Biol Trace Elem Res https://doi.org/10.1007/s12011-017-1186-9
The Protective Effect of Selenium on the Chicken Pancreas against Cadmium Toxicity via Alleviating Oxidative Stress and Autophagy Ruohan Liu 1 & Tiantian Jia 1 & Yuan Cui 1 & Hongjin Lin 1 & Shu Li 1
Received: 27 August 2017 / Accepted: 2 October 2017 # Springer Science+Business Media, LLC 2017
Abstract Cadmium (Cd) is a highly toxic heavy metal that can affect human and animal health. Selenium (Se) is an essential microelement that can protect various organs against toxic heavy metals. Although many studies have investigated the adverse effect of Cd in rats and several other animals, little is known regarding the mechanisms of Cd-induced autophagy in the chicken pancreas and the antagonistic effect of Se on Cd. In the current study, we fed chickens Se, Cd, or Se and Cd supplements to establish the Se and Cd interaction model and to measure the concentrations of Se and Cd in the chicken pancreas. The ultrastructure changes of the chicken pancreas were also observed, and we detected oxidative stress indexes in each group. The expression levels of autophagy-related genes were also examined. We found that Cd exposure could increase the concentration of Cd, the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); and the total antioxidant capacity (T-AOC) content in the chicken pancreas. The protein expression levels of dynein, Beclin1, LC3-1, LC3-2, and Atg5 were increased and that of TOR was decreased under Cd exposure conditions. However, the changes induced by Cd were significantly alleviated by Se. This study suggested that Cd could accumulate in the chicken pancreas and lead to oxidative stress and autophagy. Se was shown to antagonize Cd
All other authors have read the manuscript and agreed to submit it in its current form for consideration for publication in the Journal. * Hongjin Lin [email protected] * Shu Li [email protected] 1
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China
toxicity though reducing Cd accumulation, alleviating oxidative stress, and inhibiting autophagy. This study revealed a concrete mechanism for the Se antagonism of Cd and might provide a new clue for the detoxification of Cd poisoning. Keywords Cadmium . Selenium . Pancreas . Chickens . Oxidative stress . Autophagy
Introduction Cadmium (Cd) is a highly toxic heavy metal that can lead to acute and chronic Cd poisoning by accumulation in mammals, birds, and fish via the food chain [1, 2]. Cd can damage nerves, the liver, kidney, bones, and other organs [3–5]. The pancreas is one of the main target organs of Cd. Cd has been reported to increase the proportion of rat pancreatic α-cells and pancreatic β-cells, indicating that Cd could induce changes in pancreatic function [6]. Selenium (Se), an indispensable micronutrient in animals [7–9], is an antioxidant that can protect tissues against Cd toxicity [10]. Jamall found that Cd could decrease the activity of glutathione peroxidase (GSHPx) in the heart and that Se could alleviate this effect [11]. Se can also protect organs and tissues from oxidative damage and is beneficial to the immune system [12, 13]. Se is related to apoptosis and autophagy. Se has been shown to decrease the rate of apoptosis, restrict oxidative stress, and maintain cell survival through balancing anabolic metabolism and catabolism [10, 14, 15]. Autophagy is the chief process for recycling intracellular biomacromolecules and faulty cell components in eukaryotic cells [16]. The toxic effect of heavy metals is correlated to autophagy. It has been reported that autophagy plays a protective role in rat osteoblasts and that the ratio of LC3-II to LC3-I increases under Cd exposure conditions [17]. Gioacchino found that Cd induced autophagy in hematopoietic
Liu et al.
stem/progenitor cells [18]. Chiarelli found that Cd increased autophagy in sea urchin embryos [19]. Moreover, researchers have discovered that low doses of Cd for short periods of time increased proliferation and autophagyin proximal convoluted tubule cells [20]. Young-Ok Son reported that Cd increases autophagy by inhibiting the activity of mTOR, which is the chief regulatory factor of autophagy [21]. Researchers have found that Cd up-regulates the expression of autophagyrelated genes (such as ATG3, ATG5, ATG9, Beclin1, and P62) in LMH, while Se down-regulates the expression of autophagy-related genes. Se has been shown to regulate the expression of these genes by antagonizing Cd toxicity. In recent years, researchers have focused a great deal of attention on Cd toxicity, primarily in the liver, kidney, heart, and spleen [22–24]. The pancreas is an important organ in animals, but little research has been focused on this organ. Moreover, the protective effects of Se against Cd-induced autophagy in the pancreas remain to be determined. In the present investigation, the function of the pancreas was observed and the concentrations of Se and Cd were measured, as well as the level of oxidative stress. We determined the effects of Se against Cd-induced autophagy in chicken pancreases by ultrastructural observations and related gene expression. This experiment was conducted to provide new information on the detoxification of Cd.
