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Selenium elicited an enhanced anti-inflammatory effect in primary bovine endometrial stromal cells with high cortisol background

BMC Veterinary Research volume 20, Article number: 383 (2024) Cite this article

Abstract

Background

An elevated endogenous cortisol level due to the peripartum stress is one of the risk factors of postpartum bovine uterine infections. Selenium is a trace element that elicits anti-inflammation and antioxidation properties. This study aimed to reveal the modulatory effect of selenium on the inflammatory response of primary bovine endometrial stromal cells in the presence of high-level cortisol. The cells were subjected to lipopolysaccharide to establish cellular inflammation. The mRNA expression of toll-like receptor 4 (TLR4), proinflammatory factors, and selenoproteins was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blot and immunofluorescence.

Results

The pretreatment with sodium selenite (2 and 4 µΜ) resulted in a down-regulation of TLR4 and genes encoding proinflammatory factors, including interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase. Selenium inhibited the activation of NF-κB and the phosphorylation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase. The suppression of those genes and pathways by selenium was more significant in the presence of high cortisol level (30 ng/mL). Meanwhile the gene expression of glutathione peroxidase 1 and 4 was promoted by selenium, and was even higher in the presence of cortisol and selenium.

Conclusions

The anti-inflammatory action of selenium is probably mediated through NF-κB and MAPK, and is augmented by cortisol in primary bovine endometrial stromal cells.

Peer Review reports

Background

The postpartum reproductive disorders of dairy cows seriously affect their production performance and cause economic losses [1]. Due to the cervical laxity and the damage of endometrial epithelium after calving, cows are prone to uterine infections, such as metritis and endometritis [2, 3], which can impair the bovine fertility [4]. The tight junction between endometrial epithelial cells prevents pathogens from penetrating into the stroma. However, this protection is lost because of endometrial exfoliation, so that the pathogens are more likely to invade the stroma [5]. Escherichia coli (E. coli) is one of the main pathogenic bacteria found in the infected bovine uterine cavity, and can cause endometrial tissue damage and inflammation through their virulence factor lipopolysaccharide [6, 7].

The endometrium defends itself against pathogen invasion mainly through the innate immunity, in which the inflammatory response predominates [8]. Toll-like Receptors (TLR) are among the most studied families of pattern recognition receptors in mammals [9, 10]. Lipopolysaccharide (LPS) is an exogenous ligand for Toll-like receptor 4 (TLR4), which induces an inflammatory response manifested as the increased release of inflammatory mediators and tissue damage [11]. Both the bovine endometrial epithelial cells (BEEC) and stromal cells (BESC) express TLR [11], and the binding of LPS to TLR4-CD14-MD2 receptor complex activates the MyD88-dependent pathway, which further activates the NF-κB and MAPK intracellular signalling and promotes the transcription of genes encoding inflammatory mediators, such as interleukin 1 (IL1B), interleukin 6 (IL6), C-X-C motif chemokine ligand 8 (CXCL8), and tumour necrosis factor (TNF), and prostaglandin-endoperoxide synthase 2 (PTGS2) and inducible nitric oxide synthase 2 (NOS2) [11,12,13].

Selenium (Se) is a trace element that plays a vital role in animal health and performance [14]. The main source of Se is diet or dietary supplements [15], and the nutritional requirement of Se is 300 µg/kg dry matter (DM) for dairy cows [16]. A marked Se deficiency can be observed when Se content is less than 0.05 mg/kg DM [17]. The accumulation of Se in edible part of plants is directly dependent on soil Se concentration, and the heterogeneous distribution of Se making many parts of the world Se deficient [18, 19]. The decreased dry matter intake and the increased demand for nutrition during peripartum period makes the animal more prone to Se deficiency [20], which has been associated with the reduced fertility, and the incidence of retained placenta, mastitis, and metritis [21, 22]. Exogenous Se supplementation has been reported to reduce the incidence of reproductive diseases [21, 23]. Feeding Se-replete cows with a supranutritional Se-yeast supplement during late gestation improves their postpartum antioxidant status and immune responses [24]. In the form of selenocysteine, Se incorporates in selenoproteins such as glutathione peroxidase (GPX) superfamily. Se supplementation in the diet of calves [25] and heifers [26] is often marked with an elevated serum Se content and GPX activity. The Se-containing GPX 1–4 help protect cells from oxidative stress, inflammation, and oxidant-mediated cell death [14]. Studies have shown that Se alleviated the LPS-induced endometrial damage, inhibited TLR4-mediated activation of NF-κB pathway, and reduced the production of inflammatory mediators in mouse uterus [27]. Our laboratory also found that Se supplement relieved the E. coli-induced goat endometritis with less tissue damage and polymorphonuclear cell count in uterine secretions [28], and that Se suppressed the LPS-induced activation of NF-κB and MAPK signalling and the downstream proinflammatory gene expression in BEEC [29].

