Biometals DOI 10.1007/s10534-014-9804-x
The role of selenoprotein W in inflammatory injury in chicken immune tissues and cultured splenic lymphocyte Dong Yu • Ziwei Zhang • Haidong Yao Shu Li • Shi-Wen Xu
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Received: 27 August 2014 / Accepted: 19 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Selenoprotein W (SelW) is mainly understood in terms of its antioxidant effects in the cellular defense system. Inflammation is an important indicator of animal tissue injury, and the inflammatory cells may trigger a sophisticated and well-orchestrated inflammatory cascade, resulting in exaggerated oxidative stress. To investigate the role of SelW in inflammatory injury in chicken immune tissues and cultured splenic lymphocyte, in this report, the effects of selenium (Se) on mRNA expressions of SelW and inflammatory factors (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) in the chicken immune organs (spleen, thymus and bursa of Fabricius) and cultured splenic lymphocyte treated with sodium selenite and H2O2, or knocked down SelW with small interfering RNAs (siRNAs) were examined. The results showed that Sedeficient diets effectively decreased the mRNA expression of SelW (P \ 0.05), and induced a significantly up-regulation of COX-2, iNOS, NF-jB, PTGEs and TNF-a mRNA levels (P \ 0.05). The histopathological analysis showed that immune tissues were obviously injured in the low-Se groups. In vitro, H2O2 induced a significantly up-regulation of the D. Yu Z. Zhang H. Yao S. Li S.-W. Xu (&) College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected]; [email protected] D. Yu College of Life and Technology, Harbin Normal University, Harbin 150025, People’s Republic of China
mRNA levels of inflammation-related genes (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) in cultured splenic lymphocyte (P \ 0.05). When lymphocytes were pretreated with Se before treated with H2O2, the inflammation-related genes were significantly decreased (P \ 0.05). Silencing of SelW significantly up-regulated the inflammation-related genes (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) in cultured splenic lymphocyte (P \ 0.05). The results suggested that the expression levels of inflammatory factors (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) and SelW can be influenced by Se in birds. SelW commonly played an important role in the protection of immune organs of birds from inflammatory injury by the regulations of inflammation-related genes. Keywords Selenoprotein W Small interfering RNAs Inflammatory injury Splenic lymphocyte Chicken
Introduction Selenium (Se) is recognized as an essential nutritional trace element for organisms with important roles in many aspects of health. For example, oxidant defense (Rayman 2000), muscle metabolism (Terry et al. 2009), reproduction (Kaur and Bansal 2005), and other aspects of health (Li et al. 2010a; Schweizer et al. 2004; Martin-Romero et al. 2001), including immune
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system (Rayman 2000; McKenzie et al. 1998; Hoffmann and Berry 2008). In poultry, it has been reported that Se can regulate the immune status (Leng et al. 2003). Se-deficient diets were found to inhibit the function of immune organs (Arthur et al. 2003), the growth of bursal and thymic in chicken (Marsh et al. 1986). Se exerts its biological functions primarily through selenoproteins at their active site (Stadtman 2000). Se is incorporated into selenoproteins mainly in the form of selenocysteine (Sec) (Saito and Takahashi 2000). Approximately 30 selenoprotein families have been identified (Kryukov et al. 2003). Many of the found selenoprotein are obviously essential for normal growth, development, and metabolism of an organism (Gladyshev and Hatfield 1999; Stadtman 2002). The functions of selenoproteins are uncharacterized in the immune system, except with regards to inflammation. Selenoproteins affect inflammatory responses by regulating the oxidative state of immune cells (McCord 2000). However, little is known about the selenoproteins in birds. SelW is a small selenoprotein in that it shares the redox motif and binds glutathione (Beilstein et al. 1996), and widely present in the tissues of animals. The expression of SelW is influenced by Se status and intake. It has been shown that selenium-supplemented diets lead to an increase in the expression of SelW, and selenium-deficient diets lead to a decrease in the expression of SelW in spleen in rats and sheep (Yeh et al. 1997a, b). SelW is mainly understood in terms of its antioxidant effects in the cellular defense system (Beilstein et al. 1996; Jeong et al. 2002; Wang et al. 2010). In poultry, although the amino acid sequence of SelW (Li et al. 2011), and its expression in the gastrointestinal system (Li et al. 2010b; Gao et al. 2012), liver (Sun et al. 2011), pancreas tissue (Wang et al. 2011), muscle tissues (Zhang et al. 2012; Ruan et al. 2012), embryos neurons (Li et al. 2012), immune organs (Yu et al. 2011), and antioxidant function in myoblasts and splenic lymphocyte have been reported (Yao et al. 2013; Yu et al. 2014), little information is available on avian SelW. Inflammation is an important indicator of animal tissue injury caused by noxious physical or chemical stimuli, and is a key component of multiple pathologies (Rankin 2004). Inflammation is marked by the accumulation of a variety of inflammatory cells, and the inflammatory cells may trigger a sophisticated and well-orchestrated inflammatory cascade, resulting in
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exaggerated oxidative stress, activation of the nuclear factor-kappa B (NF-jB), production of several cytokines and overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), leading, in turn, to the release of prostaglandins and nitrates (Sciarra et al. 2007). Probably the most pivotal enzymes involve in maintaining inflammation are the inducible enzymes: iNOS and COX-2. iNOS can produce nitric oxides (NOs). NOs are well known proinflammatory mediators in the pathogenesis of inflammation, and have been implicated in the pathogenesis of many inflammatory diseases (Moncada et al. 1991). COX-2 is induced by several pro-inflammatory stimuli, such as, growth factors, cytokines and endotoxin. prostaglandin E2 (PGE2) is another important mediator of inflammation, and is synthesized by COX-2 at inflammatory sites, where it contributes to local blood flow increases, edema formation, and pain sensitization (Prescott and Fitzpatrick 2000; Hinz and Brune 2002). NF-jB is one of most important transcription factors in the inflammatory process. NF-jB represents a ubiquitously expressed family of transcription factors that participate in the regulation of diverse biological processes, including immune, inflammatory and apoptotic responses (Baud and Karin 2009). It controls the expressions of numerous genes, such as, iNOS, COX-2, tumor necrosis factor-a (TNF-a) (Baud and Karin 2009). Several studies have investigated the relationship between Se status and inflammatoryrelated genes expression. It has been reported that NFjB, COX-2 and PGE2 expression levels in Sedeficient groups are higher than Se-supplemented groups in RAW 264.7 (Zamamiri-Davis et al. 2002). Selenium supplementation resulted in the decreased mRNA and protein levels of iNOS (Kim et al. 2004), and significantly decreased the elevated expression of TNF-a in the blood lymphocytes (Chang et al. 2005). Selenite treatment to diabetic mice significantly increased pancreatic selenium content, reduce the mRNA expressions of proinflammatory cytokines TNF-a and attenuate iNOS-mediated NO production (Zeng et al. 2009). However, the precise mechanisms by which selenium and SelW expression regulate inflammatory responses in immune organs of birds are not fully understood. Herein, we first investigated the effect of Sedeficient on SelW and inflammatory-related genes (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) mRNA expression in chicken immune organs (spleen, thymus
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and bursa of Fabricius); second, we investigated the effect of Se-supplementation and SelW gene silence on inflammatory-related genes (iNOS, COX-2, NFjB, PTGEs, and TNF-a) mRNA expression in cultured splenic lymphocyte. Additionally, we investigated the protective role of SelW on inflammation induced by H2O2-mediated oxidative stress in cultured cells, and induced by Se-deficient in chicken immune organs.
