Selenium suppressed the LPS‐induced inflammation of bovine endometrial epithelial cells through NF‐κB and MAPK pathways under high cortisol background

Abstract The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti‐inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high‐level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF‐κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre‐treatment of Na2SeO3 (1, 2 and 4 μΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF‐κB and suppressed the phosphorylation of extracellular signal‐regulated kinase, P38MAPK and c‐Jun N‐terminal kinase. This inhibition of inflammation was more apparent in the presence of high‐level cortisol (30 ng/mL). These results indicated that selenium has an anti‐inflammatory effect, which is mediated via NF‐κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.


| INTRODUC TI ON
Uterine health is often compromised in cattle because postpartum contamination of the uterine lumen by bacteria is ubiquitous, and pathogenic bacteria frequently persist causing clinical diseases. 1 Postpartum metritis and endometritis are important disorders in cattle, 2,3 causing subfertility and infertility 4 and high economic losses due to the lower oestrus expression and prolonged calving intervals, resulting in involuntary culling. 5 The presence of pathogenic bacteria in the uterus causes inflammation and histological lesions of the endometrium, delays uterine involution and perturbs embryo survival. 6 The recognized uterine pathogens most often associated with clinical disease are Arcanobacterium pyogenes, Escherichia coli (E. coli), Fusobacterium necrophorum and Prevotella melaninogenicus. 1 Thus, Arcanobacterium pyogenes and E. coli are often used to induce uterine infections. 7 There is general agreement that lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, is the main pathogenic factor involved in E. coli infection. 8 Recognition of LPS by the innate immune system is critical to elicit inflammatory responses. 9 Host cells recognize LPS via a specific receptor complex comprising of Tolllike receptor 4 (TLR4), CD14 and MD-2, which leads to an inflammatory response characterized with the secretion of cytokines and chemokines. 10 Endometrial epithelial and stromal cells express the TLR4 complex. LPS stimulates the secretion of chemokines such as interleukin (IL)-8 and disrupts endocrine function by switching prostaglandin secretion to predominantly prostaglandin E 2.

| Experiment design and treatments
The sodium selenite (Na 2 SeO 3 ) used in this study is a lyophilized powder synthesized by Sigma (S5261). The powder was dissolved in DMEM/F-12, filtered and diluted to a concentration of 64 μM and was stored at −20°C. Selection of the concentration of Na 2 SeO 3 in the range of 1-64 μM was based on previous studies. 31 The physiological level of cortisol concentration in cattle ranges from 5 to 30 ng/mL. Based on a preliminary study in our laboratory, 29 a concentration of 30 ng/mL was selected as the high cortisol level. After Se pre-treatment for 12 h, the cells were challenged with LPS (1 μg/mL) and cortisol (30 ng/mL). The group were as follows: the blank control group, the LPS group, the LPS and cortisol co-treatment group, the LPS, cortisol and Se (1, 2 and 4 μM) co-treatment groups.
The experimental design was shown in Figure 1.

| Cell viability assay
The effect of Na 2 SeO 3 on BEEC viability was measured by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc.). The cells were plated into 96-well plates at a density of 1 × 10 3 cells per well and grown to 80% confluence in a 37°C, 5% CO 2 incubator.
Then the cells of Se supplement groups were treated with DMEM/ F12, which containing various concentrations of Na 2

| RNA extraction and quantitative PCR
The BEEC was seed in 6-well plates (1 × 10 6 cells/well), and the cells were grown to 80% confluence. The BEEC was collected at 3, 12 and 18 h posttreatment 30

| Western blot analysis
The protein extraction time point was based on previous reports. 29,30 The protein concentration was determined using the bicinchoninic acid protein assay kit (P0010, Beyotime). The proteins were detected using a chemiluminescence assay. The antigen-antibody complexes were visualized on horseradish peroxidase substrate (Millipore) using the ChemiScope5300Pro CCD camera (Clinx Science Instruments). The band intensity was quantified by Quantity One software (Bio-Rad).

| Immunofluorescence staining
The BEEC was plated on cover glasses in 24-well cell plates (  nucleus, the immunofluorescence signals were quantified by Image J software (National Institutes of Health).

| Statistical analysis
Each experiment was repeated three times. All data were analysed using IBM SPSS Statistics 21.0 (IBM) and were expressed as the means ± standard error of means (SEM). Statistically significant differences throughout this study were calculated by one-way anova, followed by Dunnett's test.
A two-sided p value <0.05 was considered statistically significant.

| Cell viability and LDH release
The effect of different concentrations of Na 2 SeO 3 on cell viability and LDH release was shown in Figure 2. Compared to the blank control, the LDH release increased (p < 0.05) and cell viability decreased (p < 0.01) when the Se concentration exceeded 4 μM.
The cell viability was around 85% in 8 μM Se group and only about 66% in 64 μM Se group ( Figure 3A). Furthermore, no difference was found between the control group and the group treated with 1, 2 and 4 μM Na 2 SeO 3 .

| Gene expression of NOS2, PTGS2 and proinflammatory cytokines
To determine the effect of Se on the inflammatory response of BEEC

| NFκ B activation and MAPK phosphorylation
To investigate the mechanism of Se on the inflammatory status As shown in Figure 6, compared with the LPS and cortisol cotreatment group, co-incubation with LPS, cortisol, and various con-

| DISCUSS ION
The previous study of our lab found that Se alleviated the inflammatory reaction by decreasing TNF, IL1B and IL6 expression and inhibiting NF-κB and MAPK activation in RAW264.7 macrophages stimulated with Staphylococcus aureus. 31 In this study, we found that in rats with acetic acid-induced colitis. 43 The release of cortisol is a primary adaptive mechanism in response to adverse conditions through the short-term mobilization of body reserves and modulation of inflammatory responses. 44 Parturition and dystocia causes elevated cortisol release. 45 In addition, the decreased food intake from 1-2 weeks before calving also reduces the availability of antioxidants in the body. Antioxidant supplementation during the peripartum period is especially important for cattle, 46 and the improvement of antioxidative status could be one important mechanism supporting the immune function of dairy cows during peripartum period. 47 Therefore, we investigated the effect of Se supplementation on the inflammatory response of BEEC in a high-level cortisol state. In agreement with previous reports, cortisol suppressed the inflammatory response in BEEC. 29 More importantly, we observed that Se supplement further decreased the proinflammatory gene expression and inhibited the NF-κB and MAPK activation. This coincided with the report that supplementation of antioxidants such as vitamin E and Se during pregnancy relieved the inflammation in dystocia-affected buffaloes during the immediate postpartum period. 48 In the form of selenoprotein, Se has been confirmed to inhibit inflammation through Cortisol has been verified to suppress LPS-induced inflammation in BEEC by inhibiting NF-κB and MAPK signalling pathways. 29 In addition, the anti-inflammatory effect of 4 μM Se was more pro-

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that there is no conflict of interest regarding the publication of this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.