Inflammation ( # 2013) DOI: 10.1007/s10753-013-9761-5

Selenium Inhibits LPS-Induced Pro-inflammatory Gene Expression by Modulating MAPK and NF-κB Signaling Pathways in Mouse Mammary Epithelial Cells in Primary Culture Wen Zhang,1 Runxiang Zhang,2 Tiancheng Wang,1 Haichao Jiang,1 Mengyao Guo,1 Ershun Zhou,1 Yong Sun,1 Zhengtao Yang,1 Shiwen Xu,3 Yongguo Cao,1,4 and Naisheng Zhang1,4

Abstract—Mastitis is characterized by an inflammation of the mammary gland of dairy animals and humans; this condition is one of the major causes of economic losses in dairy industries. Selenium (Se), a biological trace element, modulates the functions of many regulatory proteins in signal transduction and provides advantages for animals with inflammatory diseases, including mastitis. The current study aimed to assess the protective effects and the active mechanism of Na2SeO3 against lipopolysaccharide (LPS)-induced inflammation in mouse mammary epithelial cells (MMECs). Our results showed that LPS-induced expressions of cyclooxygenase-2 and tumor necrosis factor-α significantly decreased after Se was supplemented to Se-deficient MMECs. Na2SeO3 also suppressed LPS-induced nuclear factor-κB activation, inhibitory kappa B degradation, and ERK, JNK, and P38 phosphorylation in a dose-dependent manner. These results suggested that Se functions as an antiinflammatory agent in mastitis. KEY WORDS: selenium; mouse mammary epithelial cell; anti-inflammation; nuclear factor-κB; mitogen-activated protein kinase.

INTRODUCTION Bovine mastitis is characterized by pathological changes in udder tissues caused by mammary gland inflammation; as a result, milk yield is reduced, milk quality is compromised, and veterinary costs are increased, thereby causing worldwide economic losses in the dairy industry [1]. Cellular factors of the immune 1

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, People’s Republic of China 2 Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, 130112, Jilin, People’s Republic of China 3 Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northeast Agriculture University, Harbin, 150030, Heilongjiang, People’s Republic of China 4 To whom correspondence should be addressed at Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, People’s Republic of China. Email: [email protected]; [email protected]

system of bovine mammary glands come from two main types: mammary epithelial cells and immune cells, including macrophages, neutrophils, natural killer (NK) cells, and dendritic cells [2]. Mammary epithelial cells are the most numerous cells in the mammary gland and initiate immune response to pathogens [3, 4]. Mammary epithelial cells express mRNA for toll-like receptor (TLR) 2, TLR 4, TLR 9, and β-defensin 5, thereby contributing positively to pathogen response [5]. Mammary epithelial cells react to pathogens, and such a reaction is important to determine the outcome of an infection in the mammary gland [6, 7]. Lipopolysaccharide (LPS), a powerful bacterial virulence factor, is a common substance that triggers inflammation [8]. LPS-triggered mouse mammary epithelial cells (MMECs) result in the expression of various cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1β [2, 3]. Cyclooxygenase-2 (COX-2) expression and consequent prostaglandin E2 (PGE2) production are used as prognostic markers of inflammation and targets of therapeutic intervention in mastitis [9]. As one of the two well-studied isozymes, COX-2 is highly inducible in response to LPS and primarily associated with the

0360-3997/13/0000-0001/0 # 2013 Springer Science+Business Media New York

Zhang, Zhang, Wang, Jiang, Gou, Zhou, Sun, Yang, Xu, Cao, and Zhang biosynthesis of pro-inflammatory mediators such as PGE2 [20]. PGE2, as one of the end products of arachidonic acid, can regulate several inflammatory processes, such as vascular permeability, leukocyte infiltration, localized edema, and fever [9]. Selenium (Se), in the form of selenoproteins, is an essential trace element [10]. Se selenoproteins, such as Sedependent glutathione peroxidase (Se-GPx) and thioredoxin reductase, have an important function in the cellular antioxidant defense system [4]. Se also regulates inflammatory processes and transcription [11–13]. Nuclear factor kappa B (NF-κB) is considered as a central mediator of immune responses [14]. The NF-κB–inhibitory kappa B (IκBα) complex is located in the cytoplasm of unstimulated cells [4]. IκBα is phosphorylated and degraded, thereby releasing the heterodimer NF-κBα composed of various subunits, such as p65 and p50 [4]. NF-κB enters the nucleus and transactivates the genes encoding pro-inflammatory cytokines. TNF-α targets the liver, synchronizes with IL-6, and releases CRP [15]; by contrast, IL-1, together with TNF-α, reactivates NF-κB by stimulating IκBα phosphorylation using protein kinases [14]. Mitogen-activated protein kinase (MAPK) pathways of signal transduction, involving extracellular signalregulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases, activate COX-2 and other proinflammatory genes [16]. Previous studies also showed the modulatory effects of Se on NF-κB and MAPKs in vitro [4, 10, 12, 14]. However, the detailed mechanisms underlying the anti-inflammatory effect of Se in LPS-stimulated MMECs remain unclear. This study aimed to examine the antiinflammatory effects and the associated signaling pathways of Se supplement in LPS-stimulated MMECs.