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).
phosphate buffer with 0.85 % NaCl, pH 7.2). Single cell suspension was 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 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.
siRNA preparation and transfection Birds and diets All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. One hundred and fifty chickens (1-day-old; Weiwei Co. Ltd., Harbin, China) were randomly allocated to two groups. Low-Se group (L-Se group) was fed the Sedeficient basal diet (basal diet from Se deficiency region of Heilongjiang Province in China, containing 0.033 mg/kg Se); Control-Se group (C-Se group) was fed the medium-Se diet (diet supplemented with Se as sodium selenite, the final Se content in this groups was 0.15 mg/kg) for 55 days. The immune tissues of chicken were taken at 15, 25, 35, 45 and 55 days old. During the experiment, animals were anesthetized with sodium pentobarbital. The tissues were quickly removed, frozen immediately in liquid nitrogen and stored at -80 °C until required for RNA isolation. Cell culture and treatments Spleens were dissected from Isa brown cocks (60 days old) and were collected aseptically and placed in sterile phosphate-buffered saline (PBS, 0.1 M
The siRNA corresponding to the SelW gene was designed and synthesized by Invitrogen (Stealth RNAi). The sequence for the SelW siRNA was 50 CGGCUUCGUG GACACCGACGCCAAA-30 . A random siRNA sequence (sense 50 -CGGUCGUGGACACCGACGCCCUAAA-30 ; antisense 50 -UUUAG GGCGUCG GUGUCCACGACCG-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.
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Real-time PCR analysis
Statistical analysis
Total RNA were isolated from cells and tissue samples using Trizol reagent according to the manufacturer’s instructions (Invitrogen, China). The dried RNA pellets were resuspended in 50 ll of diethyl-pyrocarbonate-treated 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. To design primers, Primer Premier Software (PREMIER Biosoft International, USA) was used to design specific primers for iNOS, COX-2, NF-jB, PTGEs, TNF-a, SelW and b-actin based on known chicken sequences (Zhao et al. 2013). General PCRs were first performed to confirm the specificity of the primers. 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 50 9 ROX reference Dye II and 6.8 lL of PCRgrade 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 of all data was performed by using SPSS for Windows (version 13, SPSS Inc., Chicago, IL, USA). When a significant value (P \ 0.05) was obtained by oneway ANOVA, further analysis was carried out. 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, and not significant P [ 0.05.
Histopathologic examination After necropsy, tissue specimens of heart were fixed in 4 % buffered formaldehyde and routinely processed in paraffin. Thin sections (5 lm) of each tissue were sliced from each block and mounted on glass. Slides were stained with hematoxylin and eosin (H&E). Histological slides were examined under an Olympus light microscope.
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Results Histological analysis of chicken spleen, thymus and bursa of Fabricius Histological analysis, done by light microscopy, revealed that spleen tissues from the control group showed normal histological structures with regular splenic trabecula and the splenic corpuscle, and the cortical and medullar structure is clear (Fig. 1a). Spleen tissues from chickens treated with Se-deficient diets (containing 0.033 mg/kg Se) for 55 days showed that the number of lymphocytes were reduced in the red pulp, and vacuolar degeneration, mesenchymal rarefaction, and infiltration of inflammatory occured (Fig. 1b). Analysis of thymus sections demonstrated that Sedeficiency induces constriction of the thymus cortex with scant cell numbers, and an unregular morphology of lobules of thymus (Fig. 1d). In the case of Sedeficiency, the boundary between cortical and medullar structure is not clear, and vacuolation and necrosis occurred (Fig. 1d). In contrast, the thymus injury was markedly attenuated in control group chickens fed with medium-Se diets (containing 0.15 mg/kg Se) for 55 days (Fig. 1c). The bursa of Fabricius from the control group revealed that the bursae contained follicles, and the bursal follicles arranged closely, distinguished by their size and the apparent cortical and medullar structure. In the lymphocytic follicles the enlarged density of lymphocytics was evident (Fig. 1e). Compared with the control group, within the bursal plicae, the bursal follicles arranged loosely, and the number of
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Fig. 1 Hematoxylin and eosin (H&E) stained immune tissue sections of chicken (9400). a Spleen of chicken from control group, b spleen of chicken from low-Se group, c thymus of
chicken from control group, d thymus of chicken from low-Se group, e bursa of Fabricius of chicken from low-Se group, and f bursa of Fabricius of chicken from low-Se group
lymphocytes were reduced in the lymphocytic follicles, and the boundary between cortical and medullar structure is not clear in chickens treated with Sedeficient diets (containing 0.033 mg/kg Se) for 55 days (Fig. 1f).
effects of Se deficient on the mRNA levels of SelW and the corresponding inflammation genes (Bcl-2, Bax, Bak-1, caspase-3 and p53) mRNA expression in spleen were examined. As Fig. 2 shows, the iNOS, COX-2, NF-jB, PTGEs, TNF-a mRNA levels of spleen tissues were significantly increased in low-Se groups compared to control groups from 15 to 55 days (Fig. 2a–e) (P \ 0.05). In contrast, the expression of SelW mRNA was significantly decreased from 15 to 55 days (P \ 0.05). These results suggest that Se deficiency-induced chicken spleen injury possibly via downregulating Selw gene and upregulating inflammation genes expression.
