Biometals DOI 10.1007/s10534-014-9708-9

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death Dong Yu • Zi-wei Zhang • Hai-dong Yao Shu Li • Shi-wen Xu

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Received: 7 January 2014 / Accepted: 23 January 2014 Ó Springer Science+Business Media New York 2014

Abstract To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H2O2-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H2O2, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H2O2-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 lM H2O2 was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 lM H2O2. Meanwhile, H2O2 induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and

p53 and down-regulation of Bcl-2 (P \ 0.05). When lymphocytes were pretreated with Se before treated with H2O2, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P \ 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H2O2 than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H2O2. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase3 and p53 and down-regulated Bcl-2 (P \ 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress. Keywords Selenoprotein W  Small interfering RNAs  Apoptosis  Splenic lymphocyte  Chicken

Introduction D. Yu College of Life and Technology, Harbin Normal University, Harbin 150025, People’s Republic of China D. Yu  Z. Zhang  H. Yao  S. Li (&)  S. Xu (&) College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected] S. Xu e-mail: [email protected]

Selenium (Se) is an essential nutritional trace element for organisms with important roles in many aspects of health. For example, oxidant defense and immune progress (Burk and Hill 2005; Rayman 2000; Hoffmann and Berry 2008; McKenzie et al. 1998), and other aspects of health (Terry et al. 2009; Li et al. 2010a; Schweizer et al. 2004; Martin-Romero et al. 2001; Kaur and Bansal 2005; Mahmoud and Edens

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2005). Se is present in several proteins, called selenoproteins. In vivo, Se is incorporated into proteins mainly in the form of selenocysteine (Sec) (Saito and Takahashi 2000). Approximately 30 selenoproteins and 25 families of selenoprotein genes have been identified in mammals (Kryukov et al. 2003), and a variety beneficial biological functions of selenoproteins have been described (Kryukov and Gladyshev 2004; Castellano et al. 2005; Zhang et al. 2005; Papp et al. 2007). Selenoproteins play in regulating reactive oxygen species (ROS) and redox status in nearly all tissues (Hoffmann and Berry 2008). Selenoproteins are largely uncharacterized in the inmmune system, except with regards to inflammation (McCord 2000). Levels of ROS influence inflammatory responses by regulating the oxidative state of immune cells. GPXs and TRXRs are necessary for optimal function of immune cells by controlling oxidative stress and redox regulation (Hoffmann 2007). Mice with a T cell-specific deletion in tRNASec, resulting in knockout of all T-cell selenoproteins, have significantly diminished numbers of functional T cells and exhibited moderate to severe atrophy of the thymus, spleen, and lymph nodes. Furthermore, the mice have reduced antigen-specific production of immunoglobulins in vivo, implying a dysfunctional adaptive immune responses (Shrimali et al. 2008). Those properties have provided important information about the physiological roles of selenoproteins in immune system. Thus, it is crucial to characterize the function of selenoproteins. However, little is known about the relation between selenoprotein and the immune system in birds. SelW first described in 1993 (Vendeland et al. 1993). SelW is similar to the GPX family in that it shares the redox motif and binds glutathione (Beilstein et al. 1996). SelW is a small selenoprotein which selenocysteine is located in the N-terminal portion of a relatively short functional domain. The sequences of SelW are identical in rats and mice as well as monkeys and humans (Whanger 2009). Recently, the avian SelW gene (GenBank accession number GQ919055) was cloned by our group. We have reported that the structure of chicken SelW is similar to that of mammals (Li et al. 2010b). To date, the biological activity of SelW has not been identified. However, studies supported that SelW of rat and monkey muscle can bind glutathione (Beilstein et al. 1996; Gu et al. 1999) and subsequently shown to be bound to residue number 37 cysteine (Gu et al. 1999). In addition, it has

