Biol Trace Elem Res DOI 10.1007/s12011-015-0596-9
Excessive Selenium Supplementation Induced Oxidative Stress and Endoplasmic Reticulum Stress in Chicken Spleen Yachao Wang 1,2 & Li Jiang 1 & Yuanfeng Li 1 & Xuegang Luo 2,3 & Jian He 1
Received: 5 November 2015 / Accepted: 14 December 2015 # Springer Science+Business Media New York 2016
Abstract Excessive selenium (Se) intake is harmful for animals and humans. The aim of the present study was to examine the effect of long-term excessive Se supplementation on oxidative stress and endoplasmic reticulum (ER) stress-related injuries in chicken spleen. A total of 180 1-day-old chickens were randomly divided into four groups with different Se dietary contents (0.2 mg/kg Se, 5 mg/kg Se, 10 mg/kg Se, or 15 mg/kg Se) for 45 days. Then, the levels of antioxidative enzymes, GPx, SOD, and MDA as well as the expression levels of GRP78, ARF6, caspase 3, caspase 12, and Bcl 2 in the spleen were determined at days 15, 30, and 45, respectively. The results showed that excessive Se treatment decreased the activities of GPx and SOD (P < 0.05) but increased the
levels of MDA (P < 0.05) in a dose- and time-dependent manner. In addition, the ER stress genes GRP78 and ATF6 were highly expressed (P < 0.05), and the apoptosis genes caspase 3 and caspase 12 were increased, but Bcl 2 was decreased by Se treatment (P < 0.05). Correlation analysis showed that there was a high correlation between these biomarkers, which indicated that ER stress and ER stress-related apoptosis were correlated with oxidative stress. These results showed the important role of oxidative stress and ER stress in Se-related immune injuries in chicken. Keywords Selenium . Oxidative stress . Endoplasmic reticulum stress . Apoptosis . Chicken spleen
Yachao Wang and Li Jiang should be considered as co-first authors Yachao Wang and Li Jiang contributed equally to this work.
Introduction
* Xuegang Luo [email protected]
Selenium (Se) is an essential micronutrient that plays important biological roles in animals. Se deficiency may lead to exudative diathesis and muscular dystrophy [1], oxidative damages [2], liver injuries [3], and disordered immune function [4]. However, excessive intake of Se from dietary sources has been shown to induce deleterious effects in animals and human [5], such as insulin resistance in gestating rats and their offspring [6] and the pro-arrhythmic effects in rat cardiomyocytes [7]. In the animal industry, especially, widely used inorganic Se additives (such as sodium selenite) have led to highly toxic incidences in animals and the excessive release of Se to the environment. Consequently, the toxic attributes of Se have raised great interest among researchers. Oxidative stress is described as the imbalance between the generation of reactive oxygen species (ROS) and antioxidant capacity. As an important antioxidant, Se could influence the oxidative state by regulating the levels of antioxidative enzymes such as glutathione peroxidase (GPx) and superoxide
* Jian He [email protected] Yachao Wang [email protected] Li Jiang [email protected] Yuanfeng Li [email protected] 1
School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China
2
Engineering Research Center of Biomass Materials, Ministry of Education, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China
3
School of Material Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China
Wang et al.
dismutase (SOD) and the antioxidative selenoproteins [1, 8–10]. On the other hand, variations in the oxidative state trigger several types of injuries through a variety of signaling pathways, such as higher expression of apoptosis genes [1], inflammation [11, 12], and the endoplasmic reticulum (ER) stress response [13]. As indicated in previous studies, ER stress is considered to be an early or initial response of cells to stress or damage [14]. Disturbed ER homeostasis could induce the unfolded protein response (UPR) and the accumulation of unfolded proteins in the ER lumen, which triggers cell death responses [15]. In this process, the major ER chaperone, GRP78, is released from the ER stress sensors, such as inositol-requiring protein (IRE1), protein kinase RNA (PKR)like ER kinase (PERK), and activating transcription factor 6 (ATF6) [16]. The release of GRP78 triggers higher expression of apoptosis-related genes (pro-apoptotic (caspase) and antiapoptotic (Bcl-2-related family)). In animals, the relation between Se and oxidative stress, ER stress, or apoptosis have been widely reported [3, 17, 18], which shows the important roles of oxidative stress and ER stress in Se-related injuries or diseases. As an important inorganic Se additive in chicken diets, sodium selenite is widely used, and its overuse can lead to sever toxic incidences and environment pollution. Consequently, studying effects of excessive Se supplementation on chickens is important to understand the mechanism of Se toxicity. As one important target of Se, the immune system or immune function of chicken is known to be influenced by variations in Se [4, 18, 19]. However, less is known about the role of Se supplementation in the spleens of chickens. In the present study, we examined oxidative stress and ER stress markers, and the expressions of apoptosis genes in chicken spleens following treatment with different Se supplemented diets.