Materials and Methods Birds, Diets, and Tissue Collection The experiments were conducted in accordance with the animal welfare and ethical protocols of the Committee of Northeast Agriculture University. A total of 128 Hy-Line Brown laying hens (31 weeks old) were used in the experiment. The laying hens were randomly divided into the following four groups: a normal control group containing 0.2 mg/kg Se, a Se-treated (+Se) group (normal group with Na2SeO3 total Se content of 2 mg/kg), a Cd-treated (+Cd) group (normal group with 150 mg/kg CdCl2), and a Se/Cd-treated (Se+Cd) group (total Se content of 2 and 150 mg/kg CdCl2). Each treatment group consisted of 32 laying hens that were housed in battery cages and received identical standard feeding and management. Water and food were provided ad libitum. The diets of each group are listed in Table 1. On the 90th day of the experiment, eight chickens in each group were selected randomly. Following euthanasia, the pancreas tissues were quickly removed, minced in sextuplicate and stored at − 80 °C to determine the index of oxidative stress and to isolate RNA and protein. The tissue used for total RNA extraction was soaked in an RNA locker for 24 h before storage at − 80 °C.
Ultrastructural Observations The pancreas tissue specimens were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 °C, washed in the same buffer for 1 h at 4 °C, and postfixed with 1% osmium tetroxide in sodium phosphate buffer for 1 h at 4 °C. The tissues were then dehydrated in a graded series of ethanol starting at 50% for 10 min after two changes in propylene oxide. The tissue specimens were embedded in araldite. Serial sections of each pancreas were cut into ultrathin sections and stained with Mg-uranyl acetate and then lead citrate for transmission electron microscope evaluation. Determination of Se and Cd Concentrations in the Pancreas The concentrations of elements (Se and Cd) in the chicken pancreas were determined using inductively coupled plasma mass spectrometry (ICP-MS, iCAP Q, Thermo, USA). The instrumental parameters were summarized in Table 2. The element concentrations were determined on the acid digest of the samples. Briefly, 1.00 g of each sample was digested with a solution of 5 ml of HNO3 (65%) and 2 ml of H2O2 (30%) and then diluted to a final volume of 10 ml with deionized water. All of the sample solutions were clear. Samples were microwave digested according to the following method: 3 min at 1800 W at 100 °C; 10 min at 1800 W at 150 °C; and 45 min at 1800 W at 180 °C. A blank digestion was carried out in the same manner. All digested samples were diluted with ultrapure water to a final volume of 50 ml and mixed well before ICP-MS analysis. Quantification of Autophagy Gene mRNA Expression Total RNA was isolated by the TRIZOL method (Invitrogen, China). An ultraviolet spectrophotometer was used to measure the OD at 260 and 280 nm to determine sample concentration and purity. The A260/A280 ratio was above 1.6. After quantification, the expression levels of autophagy genes were determined by quantitative reverse transcription PCR using SYBR Premix ExTaq TM (Takara, China) and an ABI PRISM 7500 real-time PCR system (Applied Biosystems). The PCR primers (Table 3) were designed using Oligo Primer Analysis software (version 6.0) and synthesized by Invitrogen (Shanghai, China). The PCR reactions consisted of the following: 10 ml of 2Â SYBR Green I PCR Master Mix (TaKaRa, China), 0.4 ml of 50 Â ROX reference Dye II, 0.4 ml of each primer (10 mM), 2 ml of diluted cDNA, and 6.8 ml of PCR-grade water. The PCR program for amplification of GAPDH was 95 °C for 30 s followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. The results (fold changes) were expressed as 2 −ΔΔCt in which ΔΔCt = (Ct LC3-
The Protective Effect of Selenium on the Chicken Pancreas against Cadmium Toxicity via Alleviating... Table 1 Feeding diets Groups
Diets
Control (basic diet) +Se +Cd Se+Cd
2 mg/kg of Na2SeO3 Basic diet + 2 mg/kg of Na2SeO3 Basic diet + 150 mg/kg of CdCl2 Basic diet + 150 mg/kg of CdCl2 + 2 mg/kg of Na 2SeO3
1 −Ct GAPDH )t − (Ct LC3-1 −Ct GAPDH )c, where Ct LC3-1 and CtGAPDH are the cycle thresholds for chicken LC3-1 and GAPDH genes in the different treatment groups.