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been found that overexpression of SelW cells to be more resistant to oxidative stress. The expression of this selenoprotein plays an important role in protection of cells from oxidative stress in the cellular defense system (Beilstein et al. 1996; Jeong et al. 2002; Wang et al. 2010), suggesting a potential role as an antioxidant (Jeong et al. 2002). SelW is widely present in the tissues of animals. The highest amount of SelW expression was detected in muscle, heart (except rodents), spleen, and brain in mammals (Whanger 2009). The expression level of SelW are strongly dependent on Se status and intake. It has been shown that selenium-supplemented diets lead to a increase in the expression of SelW in spleen in rats and sheep (Yeh et al. 1997a, b), and selenium-deficient diets lead to a decrease in the expression of most selenoproteins in various tissues (Allan et al. 1999; Gu et al. 1999; Yeh et al. 1997a, b). We have reported that SelW is widely expressed in immune organs (spleen, thymus, and bursa of Fabricius) of chickens and that Sesupplementation of the feed increases SelW expression in those immune organs. However, although Se content was the highest in the spleen, the remarkable stability of the SelW mRNA level was observed in this organ during different times of dietary Se supplementation (Yu et al. 2011). These observations suggest that Se and SelW play important roles in the immune function. However, except for its amino acid sequence (Li et al. 2010b) and expression in the gastrointestinal system (Li et al. 2010c; Gao et al. 2012), liver (Sun et al. 2011), muscle tissues (Zhang et al. 2012; Ruan et al. 2012), embryos neurons (Li et al. 2012), little information is available on avian SelW. The mechanisms by which SelW expression is regulated in immune organs of birds, and the mechanisms by which it exerts its antioxidant activity in the chicken splenic lymphocytes are not fully understood. Oxidative stress may result in cell apoptosis, and Se deficient and Se excess could stimulate cell apoptosis and aggravate cell apoptosis induced by H2O2 and other inducer (Demelash et al. 2004; Guan et al. 2009). Silencing of the SelW gene increased cell apoptosis rate and susceptibility to H2O2 (Chung et al. 2009; Yao et al. 2013). Overexpression of SelW cells had a lower apoptotic cell death than control cells in the absence or presence of extracellular H2O2 (Han et al. 2012a; Jeong et al. 2002). The bcl-2 gene family play a central role in the regulation of apoptosis because its members integrate diverse survival and death signals that are

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generated outside and inside the cell. This family includes antiapoptotic members such as Bcl-2, Bcl-xl, Bcl-w, which protect cells from apoptosis, and proapoptotic members such as Bax, Bak, Bad, which act as promoters of apoptosis. It has been reported that Bcl-2 protein blocks cytochrome c release from mitochondria and therefore blocks the activation of caspases, which is pivotal for ensuing apoptotic process (Oliver et al. 2005), and p53 might share similar function and similar transcriptional regulatory pathways in apoptosis following excitotoxic stimulation (Nango et al. 2003). However, the precise mechanisms by which selenium and SelW expression regulate the apoptotic machinery remain poorly understood. Therefore, there were two aims in this study. The first was to investigate the effect of Se-supplementation and SelW gene silence on SelW and apoptosisrelated genes (Bcl-2, Bax, Bak-1, caspase-3 and p53) mRNA expression in cultured splenic lymphocyte, and the second was to verify SelW function, the influence of Se-supplementation and SelW gene silence on H2O2-mediated oxidative stress and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken.

Materials and methods Reagents RPMI 1640 medium was purchased from SigmaAldrich (St Louis, MO). Trizol reagent was purchased from Invitrogen Biotechnology Co Ltd. (Shanghai, China). The SYBR PremixScript real-time (RT)-PCR Kit II was purchased from TaKaRa (Shiga, Japan). Lymphocytes separation medium was purchased from Hao Yang Biological Manufacture Co, Ltd. (Tian Jin, China). CCK-8 assay kit was purchased from Dojindo Laboratories (Dojin, Japan).

prepared by gently pushing the splenic pulp through a sterile stainless steel mesh with a pore size of 200 lM. Cells were washed and resuspended in 5 ml sterile PBS and then layered over 5 ml lymphocytes separation medium. The splenocyte preparations were enriched by centrifuge (2,0009g) for 15 min at room temperature. Cells were recovered from the interface, resuspended, and washed twice in RPMI-1640 medium (without phenol red). The cells were suspended in RPMI-1640 medium (without phenol red) containing HEPES and 2 mM glutamine. Splenic lymphocyte density was adjusted to 1.5 9 106 cells/ml and the viability of the freshly isolated cells was always above 95 % (trypan blue exclusion test). For the monitoring of various parameters in the present investigation, cells were treated for 24 h in the absence and presence of various concentrations of H2O2 (10, 50, 100, 200, and 400 lM), and also were incubated with 10-8, 10-7 or 10-6 mol/l of Se as sodium selenite (Sigma, USA) for 6 h, and the cells were then treated with H2O2 for an additional 24 h. Assay of lymphocyte viability Cell viability was measured by a CCK-8 assay kit. Splenic lymphocytes at a concentration of 1.0 9 105 cells were seeded in a 96-well plate. After incubating chicken splenic lymphocytes with H2O2, 10 ll of CCK-8 was added, Following another 4 h of co-culture, the optical density (OD) of each well was read at 570 nm in a microplate reader (Synergy HT Multimode Reader, BioTek Instruments, USA). In addition, absorbance measurements were also recorded for the target cell control, blank control, and effector cell control. The percentage of lymphocytes activity was determined by the following equation: % lymphocytes viability = 1 - [(optical density value of test samples - optical density value of effector cell control)/optical density value of target cell control] 9 100.