Effects of Se deficient on the mRNA levels of iNOS, COX-2, NF-jB, PTGEs, TNF-a and SelW in spleen of chickens To investigate the protective role of SelW in inflammation induced by Se deficiency in spleen of chickens,
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Biometals Fig. 2 The effects of Se deficient on the mRNA levels of iNOS, COX-2, NFjB, PTGEs, TNF-a and SelW in spleen of chickens. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between L-Se group and C-Se group (*P \ 0.05)
Effects of Se deficient on the mRNA levels of iNOS, COX-2, NF-jB, PTGEs, TNF-a and SelW in thymus of chickens To determine whether or not the protective role of SelW in inflammation induced by Se deficiency in thymus of chickens is exerted by the mechanisms of downregulating inflammation genes expression, effects of Se deficient on the mRNA levels of the corresponding inflammation genes in thymus of chickens were examined. As Fig. 3 shows, the SelW mRNA levels of spleen tissues were significantly decreased in low-Se groups compared to control groups from 15 to 55 days (Fig. 3a–e) (P \ 0.05). In contrast, the expression of iNOS, COX-2, NF-jB, PTGEs, TNF-a genes in thymuses of chickens during the different days of Se deficient were significantly increased from 15 to 55 days (P \ 0.05). These observations suggest that inflammation injury induced by Se deficiency in thymus of chickens are at least in part mediated by augmen-
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tation of inflammation genes levels, and decreasing SelW expression. Effects of Se deficient on the mRNA levels of iNOS, COX-2, NF-jB, PTGEs, TNF-a and SelW in bursa of Fabricius of chickens The effects of Se deficient on the mRNA levels of the corresponding inflammation genes in bursa of Fabricius are shown in Fig. 4. The iNOS, COX-2, NF-jB, PTGEs, TNF-a mRNA levels of spleen tissues were significantly increased in low-Se groups compared to control groups from 15 to 55 days (Fig. 4a–e) (P \ 0.05). In order to determine the effects of SelW on the expression of iNOS, COX-2, NF-jB, PTGEs, TNF-a genes in bursa of Fabricius during the different days of Se deficient, the mRNA expression level of SelW genes were examined by qPCR. From Fig. 4f, qPCR results revealed that the levels of SelW mRNA were significantly decreased from 15 to 55 days (P \ 0.05).
Biometals Fig. 3 The effects of Se deficient on the mRNA levels of iNOS, COX-2, NFjB, PTGEs, TNF-a and SelW in thymus of chickens. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between L-Se group and C-Se group (*P \ 0.05)
Effects of Se on mRNA levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a in H2O2-induced lymphocyte
Effects of SelW siRNA on iNOS, COX-2, NF-jB, PTGEs and TNF-a mRNA in H2O2-induced lymphocyte
To investigate whether iNOS, COX-2, NF-jB, PTGEs and TNF-a mRNA expression were regulated by Se. Chicken splenic lymphocyte were treated with 10-8, 10-7 or 10-6 mol/L of Se for 6 h, and were then treated for 24 h in the absence and presence of 20 lM H2O2. As shown in Fig. 5, the lymphocytes with H2O2 (20 lM) resulting in a significantly increase mRNA expression of iNOS, COX-2, PTGEs and TNF-a (P \ 0.05) (Fig. 5a, b, d, e), and decrease mRNA expression of NF-jB (P \ 0.05) (Fig. 5c), 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 mRNA expression of iNOS, COX-2, NF-jB, PTGEs and TNF-a were significantly decreased (P \ 0.05), compared with the 20 lM H2O2 control, and significantly dose-dependent increased compared with the 10-8, 10-7 or 10-6 mol/L of Se group respectively (P \ 0.05).
As shown in Fig. 6, there were higher mRNA expression levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a in siRNA groups when compared with the corresponding RNAi Negative Control groups (P \ 0.05). With the increasing of transfected time, the mRNA levels of these inflammation-related genes showed an increased trend.
Discussion Se is essential for normal biological functions. The main biological function of Se is known to serve an antioxidant cativity and this is achieved by selenocysteine in the enzymatic active site, such as GPx. There have been reported that Se deficiency (0.02 ppm for 8 wk) in in vivo or absence of 10-7 M Se in in vitro results in a significant retrogression of the proliferative
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Biometals Fig. 4 The effects of Se deficient on the mRNA levels of iNOS, COX-2, NFjB, PTGEs, TNF-a and SelW in bursa of Fabricius of chickens. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between L-Se group and C-Se group (*P \ 0.05)
responses of spleen lymphocytes from C57B1/6J mice in response to stimulation with mitogen or antigen, supplementation with Se had the opposite effect (Kiremidjian-Schumacher et al. 1992). In poultry, numerous lines of previous evidence suggested that Se deficiency reduced growth and resulted in exudative diathesis and reduced the growth of lymphoid organs (Bartholomew et al. 1998), and inhibited the growth of the immune organs and reduced immune function (Marsh et al. 1986). Supplementation of Se leads to enhanced comparative metabolic and immune response of chickens, and had protective effect in cancer of the colon (Leng et al. 2003). However, Excess dietary selenium induced the decrease of relative weight, lesions, and apoptosis of bursa of Fabricius chickens (Peng et al. 2009). This result was similar to the effect of Se on cells viability of human cancer cells (Rigobello et al. 2009), NB4 cells (Cao et al. 2012), rat vascular smooth muscle cells and human prostate cancer cells (Zhong and Oberley 2001; Tang and Huang 2004) in in vitro. In the present study, compared with the control group, Se-deficient diets results in a significant disordered histological structures, and the number of lymphocytics were reduced in the lymphocytic follicles, and vacuolar degeneration, mesenchymal rarefaction, and infiltration of inflammatory occurred. These data suggest that Se-deficient
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caused inflammatory damage to immune organs of chickens. In previous studies, they reported that the presence of SelW gene was detected in various organs of mammals. Intensive researches have revealed that the expression level of SelW gene is generally sensitive to Se status in rats and sheep fed dietary supplementation with Se (Yeh et al. 1995, 1997b, 1998; Sun et al. 1998). In poultry, they have reported that SelW was expressed widely in chicken tissues, and 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. 2010b). The SelW mRNA levels were found to increase in a dosedependent manner with dietary supplementation with Se in the gastrointestinal tract tissue, liver, pancreas and skeletal muscles of chickens (Sun et al. 2011; Wang et al. 2011; Ruan et al. 2012; Zhang et al. 2012; Gao et al. 2012; Wu et al. 2012).The similar response pattern was also found in vitro that the level of SelW was regulated by the Se concentration in the medium in cultured L8 cells (Gu et al. 2002; Yeh et al. 1997c). The mRNA expression of SelW was increased when the cultured chicken myoblasts were treated with additional Se treatment, but deceased in high-Se group (Ruan et al. 2012; Wu et al. 2012). In addition, our
Biometals Fig. 5 Effect of Se on mRNA levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a 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)