cultured at 37 °C and 5 % CO2 in a humidified atmosphere. A serum-free medium DMEM/F12 (1:1) containing 10 mg/ml insulin, 1 mg/ml progesterone, 1 mg/ml hydrocortisone, 10 ng/ml EGF, 1 mg/ml prolactin, 5 mg/ml transferrin, 5 mM ascorbic acid, 1 mg/ml fatty acid-free bovine serum albumin (BSA), and 50 mg/ml gentamycin was used as the basic cell culture media in these experiments. The tissue culture plates used for these experiments were precoated with 0.012-mg/ml rat tail collagen. MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide] Assay for Cell Viability Cell viability was determined by MTT assay. In brief, 1×104 MMECs were isolated and seeded in 96-well plates. The cells were cultured to confluence and pretreated with Na2SeO3 (0, 0.25, 0.5, 1, 2, 5, and 10 μM) for 48 h at 37 °C and 5 % CO2. MTT (20 μl of 5 mg/ml) was added to each well. The cells were then incubated for another 4 h. The supernatant was removed and formazan formation was resolved with 150 μl per well of dimethyl sulfoxide. The optical density was measured at 570 nm using a microplate reader (Tecan, Durham, NC). Enzyme-Linked Immunosorbent Assay

MATERIALS AND METHODS

MMECs (1×106) were cultured to confluence and pretreated with Na2SeO3 (0, 0, 1, 5, and 10 μM) for 24 h at 37 °C and 5 % CO2 in six-well plates. These pretreated cells were then incubated in a fresh growth medium, which contained LPS (0 or 100 ng/ml) and the same concentration of Na2SeO3 as in the pretreated medium, for 24 h. Cell-free supernatants were subsequently used for the pro-inflammatory cytokine assays with a mouse ELISA kit according to the manufacturer's instructions (BioLegend, Inc., San Diego, CA, USA).

Isolation and Culture of MMECs

Western Blot Analysis

MMECs were isolated according to a previously described method [17] with slight modifications. In brief, the mammary glands from pregnant female mice were minced and digested at 37 °C for 3 h with 0.2 % (w/v) collagenase and 0.2 % (w/v) dispase. Afterward, the mammary glands were pelleted, resuspended, and sequentially filtered using 530- and 60-mm nitex filters to remove large tissue fragments and trap epithelial organoids, respectively. Single cells, including fibroblasts, endothelial cells, and red blood cells, were removed. MMECs were washed off from the nitex filter and

MMECs (1×106) used to detect COX-2 and β-actin were seeded in six-well plates and cultured to confluence. These cells were pretreated with Na2SeO3 (0, 0.25, 0.5, 1, 2, 5, and 10 μM) for 48 h and incubated in a fresh medium containing LPS (100 ng/ml) and the same Na2SeO3 concentration for 2 h. MMECs (1×106) used to detect upstream kinases were also seeded in six-well plates and cultured to confluence; afterward, these cells were pretreated with Na2SeO3 (0, 0, 1, 5, and 10 μM) for 48 h and incubated in a fresh medium containing LPS (0

Se Inhibits LPS-Induced Expression by MAPK and NF-κB Pathways or 100 ng/ml) and the same Na2SeO3 concentration for 30 min. At the end of each experiment, the cells were collected and washed twice with ice-cold PBS. The total proteins from these cells were extracted using M-PER mammalian protein extraction reagent (Thermo Scientific). The supernatants were prepared by subjecting the cell lysates to centrifugation at 14,000×g for 10 min at 4 °C and then used for analyses. The protein concentration in the cell supernatants was determined by BCA protein assay (Pierce). Total cellular proteins (30 μg) from the cell l y s a t es w er e se pa r ate d on a 1 0 % SD S – polyacrylamide gel and transblotted onto polyvinylidene fluoride membrane. The membrane was blocked with Tris-buffered saline containing 0.05 % Tween-20 and 3 % BSA. The membrane was probed with a primary antibody and then with an appropriate secondary antibody coupled to h or s e r ad i s h p er ox i d a s e . R a bb i t m on o cl on al antibodies COX-2, β-actin, IκBα, p65, p38, ERK, JNK, p-IκBα, p-p65, p-p38, p-ERK, and p-JNK were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The blots were tested by SuperSignal West Pico Chemiluminescent Substrate (Pierce) using ImageQuantTM software program (GE Amersham). Statistical Analyses Data were presented as mean±SEM. The groups were compared using one-way ANOVA and Dunnett's test. p≤0.05 was considered statistically significant.