Cell culture and treatments Cell apoptosis assay All procedures in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Spleens were dissected from Isa brown cocks (60 days old) and were collected aseptically and placed in sterile phosphatebuffered saline (PBS, 0.1 M phosphate buffer with 0.85 % NaCl, pH 7.2). Single cell suspension was

Apoptosis was determined morphologically after staining the cells with AO/EB followed by fluorescence microscopy inspection. Briefly, Lymphocytes were incubated with 10-8, 10-7 or 10-6 mol/l of Se as sodium selenite (Sigma, USA) for 6 h, and the cells were then treated with H2O2 for an additional 24 h,

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and also lymphocytes were transfected with SelW siRNA or Stealth RNAi Negative Control Duplexes for 24 h, the cells were then treated with H2O2 for 24 h, then AO/EB staining was conducted as described in the manufacturer’s instruction. In brief, 25 ll of cell suspension (106 cells/ml) mix gently with 1 ll of AO/EB solution. Each sample should be mixed just prior to microscopy and quantification. Samples must be evaluated immediately. Place 10 ll of cell suspension onto a microscopic slide, cover with a glass coverslip, and examine at least 300 cells in a fluorescence microscope using a fluorescein filter. The percentage of apoptotic cells was calculated by dividing the number of apoptotic cells (early apoptotic cells and late apoptotic cells) by the total number of AO/EB-stained cells. siRNA preparation and transfection The siRNA corresponding to the SelW gene was designed and synthesized by Invitrogen (Stealth RNAi). The sequence for the SelW siRNA was 50 -CGGCUUC GUG GACACCGACGCCAAA-30 . A random siRNA sequence (sense 50 -CGGUCGUGGACACCGACGCC CUAAA-30 ; antisense 50 -UUUAGGGCGUCGGUGU CCACGACCG-30 ) was used as a negative control and have no homology with any genes. siRNAs were designed using Prime 5 Software (Molecular Biology Insights, Inc, Cascade, CO) and were synthesized by Invitrogen Biotechnology Co Ltd. Cells were seeded into six-well plates at appropriate densities and cultured overnight. Transfections were carried out using LipofectAMINE 2000 transfection reagent (Invitrogen), following the manufacturer’s instructions. The lymphocytes were transfected with siRNA using LipofectAMINE 2000 (Invitrogen) in serum-free RPMI-1640 medium for 6 h. The medium was subsequently exchanged with RPMI-1640 medium (without phenol red), and the cells were cultured for an additional 6, 12 or 24 h, respectively. Stealth RNAi Negative Control Duplexes (Invitrogen) were used as a control. Real-time PCR analysis Total RNA was isolated from cells using Trizol reagent according to the manufacturer’s instructions (Invitrogen, China). The dried RNA pellets were resuspended in 50 ll of diethyl-pyrocarbonate-treated

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water. The concentration and purity of the total RNA were determined spectrophotometrically at 260/280 nm. First-strand cDNA was synthesized from 5 lg of total RNA using oligo dT primers and Superscript II reverse transcriptase according to the manufacturer’s instructions (Invitrogen, China). Synthesized cDNA was diluted five times with sterile water and stored at -80 °C before use. Primer Premier Software (PREMIER Biosoft International, USA) was used to design specific primers for SelW and b-actin based on known chicken sequences. Transcripts were quantified by qRT-PCR using the following primer sequence for SelW were: 50 -CTC CGCGTCACCGTGCTC-30 /50 -CACCGTCACCTCG AACCAT CCC-30 ; for b-actin were: 50 -CCGCTCT ATGAAGGCTACGC-30 /50 -CTCTCGGCTG TGGT GGTGAA-30 . The primers were designed using Prime 5 Software (Molecular Biology Insights, Inc, Cascade, CO) and were synthesized by Invitrogen Biotechnology Co Ltd. General PCRs were first performed to confirm the specificity of the primers. The PCR products were electrophoresed on 2 % agarose gels, extracted, cloned into the pMD18-T vector (TaKaRa, China), and sequenced. Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, USA). Reactions were performed in a 20-ll reaction mixture containing 10 ll of 29 SYBR Green I PCR Master Mix (TaKaRa, China), 2 ll of either diluted cDNA, 0.4 ll of each primer (10 lM), 0.4 ll of 509 ROX reference Dye II and 6.8 ll of PCR-grade water. The PCR procedure for SelW and b-actin consisted of 95 °C for 30 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 60 °C for 30 s. The melting curve analysis showed only one peak for each PCR product. Electrophoresis was performed with the PCR products to verify primer specificity and product purity. A dissociation curve was run for each plate to confirm the production of a single product. The amplification efficiency for each gene was determined using the DART-PCR program (Peirson et al. 2003). The mRNA relative abundance was calculated according to the method of Pfaffl (2001). Statistical analysis Statistical analysis of all data was performed using SPSS statistical software for Windows (version 13;