previous studies have shown that the SelW gene was also distributed widely in the immune organs of chickens fed the basal commercial diets and the Sesupplemented diets, and SelW mRNA expression is generally sensitive to Se dietary supplementation in those organs (Yu et al. 2011). Moreover, 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 was significantly increased compared with the 20 lM H2O2 control (Yu et al. 2014). However, effect of Se-deficient diets on the expression of SelW gene in the avian immune organs remains unknown until now. In this study, it was showed that the SelW gene was highly sensitive to Se-deficient in chicken immune organs, and SelW mRNA expression was significantly decreased as time-dependent in chickens fed Se-deficient diets (Figs. 2f, 3f, 4f). It is well know that Se deficient might decrease cell viability induced by H2O2 in primary cultured pig
thyrocytes (Demelash et al. 2004). In our previous investigation have shown that the cells were more sensitive to the oxidative stress induced by H2O2, and the sensitivity decreases with the increasing of Se concentration and up-regulating of SelW gene, when cells were incubated with Se before treated with H2O2 (Yu et al. 2014). Furthermore, suppression of SelW decreased lymphocyte viability, and promoted the sensitivity of lymphocyte induced by H2O2 (Yu et al. 2014). Similar results were reported that the attenuation of SelW expression due to siRNA renders the cells more sensitive to H2O2 in the cultured SelW siRNA-transfected mouse embryonic neuronal cells (Chung et al. 2009) and skeletal muscle cells (Wang et al. 2010). Overexpression of SelW markedly aggravated the viability of CHO cells and H1299 human lung cancer cell lines (Jeong et al. 2002), and CHO-K1 cells (Han et al. 2012), after treatment with H2O2. However, no report has been issued on the mechanism underlying its anti-inflammatory effects in chicken lymphocyte and immune organs. In order to further detect Se deficient induced tissues damage and host defenses such as inflammatory reactions, the mRNA expression levels of NF-jB,
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Biometals Fig. 6 Effects of SelW siRNA on mRNA levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a in H2O2induced splenic lymphocyte. Bars represent mean ± standard deviation (n = 3/group). Bars without sharing a common letter are significantly different (P \ 0.05)
COX-2, iNOS, TNF-a, and PTGEs were examined by qPCR. Furthermore, we further tested the influence of endogenous SelW on the expression levels of those inflammatory factors. In the nucleus, NF-jB induces the transcription of a large variety of target genes by binding to the cis-acting jB element. Those target genes involve normally encode cytokines (Kiemer et al. 2003), cell adhesion molecules, and inflammatory enzymes including COX-2 (Abate et al. 1998) and iNOS (Lee et al. 2000). COX-2 inhibitors reduce mucosal damage, which further indicates the role of prostaglandins in tissue damage and inflammation reactions (Zheng et al. 2012). PTGES catalyzes the isomerization of PGH2 to PGE2, and the latter plays an important role in inflammation (Liu et al. 2012), cell growth, and transformation (Sasaki et al. 2012). It has been reported that the NF-jB activation induced by monocyte-conditioned medium and TNFa were inhibited by Se at the physiological levels, and the maximum activation of NF-jB was induced at half the level of the serum Se in healthy subjects in cultured Human hepatoma cell line HuH-7 (Maehira et al. 2003). Mice fed a Se-deficient diet showed higher NOS activity levels either at basal or after isoproterenol stimulation, and then were suppressed by feeding Se-deficient diet mice with a Se-supplemented
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diet for 1 wk or by inhibition of iNOS (Natalia et al. 2007). Studies indicated that the expression of both p65 and p50 genes(components of NF-jB), and the iNOS levels as well as NO levels increased in Se deficient group mice testis while the expression of the inhibitory IjBa declined significantly. This result clearly demonstrates both enhanced NO levels and NF-jB are harmful in the progression of normal spermatogenic cycle (Sonia and Bansal 2007).It also reported that pancreatic mRNA expressions of proinflammatory cytokines TNF-a, and activity of iNOS and content of nitric oxide were significantly increased in untreated diabetes mellitus mice, and they were decreased in selenite-treated diabetes mellitus mice (Zeng et al. 2009). Studies found that low doses of selenium may protect the prostate from prostatitisinduced cancer by inhibiting NF-jB and the subsequent production of the immunosuppressive cytokine TGFb1 and pro-inflammatory factor IL-6 (Pei et al. 2010). It reported that COX-2 protein expression and PGE2 levels were higher in Se-deficient RAW 264.7 macrophages treated with lipopolysaccharide, and the NF-jB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2 (Zamamiri-Davis et al. 2002). Similar studies reported that lipopolysaccharide stimulation
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produced a proinflammatory phenotype in rat peritoneal macrophages. Se caused a greater inhibitory effect on the expression of COX-2 and iNOS, and a higher efficacy in reducing the loss of IjB-a, the increased NF-jB binding activity, the enhanced mRNA levels of TNF-a and nitrite content. Moreover, Se in vivo caused a greater inhibitory effect on prostate inflammation induced in rats by partial bladder outlet obstruction (Bonvissuto et al. 2011). In the current study, we found that the iNOS, COX-2, NF-jB, PTGEs and TNF-a mRNA levels of immune tissue were significantly increased in Low-Se groups. In vitro, H2O2 induced a significantly up-regulation of the iNOS, COX-2, NF-jB, PTGEs and TNF-a. When lymphocyte were pretreated with Se before treated with H2O2, those genes were significantly decreased (Fig. 5). Moreover, mRNA expression level of iNOS, COX-2, NF-jB, PTGEs and TNF-a were higher in siRNA groups (Fig. 6). So, in our study, the increased inflammation factors suggested that inflammatory response was induced by Se deficient and the attenuation of SelW expression, which may lead to chicken immune tissues and cultured splenic lymphocyte injury. The mechanisms underlying the actions of sodium selenite and SelW on these genes remain to be clarified further. Thus, the results of present study suggest that the antioxidant defense system could be damaged in immune organs (spleen, thymus and bursa of Fabricius) of chickens by Se deficient, and resulted in inflammatory injury. The increase in mRNA expression of inflammation factors further suggested that Se deficient caused damage to immune tissue of the chicken. siRNAinduced suppression of SelW gene in cultured chicken splenic lymphocyte resulted in an increase in mRNA expression of inflammation factors further to H2O2induced oxidative stress. Selenite supplementation significantly up-regulated SelW mRNA expression, resulted in an decrease in mRNA expression of inflammation factors to H2O2-induced oxidative stress, provides definitive evidence supporting the antioxidant function of SelW. Together, our data suggest the possibility that SelW may play an important role in the protection of immune organs of birds from inflammatory injury by the regulations of inflammation-related genes. However, so far, the specific SelW-activated induction and regulation mechanisms among inflammation-related genes are unclear, and further studies are needed.
Acknowledgments This study was supported by the International Cooperation and Exchanges NSFC (31320103920), the National Natural Science Foundation of China (31272626), the Doctoral Fund of Ministry of Education of China (20122325110018), the Doctoral Fund of Harbin Normal University of China (220601085), the Study Abroad Foundation of Heilongjiang Province (LC201031).