Cortisol is a kind of glucocorticoid substance, which is widely used for anti-inflammation and immunosuppression purposes [30]. The periparturient cattle endure a series of stressors and have high endogenous cortisol concentrations [31, 32]. The maternal cortisol level increases 3- to 4-fold during calving, and even 5- to 7-fold in cows under metabolic stress [33]. A comparatively high cortisol concentration suppresses innate immune function and increases the risk of uterine infections [34]. Our laboratory has reported that cortisol inhibited endometrial inflammation through NF-κB and MAPK pathways both in vivo and in vitro [35,36,37]. In the presence of high cortisol, Se has been found to relieve endometrial inflammation and promote tissue repair in E. coli-induced endometritis, and has been reported to ameliorate inflammation in LPS-stimulated BEEC [29, 35, 36]. However, as to the stromal cells with high cortisol background, neither the anti-inflammatory activity of Se nor the mechanism of this process has been reported.

Compared with epithelial cells, the stromal cells are more abundant and closer to the vascular system, so the inflammatory response of stromal cells is no less important than that of epithelial cells [38, 39]. Previously, we have revealed the anti-inflammatory effect of Se in BEEC with high cortisol background [29]. Here we used the primary BESC to determine whether there is a similar effect of Se with the presence of high cortisol. We hypothesized that Se protects BESC from LPS-induced inflammatory response with high cortisol background, and the underlying mechanism involves the NF-κB and MAPK pathways, as well as selenoproteins.

Results

Vimentin identification in primary BESC

As shown in Fig. 1a, the cells were stained positive for vimentin, and were mainly spindle or polygonal in shape. The cytoplasm of the positive cells was dark brown, whereas the nucleus was not stained. The purity of the primary BESC was more than 95%. These cells were confirmed to be the primary BESC and can be used for subsequent experiments.

Fig. 1
figure 1

Vimentin identification in primary bovine endometrial stromal cells. The immunocytochemistry of vimentin (a) and IgG1 (b) were presented. Scale bar represented 100 μm

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Se inhibited the LPS-induced TLR4 and proinflammatory gene expression

After LPS stimulation for 12 and 24 h, the gene expression of TNF, IL1B, IL6, CXCL8, NOS2, PTGS2, and TLR4 was upregulated (p < 0.01) in various degrees in BESC (Fig. 2). Except for NOS2 and TLR4, the rises of these genes were greater at 24 h than those at 12 h. Among them, the IL1B and IL6 increased (p < 0.01) about 60-fold at 24 h. Se supplement of both 2 and 4 µM down-regulated (p < 0.01) the LPS-induced expression of these genes. However, no difference was found between the two Se supplement groups. Se alone had no effect (p > 0.01) on these gene expression compared to the blank control.

Fig. 2
figure 2

The effect of selenium on LPS-induced proinflammatory gene expression in primary BESC. The changes in IL1B (a), IL6 (b), CXCL8 (c), NOS2 (d), PTGS2 (e), TNF (f), and TLR4 (g) were detected. The cells were divided into five groups. In Na2SeO3 (4 µM) group, the cells were pretreated with 4 µM Na2SeO3 for 12 h. In LPS + Se groups, the cells were pretreated with Na2SeO3 (2 or 4 µM) for 12 h, followed by 1 µg/mL LPS stimulation. In the LPS group, the cells were stimulated with LPS without any pretreatment. After LPS stimulation for 12–24 h, the cells were subjected to RNA extraction and the subsequent quantitative PCR detection. BESC, bovine endometrial stromal cells. LPS, lipopolysaccharide. Na2SeO3, sodium selenite. The data were presented as means ± SEM (n = 3). *p < 0.05, **p < 0.01 versus the control group. # p < 0.05, ## p < 0.01 versus the LPS group

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Se inhibited the LPS-induced MYD88 expression and activation of NF-κB and MAPK pathways

LPS stimulation caused an increase (p < 0.01) in MYD88 protein level (Fig. 3b), as well as the elevated (p < 0.05) phosphorylation levels of P65 and IκBα (Fig. 3c and d), and ERK, JNK, P38, and MEK1/2 (Fig. 3e-i), indicating the MyD88-dependent activations of NF-κB and MAPK. Compared with LPS groups, the phosphorylation levels of MYD88 and the key proteins in NF-κB and MAPK pathway decreased (p < 0.05) in cells cotreated with LPS and Se (2 and 4 µM). No difference was found between the two cotreatment groups, or between the control group and the Se (4 µM) group.