Measurement of Reactive Oxygen Stress in the Pancreas The procedures for measuring reactive oxygen stress were conducted according to kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The reactive oxygen species were total superoxide dismutase (T-SOD) (U/mg prot), catalase (CAT) (U/mg prot), malondialdehyde (MDA) (nmol/mg prot), glutathione peroxidase (GSH-Px) (U/mg prot), and total antioxidant capacity (T-AOC) (U/mg prot).
Ultrastructural Observations The effects of Se and Cd on the histopathology of the pancreas are shown in Fig. 1. No obvious ultrastructural changes were observed in the control group and in the +Se group. The nuclei have a distinct outline, and the structure of cytoplasmic mitochondria is clear and complete. The +Cd group showed abnormal ultrastructures with visible autophagy (red arrows). The vesicles increased, and the mitochondria swelled and dissolved into vacuoles. However, the pathological changes in the Se+Cd group were less serious than those in the +Cd group. Reactive Oxygen Stress in the Pancreas
Data Analysis All data analysis is described in SPSS for Windows (SPSS, Chicago, IL, USA). Data are expressed as the mean±standard deviation. All histograms were drawn using the GraphPad Prism 5 for Windows. P < 0.05 was considered statistically significant.
Oxidative stress indexes were measured using Reagent Kits (Nanjing Jiancheng Bioengineering Institute, China). The activities of T-SOD, CAT, MDA, GSH-Px and T-AOC, and the MDA content in each group are shown in Table 5. The Expression of Autophagy-Related Genes The effects of Se and Cd on the mRNA expression levels of autophagy-related genes (dynein, Beclin 1, LC3-1, LC3-2, TOR, and Atg5) in chicken pancreases were examined by real-time PCR (Fig. 2). The +Se group showed no significant
Results Determination of Se and Cd Concentrations in the Pancreas
Table 3
ICP-MS analytical technology was used to analyze the concentrations of Se and Cd in the chicken pancreases. The data are presented in Table 4. Table 2 Instrumental parameters for the ICPMS
Parameters Frequency (MHz) Reflect power (kW) Sampling depth (mm) Torch-H (mm) Torch-V (mm) Carrier gas (L/min) Nebulizer pump (rpm) S/C temperature (°C) Oxide ions (156/140) Doubly charged (70/140)
27.12 1.55 5.0 0.01 − 0.39 1.05 40 2.7 < 2.0% < 3.0%
The primers used for quantitative RT-PCR PCR primers
LC3-I LC3-II ATG5 TOR Beclin1 Dynein GADPH
Forward 5′-TTACACCCATATCAGATTCTTG-3′ Reverse 5′-ATTCCAACCTGTCCCTCA-3′ Forward 5′-AGTGAAGTGTAGCAGGATGA-3′ Reverse 5′-AAGCCTTGTGAACGAGAT-3′ Forward 5′-GGCACCGACCGATTTAGT-3′ Reverse 5′-GCTGATGGGTTTGCTTTT-3′ Forward 5′-GGACTCTTCCCTGCTGGCTAA-3′ Reverse 5′-TACGGGTGCCCTGGTTCTG-3′ Forward 5′-CGACTGGAGCAGGAAGAAG-3′ Reverse 5′-TCTGAGCATAACGCATCTGG-3’ Forward 5′-CGGCTTGACCTATGGAATCT-3′ Reverse 5′-CATCACTGCGAGGAACTGC-3′ Forward 5′-AGAACATCATCCCAGCGT-3′ Reverse 5′-AGCCTTCACTACCCTCTTG -3′
Liu et al. Table 4
Determination of Se and Cd content in the pancreas Se (μg/kg)
Cd (μg/kg)
Control
225.538 ± 16.352
32.938 ± 4.108
Se
375.113 ± 90.726
Se+Cd Cd
278.655 ± 23.038*a 203.283 ± 22.531*b
15.390 ± 2.975 28,504.870 ± 2514.480*a 20,830.847 ± 188.195*b
*Means a significant different from the control group (P < 0.05), while the different capital letter means a significant different from the adjacent data in same column
changes compared with the control group (P > 0.05). Compared with the control group, there is a significant increase in the mRNA expression levels of dynein, Beclin-1, LC3-I, LC3-II, and Atg5, but the expression level of TOR was decreased (P < 0.05). Dynein, Beclin-1, LC3-I, LC3-II, and Atg5 expression levels in the +Cd group were higher than in the Se+Cd group. However, the expression of TOR in the +Cd group was lower than in the Se+Cd group (P < 0.05).
