E2inhibited the expression of TLR2/4mRNA and protein, and reduced secretion of inflammatorycytokines and chemokine induced by TLR2/4.Promptde to the regulate of E2can be carried byTLRs, this role is mainly inhibiting by induced cytokines and inflammatory chemokines ofTLRs.Before and after the hormone treatment, the TLR2/4ligand LTA/LPS was used tostimulate the cells,study the role of P around the inflammatory substances stimulate the cells.The result showed that: Add to LTA/LPS, the expression levels of TLR2/4mRNA and proteinwere significantly increased. Secretion levels of cytokines and inflammatory chemokines thatinduced by TLR2/4were significantly increased. P promote the expression of TLR2/4mRNAand protein, and increase secretion of inflammatory cytokines and chemokine induced byTLR2/4. Prompted to regulation of P can be adjusted by the expression and biological activitychanges of TLRs.According to the publishing sequence in GenBank, primers and TaqMan probe weredesigned, The method of fluorescent quantitation RT-PCR was established to detect the mRNAexpression of TLR2and TLR4. Respectively, quantitative detection the mRNA expression ofTLR2and TLR4by add E2+LTA/LPS, LTA/LPS+E2, P+LTA/LPS, LTA/LPS+P. It wasfound that, TLR2and/or TLR4mRNA experession were E2,P,LTA/LPS,LTA/LPS+E2,E2+LTA/LPS, P+LTA/LPS, P+LTA/LPS.This study further confirmed the regulation ofTLR2,TLR4expression from E2and P by the method of quantitative. Establish a rapid andstable quantitative detection method of TLR2and TLR4, this detection method have highlyspecific, sensitive.Our current studies suggest a role for TLR2/4in the endometrium. Recognition of LTA andLPS by TLR2/4and the resulting production of inflammatory and antiviral cytokines and chemokines may be pivotal in the protection of the endometrium from pathogens. The cyclicregulation of TLR2/4in the endometrium may allow protection against pathogens whilemaintaining a system of tolerance toward the embryo and preventing extensive tissue damagefrom the inflammatory response.. Thus, the influence of steroid hormones on TLR2/4function,specifically the suppression of cytokines and chemokines produced upon stimulation of TLR2/4by LTA and LPS, may be critical in the regulation, maintenance, and defense of theendometrium.