The Effect Of XBP1s In Escherichia Coli LPS-induced Inflammatory Response In Goat Endometrial Epithelial Cells

Bacterial contamination of the uterus and tissue degradation in animals after delivery leads to an increased inflammatory environment in the postpartum uterus,causing the development of endometritis and other uterine diseases.E.coli is the most prevalent pathogen isolated from the uterus of ruminants with endometritis,and lipopolysaccharide(LPS),a structural component of the E.coli cell wall,can cause endometrial inflammation by initiating an immune response in endometrial cells.Endometritis in animals has a high incidence and complex etiology,and there are no effective prevention and treatment methods,therefore,it is important to investigate the pathogenesis of endometritis in animals.A variety of physiological or pathological factors can induce an imbalance in endoplasmic reticulum homeostasis,causing Endoplasmic reticulum stress(ERS)and activating the Unfolded protein reaction(UPR)in response to it.It has been demonstrated that ERS is coupled to intracellular inflammatory response signaling pathways via the UPR,and XBP1 s,as key transcription factors downstream of the UPR,is involved in the pathological processes of multiple inflammatory diseases in the body,but the exact mechanism of ERSinduced activation of XBP1 s in the endometrial inflammatory response in ruminants is still not elucidated.In this study,we used E.coli LPS-treated goat endometrial epithelial cells(g EECs)to construct an in vitro inflammatory response model for endometrial cells,and investigated the effects of XBP1 s on LPS-induced inflammatory responses by cellular immunofluorescence,RT-q PCR,Western blot,and lentiviral interference cell line construction.The main results were as follows:(1)LPS activated ERS and the splicing of XBP1s in g EECs.LPS treatment upregulated the expression of GRP78,a marker gene of ERS,and promoted the phosphorylation of IRE1αprotein as well as the splicing and nucleation of XBP1 s.(2)LPS activates IRE1α-mediated XBP1s splicing via TLR4 and regulates inflammatory responses.Inhibition of TLR4 activity using TAK-242 was found to inhibit LPS-induced expression of the ERS marker gene GRP78 and phosphorylation of IRE1α protein,and inhibit XBP1 s splicing and nucleation;inhibition of IRE1α activity using 4μ8C inhibited LPSinduced XBP1 s splicing and nucleation;in addition,inhibition of IRE1α activity can promote LPS-induced expression and secretion of inflammatory cytokines such as IL-6,IL-8 and IL-1β,and promoted the phosphorylation of NF-κB p65 and activation of NLRP3 inflammatory vesicles.(3)Inhibition of XBP1s expression can promote LPS-induced inflammatory responses in g EECs.A goat XBP1 s lentivirus-interfering cell line was constructed,and it was found that inhibition of XBP1 s expression could promote LPS-induced expression and secretion of inflammatory cytokines such as IL-6,IL-8 and IL-1β,and promote LPS-induced phosphorylation and nucleation of NF-κB p65,in addition,inhibition of XBP1 s expression also promoted LPS-induced activation of NLRP3 inflammatory vesicles.In summary,this study found that in the LPS-induced inflammatory response of g EECs,LPS-TLR4 interacts to generate an intracellular signaling cascade that activates ERS and XBP1 s splicing,and XBP1 s,as a key transcription factor downstream of UPR,influences the LPS-induced inflammatory response of g EECs by regulating the NF-κB pathway and NLRP3 inflammatory vesicle pathway.It partially reveals the molecular regulatory mechanism of XBP1 s on LPS-induced inflammatory response in g EECs,and provides a theoretical basis for further study of the mechanism related to endometritis in goats.