The Effect Of Selenium On The Inflammatory Response Of BEEC Under High Cortisol Background

Uterine health is often compromised in cattle after parturition because postpartum contamination of the uterine lumen by bacteria is ubiquitous,and pathogenic bacteria frequently persist causing subfertility and infertility and high economic losses.Escherichia coli(E.coli)is the most common endometritis pathogens in dairy cattle and lipopolysaccharides(LPS)is the major virulence factor of E.coli.LPS binds to Toll-like Receptor 4(TLR4)on endometrial cells,leading to an inflammatory response characterized with the secretion of cytokines and the activation of the nuclear transcription factor kappa B(NF-κB)and mitogen-activated protein kinase(MAPK)signalling pathways.Selenium(Se)is an important essential trace element that mainly functions in the form of selenoprotein,and plays a critical role in the anti-inflammatory process and the antioxidant defense system.Selenium supplementation can prevent a variety of obstetric diseases,such as miscarriage,preterm labor,retained placenta and endometritis.Studies have shown that adding selenium supplements to forage can alleviate postpartum inflammation in buffaloes.Cortisol(COR),an endogenous glucocorticoid that plays an important role in the regulation of normal body function,which concentrations are higher in postpartum cattle due to stress.However,the regulatory mechanism of selenium supplementation on the inflammatory response of bovine endometrium under high cortisol background has not been clarified.This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol.The primary bovine endometrial epithelial cells(BEEC)were subjected to LPS to establish cellular inflammation model.This study helps provide theoretical foundation for selenium supplementation in the prevention and treatment of bovine endometritis during perinatal period.1.The effect of Se on LPS-induced NF-κB and MAPK activation and cytokines mRNA expression in BEEC.The cell viability was measured by CCK-8 assay and the cell cytotoxicity was detected by LDH assay after BEEC were treated with various concentrations of Se(1,2,4,8,16,32,and 64μmol/L)for 12h.The results indicated that cell viability decreased(p<0.01)and LDH release were increased(p<0.05)when the selenium concentration exceeded 4 μmol/L compared to the control group.To determine the effect of Se on the inflammatory response of BEEC induced by LPS,we examined the mRNA expression of cytokines and the proteins level of NF-κB and MAPK signaling pathways.The results showed that,as compared with control group,the mRNA expression of cytokines was increased after 1.0μg/mL LPS treatment BEEC for 3,12 and 18 h(p<0.05 or p<0.01)and the NF-κB and MAPK pathways were activated by 1.0 μg/mL LPS treatment BEEC for 30 min,and none gene expression(p>0.05)and proteins level(p>0.05)were affected by 4 μmol/L Se treatment alone;Compared with the LPS group,Se decreased(p<0.05 or p<0.01)TNF-α,IL1-β,IL-6,CXCL8,PTGS2 and NOS2 mRNA expression at the indicated time points,and Se supplement down regulated(p<0.05 or p<0.01)the ratio of p-p65/p65,p-IκBα/IκBα and the phosphorylation of ERK1/2,p38 and JNK in BEEC.These results indicated that Se inhibited the activation of NF-κB and MAPK pathway and the gene expression of pro-inflammatory mediators and inflammatory factors in LPS-stimulated BEEC.2.The effect of Se on LPS-induced NF-κB and MAPK activation and cytokines mRNA expression of BEEC under high cortisol background.In order to explore the mechanism of Se regulation on the inflammatory response in BEEC under high cortisol background,the cells were co-treated with Se,COR and LPS.The groups were as follows:the control group,the LPS(1.0 μg/mL)treatment group,the LPS and COR(30 ng/mL)co-treatment group,LPS and COR and Se(1,2 and 4 μmol/L)co-treatment group and COR(30 ng/mL)and Se(4 μmol/L)co-treatment group.Of all treatments,the group containing Se was pretreated for 12h.The results showed that,the mRNA expression of cytokines and the phosphorylation of proteins in LPS group were higher than those in control group(p<0.01).Compared with control group,co-treatment with COR and Se did not cause any change(p>0.05)in the mRNA expression of cytokines and LPS stimulation resulted in a increase(p<0.01)of cytokines mRNA expression and activation of NF-κB/MAPK pathways(p<0.01).Compared with LPS group,the gene expression of cytokines in the COR and LPS co-treatment group decreased(p<0.05 or p<0.01),and the phosphorylation level of proteins decreased(p<0.05 or p<0.01).Compared with LPS and COR co-treatment groups,the mRNA expression of pro-inflammatory mediators(PTGS2 and NOS2)and inflammatory factors(TNF-α,IL-1β,IL-6,and CXCL8)mRNA in LPS,COR and Se co-treatment groups decreased(p<0.05 or p<0.01),and Se supplement further down regulated the level of protein phosphorylation decreased(p<0.05 orp<0.01).The results of immunofluorescence indicated that the level of p65 in nucleus of LPS group was higher(p<0.01)than that in control group,COR group,Se group,and COR and Se co-treatment group.Nucleus p65 in the COR and Se co-treatment group was less than that in COR group(p<0.05)and Se group(p<0.01).In conclusion,Se and COR have no effect on gene expression and NF-κB/MAPK pathways of BEEC.Se inhibited the LPS-induced inflammatory response by suppressing NF-κB and MAPK in BEEC,and this inhibitive property of Se was augmented with high cortisol background.