Materials and Methods Animal Care and Experimental Design All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Southwest University of Science and Technology. A total of 180 chickens (1-day-old, laying hens, Mianyang, China) were randomly divided into four groups (45 chickens per group). Each group was separated into five pens (nine chickens per pen). The chickens were maintained on a basic diet (control group) containing 0.2 mg/kg Se, a low supplemented diet (L group) containing 5 mg/kg Se, a medium supplemented diet (M group) containing 10 mg/kg Se, or a high supplemented diet (H group) containing 15 mg/kg Se for 15, 30, or 45 days. Over the entire experimental period, the chickens were allowed ad libitum consumption of food and water. Chickens were killed
at 15, 30, and 45 days. Following euthanasia with sodium pentobarbital, the spleens were quickly removed. The tissues were rinsed with ice-cold sterile deionized water, frozen immediately in liquid nitrogen, and stored at −80 °C until required. Measurement of Antioxidative Enzymes Spleen samples were homogenized on ice in physiological saline and centrifuged at 700×g to collect supernatants for biochemical assays. Levels of malondialdehyde (MDA) were determined using the thiobarbituric acid assay [20] (MDA detection kit A003, Nanjing Jiancheng Bioengineering Institute). GPx (glutathione peroxidase (GSH-PX) assay kit A005, Nanjing Jiancheng Bioengineering Institute) and SOD (superoxide dismutase (SOD) assay kit (WST-1 method) A001-3, Nanjing Jiancheng Bioengineering Institute) activities were determined by a previously described method from Yao [1], according to the protocol of the kit manufacturer. Protein concentrations of samples were measured using the Bradford method [21]. Quantitative Real-Time PCR Analysis Total RNA was isolated from tissue samples using Trizol reagent according to the manufacturer ’s instructions (Invitrogen, Shanghai, China). The concentration and purity of total RNA were determined spectrophotometrically at 260/ 280 nm using the GeneQuant 1300. The procedure for reverse transcription was according to the manufacturer’s instructions (Invitrogen, Shanghai, China). Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, Foster City, CA). Specific primers for ATF6, Bcl 2, caspase 3, caspase 12, GRP78, and GADPH were based on known chickens sequences (Table 1). PCR reactions were similar to those described by Yao [2]. The mRNA relative abundance was calculated according to the method of Pfaffl [22]. Sections for Electron Microscopy The detection of ultrastructure was similar to a previous study by Shao [23]. The collected cells were fixed immediately in 2.5 % glutaraldehyde in phosphate-buffered saline (v/v, pH 7.2), post-fixed in 1 % osmium tetroxide (v/v) and stained with 4.8 % uranyl acetate following dehydration. The samples were washed in propylene oxide and impregnated with epoxy resins. The semi-fine sections were contrasted with uranyl acetate and lead citrate for study via microscopy. The microphotographs were taken with a transmission electron microscope (TEM).
Excessive Se Supplementation in Chicken Spleen Table 1 Primers used for realtime PCR
Gene
Forward primer
Reverse primer
GRP 78
5′-GAATCGGCTAACACCAGAGGA-3′
5′-CGCATAGCTCTCCAGCTCATT-3′
ATF6
5′-CGTCGTCTGAACCACTTACTGA-3′
5′-CCTTCTTTCCTAACAGCCACAC-3′
Caspase 12 Caspase 3
5′-AATAGTGGGCATCTGGGTCA-3′ 5′-CATCT GCATCCGTGCCTGA-3′
5′-CGGTGTGATTTAGACCCGTAAGAC-3′ 5′-CTCTCGG CTGTGGTGGTGAA-3′
Bcl-2
5′-ATCGTCGCCTTCTTCGAGTT-3′
5′-ATCCCATCCTCCGTTGTCCT-3′
GADPH
5′-AGAACATCATCCCAGCGT-3′
5′-AGCCTTCACTACCCTCTTG-3′
Statistical Analysis Statistical analysis of all data was performed using SPSS for Windows (version 13; SPSS Inc., Chicago, IL). All data were checked for normal distribution and equal variance. The differences between the treatment groups were assessed using one-way ANOVA. The data were expressed as the mean ± standard deviation. Differences were considered to be significant at P < 0.05. Pearson’s correlation coefficients were calculated to determine correlations between biomarkers.
The Effect of Se Supplementation on Apoptosis in Chicken Spleen In the present study, we examined the expression of the proapoptosis genes caspase 3 and caspase 12 as well as the antiapoptosis gene Bcl 2. The results (Fig. 3) showed that Se supplementation increased the expression of caspase 3 and caspase 12 but decreased the levels of Bcl 2 (P < 0.05) in a time- and dose-dependent manner. We conclude that Se treatment induced the occurrence of apoptosis. In the present study, caspase 12 and Bcl 2 were apoptosis genes downstream
Results The Effect of Se Supplementation on Oxidative Stress in Chicken Spleen In the present study, we examined the activities of GPx and SOD and the levels of MDA as biomarkers of oxidative stress. The results (Fig. 1) showed that Se treatment decreased the activities of GPx and SOD (P < 0.05) but increased the levels of MDA (P < 0.05). From the results, we can see that the effect of Se on these biomarkers was dose and time dependent. At 45 days, especially, GPx and SOD activities and MDA levels were significantly influenced by Se treatment. These results showed that long-term high-level Se treatment induced oxidative stress in chicken spleen.