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SPSS Inc, Chicago, IL, USA). When a significant value (P \ 0.05) was obtained by one-way analysis of variance, further analysis was done. All data showed a normal distribution and passed equal variance testing. Differences between means were assessed by Tukey’s honestly significant difference test for post hoc multiple comparisons. Data are expressed as mean ± standard deviation. Differences were considered to be significant at P \ 0.05, P \ 0.01, P \ 0.001, and not significant P [ 0.05.

Results Effects of Se on H2O2-induced chicken splenic lymphocyte viability The effect of an exogenous oxidant, cells were treated with H2O2, on cell viability of cultured of chicken splenic lymphocyte. Figure 1a shows that the viability of splenic lymphocyte significantly decreases as a function of increasing H2O2 concentration (P \ 0.01). When cells were incubated for 24 h in the presence of 50 lM H2O2, approximately 50 % of the treated cells survived. However, when the concentration of H2O2 was elevated to 400 lM, less than 6 % of the cells survived. Figure 1b shows that the effect of Se on H2O2induced cell viability was dose-dependent. When cells were incubated for 24 h in the presence of 20 lM H2O2, approximately 60 % of the treated cells survived. However, the cell viability increases as a function of increasing Se concentration, when cells were incubated with 10-8, 10-7 or 10-6 mol/l of Se as

Fig. 1 The cell viability of cultured of chicken splenic lymphocyte. a The cell viability of cultured of chicken splenic lymphocyte to H2O2. b The effect of Se on H2O2-induced

sodium selenite for 6 h, and the cells were then treated with 20 lM H2O2 for an additional 24 h, compared with the 20 lM H2O2 control group. Effects of Se on H2O2-induced chicken splenic lymphocyte apoptosis To estimate the role of Se in H2O2-induced chicken splenic lymphocyte apoptosis. Using AO/EB staining method, we further monitored the extent of the H2O2induced cells apoptosis. As shown in Figs. 2 and 7a, the population of AO/EB-stained apoptotic cells was significantly increased in the presence of 20 lM H2O2 (P \ 0.01), compared with the control, approximately 40 % of the treated cells apoptosis. However, amount of apoptotic cells decreases as a function of increasing Se concentration (P \ 0.01), when cells were pretreated with 10-8, 10-7 or 10-6 mol/l of selenite for 6 h, and then treated with 20 lM H2O2 for an additional 24 h, compared with the 20 lM H2O2 control group. Effects of Se on mRNA levels of Bcl-2, Bax, Bak-1, caspase-3, p53 and SelW in H2O2-induced lymphocyte To investigate whether SelW mRNA expression was regulated by Se. Chicken splenic lymphocyte were treated with various concentrations of Se for 6 h, and were then treated for 24 h in the absence and presence of 20 lM H2O2. SelW mRNA expression was significantly increased as dose-dependent in cells treated with 10-8, 10-7 or 10-6 mol/l of Se (P \ 0.05), compared with the control. When cells

chicken splenic lymphocyte viability. Each bar represents mean ± SD (n = 5). Bars with asterisks are statistically significantly different from control (**P \ 0.01)