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The role of selenoprotein W in inflammatory injury in chicken immune tissues and cultured splenic lymphocyte Dong Yu • Ziwei Zhang • Haidong Yao Shu Li • Shi-Wen Xu
•
Received: 27 August 2014 / Accepted: 19 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Selenoprotein W (SelW) is mainly understood in terms of its antioxidant effects in the cellular defense system. Inflammation is an important indicator of animal tissue injury, and the inflammatory cells may trigger a sophisticated and well-orchestrated inflammatory cascade, resulting in exaggerated oxidative stress. To investigate the role of SelW in inflammatory injury in chicken immune tissues and cultured splenic lymphocyte, in this report, the effects of selenium (Se) on mRNA expressions of SelW and inflammatory factors (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) in the chicken immune organs (spleen, thymus and bursa of Fabricius) and cultured splenic lymphocyte treated with sodium selenite and H2O2, or knocked down SelW with small interfering RNAs (siRNAs) were examined. The results showed that Sedeficient diets effectively decreased the mRNA expression of SelW (P \ 0.05), and induced a significantly up-regulation of COX-2, iNOS, NF-jB, PTGEs and TNF-a mRNA levels (P \ 0.05). The histopathological analysis showed that immune tissues were obviously injured in the low-Se groups. In vitro, H2O2 induced a significantly up-regulation of the D. Yu Z. Zhang H. Yao S. Li S.-W. Xu (&) College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected]; [email protected] D. Yu College of Life and Technology, Harbin Normal University, Harbin 150025, People’s Republic of China
mRNA levels of inflammation-related genes (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) in cultured splenic lymphocyte (P \ 0.05). When lymphocytes were pretreated with Se before treated with H2O2, the inflammation-related genes were significantly decreased (P \ 0.05). Silencing of SelW significantly up-regulated the inflammation-related genes (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) in cultured splenic lymphocyte (P \ 0.05). The results suggested that the expression levels of inflammatory factors (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) and SelW can be influenced by Se in birds. SelW commonly played an important role in the protection of immune organs of birds from inflammatory injury by the regulations of inflammation-related genes. Keywords Selenoprotein W Small interfering RNAs Inflammatory injury Splenic lymphocyte Chicken
Introduction Selenium (Se) is recognized as an essential nutritional trace element for organisms with important roles in many aspects of health. For example, oxidant defense (Rayman 2000), muscle metabolism (Terry et al. 2009), reproduction (Kaur and Bansal 2005), and other aspects of health (Li et al. 2010a; Schweizer et al. 2004; Martin-Romero et al. 2001), including immune
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system (Rayman 2000; McKenzie et al. 1998; Hoffmann and Berry 2008). In poultry, it has been reported that Se can regulate the immune status (Leng et al. 2003). Se-deficient diets were found to inhibit the function of immune organs (Arthur et al. 2003), the growth of bursal and thymic in chicken (Marsh et al. 1986). Se exerts its biological functions primarily through selenoproteins at their active site (Stadtman 2000). Se is incorporated into selenoproteins mainly in the form of selenocysteine (Sec) (Saito and Takahashi 2000). Approximately 30 selenoprotein families have been identified (Kryukov et al. 2003). Many of the found selenoprotein are obviously essential for normal growth, development, and metabolism of an organism (Gladyshev and Hatfield 1999; Stadtman 2002). The functions of selenoproteins are uncharacterized in the immune system, except with regards to inflammation. Selenoproteins affect inflammatory responses by regulating the oxidative state of immune cells (McCord 2000). However, little is known about the selenoproteins in birds. SelW is a small selenoprotein in that it shares the redox motif and binds glutathione (Beilstein et al. 1996), and widely present in the tissues of animals. The expression of SelW is influenced by Se status and intake. It has been shown that selenium-supplemented diets lead to an increase in the expression of SelW, and selenium-deficient diets lead to a decrease in the expression of SelW in spleen in rats and sheep (Yeh et al. 1997a, b). SelW is mainly understood in terms of its antioxidant effects in the cellular defense system (Beilstein et al. 1996; Jeong et al. 2002; Wang et al. 2010). In poultry, although the amino acid sequence of SelW (Li et al. 2011), and its expression in the gastrointestinal system (Li et al. 2010b; Gao et al. 2012), liver (Sun et al. 2011), pancreas tissue (Wang et al. 2011), muscle tissues (Zhang et al. 2012; Ruan et al. 2012), embryos neurons (Li et al. 2012), immune organs (Yu et al. 2011), and antioxidant function in myoblasts and splenic lymphocyte have been reported (Yao et al. 2013; Yu et al. 2014), little information is available on avian SelW. Inflammation is an important indicator of animal tissue injury caused by noxious physical or chemical stimuli, and is a key component of multiple pathologies (Rankin 2004). Inflammation is marked by the accumulation of a variety of inflammatory cells, and the inflammatory cells may trigger a sophisticated and well-orchestrated inflammatory cascade, resulting in
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exaggerated oxidative stress, activation of the nuclear factor-kappa B (NF-jB), production of several cytokines and overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), leading, in turn, to the release of prostaglandins and nitrates (Sciarra et al. 2007). Probably the most pivotal enzymes involve in maintaining inflammation are the inducible enzymes: iNOS and COX-2. iNOS can produce nitric oxides (NOs). NOs are well known proinflammatory mediators in the pathogenesis of inflammation, and have been implicated in the pathogenesis of many inflammatory diseases (Moncada et al. 1991). COX-2 is induced by several pro-inflammatory stimuli, such as, growth factors, cytokines and endotoxin. prostaglandin E2 (PGE2) is another important mediator of inflammation, and is synthesized by COX-2 at inflammatory sites, where it contributes to local blood flow increases, edema formation, and pain sensitization (Prescott and Fitzpatrick 2000; Hinz and Brune 2002). NF-jB is one of most important transcription factors in the inflammatory process. NF-jB represents a ubiquitously expressed family of transcription factors that participate in the regulation of diverse biological processes, including immune, inflammatory and apoptotic responses (Baud and Karin 2009). It controls the expressions of numerous genes, such as, iNOS, COX-2, tumor necrosis factor-a (TNF-a) (Baud and Karin 2009). Several studies have investigated the relationship between Se status and inflammatoryrelated genes expression. It has been reported that NFjB, COX-2 and PGE2 expression levels in Sedeficient groups are higher than Se-supplemented groups in RAW 264.7 (Zamamiri-Davis et al. 2002). Selenium supplementation resulted in the decreased mRNA and protein levels of iNOS (Kim et al. 2004), and significantly decreased the elevated expression of TNF-a in the blood lymphocytes (Chang et al. 2005). Selenite treatment to diabetic mice significantly increased pancreatic selenium content, reduce the mRNA expressions of proinflammatory cytokines TNF-a and attenuate iNOS-mediated NO production (Zeng et al. 2009). However, the precise mechanisms by which selenium and SelW expression regulate inflammatory responses in immune organs of birds are not fully understood. Herein, we first investigated the effect of Sedeficient on SelW and inflammatory-related genes (iNOS, COX-2, NF-jB, PTGEs, and TNF-a) mRNA expression in chicken immune organs (spleen, thymus
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and bursa of Fabricius); second, we investigated the effect of Se-supplementation and SelW gene silence on inflammatory-related genes (iNOS, COX-2, NFjB, PTGEs, and TNF-a) mRNA expression in cultured splenic lymphocyte. Additionally, we investigated the protective role of SelW on inflammation induced by H2O2-mediated oxidative stress in cultured cells, and induced by Se-deficient in chicken immune organs.