The Effect of Se Supplementation on ER Stress in Chicken Spleen In the present study, we measured the expression levels of an ER stress marker, GRP78, and an ER stress sensor gene, ATF6. The results (Fig. 2) showed that excessive Se treatment increased the expression of GRP78 and ATF6 (P < 0.05). GRP78 levels were highly (10 to 40 times) enhanced by Se treatment (P < 0.05). Consistent with the higher expression of GRP78, ATF6 was also induced by Se treatment. Thus, Se treatment induced ER stress in chicken spleen. Similar to oxidative stress, the effect of Se on ER stress genes was also observed to occur in a dose- and time-dependent manner.
Fig. 1 The effect of Se supplement on the oxidative stress in chicken spleen. Bars without a shared common letter were significantly different (P < 0.05). The data are expressed as the means ± SD, n = 5
Wang et al.
Fig. 2 The effect of Se supplement on the ER stress in chicken spleen. Bars without a shared common letter were significantly different (P < 0.05). The data are expressed as the means ± SD, n = 5
of ER stress, and caspase 3 was the apoptosis executor. Thus, excessive Se was associated with ER stress-related apoptosis.
The Correlation Between Detected Biomarkers In the present study, we also analyzed the correlation between detected biomarkers. The correlation results (Table 2) showed that the correlation between detected biomarkers was high. There was a high positive correlation between GPx, SOD, and Bcl 2 but a negative correlation between antioxidative enzymes and other biomarkers. In addition, except for GPx, SOD and Bcl2, a high positive correlation exists between the other detected biomarkers. The results showed that higher expression of ER stress and apoptosis genes was significantly correlated with oxidative stress levels.
Ultrastructural Changes In this part, we collected the spleen from control and the high Se groups from 45 days. Spleen tissues from the control group after 45 days showed normal ultrastructure (Fig. 4a, b). The nuclei and organelles were clear and intact. However, after excessive Se treatment (15 mg/kg) for 45 days, the ultrastructure of the spleen was damaged. The results (Fig. 4c–f) showed the typical cell apoptosis structures such as chromatin edges and nucleus shrinkage (Fig. 4c, d) and chromatin condensation (Fig. 4e, f). Consistent with the expression of apoptosis genes, the ultrastructure of the spleen showed typical apoptosis damage.
Fig. 3 The effect of Se supplement on the apoptosis in chicken spleen. Bars without a shared common letter were significantly different (P < 0.05). The data are expressed as the means ± SD, n = 5
Discussion The important role of Se has been widely recognized due to the essential functions of Se and the negative effects of Se deficiency in animals and humans. As a consequence, inorganic or organic Se is widely added into diets, especially in the breeding industry. In addition, some human activities such as coal mining and combustion, metal mining, oil industry, or agricultural irrigation also contribute to the excessive release of Se into the environment [24]. Because of this use of Se, Se toxicity or Se pollution has recently attracted the attention of many researchers. Excessive intake of Se from dietary sources has been shown to induce deleterious health effects in animals and humans [5]. However, the exact mechanism requires further studied. In the present study, we examined the effect of supplemented Se levels on oxidative stress, ER stress, and apoptosis in chicken spleen. The results showed that excessive Se intake induced lower GPx and SOD activities, but higher levels of MDA and ER stress markers, and ER stress-related apoptosis in a time and dose dependent manner. Thus, Se toxicity in chicken spleen was related to oxidative stress and ER stress.