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Control

10-6MSe+H2O2

10-7MSe+H2O2

10-8MSe+H2O2

Fig. 2 Effects of Se on H2O2-induced chicken splenic lymphocyte apoptosis (9400). Chicken splenic lymphocyte were pretreated with 10-8, 10-7 or 10-6 mol/l of Se as sodium selenite for 6 h, and then treated with 20 lM H2O2 for an

additional 24 h. White arrows, blue arrows, yellow arrows and red arrows, examples of green live cells, green early apoptotic cells, orange later apoptotic cells and red necrotic cells, respectively. (Color figure online)

were incubated for 24 h in the presence of 20 lM H2O2, SelW mRNA expression was significantly decreased (P \ 0.05), compared with the control. Lymphocyte were treated with 10-8, 10-7 or 10-6 mol/l of Se for 6 h, and the cells were then treated with 20 lM H2O2 for an additional 24 h, SelW mRNA expression was significantly increased (P \ 0.05), compared with the 20 lM H2O2 control (Fig. 3g). As shown in Fig. 3, the lymphocytes with H2O2 (20 lM) resulting in a significantly increase the Bax/ Bcl-2 ratio and mRNA expression of Bax, Bak-1, caspase-3 and p53 (P \ 0.05), and decrease mRNA expression of Bcl-2 (P \ 0.05), compared with the control. When pretreated lymphocyte with 10-8, 10-7 or 10-6 mol/l of Se for 6 h before treated with 20 lM H2O2, the Bax/Bcl-2 ratio and mRNA expression of Bax, Bak-1, caspase-3 and p53 were significantly decreased (P \ 0.05), and Bcl-2 expression were significantly increased (P \ 0.05), compared with

the 20 lM H2O2 control, and significantly dosedependent increased compared with the 10-8, 10-7 or 10-6 mol/l of Se group respectively. In contrast, Bcl-2 expression were decreased (P \ 0.05).

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Transfection efficiency of SelW knockdown Transfection efficiency for lymphocyte was optimized on the basis of cellular uptake of BAFRFO (as the indicator of transfection efficiency). A strong intracellular red fluorescent signal was observed within the cytoplasm of lymphocytes when cells successfully transfected. The efficiency was assessed by calculating the proportion of cells with visible red fluorescent in the total number of cells (nuclei) per field. After the SelW silencing, no significant differences in phenotype of lymphocytes were observed under light microscope (data not shown). Effects of the SelW knockdown on the mRNA expression levels of SelW were verified by the qPCR. Either SelW siRNA or Stealth RNAi

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Fig. 3 Effect of Se on mRNA levels of Bcl-2, Bax, Bak-1, caspase-3, p53 and SelW in cultured chicken splenic lymphocyte. Bars represent mean ± standard deviation (n = 3/group).

Bars with asterisks are statistically significantly different from H2O2-control (*P \ 0.05). Bars without sharing a common letter are significantly different (P \ 0.05)

Negative Control Duplexes were transfected into the cultures of chicken splenic lymphocyte. Compared with the control group (Fig. 8g). 6, 12 and 24 h after transfection, the remarkable stability of the SelW mRNA level was observed in the Stealth RNAi Negative Control Duplexes group (P [ 0.05), and the levels of SelW mRNA expression were effectively attenuated by SelW siRNA (P \ 0.05). When cells

were transfected with SelW siRNA for 24 h, the levels of SelW mRNA expression were effectively attenuated approximately 60 % by SelW siRNA (P \ 0.05). Because siRNA resulted in depletion of SelW expression levels by more than 60 % after transfection for 24 h, this efficient siRNA sequence was selected for the subsequent experiments. The images (Fig. 4) indicated that the majority of lymphocytes ([95 %) was

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Fig. 4 Assessment of transfection efficiency of SelW knockdown. a Bright field image of lymphocytes 24 h after incubated. b 24 h after transfection, fluorescence signal of DAPI (blue) in lymphocytes. c 24 h after transfection, fluorescence signal of

BAFRFO (red) in lymphocytes. d Merged picture of lymphocytes in c with image of these cells counterstained with DAPI. (Color figure online)

effectively transfected at 24 h where strong fluorescent was localized in cytoplasm of lymphocytes. Effects of SelW siRNA on H2O2-induced splenic lymphocyte viability Figure 5 shows that the SelW siRNA transfected chicken splenic lymphocytes were more sensitive to exogenous oxidant than those obtained with Stealth RNAi Negative Control Duplexes. In the presence of 10 lM H2O2, 58 % of siRNA-transfected cells were viable compared with of 79 % control cells. The cells treated with 50 lM H2O2, 40 % of siRNA-transfected cells were viable compared with 65 % of control cells. Effects of SelW siRNA on H2O2-induced chicken splenic lymphocyte apoptosis As shown in Figs. 6 and 7b, the population of AO/EBstained apoptotic cells was higher in SelW siRNA

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Fig. 5 The effects of SelW siRNA on H2O2-induced splenic lymphocytes viability. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between SelW siRNA and RNAi Negative Control Duplexes cells (*P \ 0.05)

transfected cells compared with the control cells, even before the addition of H2O2. When these cells were treated with H2O2, the number of AO/EB-stained apoptotic cells significantly increased (P \ 0.05). The