Fig. 3
figure 3

The effect of selenium on MyD88/NF-κB and MAPK pathways in LPS-stimulated primary BESC. The changes in MYD88 protein (a, b) and key protein phosphorylation of NF-κB (a, c, d) and MAPK (e to i) pathways were detected by Western blot. The cells were pretreated with 2 or 4 µM Na2SeO3 for 12 h, followed by 1 µg/mL LPS stimulation. The cells were collected at 45–60 min post stimulation to detect MyD88/NF-κB or MAPK related proteins, respectively. BESC, bovine endometrial stromal cells. LPS, lipopolysaccharide. Na2SeO3, sodium selenite. The data were presented as means ± SEM (n = 3). *p < 0.05, **p < 0.01 versus the control group. # p < 0.05, ## p < 0.01 versus the LPS group. The membranes were cut prior to hybridisation with antibodies. The original blots were presented in Additional file 2

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Se inhibited TLR4, MYD88, and proinflammatory genes at high cortisol level

As shown in Fig. 4, LPS stimulated (p < 0.01) the expression of mRNAs encoding TLR4, MYD88, and the inflammatory mediators in BESC. Compared with the LPS group, cotreatment of cortisol and LPS decreased (p < 0.01) these gene expressions. On the basis of cortisol and LPS, Se supplement further down-regulated (p < 0.01) the expression of these genes except IL1B, IL6, and CXCL8 at 12 h, and TLR4 and CXCL8 at 24 h (p > 0.05).

Fig. 4
figure 4

Selenium inhibited the LPS-induced proinflammatory gene expression in BESC at high cortisol level. The changes in IL1B (a), IL6 (b), CXCL8 (c), NOS2 (d), PTGS2 (e), TNF (f), TLR4 (g), and MYD88 (h) expression were determined. The cells were divided into six groups. In selenium treatment groups (COR + Se group and LPS + COR + Se group), the cells were pretreated with Na2SeO3 (2 or 4 µM) for 12 h, followed by the addition of 30 ng/mL cortisol and/or 1 µg/mL LPS. After LPS and/or cortisol treatment for 12–24 h, the cells were collected for RNA extraction and the subsequent quantitative PCR detection. BESC, bovine endometrial stromal cells. COR, cortisol. LPS, lipopolysaccharide. Na2SeO3, sodium selenite. The data were presented as means ± SEM (n = 3). *p < 0.05, **p < 0.01 versus the control group, +p < 0.05, ++p < 0.01 versus the LPS group, #p < 0.05, ##p < 0.01 versus the LPS + COR group

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Se inhibited MYD88 expression and the activation of NF-κB and MAPK under high cortisol level

As shown in Fig. 5, cortisol reduced (p < 0.01) the LPS-induced MYD88 protein expression, and the key protein phosphorylation in NF-κB and MAPK pathways. Compared with the LPS and cortisol cotreatment (LPS + COR) group, the addition of Se (2 and 4 µM) further reduced (p < 0.05) the phosphorylation levels of key proteins in both pathways (p < 0.05). The reduction in MYD88 level was observed by Se supplementation (p < 0.01). This result suggested that Se can inhibit MyD88/NF-κB and MAPK pathway in BESC at high cortisol level.

Fig. 5
figure 5

Selenium inhibited MyD88/NF-κB and MAPK activations in primary BESC at high cortisol level. The changes in MYD88 protein (a, b), and the key protein phosphorylation in NF-κB (a, c, d) and MAPK (e to i) pathways were measured. The cells were pretreated with 2 or 4 µM Na2SeO3 for 12 h, followed by 1 µg/mL LPS and/or 30ng/mL cortisol treatment. The cells were collected at 45–60 min post treatment to detect MyD88/NF-κB or MAPK related proteins, respectively. BESC, bovine endometrial stromal cells. COR, cortisol. LPS, lipopolysaccharide. Na2SeO3, sodium selenite. The data were presented as means ± SEM (n = 3). *p < 0.05, **p < 0.01 versus the control group. +p < 0.05, ++p < 0.01 versus the LPS group. # p < 0.05, ## p < 0.01 versus the LPS + COR group. The membranes were cut prior to hybridisation with antibodies. The original blots were presented in Additional file 2

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Se inhibited the LPS-induced P65 nuclear translocation at high cortisol level

To verify the inhibition of Se on the LPS-induced NF-κB activation at high cortisol level, we used immunofluorescence staining to detect P65 nuclear translocation. As depicted in Fig. 6, LPS stimulated P65 to enter the nucleus (p < 0.01). Both Se and cortisol reduced (p < 0.05) the amount of nuclear P65 in LPS-stimulated BESC. Compared with LPS + COR group, Se supplement further prevented (p < 0.05) P65 from entering the nucleus.