Discussion Se is an essential micronutrient [25]. Various studies have shown that Se can protect tissue against Cd toxicity and reduce Cd accumulation in organs [26]. Previous research has shown that Cd exposure could trigger severe damage in Fig. 1 The effects of Se and Cd on the histopathology of the pancreas. a Pancreas in the control group. b Pancreas in the +Se group. c Pancreas in the Se+ Cd group. d Pancreas in the +Cd group. No obvious ultrastructural changes were observed in the control group and the +Se group. The +Cd group showed abnormal ultrastructures with visible autophagosomes (red arrows). The pathological changes in the Se+Cd group were less severe than those in the +Cd group
chicken spleens, livers, kidneys, and brains and that Se could antagonize Cd toxicity [24, 27, 28]. Furthermore, Cd has been shown to accumulate in the pancreas and adversely affect animal health and this Cd-induced response can be alleviated by Se [29]. Our data showed that Cd concentrations in the +Cd group were higher than in the control group and that the Cd concentrations in the Se+Cd group were much lower than that in the +Cd group. These results suggested that Se could antagonize Cd toxicity by reducing Cd accumulation in the chicken pancreas. Se is a necessary component of GSH-Px. Se deficiency may trigger a decrease in GSH-Px activity. Excess Se intake has been reported to inhibit GSH-Px activity and cause damage to multiple aspects of the body [30, 31]. B.I. Ognjanović notes that chronic Cd poisoning decreases SOD and GSH-Px activities in rat livers and kidneys, but the activity of CAT decreases in the liver and shows no remarkable change in the kidney. Se reduces the content of Cd in tissues by forming compounds and antagonizing Cd-induced oxidative stress [14]. In piglet Sertoli cells, a significant decrease in SOD, T-AOC, and GSH-Px activities and a significant increase in MDA levels were observed when the Cd content in the cells was 10 mg/kg [32]. Consistent with this research, our study revealed decreases in the +Cd group in antioxidant activity markers (CAT, SOD, GSH-Px, and T-AOC) and an increase in MDA levels. However, these Cd-induced changes were relieved in the Se+Cd group. Our study revealed that Se could antagonize Cd toxicity by reducing oxidative stress in the chicken pancreas.
The Protective Effect of Selenium on the Chicken Pancreas against Cadmium Toxicity via Alleviating... Table 5 Reactive oxygen indexes T-SOD (U/mg prot) CAT (U/mg prot) MDA (nmol/mg prot) GSH-Px (U/mg prot) T-AOC (U/mg prot)
Control
Se
Cd
Se+Cd
126.34 ± 2.69 17.59 ± 3.71 2.42 ± 1.30 18.03 ± 0.66 6.63 ± 2.59
133.78 ± 4.77 18.40 ± 2.35 2.59 ± 1.14 19.83 ± 0.98 7.00 ± 0.99
41.71 ± 1.48*a 11.26 ± 3.43*a 3.40 ± 0.75*a 9.51 ± 0.96*a 4.61 ± 1.64*a
48.279 ± 3.10*b 14.20 ± 3.33*b 2.63 ± 1.84* b 15.04 ± 2.56*b 5.30 ± 3.44*b
*Means a significant different from the control group (P < 0.05), while the different capital letter means a significant different from the adjacent data in same row
In recent years, more scholars have focused on autophagy [33]. The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, which plays a central role in the negative regulation of autophagy. Moreover, mTOR regulates autophagy under oxidative stress via the activation of Akt [34]. Several researchers have found that during starvation, mTOR, a nutrient-responsive kinase, was inhibited and that this process induced autophagy [35]. Wilson found that mTOR signal transduction pathway has a significant effect
on regulating autophagy in type II diabetes mellitus [36]. Beclin-1, the earliest known tumor suppressor gene associated with autophagy, participates in the regulation of autophagy and has an important role in development [37]. LC3-I, LC3-II, and Atg5 participate in autophagosome formation. LC3-I can convert into LC3-II during autophagy [38]. Atg5 forms a complex with the multimeric protein and promotes LC3 recruitment to autophagosomes by association with autophagosome membranes [39]. The effect of Se
Fig. 2 The effects of Se and Cd on mRNA and protein expression of autophagy-related genes (dynein, Beclin 1, LC3-1, LC3-2, TOR, and Atg5) in the chicken pancreas. Each value represents the mean±SD
(n = 5/group). Means with different letters represent statistically significant values (P < 0.05); the bars with a common letter are not significantly different (P > 0.05)
Liu et al.