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Control (-)siRNA

Control (+)siRNA

50uMH2O2(-)siRNA

50uMH2O2(+)siRNA

Fig. 6 Effect of SelW suppression on H2O2-induced cell apoptosis (9400). Lymphocytes were transfected with SelW siRNA or Stealth RNAi Negative Control Duplexes, the cells were incubated for 24 h, and then treated with H2O2 for 24 h,

then stained with AO/EB for cell apoptosis assay. White arrows, blue arrows, yellow arrows and red arrows, examples of green live cells, green early apoptotic cells, orange later apoptotic cells and red necrotic cells, respectively. (Color figure online)

extent of exogenous oxidant-induced cell apoptosis was approximately twofold higher in SelW siRNA transfected cells compared with the Stealth RNAi Negative Control Duplexes transfected cells in 24 h, suggesting that the attenuation of SelW expression due to siRNA renders the chicken splenic lymphocyte more sensitive to H2O2.

Discussion

Effects of SelW siRNA on Bcl-2, Bax, Bak-1, caspase-3 and p53 mRNA in H2O2-induced lymphocyte As shown in Fig. 8, there were higher mRNA expression levels of Bax, Bak-1, caspase-3 and p53, and lower mRNA expression level of Bcl-2 in siRNA groups when compared with the corresponding RNAi Negative Control groups (P \ 0.05). The ratio of Bax/ Bcl-2 mRNA levels was also higher in siRNA groups (P \ 0.05). With the increasing of transfected time, the Bax/Bcl-2 ratio and mRNA levels of these apoptosis related genes showed an increased trend.

The main biological function of antioxidant activity of Se results from selenocysteine in the enzymatic active site, such as GPx (Rotruck et al. 1973). In poultry, numerous lines of previous evidence suggested that Se deficiency reduced growth and resulted in exudative diathesis (Bartholomew et al. 1998), and inhibited the function of neutrophils, NK cells, B cells, and T cells (Kiremidjian-Schumacher et al. 1992), and reduced the growth of lymphoid organs (Marsh et al. 1986). Supplementation of Se leads to enhanced comparative metabolic and immune response of chickens (Leng et al. 2003). However, both excess and deficiency of Se supply led to impaired growth. There has been reported that after 18 h treatment of human cancer cells with 0.05 lM selenite, cell viability reached a maximum with respect to the control. However, concentrations higher than 2 lM resulted in a marked decrease in cell proliferation (Rigobello et al. 2009). 100 nM selenite promoted cell viability and alleviate

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Fig. 7 The AO/EB-stained cells were analyzed as apoptotic cell death using fluorescence microscopy. The percentage of apoptotic cells was calculated by dividing the number of the apoptotic cells by the total number of cells. a Effect of Se on H2O2-induced cell apoptosis. b Effect of SelW suppression on

H2O2-induced cell apoptosis. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different (*P \ 0.05; **P \ 0.01)

the apoptosis, however, treatment with 1,000 nM selenite, resulted in a marked decrease in cell viability (Du et al. 2010). It was also reported that the viability of NB4 cells were not significantly affected by the treatment with 2 lmol/l Na2SeO3, However, treated by 5, 10, and 20 lmol/l Na2SeO3, respectively, showed a Na2SeO3 concentration-dependent increasingly decreased cell viability (Cao et al. 2006). This result was similar to the effect of Se on cells viability of rat vascular smooth muscle cells and human prostate cancer cells (Tang and Huang 2004; Zhong and Oberley 2001). In the present study, as demonstrated by CCK-8 experiments, the viability of splenic lymphocyte significantly decreases with the increasing of H2O2 (Fig. 1a). The effect of Se on cell viability of splenic lymphocyte was dose-dependent. When cells were incubated with 20 lM H2O2 for 24 h, approximately 60 % of the treated cells survived. However, when cells were pretreated with 10-8, 10-7 or 10-6 mol/l of Se as sodium selenite for 6 h before treated with H2O2, the cell viability were markedly increased (Fig. 1b). In previous studies, they reported that SelW was expressed widely in chicken tissues. Among them, the predominant expression was in the nervous tissues, muscle tissues, gizzard, blood vessel, and cartilaginous tissues, and the weak expression was in the pancreas, testis, ovary, kidney, and veins (Li et al. 2010c). We have reported that the SelW gene was distributed widely in the immune organs of chickens fed the basal commercial diets and the Se-