Fig. 6
figure 6

Selenium prevented the P65 nuclear translocation in primary BESC at high cortisol level. The cells were pretreated with 4 µM Na2SeO3 for 12 h, followed by 1 µg/mL LPS and/or 30ng/mL cortisol treatment for 45 min. Then the cells were subjected to immunofluorescence assay and were observed using a confocal microscope. (a) Visualization of P65 translocation from the cytoplasm into the nucleus. (b) Quantification above background levels of nuclear P65 intensity using the Image J software. BESC, bovine endometrial stromal cells. COR, cortisol. LPS, lipopolysaccharide. Na2SeO3, sodium selenite. Scale bar represented 10 μm. The data were presented as means ± SEM (n = 3). *p < 0.05 and **p < 0.01 versus the control group. +p < 0.05 and + + p < 0.01 versus the LPS group. #p < 0.05 and ##p < 0.01 versus the LPS + COR group

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Se promoted GPX1 and GPX4 expression

Se acts as selenoproteins. Therefore, we examined the effect of Se on the gene expression of GPX1 and GPX4 (Fig. 7). As expected, Se supplement up-regulated (p < 0.05) GPX1 and GPX4 expression with or without the presence of LPS, and the increment was more pronounced at 24 h (5- to 8-fold) than that at 12 h (around 2-fold). The increase amplitude of GPX is more obvious (p < 0.01) in cells treated with 4 µM Se than those treated with 2 µM Se. The presence of LPS seemed to reduce (p < 0.05) GPX4 expression because of the differential expression between the LPS group and the LPS and Se cotreatment (LPS + Se) groups, and between the Se + COR group and the LPS, COR, and Se cotreatment (LPS + COR + Se) groups. It seemed to suggest that LPS negatively impact GPX4 expression. Surprisingly, the GPX1 and GPX4 expression was ever higher (p < 0.05) in Se + COR group than that in Se group, and in the LPS + COR + Se group than that in the LPS + Se group.

Fig. 7
figure 7

Selenium promoted GPX1 (a, b) and GPX4 (c, d) expression in primary BESC. The cells were pretreated with 2 or 4 µM Na2SeO3 for 12 h, followed by the treatment of LPS and/or cortisol. After treatment for 12 (a, c) or 24 h (b, d), the cells were collected for RNA extraction and the subsequent quantitative PCR detection. BESC, bovine endometrial stromal cells. COR, cortisol. GPX, glutathione peroxidase. LPS, lipopolysaccharide. Na2SeO3, sodium selenite. The data were presented as means ± SEM (n = 3). Different letters indicate statistically significant differences (p < 0.05) among the treatment groups. The asterisks showed significant difference between the two Se concentrations with similar treatment (**p < 0.01)

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Discussion

The innate immunity exerts fast, non-specific defense against unfamiliar pathogens [9]. Cattle with uterine disease present a typical innate immune response with increased expression of genes encoding inflammatory cytokines (IL1A, IL1B, IL6, TNF and IL12A), chemokines (CXCL5 and CXCL8), and prostaglandin synthesis enzymes in the endometrium [8]. LPS has been observed to stimulate the proinflammatory factors including IL-6, TNFα, and nitric oxide in bovine endometrial explants [40], and to induce the degradation of IκB and nuclear translocation of NF-κB P65, as well as the fast phosphorylation of mitogen-activated protein kinas proteins, such as JNK, MEK, ERK1/2, and P38 in bovine endometrial cells [11, 41]. The NF-κB pathway activates downstream inflammatory mediators, including IL-1β, IL-6, TNF-α, COX-2, iNOS and IL-8 [42, 43]. The MAPK pathways have been reported to regulate the expression of TNF-α [44]. Ding et al. investigated the whole-transcriptomic gene changes of stromal cells treated with LPS, and revealed the enrichment of differentially expressed genes in immune-related pathways such as TNF signaling pathway, leukocyte-mediated immunity, IL-1β secretion, and NF-κB signaling [45]. In our experiments, LPS stimulated the expression of TLR4, MYD88, TNF, NOS2, IL1B, IL6, CXCL8, and PTGS2, and the key protein phosphorylation of NF-κB and MAPK signalling pathways in BESC. These results were consistent with the studies described above.