Excessive Se Supplementation in Chicken Spleen Table 2 The correlation between detected biomarkers
GPx GPx
SOD
MDA
GRP78
ATF6
Caspase 3
Caspase 12
Bcl 2
0.97
−0.95
−0.91
−0.93
−0.95
−0.98
0.96
−0.93
−0.93
−0.95
−0.95
−0.98
0.94
0.89
0.86 0.93
0.96 0.88
0.94 0.95
−0.94 −0.88
SOD
0.97
MDA GRP78
−0.95 −0.91
−0.93 −0.93
0.89
ATF6
−0.93
−0.95
0.86
0.93
Caspase 3
−0.95
−0.95
0.96
0.88
0.89
Caspase 12 Bcl 2
−0.98 0.96
−0.98 0.94
0.94 −0.94
0.95 −0.88
0.98 −0.89
As indicated in previous studies, Se is an important antioxidant in animals and humans. On the one hand, Se is the central component for the antioxidative enzymes GPx and the thioredoxin reductases (Txnrd). On the other hand, Se primarily performs its biological functions by incorporating into selenoproteins (such as the antioxidative selenoprotein, Selw, Seln, and Selt [2]) as the amino acid selenocysteine (Sec). Dietary Se could influence the levels of these antioxidative enzymes or selenoproteins in chicken tissues and induce different types of injuries [1, 4, 25]. Se deficiency suppresses chicken immune function by regulating the levels of Fig. 4 The effect of Se supplementation on the chicken spleen ultrastructure. 6000 ×. a, b The spleen ultrastructure of the control group at 45 days. c–f The spleen ultrastructure of the H Se group at 45 days
0.89 0.95 −0.98
0.98
−0.89
0.95
−0.98 −0.95
−0.95
several antioxidative selenoproteins [26]. In contrast, Se decreased the immune injuries in spleen cells exposed to Cd [18], preserves the antioxidant capacity in the erythrocytes of rat [27], and supports the interferon-γ and IL-6 immune response pathways in mice by influencing selenoproteins [28]. Therefore, oxidative-reductive signals play a central role in the process of immune diseases, injuries, or protection that relate to Se. In the present study, following treatment with different levels of Se, we collected spleens on days 15, 30, and 45. The results show that excessive Se intake decreased the activities of GPx and SOD, which was followed by higher
Wang et al.
MDA levels. The effects of Se on these biomarkers occurred in a time- and dose-dependent manner, which indicated that long-term intake of high levels of Se induced sever oxidative damage in the chicken immune system. This effect was similar to excessive Se treatment on erythrocytes in rat [27]. The present study also showed that disordered oxidativereductive signals were involved in Se-induced immune toxicity in chicken. Thus, together with previous reports, we can see that the nutrition dose of Se could mediate and alleviate immune injuries by preserving the antioxidant capacity of the immune system. However, excessive Se worsens oxidative injuries and influences normal immune function. Thus, the immune-regulating role of Se was closely related to oxidative-reductive signals. Oxidative stress could trigger several types of physical or pathological signal cascades, such as the inflammation response, cytokine responses, apoptosis, and ER stress. Among these signaling pathways, ER stress may be one early or initial response of cells to stress or damage [14]. ER stress is involved in several types of cellular processes, including cell death, cell protection, signal regulation, disease development, or chemical toxicity [29–31]. Several reports have indicated the involvement of ER stress in the process of Se-related injuries. Yao showed that ER stress and oxidative injuries played important roles in chicken liver apoptosis induced by Se deficiency [3]. ER stress is also associated with cardiac malfunction induced by Se deficiency in a rat model [32]. In addition, Liu showed that Se can ameliorate the effects of Cd induced oxidative stress and ER stress in chicken kidney [17]. Similar to these reports, the present study showed that excessive Se supplementation also induced higher expression of ER stress markers, GRP78, and the ER stress-related signal cascade gene, ATF6. It is known that the release of GRP78 from IRE1, PERK, and ATF6 could induce downstream signal cascades, such as increases in the apoptosis gene CHOP, caspase 12, and cell death pathways [16]. Therefore, higher expression of the GRP78 gene was one indicator of ER stress. In the present study, we also detected changes in ER stress-related apoptosis genes, such as caspase 12, the apoptosis executor caspase 3, and the antiapoptosis gene Bcl 2. Excessive Se treatment induced higher expression of caspase 12 and caspase 3 but lower Bcl 2. So ER stress and ER stress-related apoptosis was induced by excessive Se supplementation in chicken spleen. The present study supported the idea that ER stress-related cellular injuries were related to Se toxicity. The interaction between oxidative stress and ER stress has been widely reported [16, 33]. ROS plays a central role in the crosstalk signal between ER stress and oxidative injuries [33, 34]. In the present study, we found that ER stress-related injuries also showed higher levels following increased oxidative stress. In addition, Pearson’s correlation results showed that there exists a highly positive correlation between ER stress markers, apoptosis, and MDA levels, but a negative
correlation with GPx and SOD activities. In the present study, oxidative stress and ER stress showed a high correlation, which was similar to previous reports. Thus, both oxidative stress and ER stress play important roles in immune injuries induced Se toxicity. In summary, the results showed that excessive Se intake induced chicken immune injuries. Excessive Se exposure induced higher oxidative stress and ER stress-related apoptosis. In this process, there is a high positive correlation between oxidative stress and ER stress, which showed the important role of oxidative stress and ER stress in Se-related immune injuries. Acknowledgments The present work was financially supported by Southwest University of Science and Technology (15zx7121) and Mianyang Science and Technology Project (14 N043). The authors thank the Elsevier English Language Editing System to correct grammatical, spelling, and other common errors. Compliance with ethical standards Conflict of Interest The authors declare that they have no competing interests.