supplemented diets (Yu et al. 2011). Numerous studies have shown that SelW levels increase in response to Se dietary supplementation, and a significant increase of SelW expression level in the tissues was observed in rats and sheep fed dietary supplementation with Se (Yeh et al. 1995, 1997a, b, 1998; Sun et al. 1998). There have been reported that the SelW mRNA levels in the gastrointestinal tract tissue, liver and skeletal muscles of chickens were found to increase in a time-dependent manner with increasing feeding time (Ruan et al. 2012; Wu et al. 2012; Gao et al. 2012; Sun et al. 2011). The response pattern was also found in vitro that the level of SelW in cultured L8 cells was regulated by the Se concentration in the medium (Yeh et al. 1997c; Gu et al. 2002). The sodium selenite medium enhanced the mRNA expression of SelW, the level of SelW mRNA increased when chicken myoblasts were treated with additional Se treatment, but deceased in high-Se group (Ruan et al. 2012; Wu et al. 2012). However, effect of Se concentration in the medium on the level of SelW in cultured chicken splenic lymphocyte remained unknown until now. In this study, it was showed that the SelW gene was highly inducible by Se concentration in the medium in cultured chicken splenic lymphocyte. SelW mRNA expression was significantly increased as dose-dependent in cells treated with 10-8, 10-7 or 10-6 mol/l of Se. When lymphocyte were pretreated with 10-8, 10-7 or 10-6 mol/l of Se before treated with H2O2, SelW mRNA expression

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Fig. 8 Effects of SelW siRNA on mRNA levels of Bcl-2, Bax, Bak-1, caspase-3 and p53 in H2O2-induced splenic lymphocyte and SelW mRNA levels in splenic lymphocyte. Bars represent mean ± standard deviation (n = 3/group). Bars without

sharing a common letter are significantly different (P \ 0.05). Bars with asterisks are statistically significantly different from control (***P \ 0.001)

was significantly increased compared with the 20 lM H2O2 control (Fig. 3g). It is well know that oxidative stress may result in cell apoptosis, and Se deficient might increase cell apoptosis induced by H2O2 in primary cultured pig thyrocytes (Demelash et al. 2004). The effect of selenium on cell apoptosis dependents its concentration (Cao et al. 2006). Generally, Se adequate could inhibit cell apoptosis induced (Zhou et al. 2009). Se excess could stimulate cell apoptosis and aggravate cell apoptosis induced by other inducer (Guan et al. 2009). In our investigation, as shown in Figs. 2 and 7a, the population of AO/EB-stained apoptosis cells was significantly increased in the presence of 20 lM H2O2,

compared with the control, approximately 40 % of the treated cells apoptosis. However, amount of apoptosis cells decreases with the increasing of Se concentration and up-regulating of SelW gene, when cells were incubated with 10-8, 10-7 or 10-6 mol/l of Se before treated with H2O2. Furthermore, we further tested the influence of endogenous SelW on H2O2-induced cell viability and cell apoptosis. SelW mRNA levels were downregulated by 56.5 % in SelW-depleted chicken splenic lymphocytes, suppression of SelW decreased lymphocyte viability, and promoted cells apoptosis induced by H2O2 (Figs. 6, 7b), which further demonstrated that the attenuation of SelW expression due to siRNA

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renders the lymphocyte more sensitive to H2O2, and suggested that SelW played an important role in protecting lymphocyte against H2O2-induced oxidative stress. Similar result was reported that suppression of SelW in the cultured SelW siRNA-transfected mouse embryonic neuronal cells promoted H2O2induced apoptosis cell death at a higher frequency than in the control cells (Chung et al. 2009). Overexpression of SelW markedly aggravated the viability of CHO cells and H1299 human lung cancer cell lines after treatment with H2O2 (Jeong et al. 2002). Studies also suggested that the expression of SelW was significant increased in chicken SelW transfected CHO-K1 cells (Han et al. 2012b), and overexpression of SelW resulted in a markedly decrease in sensitivity to H2O2-induced oxidative stress and had a lower apoptotic cell death in AO/EB and TUNEL assays, and had higher cell viability than wild-type cells (Han et al. 2012a). Depletion of SelW by RNA interference in the mouse skeletal muscle cell line C2C12 (C3H), the viability of the SelW siRNA-transfected group decreased significantly by 21.5 % compared with the blank control, and 34.6 % of SelW siRNA-transfected cells were apoptotic as compared with non-SelWdepleted cells (Wang et al. 2010). However, it is first time we observed that suppression of SelW gene aggravated chicken splenic lymphocyte apoptosis induced by H2O2. It has been reported that Se excess could aggravate cell apoptosis, and showed down-regulation for bcl-2 and up-regulation of bax and p53 in cultured cortical neurons (Xiao et al. 2006). Treatment of MCF-7 cells with a synthetic selenadiazole derivative resulted in rapid depletion of mitochondrial membrane potential and release of cytochrome c through up-regulation of Bax and down-regulation of Bcl-xl expression, and up-regulated the expression levels of total p53. Moreover, Silencing of p53 activation with RNA interference effectively blocked the induced caspase activation (Chen et al. 2009). It has been reported that human Bcl-2 can promote cell survival through protein–protein interactions with other Bcl-2 related family members, such as the death suppressors Bcl-xl, or the death agonists Bax, Bak, and with the increasing of apoptotic cell death, the pro-apoptotic activity of Bak-1 is broadly distributed (Hockenbery et al. 1993). It suggested that modulated bcl-2 expression is a determinant of life and death in normal mice lymphocytes (Strasser et al. 1991). Selenium could partly