deficiency in rats has been reported by Liu who indicated that Se deficiency induced an increase in the expression of LC3-I, LC3-II, and Atg5, resulting in the promotion of autophagy [40]. YJ Wang reported that autophagy and the expression levels of Beclin1 and LC3-II increased with Cd exposure [41]. Dynein is a family of cytoskeletal motor proteins. Several researchers have indicated that dynein plays an important role in transporting autophagosomes to lysosomes in the process of autophagosome-lysosome fusion [42, 43]. Ravikumar showed an increase in autophagosome marker levels and impaired autophagosome-lysosome fusion in both cell culture and mouse models [44]. In this study, the expression of Beclin1, LC3-I, LC3-II, Atg5, and dynein genes were increased and mTOR was decreased. Furthermore, the changes in autophagy-related genes in the +Cd group were reduced in the Se+Cd group, suggesting that Se reduced Cd-induced autophagy. The histopathology of the pancreas was also examined and revealed that Cd increased autophagy in the chicken pancreas and that Se co-treatment exerted a protective effect against Cd. In conclusion, Cd could accumulate in the chicken pancreas and lead to oxidative stress and autophagy. Se could antagonize Cd toxicity in the chicken pancreas by reducing Cd accumulation, alleviating oxidative stress, and inhibiting autophagy. This study showed a concrete mechanism by which Se antagonizes Cd and might provide a new clue to the detoxification of Cd poisoning.
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Acknowledgements and Grant Support The authors extend their sincere thanks to the Student Innovation Practical Training (No.201610224044) for supporting this research.
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The Protective Effect of Selenium on the Chicken Pancreas against Cadmium Toxicity via Alleviating Oxidative Stress and Autophagy Ruohan Liu 1 & Tiantian Jia 1 & Yuan Cui 1 & Hongjin Lin 1 & Shu Li 1
Received: 27 August 2017 / Accepted: 2 October 2017 # Springer Science+Business Media, LLC 2017
Abstract Cadmium (Cd) is a highly toxic heavy metal that can affect human and animal health. Selenium (Se) is an essential microelement that can protect various organs against toxic heavy metals. Although many studies have investigated the adverse effect of Cd in rats and several other animals, little is known regarding the mechanisms of Cd-induced autophagy in the chicken pancreas and the antagonistic effect of Se on Cd. In the current study, we fed chickens Se, Cd, or Se and Cd supplements to establish the Se and Cd interaction model and to measure the concentrations of Se and Cd in the chicken pancreas. The ultrastructure changes of the chicken pancreas were also observed, and we detected oxidative stress indexes in each group. The expression levels of autophagy-related genes were also examined. We found that Cd exposure could increase the concentration of Cd, the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); and the total antioxidant capacity (T-AOC) content in the chicken pancreas. The protein expression levels of dynein, Beclin1, LC3-1, LC3-2, and Atg5 were increased and that of TOR was decreased under Cd exposure conditions. However, the changes induced by Cd were significantly alleviated by Se. This study suggested that Cd could accumulate in the chicken pancreas and lead to oxidative stress and autophagy. Se was shown to antagonize Cd
All other authors have read the manuscript and agreed to submit it in its current form for consideration for publication in the Journal. * Hongjin Lin [email protected] * Shu Li [email protected] 1
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China
toxicity though reducing Cd accumulation, alleviating oxidative stress, and inhibiting autophagy. This study revealed a concrete mechanism for the Se antagonism of Cd and might provide a new clue for the detoxification of Cd poisoning. Keywords Cadmium . Selenium . Pancreas . Chickens . Oxidative stress . Autophagy