Se has a biphasic dose-response, with toxicity at high doses but favorable properties at very low doses [14]. An adequate supply of Se contributes to the reduced risk of cancer, auto-immune diseases, and subfertility, whereas an over-supply of Se increases the risk of endocrine disruption, mental disorders, and cancer [46]. Supra-nutritional doses of Se-containing compounds can be applied as a chemotherapeutic agent to induce oxidation and apoptosis of cancer cells [47, 48]. Se supplementation is critical to the last 60 days of gestation, when the fetus was completely dependent on the dam for its supply of nutrient [49]. In Se-deficient states, cows sacrifice their available Se to ensure adequate Se intake for their calf [50]. The rate of adequate Se in the blood of cattle is 0.08 ~ 0.16 mg/L [51]. Here the Se concentration in basal medium was measured to be 45.63 µg/L, which was between upper limit of Se deficiency and the lower limit of Se sufficiency [52]. After supplementation with 2 and 4 µM Se, and concentration reached Se sufficient levels [53, 54].

The inflammatory status can be related with Se concentration. According to Prabhu et al., the Se-deficient RAW 264.7 macrophage cell line represented with an upregulation of NF-κB and iNOS. Upon LPS stimulation, these cells showed higher iNOS expression and nitric oxide production than the Se-supplemented cells [55]. In porcine brain, dietary Se deficiency activated the iNOS/NF-κB pathway and the downstream inflammatory cytokines, leading to inflammatory lesions [56]. Se supplement has been widely observed to relieve the inflammatory disease process in various tissue types by inhibiting related pathways such as NF-κB and MAPK [57]. Se attenuated TLR4 and its downstream signaling pathways in mouse endometrium and mouse endometrial epithelial cells under LPS stimulation [58]. These results were similar to what we reported in epithelial cells [29]. Here, we showed that 2 and 4 µM Se down-regulated the LPS-induced expression of TLR4 and MYD88, the activation of NF-κB and MAPK pathway, and the downstream proinflammatory mediators, suggesting that Se inhibited the MyD88/NF-κB and MAPK signalling pathways. Se pretreatment dose-dependently down-regulated the rat alveolar TNF and IL1B expression [59], and the LPS-induced phosphorylation of ERK1/2, P38, and JNK in mouse mammary epithelial cells [60]. However, in our previous study, we did not observe any dose-dependent suppression of Se on inflammatory factors and NF-κB and MAPK pathways in epithelial cells [29]. Similarly in this experiment, there was no difference in Se anti-inflammation between 2 and 4 µM Se, which could be related to the narrow range of Se concentration used in our study. The condition of Se deficiency was not investigated in this experiment.

Being absorbed in small intestine, Se is reduced to hydrogen selenide through a chain of reduction reactions related with glutathione and glutathione reductase, then becomes an active Se source in selenoprotein synthesis [27]. The synthesized selenoproteins are transported through bloodstream to different tissues and organs [61, 62]. The GPX is among the well characterized selenoprotein enzymes related to immune functions. GPX1 is the most abundant and ubiquitously expressed GPX, and GPX4 is well known to protect against lipid peroxidation [63, 64]. GPX1 and GPX4 were found to be increased in human lymphocytes upon supplementation of sodium selenite [65]. Se supplementation, in the form of selenomethionine, has been reported to upregulate GPX1 and GPX4 levels in bovine endometrial cells [66]. Similarly, we observed the increased gene expression of GPX1 and GPX4 in BESC pretreated with Se. The GPX1 and GPX4 activities has been observed to increase with the increasing selenomethionine concentration (0 to 200 nM) [67]. One study detected a dose-dependent upregulation of the GPX1 level after Se nanoparticles treatment [68]. Consistently, we observed a higher expression of GPX in cells treated with 4 µM Se than those treated with 2 µM Se. The cytosolic form of GPX4 has been proved to suppress IL-1-driven NF-κB activation and leukotriene biosynthesis [69]. GPX1 and GPX4 dampen phosphorylation cascades predominantly via prevention of inactivation of phosphatases by H2O2 or lipid hydroperoxides [14]. By controlling NF-κB, GPX indirectly regulates the expression of cyclooxygenases and lipoxygenases via MAPK and PTGS2 [14]. Considering the above reports, we proposed that the anti-inflammatory effect of Se could be mediated through GPX1 and GPX4 in BESC. Our results show that LPS stimulation reduced the expression of GPX4, but had no influence on GPX1. There are in vitro studies reporting no change in GPX1 and GPX4 gene expression, but an upregulation of their protein levels in a bovine endometrial cell line [66] and in mouse macrophages [70] stimulated with LPS. But conversely, LPS has been reported to reduce the expression of GPX1 gene in macrophages [71] and mouse uteri [72] and GPX4 protein in rat myocardial tissue [73]. In our result, it is noteworthy that the GPX1 and GPX4 expression was further upregulated by Se in the presence of cortisol. Whether and how cortisol affects Se or selenoproteins was rarely reported. In zebrafish exposed to predation stress, the GPX1 expression in gut was found to be increased, however, the animal’s cortisol level unchanged [74].