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).
phosphate buffer with 0.85 % NaCl, pH 7.2). Single cell suspension was 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 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.
siRNA preparation and transfection Birds and diets All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. One hundred and fifty chickens (1-day-old; Weiwei Co. Ltd., Harbin, China) were randomly allocated to two groups. Low-Se group (L-Se group) was fed the Sedeficient basal diet (basal diet from Se deficiency region of Heilongjiang Province in China, containing 0.033 mg/kg Se); Control-Se group (C-Se group) was fed the medium-Se diet (diet supplemented with Se as sodium selenite, the final Se content in this groups was 0.15 mg/kg) for 55 days. The immune tissues of chicken were taken at 15, 25, 35, 45 and 55 days old. During the experiment, animals were anesthetized with sodium pentobarbital. The tissues were quickly removed, frozen immediately in liquid nitrogen and stored at -80 °C until required for RNA isolation. Cell culture and treatments Spleens were dissected from Isa brown cocks (60 days old) and were collected aseptically and placed in sterile phosphate-buffered saline (PBS, 0.1 M
The siRNA corresponding to the SelW gene was designed and synthesized by Invitrogen (Stealth RNAi). The sequence for the SelW siRNA was 50 CGGCUUCGUG GACACCGACGCCAAA-30 . A random siRNA sequence (sense 50 -CGGUCGUGGACACCGACGCCCUAAA-30 ; antisense 50 -UUUAG GGCGUCG GUGUCCACGACCG-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.
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Real-time PCR analysis
Statistical analysis
Total RNA were isolated from cells and tissue samples using Trizol reagent according to the manufacturer’s instructions (Invitrogen, China). The dried RNA pellets were resuspended in 50 ll of diethyl-pyrocarbonate-treated 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. To design primers, Primer Premier Software (PREMIER Biosoft International, USA) was used to design specific primers for iNOS, COX-2, NF-jB, PTGEs, TNF-a, SelW and b-actin based on known chicken sequences (Zhao et al. 2013). General PCRs were first performed to confirm the specificity of the primers. 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 50 9 ROX reference Dye II and 6.8 lL of PCRgrade 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 of all data was performed by using SPSS for Windows (version 13, SPSS Inc., Chicago, IL, USA). When a significant value (P \ 0.05) was obtained by oneway ANOVA, further analysis was carried out. 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, and not significant P [ 0.05.
Histopathologic examination After necropsy, tissue specimens of heart were fixed in 4 % buffered formaldehyde and routinely processed in paraffin. Thin sections (5 lm) of each tissue were sliced from each block and mounted on glass. Slides were stained with hematoxylin and eosin (H&E). Histological slides were examined under an Olympus light microscope.
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Results Histological analysis of chicken spleen, thymus and bursa of Fabricius Histological analysis, done by light microscopy, revealed that spleen tissues from the control group showed normal histological structures with regular splenic trabecula and the splenic corpuscle, and the cortical and medullar structure is clear (Fig. 1a). Spleen tissues from chickens treated with Se-deficient diets (containing 0.033 mg/kg Se) for 55 days showed that the number of lymphocytes were reduced in the red pulp, and vacuolar degeneration, mesenchymal rarefaction, and infiltration of inflammatory occured (Fig. 1b). Analysis of thymus sections demonstrated that Sedeficiency induces constriction of the thymus cortex with scant cell numbers, and an unregular morphology of lobules of thymus (Fig. 1d). In the case of Sedeficiency, the boundary between cortical and medullar structure is not clear, and vacuolation and necrosis occurred (Fig. 1d). In contrast, the thymus injury was markedly attenuated in control group chickens fed with medium-Se diets (containing 0.15 mg/kg Se) for 55 days (Fig. 1c). The bursa of Fabricius from the control group revealed that the bursae contained follicles, and the bursal follicles arranged closely, distinguished by their size and the apparent cortical and medullar structure. In the lymphocytic follicles the enlarged density of lymphocytics was evident (Fig. 1e). Compared with the control group, within the bursal plicae, the bursal follicles arranged loosely, and the number of
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Fig. 1 Hematoxylin and eosin (H&E) stained immune tissue sections of chicken (9400). a Spleen of chicken from control group, b spleen of chicken from low-Se group, c thymus of
chicken from control group, d thymus of chicken from low-Se group, e bursa of Fabricius of chicken from low-Se group, and f bursa of Fabricius of chicken from low-Se group
lymphocytes were reduced in the lymphocytic follicles, and the boundary between cortical and medullar structure is not clear in chickens treated with Sedeficient diets (containing 0.033 mg/kg Se) for 55 days (Fig. 1f).
effects of Se deficient on the mRNA levels of SelW and the corresponding inflammation genes (Bcl-2, Bax, Bak-1, caspase-3 and p53) mRNA expression in spleen were examined. As Fig. 2 shows, the iNOS, COX-2, NF-jB, PTGEs, TNF-a mRNA levels of spleen tissues were significantly increased in low-Se groups compared to control groups from 15 to 55 days (Fig. 2a–e) (P \ 0.05). In contrast, the expression of SelW mRNA was significantly decreased from 15 to 55 days (P \ 0.05). These results suggest that Se deficiency-induced chicken spleen injury possibly via downregulating Selw gene and upregulating inflammation genes expression.