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Excessive Selenium Supplementation Induced Oxidative Stress and Endoplasmic Reticulum Stress in Chicken Spleen Yachao Wang 1,2 & Li Jiang 1 & Yuanfeng Li 1 & Xuegang Luo 2,3 & Jian He 1
Received: 5 November 2015 / Accepted: 14 December 2015 # Springer Science+Business Media New York 2016
Abstract Excessive selenium (Se) intake is harmful for animals and humans. The aim of the present study was to examine the effect of long-term excessive Se supplementation on oxidative stress and endoplasmic reticulum (ER) stress-related injuries in chicken spleen. A total of 180 1-day-old chickens were randomly divided into four groups with different Se dietary contents (0.2 mg/kg Se, 5 mg/kg Se, 10 mg/kg Se, or 15 mg/kg Se) for 45 days. Then, the levels of antioxidative enzymes, GPx, SOD, and MDA as well as the expression levels of GRP78, ARF6, caspase 3, caspase 12, and Bcl 2 in the spleen were determined at days 15, 30, and 45, respectively. The results showed that excessive Se treatment decreased the activities of GPx and SOD (P < 0.05) but increased the
levels of MDA (P < 0.05) in a dose- and time-dependent manner. In addition, the ER stress genes GRP78 and ATF6 were highly expressed (P < 0.05), and the apoptosis genes caspase 3 and caspase 12 were increased, but Bcl 2 was decreased by Se treatment (P < 0.05). Correlation analysis showed that there was a high correlation between these biomarkers, which indicated that ER stress and ER stress-related apoptosis were correlated with oxidative stress. These results showed the important role of oxidative stress and ER stress in Se-related immune injuries in chicken. Keywords Selenium . Oxidative stress . Endoplasmic reticulum stress . Apoptosis . Chicken spleen
Yachao Wang and Li Jiang should be considered as co-first authors Yachao Wang and Li Jiang contributed equally to this work.
Introduction
* Xuegang Luo [email protected]
Selenium (Se) is an essential micronutrient that plays important biological roles in animals. Se deficiency may lead to exudative diathesis and muscular dystrophy [1], oxidative damages [2], liver injuries [3], and disordered immune function [4]. However, excessive intake of Se from dietary sources has been shown to induce deleterious effects in animals and human [5], such as insulin resistance in gestating rats and their offspring [6] and the pro-arrhythmic effects in rat cardiomyocytes [7]. In the animal industry, especially, widely used inorganic Se additives (such as sodium selenite) have led to highly toxic incidences in animals and the excessive release of Se to the environment. Consequently, the toxic attributes of Se have raised great interest among researchers. Oxidative stress is described as the imbalance between the generation of reactive oxygen species (ROS) and antioxidant capacity. As an important antioxidant, Se could influence the oxidative state by regulating the levels of antioxidative enzymes such as glutathione peroxidase (GPx) and superoxide
* Jian He [email protected] Yachao Wang [email protected] Li Jiang [email protected] Yuanfeng Li [email protected] 1
School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China
2
Engineering Research Center of Biomass Materials, Ministry of Education, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China
3
School of Material Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan 621010, China
Wang et al.
dismutase (SOD) and the antioxidative selenoproteins [1, 8–10]. On the other hand, variations in the oxidative state trigger several types of injuries through a variety of signaling pathways, such as higher expression of apoptosis genes [1], inflammation [11, 12], and the endoplasmic reticulum (ER) stress response [13]. As indicated in previous studies, ER stress is considered to be an early or initial response of cells to stress or damage [14]. Disturbed ER homeostasis could induce the unfolded protein response (UPR) and the accumulation of unfolded proteins in the ER lumen, which triggers cell death responses [15]. In this process, the major ER chaperone, GRP78, is released from the ER stress sensors, such as inositol-requiring protein (IRE1), protein kinase RNA (PKR)like ER kinase (PERK), and activating transcription factor 6 (ATF6) [16]. The release of GRP78 triggers higher expression of apoptosis-related genes (pro-apoptotic (caspase) and antiapoptotic (Bcl-2-related family)). In animals, the relation between Se and oxidative stress, ER stress, or apoptosis have been widely reported [3, 17, 18], which shows the important roles of oxidative stress and ER stress in Se-related injuries or diseases. As an important inorganic Se additive in chicken diets, sodium selenite is widely used, and its overuse can lead to sever toxic incidences and environment pollution. Consequently, studying effects of excessive Se supplementation on chickens is important to understand the mechanism of Se toxicity. As one important target of Se, the immune system or immune function of chicken is known to be influenced by variations in Se [4, 18, 19]. However, less is known about the role of Se supplementation in the spleens of chickens. In the present study, we examined oxidative stress and ER stress markers, and the expressions of apoptosis genes in chicken spleens following treatment with different Se supplemented diets.