Cortisol dampens NF-κB activation and MAPK phosphorylation, thereby inhibiting inflammation in bovine endometrial epithelial and stromal cells, as well as the goat endometrium [35,36,37]. Previously, we found that in the presence of cortisol, Se exerted a greater inhibition on BEEC inflammation than Se treatment alone [29]. Comparably in stromal cells, the addition of Se further down-regulated the expression of inflammatory genes and reduced the phosphorylation of the key proteins of the two pathways as compared with the LPS + COR cotreatment group. This result suggested that the sites of action of COR and Se, although both related with NF-κB and MAPK, may differ. In addition, given that Se acts through selenoproteins, and that the cotreatment of Se and cortisol further up-regulated GPX1 and GPX4 expression, we speculate that cortisol facilitated Se uptake and utilisation by BESC. However, whether cortisol affect selenoprotein synthesis requires further investigation.

Conclusion

Se inhibited the LPS-induced inflammatory response by suppressing NF-κB and MAPK in BESC, and this inhibition was more apparent in the presence of high-level cortisol. Meanwhile Se promoted the expression of GPX1 and GPX4, and their expression was further enhanced under COR treatment. This anti-inflammatory effect of Se with high cortisol background may be related to GPX1 and GPX4.

Methods

Cell culture

The uteri at ovarian stage I (days 1 ~ 4 of the estrous cycle) were collected from cows at a local abattoir to prepare the primary BESC. One uterus was collected for each batch of the cells, and no fewer than three batches of cells were employed for each experiment. Only the tissue with no obvious pathological change from cows free from reproductive diseases was collected. The collected uterus was rinsed with sterile saline and taken back to the laboratory in an ice box filled with iodophor solution. Then, the uterine tissue was cleaned and opened on a clean bench. The endometrium was clipped and cleaned with phosphate-buffered saline (pH values from 7.2 to 7.4) containing 5% dual antibiotic (50 U/mL penicillin/streptomycin). The endometrium was cut into mincemeat-like pieces then rinsed, and configured with 0.4% collagenase II solution (C6885, Sigma, USA) (the volume ratio of endometrial tissue to solution, 1: 1.5) to digest for 60 min. The digested mixture was filtered and the filtrate was centrifuged at 290×g for 5 min to remove the supernatant. After resuspension with the complete medium containing 3% dual antibiotic, 15% fetal bovine serum (A3161002CC, Gibco, Australia), L-glutamine (G8540-100G, Sigma, USA), DMEM-F12 (D8900, Gibco, USA), the cells were inoculated in the 25cm2 cell culture flasks. The cells were then cultured in a 37 °C, 5% CO2 cell culture incubator (Thermo Fisher, USA) with 12-hourly fluid changes until the cells were fully grown. Compared to the epithelial cells, stromal cells adhere to the wall faster (around 12 h), and are more sensitive to trypsin (0458-50G, Amresco, USA). Therefore, we purified the stromal cells by controlling the digestion time (30 ~ 40 s) of trypsin to obtain primary BESC for subsequent experiments. The stromal cells were identified by detecting intracellular vimentin using immunocytochemistry. The primary and secondary antibodies were the Vimentin Antibody (E-5) (sc-373717, Santa Cruz Biotechnology, Inc., USA) and the mouse IgG1 (SC-3877 Santa Cruz Biotechnology, Inc., USA), respectively.

Treatment design

The effect of sodium selenite (Na2SeO3, S5261, Sigma, USA) on BESC viability was determined by a Cell Counting Kit-8 (A311-02-AA, Dojindo Molecular Technologies, Inc, China). The cytotoxicity of Na2SeO3 was measured by a lactate dehydrogenase assay kit (A020-2-2, Jiancheng Bioengineering Research Institute, China). As a result, the concentrations of 2 and 4 µM Na2SeO3 showed little influence on BESC viability (data unpublished, see Additional file 1), and were used in subsequent experiments.