Introduction Cadmium (Cd) is a highly toxic heavy metal that can lead to acute and chronic Cd poisoning by accumulation in mammals, birds, and fish via the food chain [1, 2]. Cd can damage nerves, the liver, kidney, bones, and other organs [3–5]. The pancreas is one of the main target organs of Cd. Cd has been reported to increase the proportion of rat pancreatic α-cells and pancreatic β-cells, indicating that Cd could induce changes in pancreatic function [6]. Selenium (Se), an indispensable micronutrient in animals [7–9], is an antioxidant that can protect tissues against Cd toxicity [10]. Jamall found that Cd could decrease the activity of glutathione peroxidase (GSHPx) in the heart and that Se could alleviate this effect [11]. Se can also protect organs and tissues from oxidative damage and is beneficial to the immune system [12, 13]. Se is related to apoptosis and autophagy. Se has been shown to decrease the rate of apoptosis, restrict oxidative stress, and maintain cell survival through balancing anabolic metabolism and catabolism [10, 14, 15]. Autophagy is the chief process for recycling intracellular biomacromolecules and faulty cell components in eukaryotic cells [16]. The toxic effect of heavy metals is correlated to autophagy. It has been reported that autophagy plays a protective role in rat osteoblasts and that the ratio of LC3-II to LC3-I increases under Cd exposure conditions [17]. Gioacchino found that Cd induced autophagy in hematopoietic
Liu et al.
stem/progenitor cells [18]. Chiarelli found that Cd increased autophagy in sea urchin embryos [19]. Moreover, researchers have discovered that low doses of Cd for short periods of time increased proliferation and autophagyin proximal convoluted tubule cells [20]. Young-Ok Son reported that Cd increases autophagy by inhibiting the activity of mTOR, which is the chief regulatory factor of autophagy [21]. Researchers have found that Cd up-regulates the expression of autophagyrelated genes (such as ATG3, ATG5, ATG9, Beclin1, and P62) in LMH, while Se down-regulates the expression of autophagy-related genes. Se has been shown to regulate the expression of these genes by antagonizing Cd toxicity. In recent years, researchers have focused a great deal of attention on Cd toxicity, primarily in the liver, kidney, heart, and spleen [22–24]. The pancreas is an important organ in animals, but little research has been focused on this organ. Moreover, the protective effects of Se against Cd-induced autophagy in the pancreas remain to be determined. In the present investigation, the function of the pancreas was observed and the concentrations of Se and Cd were measured, as well as the level of oxidative stress. We determined the effects of Se against Cd-induced autophagy in chicken pancreases by ultrastructural observations and related gene expression. This experiment was conducted to provide new information on the detoxification of Cd.
Materials and Methods Birds, Diets, and Tissue Collection The experiments were conducted in accordance with the animal welfare and ethical protocols of the Committee of Northeast Agriculture University. A total of 128 Hy-Line Brown laying hens (31 weeks old) were used in the experiment. The laying hens were randomly divided into the following four groups: a normal control group containing 0.2 mg/kg Se, a Se-treated (+Se) group (normal group with Na2SeO3 total Se content of 2 mg/kg), a Cd-treated (+Cd) group (normal group with 150 mg/kg CdCl2), and a Se/Cd-treated (Se+Cd) group (total Se content of 2 and 150 mg/kg CdCl2). Each treatment group consisted of 32 laying hens that were housed in battery cages and received identical standard feeding and management. Water and food were provided ad libitum. The diets of each group are listed in Table 1. On the 90th day of the experiment, eight chickens in each group were selected randomly. Following euthanasia, the pancreas tissues were quickly removed, minced in sextuplicate and stored at − 80 °C to determine the index of oxidative stress and to isolate RNA and protein. The tissue used for total RNA extraction was soaked in an RNA locker for 24 h before storage at − 80 °C.