for 24 h. TNF-α in the cell culture supernatant increased significantly after the cells were stimulated by LPS in the LPS groups (Fig. 2a). The results showed that Se decreased TNF-α expression in the LPS-induced MMECs in a dose-dependent manner. LPS significantly increased IL-6 and IL-1β expression in the cell culture supernatant in the four groups stimulated with LPS compared with the control group; however, Se supplementation did not affect LPS-induced IL-6 or IL1β expression (Fig. 2b, c). Se Inhibited LPS-Induced COX-2 Expression The effect of Se on LPS-induced COX-2 expression was investigated. MMECs were treated with 0 to 10 μM Se for 48 h and then stimulated with LPS (100 ng/ml) for 2 h. Figure 3 shows that the COX-2 expression of the cell lysates was analyzed by Western blot. Se supplement decreased LPS-induced COX-2 expression dose dependently. COX-2 expression also decreased significantly at ≥0.25 μM Se. At 10 μM Se, COX-2 was barely expressed. Se Suppressed LPS-Induced NF-κB Pathway NF-κB is present in the cytosol and binds to IκBα. Upon activation by LPS, IκBα is phosphorylated by IκBα kinase and becomes degraded; the released NF-κB is then translocated into the nucleus and enhances the transcription of pro-inflammatory genes. To examine whether or not the NF-κB pathway mediates the Seinhibited inflammatory response, we determined NF-κB and IκBα protein levels by Western blot. Figure 4 shows

RESULTS Se did not Affect Cell Viability The potential cytotoxicity of Na2SeO3 was evaluated by MTT assay. Figure 1 shows that the cell viability was not affected when the cells were exposed to 0.25–10 μM of Na2SeO3 for 48 h compare to 0. This result indicated that the cell viability was not affected by Na2SeO3 at 0.25 to 10 μM. Se Inhibited LPS-Induced TNF-α Expression in DoseDependent Effect To analyze the anti-inflammatory effects of Se, we determined TNF-α, IL-6, and IL-1β in the supernatant of MMECs treated with 1, 5, and 10 μM Se for 48 h and LPS

Fig. 1. Effects of Se on the cell viability of MMECs. Cells were treated with Se (0, 0.25, 0.5, 1, 2, 5, and 10 μM) for 48 h. Cell viability was determined by MTT assay. The values are presented as means±SEM (n=8).

Zhang, Zhang, Wang, Jiang, Gou, Zhou, Sun, Yang, Xu, Cao, and Zhang

Fig. 3. Effect of Se on COX-2 expression in LPS-induced MMECs. After pretreatment with different concentrations of Se (0, 0.25, 1, 2, 5, and 10 μM) for 48 h, cells were stimulated with LPS (100 ng/ml) for 2 h. Total cellular protein (30 μg) were analyzed by Western blot. β-actin was used as a internal control. The values are presented as means±SEM (n=3).

that the levels of p65 and IκBα phosphorylation in the NF-κB signal pathway significantly increased in the LPS group, but these levels decreased in the groups pretreated with Se. Se Suppressed LPS-Induced MAPK Pathway MAPK pathways are activated by LPS. p38, JNK, and ERK phosphorylation affected by Se was analyzed by Western blot. Figure 5 shows that LPS-stimulated MMECs increased the phosphorylation of the three MAPKs. The activation of p38, JNK, and ERK peaked at 15 to 30 min after LPS stimulation (data not shown). Se pretreatment markedly inhibited the LPS-induced increase in P38, ERK, and JNK phosphorylation in a dose-dependent manner.

DISCUSSION

Fig. 2. Effects of Se on the secretion of a TNF-α, b IL-6, and c IL-1β in LPS-induced MMECs. Cells were pretreated with different concentrations of Se (0, 0, 1, 5, and 10 μM) for 24 h and LPS (100 ng/ml) for 24 h. The levels of TNF-α, IL-6, and IL-1β in cell supernatants were detected by ELISA. The values are presented as means±SEM (n=3). ##p