The current study was divided into two parts. First, we aimed to explore the effect of Se on BESC inflammation by detecting the key proteins of NF-κB and MAPK pathways and the proinflammatory genes. The cells were pretreated with Se for 12 h. Then 1 µg/mL LPS (L2880, O55:B4, Sigma, USA) was added to the medium to stimulate the cells. RNA and protein were extracted to detect the relevant inflammatory genes and the pathway proteins, respectively. Next, we observed the effect of Se on the inflammatory response of BESC at high cortisol (H0888, Sigma, USA) levels. The concentration of 30 ng/mL cortisol were selected to mimic high cortisol background [29, 36]. The cells were pretreated with Se for 12 h, followed by LPS and cortisol treatment. The experimental groupings were as follows: the blank control group, the LPS group, the LPS + COR group, and the LPS + COR + Se groups (2 or 4 µM Se).

RNA extraction and quantitative PCR

The BESC was inoculated in the six-well plates (1 × 106 cells/well), and the above experimental treatments were carried out when the cells reached 60%~70% confluence. The BESC was collected at 12 and 24 h. Total RNA from the cells was extracted using a MagicPure 32 Totai RNA Kit (Z-EC521-S1-32, TransGen Biotech, China), and was quantified using a NanoDrop 2000 (Thermo, USA). The A260/280 ratio of each sample was between 1.8 and 2.1. The obtained RNA was converted to cDNA with a TransScript Uni ALL-in-One First-Strand cDNA Synthesis SuperMix for qPCR (#AU341-02-V2, TransGen Biotech, China), and the reverse transcription system included 4 µL of 5×All Mix, 1 µL of Remover, and 15 µL of RNA + ddH2O, 20 µL in total. QPCR was performed using a CFX 96 Real-Time PCR Detection System (BIO-RAD, USA) with a 20 µL amplification system, containing 4 µL of cDNA, 10 µL SYBR, 5 µL of ddH2O, and 0.5 µL of each primer. The sequences of the primers were presented in Table 1. The expression of each gene was normalized against the housekeeping gene actin-β (ACTB). The 2−△△Ct method was used to calculate the relative expression of the genes.

Table 1 The primer sequences for gene amplification
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Protein extraction and western blot

The BESC was inoculated in the six-well plates (1 × 106 cells/well), cell treatment was carried out when the cells grew to 80% confluence. After 45–60 min treatment, cells were lysed with the radioimmunoprecipitation assay buffer (C1053, APPLYGEN, China) containing a mixture of protease inhibitors (P1260, APPLYGEN, China) and protein phosphatase inhibitors (P1265, APPLYGEN, China) to obtain protein samples. The protein concentration was determined by a bicinchoninic acid protein assay kit (P0010, Beyotime, China). Each protein sample was mixed with 1/4 volume of 4×SDS-PAGE loading buffer (P1015, Solarbio, China), followed by a 100 °C water bath for 10 min to obtain the final sample. The protein sample of 20 ~ 30 ng was loaded on 8 ~ 10% SDS-polyacrylamide gels for separation, and was then transferred to the polyvinylidene difluoride membranes (HPVH00010, Millipore, Germany). The membranes were cut prior to hybridisation with antibodies. After being soaked with 5% non-fat milk (0.05% Tween, 20% TBST) for 1.5 h at room temperature to block the non-specific binding, the membrane was incubated with 1:1000 dilution of the primary antibody in dilution buffer (Abs954, Absin, China) overnight at 4 °C. Antibodies specific for MYD88 (# 4283), p-P65 (# 3033), P65 (# 8242), p-IκBα (# 2859), IκBα (# 4812), p-ERK1/2 (# 4370), ERK1/2 (# 4695), p-P38 (# 4511), P38 (# 8690), p-JNK (# 4668), JNK (# 9258), GAPDH (# 8884), and the HRP-conjugated goat anti-rabbit secondary antibody (# 7074) were purchased from Cell Signalling Technology (Danvers, MA, USA). Antibodies for MEK1/2 (AF6385) and p-MEK1/2 (AF8035) were purchased from Affinity Biosciences. Then the membrane was incubated with 1:2000 dilution of HRP-conjugated goat anti-rabbit secondary antibody in 5% non-fat milk for 2 h at room temperature. The chemiluminescent signal was developed using the ECL reagents (1810202, Thermo Scientific, USA), and the image was captured by a ChemiScope5300Pro CCD camera (Clinx Science Instruments, China). The band intensity was quantified by the Quantity One software (Bio-Rad, USA).