Ultrastructural Observations The pancreas tissue specimens were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 °C, washed in the same buffer for 1 h at 4 °C, and postfixed with 1% osmium tetroxide in sodium phosphate buffer for 1 h at 4 °C. The tissues were then dehydrated in a graded series of ethanol starting at 50% for 10 min after two changes in propylene oxide. The tissue specimens were embedded in araldite. Serial sections of each pancreas were cut into ultrathin sections and stained with Mg-uranyl acetate and then lead citrate for transmission electron microscope evaluation. Determination of Se and Cd Concentrations in the Pancreas The concentrations of elements (Se and Cd) in the chicken pancreas were determined using inductively coupled plasma mass spectrometry (ICP-MS, iCAP Q, Thermo, USA). The instrumental parameters were summarized in Table 2. The element concentrations were determined on the acid digest of the samples. Briefly, 1.00 g of each sample was digested with a solution of 5 ml of HNO3 (65%) and 2 ml of H2O2 (30%) and then diluted to a final volume of 10 ml with deionized water. All of the sample solutions were clear. Samples were microwave digested according to the following method: 3 min at 1800 W at 100 °C; 10 min at 1800 W at 150 °C; and 45 min at 1800 W at 180 °C. A blank digestion was carried out in the same manner. All digested samples were diluted with ultrapure water to a final volume of 50 ml and mixed well before ICP-MS analysis. Quantification of Autophagy Gene mRNA Expression Total RNA was isolated by the TRIZOL method (Invitrogen, China). An ultraviolet spectrophotometer was used to measure the OD at 260 and 280 nm to determine sample concentration and purity. The A260/A280 ratio was above 1.6. After quantification, the expression levels of autophagy genes were determined by quantitative reverse transcription PCR using SYBR Premix ExTaq TM (Takara, China) and an ABI PRISM 7500 real-time PCR system (Applied Biosystems). The PCR primers (Table 3) were designed using Oligo Primer Analysis software (version 6.0) and synthesized by Invitrogen (Shanghai, China). The PCR reactions consisted of the following: 10 ml of 2Â SYBR Green I PCR Master Mix (TaKaRa, China), 0.4 ml of 50 Â ROX reference Dye II, 0.4 ml of each primer (10 mM), 2 ml of diluted cDNA, and 6.8 ml of PCR-grade water. The PCR program for amplification of GAPDH was 95 °C for 30 s followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. The results (fold changes) were expressed as 2 −ΔΔCt in which ΔΔCt = (Ct LC3-
The Protective Effect of Selenium on the Chicken Pancreas against Cadmium Toxicity via Alleviating... Table 1 Feeding diets Groups
Diets
Control (basic diet) +Se +Cd Se+Cd
2 mg/kg of Na2SeO3 Basic diet + 2 mg/kg of Na2SeO3 Basic diet + 150 mg/kg of CdCl2 Basic diet + 150 mg/kg of CdCl2 + 2 mg/kg of Na 2SeO3
1 −Ct GAPDH )t − (Ct LC3-1 −Ct GAPDH )c, where Ct LC3-1 and CtGAPDH are the cycle thresholds for chicken LC3-1 and GAPDH genes in the different treatment groups.
Measurement of Reactive Oxygen Stress in the Pancreas The procedures for measuring reactive oxygen stress were conducted according to kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The reactive oxygen species were total superoxide dismutase (T-SOD) (U/mg prot), catalase (CAT) (U/mg prot), malondialdehyde (MDA) (nmol/mg prot), glutathione peroxidase (GSH-Px) (U/mg prot), and total antioxidant capacity (T-AOC) (U/mg prot).
Ultrastructural Observations The effects of Se and Cd on the histopathology of the pancreas are shown in Fig. 1. No obvious ultrastructural changes were observed in the control group and in the +Se group. The nuclei have a distinct outline, and the structure of cytoplasmic mitochondria is clear and complete. The +Cd group showed abnormal ultrastructures with visible autophagy (red arrows). The vesicles increased, and the mitochondria swelled and dissolved into vacuoles. However, the pathological changes in the Se+Cd group were less serious than those in the +Cd group. Reactive Oxygen Stress in the Pancreas
Data Analysis All data analysis is described in SPSS for Windows (SPSS, Chicago, IL, USA). Data are expressed as the mean±standard deviation. All histograms were drawn using the GraphPad Prism 5 for Windows. P < 0.05 was considered statistically significant.