Effects of Se deficient on the mRNA levels of iNOS, COX-2, NF-jB, PTGEs, TNF-a and SelW in spleen of chickens To investigate the protective role of SelW in inflammation induced by Se deficiency in spleen of chickens,
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Biometals Fig. 2 The effects of Se deficient on the mRNA levels of iNOS, COX-2, NFjB, PTGEs, TNF-a and SelW in spleen of chickens. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between L-Se group and C-Se group (*P \ 0.05)
Effects of Se deficient on the mRNA levels of iNOS, COX-2, NF-jB, PTGEs, TNF-a and SelW in thymus of chickens To determine whether or not the protective role of SelW in inflammation induced by Se deficiency in thymus of chickens is exerted by the mechanisms of downregulating inflammation genes expression, effects of Se deficient on the mRNA levels of the corresponding inflammation genes in thymus of chickens were examined. As Fig. 3 shows, the SelW mRNA levels of spleen tissues were significantly decreased in low-Se groups compared to control groups from 15 to 55 days (Fig. 3a–e) (P \ 0.05). In contrast, the expression of iNOS, COX-2, NF-jB, PTGEs, TNF-a genes in thymuses of chickens during the different days of Se deficient were significantly increased from 15 to 55 days (P \ 0.05). These observations suggest that inflammation injury induced by Se deficiency in thymus of chickens are at least in part mediated by augmen-
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tation of inflammation genes levels, and decreasing SelW expression. Effects of Se deficient on the mRNA levels of iNOS, COX-2, NF-jB, PTGEs, TNF-a and SelW in bursa of Fabricius of chickens The effects of Se deficient on the mRNA levels of the corresponding inflammation genes in bursa of Fabricius are shown in Fig. 4. The iNOS, COX-2, NF-jB, PTGEs, TNF-a mRNA levels of spleen tissues were significantly increased in low-Se groups compared to control groups from 15 to 55 days (Fig. 4a–e) (P \ 0.05). In order to determine the effects of SelW on the expression of iNOS, COX-2, NF-jB, PTGEs, TNF-a genes in bursa of Fabricius during the different days of Se deficient, the mRNA expression level of SelW genes were examined by qPCR. From Fig. 4f, qPCR results revealed that the levels of SelW mRNA were significantly decreased from 15 to 55 days (P \ 0.05).
Biometals Fig. 3 The effects of Se deficient on the mRNA levels of iNOS, COX-2, NFjB, PTGEs, TNF-a and SelW in thymus of chickens. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between L-Se group and C-Se group (*P \ 0.05)
Effects of Se on mRNA levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a in H2O2-induced lymphocyte
Effects of SelW siRNA on iNOS, COX-2, NF-jB, PTGEs and TNF-a mRNA in H2O2-induced lymphocyte
To investigate whether iNOS, COX-2, NF-jB, PTGEs and TNF-a mRNA expression were regulated by Se. Chicken splenic lymphocyte were treated with 10-8, 10-7 or 10-6 mol/L of Se for 6 h, and were then treated for 24 h in the absence and presence of 20 lM H2O2. As shown in Fig. 5, the lymphocytes with H2O2 (20 lM) resulting in a significantly increase mRNA expression of iNOS, COX-2, PTGEs and TNF-a (P \ 0.05) (Fig. 5a, b, d, e), and decrease mRNA expression of NF-jB (P \ 0.05) (Fig. 5c), 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 mRNA expression of iNOS, COX-2, NF-jB, PTGEs and TNF-a were significantly decreased (P \ 0.05), compared with the 20 lM H2O2 control, and significantly dose-dependent increased compared with the 10-8, 10-7 or 10-6 mol/L of Se group respectively (P \ 0.05).
As shown in Fig. 6, there were higher mRNA expression levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a in siRNA groups when compared with the corresponding RNAi Negative Control groups (P \ 0.05). With the increasing of transfected time, the mRNA levels of these inflammation-related genes showed an increased trend.
Discussion Se is essential for normal biological functions. The main biological function of Se is known to serve an antioxidant cativity and this is achieved by selenocysteine in the enzymatic active site, such as GPx. There have been reported that Se deficiency (0.02 ppm for 8 wk) in in vivo or absence of 10-7 M Se in in vitro results in a significant retrogression of the proliferative
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Biometals Fig. 4 The effects of Se deficient on the mRNA levels of iNOS, COX-2, NFjB, PTGEs, TNF-a and SelW in bursa of Fabricius of chickens. Results are from at least three independent experiments. Data are represented as mean ± SD. Bars with asterisks are statistically significantly different between L-Se group and C-Se group (*P \ 0.05)
responses of spleen lymphocytes from C57B1/6J mice in response to stimulation with mitogen or antigen, supplementation with Se had the opposite effect (Kiremidjian-Schumacher et al. 1992). In poultry, numerous lines of previous evidence suggested that Se deficiency reduced growth and resulted in exudative diathesis and reduced the growth of lymphoid organs (Bartholomew et al. 1998), and inhibited the growth of the immune organs and reduced immune function (Marsh et al. 1986). Supplementation of Se leads to enhanced comparative metabolic and immune response of chickens, and had protective effect in cancer of the colon (Leng et al. 2003). However, Excess dietary selenium induced the decrease of relative weight, lesions, and apoptosis of bursa of Fabricius chickens (Peng et al. 2009). This result was similar to the effect of Se on cells viability of human cancer cells (Rigobello et al. 2009), NB4 cells (Cao et al. 2012), rat vascular smooth muscle cells and human prostate cancer cells (Zhong and Oberley 2001; Tang and Huang 2004) in in vitro. In the present study, compared with the control group, Se-deficient diets results in a significant disordered histological structures, and the number of lymphocytics were reduced in the lymphocytic follicles, and vacuolar degeneration, mesenchymal rarefaction, and infiltration of inflammatory occurred. These data suggest that Se-deficient
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caused inflammatory damage to immune organs of chickens. In previous studies, they reported that the presence of SelW gene was detected in various organs of mammals. Intensive researches have revealed that the expression level of SelW gene is generally sensitive to Se status in rats and sheep fed dietary supplementation with Se (Yeh et al. 1995, 1997b, 1998; Sun et al. 1998). In poultry, they have reported that SelW was expressed widely in chicken tissues, and 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. 2010b). The SelW mRNA levels were found to increase in a dosedependent manner with dietary supplementation with Se in the gastrointestinal tract tissue, liver, pancreas and skeletal muscles of chickens (Sun et al. 2011; Wang et al. 2011; Ruan et al. 2012; Zhang et al. 2012; Gao et al. 2012; Wu et al. 2012).The similar response pattern was also found in vitro that the level of SelW was regulated by the Se concentration in the medium in cultured L8 cells (Gu et al. 2002; Yeh et al. 1997c). The mRNA expression of SelW was increased when the cultured chicken myoblasts were treated with additional Se treatment, but deceased in high-Se group (Ruan et al. 2012; Wu et al. 2012). In addition, our
Biometals Fig. 5 Effect of Se on mRNA levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a 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)