Immunofluorescence staining

The cells were inoculated in a 24-well plate (1 × 104 cells/well) containing cover glasses. The cell treatment was carried out when the BESC had grown to 30%~40% confluence. After 45 min treatment, the cells were fixed with 4% paraformaldehyde (BL539A, Biosharp, China) for 15 min at room temperature. Then the sample was incubated with TBST containing 0.4% Triton X-100 (ST797, Beyotime, China) for 15 min at room temperature to permeabilize the cells. After 1.5 h incubation with TBST containing 5% Albumin Bovine V (#A8020, Solarbio, China) at room temperature, the cells were incubated with 1:250 dilution of primary antibody (NF-κB P65) in the antibody diluent overnight at 4°C, and with the subsequent FITC-conjugated secondary antibody (A0423, Beyotime, China) at room temperature for 1.5 h. The cell nuclei were stained using DAPI (SD8495, Beyotime Biotechnology, China) at room temperature for 15 min. Finally, the cell slides were removed and placed on the slide, and the slides were sealed after dropping fluorescent anti-quenching solution. The fluorescent distribution of BESC was visualized and captured using a confocal microscopy (Leica TCS SP8 STED, Leica Corporation, Germany). The immunofluorescence signals were quantified by the Image J software (National Institutes of Health, USA).

Statistical analysis

All experiments were repeated at least three times. The SPSS 25.0 (IBM, USA) program was applied for data analysis. Statistically significant differences were calculated by one-way ANOVA, followed by Dunnett’s test. The result was expressed as the means ± standard error of means (SEM). A two- sided p value of < 0.05 was considered statistically significant.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BEEC:

Bovine endometrial epithelial cells

BESC:

Bovine endometrial stromal cells

CXCL8:

C-X-C motif chemokine ligand 8

COR:

Cortisol

DM:

Dry matter

E. coli:

Escherichia coli

GPX:

Glutathione peroxidase

IL1B:

Interleukin 1

IL6:

Interleukin 6

LPS:

Lipopolysaccharide

NOS2:

Inducible nitric oxide synthase2

PTGS2:

Prostaglandin-endoperoxide synthase 2

Se:

Selenium

TLR:

Toll-like Receptors

TLR4:

Toll-like receptor 4

TNF:

Tumour necrosis factor

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Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (NO: 32072937, 31802253, 32102735), the International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions (No: 8), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21_3279), the earmarked fund for Jiangsu Agricultural Industry Technology System (JATS[2023]456), the National Key R&D Program of China (2023YFD1801100), the Natural Science Foundation of Jiangsu Province (NO: BK20210808), the 111 Project (D18007), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funding bodies were not involved in the study design, data analysis, interpretation of data, or in writing the manuscript.

Author information

Author notes

  1. Luying Cui and Min Zhang contributed equally to this work.

Authors and Affiliations

  1. College of Veterinary Medicine, Yangzhou University, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou, Jiangsu, PR China

    Luying Cui, Min Zhang, Fangling Zheng, Changning Yuan, Zhihao Wang, Shangfei Qiu, Xia Meng, Junsheng Dong, Kangjun Liu, Long Guo, Heng Wang & Jianji Li

  2. Joint International Research Laboratory of Agriculture and Agriproduct Safety of the Ministry of Education, Yangzhou, Jiangsu, PR China

    Luying Cui, Min Zhang, Fangling Zheng, Changning Yuan, Zhihao Wang, Shangfei Qiu, Xia Meng, Junsheng Dong, Kangjun Liu, Long Guo, Heng Wang & Jianji Li

  3. International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, Jiangsu, PR China

    Luying Cui, Min Zhang, Fangling Zheng, Changning Yuan, Zhihao Wang, Shangfei Qiu, Xia Meng, Junsheng Dong, Kangjun Liu, Long Guo, Heng Wang & Jianji Li

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Contributions

L.C.: funding acquisition, supervision, writing-original draft, writing-review and editing. M.Z.: data curation, formal analysis, methodology, writing-original draft. F.Z., Z.W., S.Q. and X.M.: data curation. CY.: funding acquisition, data curation. J.D. and H.W. : funding acquisition. K.L. and L.G.: methodology. J.L.: conceptualization, funding acquisition, supervision. All authors have read and approved the final manuscript, and agreed to be accountable for its contents.

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Correspondence to Heng Wang or Jianji Li.

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12917_2024_4240_MOESM1_ESM.pdf

Supplementary Material 1: The effect of Na2SeO3 on the viability and LDH release of primary bovine endometrial stromal cells

Supplementary Material 2: The raw blot images

Supplementary Material 3: The raw image of immunocytochemistry

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Cui, L., Zhang, M., Zheng, F. et al. Selenium elicited an enhanced anti-inflammatory effect in primary bovine endometrial stromal cells with high cortisol background. BMC Vet Res 20, 383 (2024). https://doi.org/10.1186/s12917-024-04240-3

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Keywords

BMC Veterinary Research

ISSN: 1746-6148

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