Materials and Methods Animal Care and Experimental Design All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Southwest University of Science and Technology. A total of 180 chickens (1-day-old, laying hens, Mianyang, China) were randomly divided into four groups (45 chickens per group). Each group was separated into five pens (nine chickens per pen). The chickens were maintained on a basic diet (control group) containing 0.2 mg/kg Se, a low supplemented diet (L group) containing 5 mg/kg Se, a medium supplemented diet (M group) containing 10 mg/kg Se, or a high supplemented diet (H group) containing 15 mg/kg Se for 15, 30, or 45 days. Over the entire experimental period, the chickens were allowed ad libitum consumption of food and water. Chickens were killed
at 15, 30, and 45 days. Following euthanasia with sodium pentobarbital, the spleens were quickly removed. The tissues were rinsed with ice-cold sterile deionized water, frozen immediately in liquid nitrogen, and stored at −80 °C until required. Measurement of Antioxidative Enzymes Spleen samples were homogenized on ice in physiological saline and centrifuged at 700×g to collect supernatants for biochemical assays. Levels of malondialdehyde (MDA) were determined using the thiobarbituric acid assay [20] (MDA detection kit A003, Nanjing Jiancheng Bioengineering Institute). GPx (glutathione peroxidase (GSH-PX) assay kit A005, Nanjing Jiancheng Bioengineering Institute) and SOD (superoxide dismutase (SOD) assay kit (WST-1 method) A001-3, Nanjing Jiancheng Bioengineering Institute) activities were determined by a previously described method from Yao [1], according to the protocol of the kit manufacturer. Protein concentrations of samples were measured using the Bradford method [21]. Quantitative Real-Time PCR Analysis Total RNA was isolated from tissue samples using Trizol reagent according to the manufacturer ’s instructions (Invitrogen, Shanghai, China). The concentration and purity of total RNA were determined spectrophotometrically at 260/ 280 nm using the GeneQuant 1300. The procedure for reverse transcription was according to the manufacturer’s instructions (Invitrogen, Shanghai, China). Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, Foster City, CA). Specific primers for ATF6, Bcl 2, caspase 3, caspase 12, GRP78, and GADPH were based on known chickens sequences (Table 1). PCR reactions were similar to those described by Yao [2]. The mRNA relative abundance was calculated according to the method of Pfaffl [22]. Sections for Electron Microscopy The detection of ultrastructure was similar to a previous study by Shao [23]. The collected cells were fixed immediately in 2.5 % glutaraldehyde in phosphate-buffered saline (v/v, pH 7.2), post-fixed in 1 % osmium tetroxide (v/v) and stained with 4.8 % uranyl acetate following dehydration. The samples were washed in propylene oxide and impregnated with epoxy resins. The semi-fine sections were contrasted with uranyl acetate and lead citrate for study via microscopy. The microphotographs were taken with a transmission electron microscope (TEM).
Excessive Se Supplementation in Chicken Spleen Table 1 Primers used for realtime PCR
Gene
Forward primer
Reverse primer
GRP 78
5′-GAATCGGCTAACACCAGAGGA-3′
5′-CGCATAGCTCTCCAGCTCATT-3′
ATF6
5′-CGTCGTCTGAACCACTTACTGA-3′
5′-CCTTCTTTCCTAACAGCCACAC-3′
Caspase 12 Caspase 3
5′-AATAGTGGGCATCTGGGTCA-3′ 5′-CATCT GCATCCGTGCCTGA-3′
5′-CGGTGTGATTTAGACCCGTAAGAC-3′ 5′-CTCTCGG CTGTGGTGGTGAA-3′
Bcl-2
5′-ATCGTCGCCTTCTTCGAGTT-3′
5′-ATCCCATCCTCCGTTGTCCT-3′
GADPH
5′-AGAACATCATCCCAGCGT-3′
5′-AGCCTTCACTACCCTCTTG-3′
Statistical Analysis Statistical analysis of all data was performed using SPSS for Windows (version 13; SPSS Inc., Chicago, IL). All data were checked for normal distribution and equal variance. The differences between the treatment groups were assessed using one-way ANOVA. The data were expressed as the mean ± standard deviation. Differences were considered to be significant at P < 0.05. Pearson’s correlation coefficients were calculated to determine correlations between biomarkers.
The Effect of Se Supplementation on Apoptosis in Chicken Spleen In the present study, we examined the expression of the proapoptosis genes caspase 3 and caspase 12 as well as the antiapoptosis gene Bcl 2. The results (Fig. 3) showed that Se supplementation increased the expression of caspase 3 and caspase 12 but decreased the levels of Bcl 2 (P < 0.05) in a time- and dose-dependent manner. We conclude that Se treatment induced the occurrence of apoptosis. In the present study, caspase 12 and Bcl 2 were apoptosis genes downstream
Results The Effect of Se Supplementation on Oxidative Stress in Chicken Spleen In the present study, we examined the activities of GPx and SOD and the levels of MDA as biomarkers of oxidative stress. The results (Fig. 1) showed that Se treatment decreased the activities of GPx and SOD (P < 0.05) but increased the levels of MDA (P < 0.05). From the results, we can see that the effect of Se on these biomarkers was dose and time dependent. At 45 days, especially, GPx and SOD activities and MDA levels were significantly influenced by Se treatment. These results showed that long-term high-level Se treatment induced oxidative stress in chicken spleen.