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block the apoptosis of chondrocytes through increasing Bax mRNA expression, and decreasing the Bax/ Bcl-2 ratio (Chen et al. 2006). The knockout down of SelW up-regulated Bax and caspase-3 and downregulated Bcl-2 in chicken embryonic myoblasts mediated by exogenous H2O2 (Yao et al. 2013). Overexpression of SelW cells had a lower levels of caspase-3 and caspase-8 mRNA than wild-type cells (Han et al. 2012a). In the present study, we found that H2O2 induced a significantly up-regulation of the Bax/ Bcl-2 ratio and Bax, Bak-1, caspase-3 and p53,and down-regulation of Bcl-2. When pretreated lymphocyte with Se before treated with H2O2, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased (Fig. 3). Moreover, the Bax/ Bcl-2 ratio and mRNA expression level of Bax, Bak-1, caspase-3 and p53 were higher in siRNA groups (Fig. 8). The mechanisms underlying the actions of sodium selenite and SelW on these genes remain to be clarified further. The physiological function of SelW is not well understood until today. In previous studies, using primary cultured neuronal cells to an exogenous oxidant, they found that the viability of neuronal cells decreases as a function of increasing H2O2 concentration, and suggested that the role of SelW in the cellular defense against oxidative damage in neurons (Chung et al. 2009). It has reported that SelW protects the development of myoblasts from oxidative stress (Loflin et al. 2006). The function of SelW was also investigated by cloning the corresponding cDNA from mouse brain and expressing it in CHO cells and H1299 human lung cancer cells, and they found that overexpression of SelW markedly reduced the sensitivity of both cell lines to H2O2 cytotoxicity, and ameliorated the oxidative stress induced damage in C6 rat glial cells (Jeong et al. 2002). It also reported that SelW serves as an antioxidant in chicken myoblasts (Yao et al. 2013). These results suggest that SelW plays a crucial role in the cellular defense against oxidative stress. However, it is unknown whether SelW plays important roles in the immune function of birds. In the present study, we investigated the role of SelW in the cellular defense against oxidative damage in cultured chicken splenic lymphocyte. It was showed that the SelW gene mRNA expression was highly inducible by Se concentration in the medium in cultured chicken splenic lymphocyte. The number of AO/EB-positive cells could be alleviated obviously when cells were

Biometals

pretreated with sodium selenite before exposure to H2O2. Moreover, the suppression of SelW gene by siRNA could aggravate lymphocyte apoptosis, and the population of AO/EB-positive cells was about 88 % higher in SelW siRNA-transfected cells compared with the control cells. In both cases, we noticed the down-regulation in Bcl-2 expression and up-regulation in Bax, Bak-1, caspase-3 and p53 genes. Thus, the results of present study suggest that the siRNA-induced suppression of SelW gene in cultured chicken splenic lymphocyte resulted in an increase in sensitivity to H2O2-induced oxidative stress. Selenite supplementation significantly up-regulated SelW mRNA expression, resulted in an decrease in sensitivity to H2O2-induced oxidative stress, provides definitive evidence supporting the antioxidant function of SelW. Together, our data suggest the possibility that SelW may play a important role in protecting cells from oxidative stress-induced cell damage in splenic lymphocyte of birds. Acknowledgments This study was supported by the National Natural Science Foundation of China (30871902), the Science Foundation of the Education Department of Heilongjiang Province (11551030).

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