previous studies have shown that the SelW gene was also distributed widely in the immune organs of chickens fed the basal commercial diets and the Sesupplemented diets, and SelW mRNA expression is generally sensitive to Se dietary supplementation in those organs (Yu et al. 2011). Moreover, 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 was significantly increased compared with the 20 lM H2O2 control (Yu et al. 2014). However, effect of Se-deficient diets on the expression of SelW gene in the avian immune organs remains unknown until now. In this study, it was showed that the SelW gene was highly sensitive to Se-deficient in chicken immune organs, and SelW mRNA expression was significantly decreased as time-dependent in chickens fed Se-deficient diets (Figs. 2f, 3f, 4f). It is well know that Se deficient might decrease cell viability induced by H2O2 in primary cultured pig
thyrocytes (Demelash et al. 2004). In our previous investigation have shown that the cells were more sensitive to the oxidative stress induced by H2O2, and the sensitivity decreases with the increasing of Se concentration and up-regulating of SelW gene, when cells were incubated with Se before treated with H2O2 (Yu et al. 2014). Furthermore, suppression of SelW decreased lymphocyte viability, and promoted the sensitivity of lymphocyte induced by H2O2 (Yu et al. 2014). Similar results were reported that the attenuation of SelW expression due to siRNA renders the cells more sensitive to H2O2 in the cultured SelW siRNA-transfected mouse embryonic neuronal cells (Chung et al. 2009) and skeletal muscle cells (Wang et al. 2010). Overexpression of SelW markedly aggravated the viability of CHO cells and H1299 human lung cancer cell lines (Jeong et al. 2002), and CHO-K1 cells (Han et al. 2012), after treatment with H2O2. However, no report has been issued on the mechanism underlying its anti-inflammatory effects in chicken lymphocyte and immune organs. In order to further detect Se deficient induced tissues damage and host defenses such as inflammatory reactions, the mRNA expression levels of NF-jB,
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Biometals Fig. 6 Effects of SelW siRNA on mRNA levels of iNOS, COX-2, NF-jB, PTGEs and TNF-a in H2O2induced splenic lymphocyte. Bars represent mean ± standard deviation (n = 3/group). Bars without sharing a common letter are significantly different (P \ 0.05)
COX-2, iNOS, TNF-a, and PTGEs were examined by qPCR. Furthermore, we further tested the influence of endogenous SelW on the expression levels of those inflammatory factors. In the nucleus, NF-jB induces the transcription of a large variety of target genes by binding to the cis-acting jB element. Those target genes involve normally encode cytokines (Kiemer et al. 2003), cell adhesion molecules, and inflammatory enzymes including COX-2 (Abate et al. 1998) and iNOS (Lee et al. 2000). COX-2 inhibitors reduce mucosal damage, which further indicates the role of prostaglandins in tissue damage and inflammation reactions (Zheng et al. 2012). PTGES catalyzes the isomerization of PGH2 to PGE2, and the latter plays an important role in inflammation (Liu et al. 2012), cell growth, and transformation (Sasaki et al. 2012). It has been reported that the NF-jB activation induced by monocyte-conditioned medium and TNFa were inhibited by Se at the physiological levels, and the maximum activation of NF-jB was induced at half the level of the serum Se in healthy subjects in cultured Human hepatoma cell line HuH-7 (Maehira et al. 2003). Mice fed a Se-deficient diet showed higher NOS activity levels either at basal or after isoproterenol stimulation, and then were suppressed by feeding Se-deficient diet mice with a Se-supplemented
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diet for 1 wk or by inhibition of iNOS (Natalia et al. 2007). Studies indicated that the expression of both p65 and p50 genes(components of NF-jB), and the iNOS levels as well as NO levels increased in Se deficient group mice testis while the expression of the inhibitory IjBa declined significantly. This result clearly demonstrates both enhanced NO levels and NF-jB are harmful in the progression of normal spermatogenic cycle (Sonia and Bansal 2007).It also reported that pancreatic mRNA expressions of proinflammatory cytokines TNF-a, and activity of iNOS and content of nitric oxide were significantly increased in untreated diabetes mellitus mice, and they were decreased in selenite-treated diabetes mellitus mice (Zeng et al. 2009). Studies found that low doses of selenium may protect the prostate from prostatitisinduced cancer by inhibiting NF-jB and the subsequent production of the immunosuppressive cytokine TGFb1 and pro-inflammatory factor IL-6 (Pei et al. 2010). It reported that COX-2 protein expression and PGE2 levels were higher in Se-deficient RAW 264.7 macrophages treated with lipopolysaccharide, and the NF-jB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2 (Zamamiri-Davis et al. 2002). Similar studies reported that lipopolysaccharide stimulation
Biometals
produced a proinflammatory phenotype in rat peritoneal macrophages. Se caused a greater inhibitory effect on the expression of COX-2 and iNOS, and a higher efficacy in reducing the loss of IjB-a, the increased NF-jB binding activity, the enhanced mRNA levels of TNF-a and nitrite content. Moreover, Se in vivo caused a greater inhibitory effect on prostate inflammation induced in rats by partial bladder outlet obstruction (Bonvissuto et al. 2011). In the current study, we found that the iNOS, COX-2, NF-jB, PTGEs and TNF-a mRNA levels of immune tissue were significantly increased in Low-Se groups. In vitro, H2O2 induced a significantly up-regulation of the iNOS, COX-2, NF-jB, PTGEs and TNF-a. When lymphocyte were pretreated with Se before treated with H2O2, those genes were significantly decreased (Fig. 5). Moreover, mRNA expression level of iNOS, COX-2, NF-jB, PTGEs and TNF-a were higher in siRNA groups (Fig. 6). So, in our study, the increased inflammation factors suggested that inflammatory response was induced by Se deficient and the attenuation of SelW expression, which may lead to chicken immune tissues and cultured splenic lymphocyte injury. The mechanisms underlying the actions of sodium selenite and SelW on these genes remain to be clarified further. Thus, the results of present study suggest that the antioxidant defense system could be damaged in immune organs (spleen, thymus and bursa of Fabricius) of chickens by Se deficient, and resulted in inflammatory injury. The increase in mRNA expression of inflammation factors further suggested that Se deficient caused damage to immune tissue of the chicken. siRNAinduced suppression of SelW gene in cultured chicken splenic lymphocyte resulted in an increase in mRNA expression of inflammation factors further to H2O2induced oxidative stress. Selenite supplementation significantly up-regulated SelW mRNA expression, resulted in an decrease in mRNA expression of inflammation factors to H2O2-induced oxidative stress, provides definitive evidence supporting the antioxidant function of SelW. Together, our data suggest the possibility that SelW may play an important role in the protection of immune organs of birds from inflammatory injury by the regulations of inflammation-related genes. However, so far, the specific SelW-activated induction and regulation mechanisms among inflammation-related genes are unclear, and further studies are needed.
Acknowledgments This study was supported by the International Cooperation and Exchanges NSFC (31320103920), the National Natural Science Foundation of China (31272626), the Doctoral Fund of Ministry of Education of China (20122325110018), the Doctoral Fund of Harbin Normal University of China (220601085), the Study Abroad Foundation of Heilongjiang Province (LC201031).
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