The Effect of Se Supplementation on ER Stress in Chicken Spleen In the present study, we measured the expression levels of an ER stress marker, GRP78, and an ER stress sensor gene, ATF6. The results (Fig. 2) showed that excessive Se treatment increased the expression of GRP78 and ATF6 (P < 0.05). GRP78 levels were highly (10 to 40 times) enhanced by Se treatment (P < 0.05). Consistent with the higher expression of GRP78, ATF6 was also induced by Se treatment. Thus, Se treatment induced ER stress in chicken spleen. Similar to oxidative stress, the effect of Se on ER stress genes was also observed to occur in a dose- and time-dependent manner.
Fig. 1 The effect of Se supplement on the oxidative stress in chicken spleen. Bars without a shared common letter were significantly different (P < 0.05). The data are expressed as the means ± SD, n = 5
Wang et al.
Fig. 2 The effect of Se supplement on the ER stress in chicken spleen. Bars without a shared common letter were significantly different (P < 0.05). The data are expressed as the means ± SD, n = 5
of ER stress, and caspase 3 was the apoptosis executor. Thus, excessive Se was associated with ER stress-related apoptosis.
The Correlation Between Detected Biomarkers In the present study, we also analyzed the correlation between detected biomarkers. The correlation results (Table 2) showed that the correlation between detected biomarkers was high. There was a high positive correlation between GPx, SOD, and Bcl 2 but a negative correlation between antioxidative enzymes and other biomarkers. In addition, except for GPx, SOD and Bcl2, a high positive correlation exists between the other detected biomarkers. The results showed that higher expression of ER stress and apoptosis genes was significantly correlated with oxidative stress levels.
Ultrastructural Changes In this part, we collected the spleen from control and the high Se groups from 45 days. Spleen tissues from the control group after 45 days showed normal ultrastructure (Fig. 4a, b). The nuclei and organelles were clear and intact. However, after excessive Se treatment (15 mg/kg) for 45 days, the ultrastructure of the spleen was damaged. The results (Fig. 4c–f) showed the typical cell apoptosis structures such as chromatin edges and nucleus shrinkage (Fig. 4c, d) and chromatin condensation (Fig. 4e, f). Consistent with the expression of apoptosis genes, the ultrastructure of the spleen showed typical apoptosis damage.
Fig. 3 The effect of Se supplement on the apoptosis in chicken spleen. Bars without a shared common letter were significantly different (P < 0.05). The data are expressed as the means ± SD, n = 5
Discussion The important role of Se has been widely recognized due to the essential functions of Se and the negative effects of Se deficiency in animals and humans. As a consequence, inorganic or organic Se is widely added into diets, especially in the breeding industry. In addition, some human activities such as coal mining and combustion, metal mining, oil industry, or agricultural irrigation also contribute to the excessive release of Se into the environment [24]. Because of this use of Se, Se toxicity or Se pollution has recently attracted the attention of many researchers. Excessive intake of Se from dietary sources has been shown to induce deleterious health effects in animals and humans [5]. However, the exact mechanism requires further studied. In the present study, we examined the effect of supplemented Se levels on oxidative stress, ER stress, and apoptosis in chicken spleen. The results showed that excessive Se intake induced lower GPx and SOD activities, but higher levels of MDA and ER stress markers, and ER stress-related apoptosis in a time and dose dependent manner. Thus, Se toxicity in chicken spleen was related to oxidative stress and ER stress.
Excessive Se Supplementation in Chicken Spleen Table 2 The correlation between detected biomarkers
GPx GPx
SOD
MDA
GRP78
ATF6
Caspase 3
Caspase 12
Bcl 2
0.97
−0.95
−0.91
−0.93
−0.95
−0.98
0.96
−0.93
−0.93
−0.95
−0.95
−0.98
0.94
0.89
0.86 0.93
0.96 0.88
0.94 0.95
−0.94 −0.88
SOD
0.97
MDA GRP78
−0.95 −0.91
−0.93 −0.93
0.89
ATF6
−0.93
−0.95
0.86
0.93
Caspase 3
−0.95
−0.95
0.96
0.88
0.89
Caspase 12 Bcl 2
−0.98 0.96
−0.98 0.94
0.94 −0.94
0.95 −0.88
0.98 −0.89
As indicated in previous studies, Se is an important antioxidant in animals and humans. On the one hand, Se is the central component for the antioxidative enzymes GPx and the thioredoxin reductases (Txnrd). On the other hand, Se primarily performs its biological functions by incorporating into selenoproteins (such as the antioxidative selenoprotein, Selw, Seln, and Selt [2]) as the amino acid selenocysteine (Sec). Dietary Se could influence the levels of these antioxidative enzymes or selenoproteins in chicken tissues and induce different types of injuries [1, 4, 25]. Se deficiency suppresses chicken immune function by regulating the levels of Fig. 4 The effect of Se supplementation on the chicken spleen ultrastructure. 6000 ×. a, b The spleen ultrastructure of the control group at 45 days. c–f The spleen ultrastructure of the H Se group at 45 days
0.89 0.95 −0.98
0.98
−0.89
0.95
−0.98 −0.95
−0.95
several antioxidative selenoproteins [26]. In contrast, Se decreased the immune injuries in spleen cells exposed to Cd [18], preserves the antioxidant capacity in the erythrocytes of rat [27], and supports the interferon-γ and IL-6 immune response pathways in mice by influencing selenoproteins [28]. Therefore, oxidative-reductive signals play a central role in the process of immune diseases, injuries, or protection that relate to Se. In the present study, following treatment with different levels of Se, we collected spleens on days 15, 30, and 45. The results show that excessive Se intake decreased the activities of GPx and SOD, which was followed by higher
Wang et al.
MDA levels. The effects of Se on these biomarkers occurred in a time- and dose-dependent manner, which indicated that long-term intake of high levels of Se induced sever oxidative damage in the chicken immune system. This effect was similar to excessive Se treatment on erythrocytes in rat [27]. The present study also showed that disordered oxidativereductive signals were involved in Se-induced immune toxicity in chicken. Thus, together with previous reports, we can see that the nutrition dose of Se could mediate and alleviate immune injuries by preserving the antioxidant capacity of the immune system. However, excessive Se worsens oxidative injuries and influences normal immune function. Thus, the immune-regulating role of Se was closely related to oxidative-reductive signals. Oxidative stress could trigger several types of physical or pathological signal cascades, such as the inflammation response, cytokine responses, apoptosis, and ER stress. Among these signaling pathways, ER stress may be one early or initial response of cells to stress or damage [14]. ER stress is involved in several types of cellular processes, including cell death, cell protection, signal regulation, disease development, or chemical toxicity [29–31]. Several reports have indicated the involvement of ER stress in the process of Se-related injuries. Yao showed that ER stress and oxidative injuries played important roles in chicken liver apoptosis induced by Se deficiency [3]. ER stress is also associated with cardiac malfunction induced by Se deficiency in a rat model [32]. In addition, Liu showed that Se can ameliorate the effects of Cd induced oxidative stress and ER stress in chicken kidney [17]. Similar to these reports, the present study showed that excessive Se supplementation also induced higher expression of ER stress markers, GRP78, and the ER stress-related signal cascade gene, ATF6. It is known that the release of GRP78 from IRE1, PERK, and ATF6 could induce downstream signal cascades, such as increases in the apoptosis gene CHOP, caspase 12, and cell death pathways [16]. Therefore, higher expression of the GRP78 gene was one indicator of ER stress. In the present study, we also detected changes in ER stress-related apoptosis genes, such as caspase 12, the apoptosis executor caspase 3, and the antiapoptosis gene Bcl 2. Excessive Se treatment induced higher expression of caspase 12 and caspase 3 but lower Bcl 2. So ER stress and ER stress-related apoptosis was induced by excessive Se supplementation in chicken spleen. The present study supported the idea that ER stress-related cellular injuries were related to Se toxicity. The interaction between oxidative stress and ER stress has been widely reported [16, 33]. ROS plays a central role in the crosstalk signal between ER stress and oxidative injuries [33, 34]. In the present study, we found that ER stress-related injuries also showed higher levels following increased oxidative stress. In addition, Pearson’s correlation results showed that there exists a highly positive correlation between ER stress markers, apoptosis, and MDA levels, but a negative
correlation with GPx and SOD activities. In the present study, oxidative stress and ER stress showed a high correlation, which was similar to previous reports. Thus, both oxidative stress and ER stress play important roles in immune injuries induced Se toxicity. In summary, the results showed that excessive Se intake induced chicken immune injuries. Excessive Se exposure induced higher oxidative stress and ER stress-related apoptosis. In this process, there is a high positive correlation between oxidative stress and ER stress, which showed the important role of oxidative stress and ER stress in Se-related immune injuries. Acknowledgments The present work was financially supported by Southwest University of Science and Technology (15zx7121) and Mianyang Science and Technology Project (14 N043). The authors thank the Elsevier English Language Editing System to correct grammatical, spelling, and other common errors. Compliance with ethical standards Conflict of Interest The authors declare that they have no competing interests.
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