Oxidative stress indexes were measured using Reagent Kits (Nanjing Jiancheng Bioengineering Institute, China). The activities of T-SOD, CAT, MDA, GSH-Px and T-AOC, and the MDA content in each group are shown in Table 5. The Expression of Autophagy-Related Genes The effects of Se and Cd on the mRNA expression levels of autophagy-related genes (dynein, Beclin 1, LC3-1, LC3-2, TOR, and Atg5) in chicken pancreases were examined by real-time PCR (Fig. 2). The +Se group showed no significant
Results Determination of Se and Cd Concentrations in the Pancreas
Table 3
ICP-MS analytical technology was used to analyze the concentrations of Se and Cd in the chicken pancreases. The data are presented in Table 4. Table 2 Instrumental parameters for the ICPMS
Parameters Frequency (MHz) Reflect power (kW) Sampling depth (mm) Torch-H (mm) Torch-V (mm) Carrier gas (L/min) Nebulizer pump (rpm) S/C temperature (°C) Oxide ions (156/140) Doubly charged (70/140)
27.12 1.55 5.0 0.01 − 0.39 1.05 40 2.7 < 2.0% < 3.0%
The primers used for quantitative RT-PCR PCR primers
LC3-I LC3-II ATG5 TOR Beclin1 Dynein GADPH
Forward 5′-TTACACCCATATCAGATTCTTG-3′ Reverse 5′-ATTCCAACCTGTCCCTCA-3′ Forward 5′-AGTGAAGTGTAGCAGGATGA-3′ Reverse 5′-AAGCCTTGTGAACGAGAT-3′ Forward 5′-GGCACCGACCGATTTAGT-3′ Reverse 5′-GCTGATGGGTTTGCTTTT-3′ Forward 5′-GGACTCTTCCCTGCTGGCTAA-3′ Reverse 5′-TACGGGTGCCCTGGTTCTG-3′ Forward 5′-CGACTGGAGCAGGAAGAAG-3′ Reverse 5′-TCTGAGCATAACGCATCTGG-3’ Forward 5′-CGGCTTGACCTATGGAATCT-3′ Reverse 5′-CATCACTGCGAGGAACTGC-3′ Forward 5′-AGAACATCATCCCAGCGT-3′ Reverse 5′-AGCCTTCACTACCCTCTTG -3′
Liu et al. Table 4
Determination of Se and Cd content in the pancreas Se (μg/kg)
Cd (μg/kg)
Control
225.538 ± 16.352
32.938 ± 4.108
Se
375.113 ± 90.726
Se+Cd Cd
278.655 ± 23.038*a 203.283 ± 22.531*b
15.390 ± 2.975 28,504.870 ± 2514.480*a 20,830.847 ± 188.195*b
*Means a significant different from the control group (P < 0.05), while the different capital letter means a significant different from the adjacent data in same column
changes compared with the control group (P > 0.05). Compared with the control group, there is a significant increase in the mRNA expression levels of dynein, Beclin-1, LC3-I, LC3-II, and Atg5, but the expression level of TOR was decreased (P < 0.05). Dynein, Beclin-1, LC3-I, LC3-II, and Atg5 expression levels in the +Cd group were higher than in the Se+Cd group. However, the expression of TOR in the +Cd group was lower than in the Se+Cd group (P < 0.05).
Discussion Se is an essential micronutrient [25]. Various studies have shown that Se can protect tissue against Cd toxicity and reduce Cd accumulation in organs [26]. Previous research has shown that Cd exposure could trigger severe damage in Fig. 1 The effects of Se and Cd on the histopathology of the pancreas. a Pancreas in the control group. b Pancreas in the +Se group. c Pancreas in the Se+ Cd group. d Pancreas in the +Cd group. No obvious ultrastructural changes were observed in the control group and the +Se group. The +Cd group showed abnormal ultrastructures with visible autophagosomes (red arrows). The pathological changes in the Se+Cd group were less severe than those in the +Cd group
chicken spleens, livers, kidneys, and brains and that Se could antagonize Cd toxicity [24, 27, 28]. Furthermore, Cd has been shown to accumulate in the pancreas and adversely affect animal health and this Cd-induced response can be alleviated by Se [29]. Our data showed that Cd concentrations in the +Cd group were higher than in the control group and that the Cd concentrations in the Se+Cd group were much lower than that in the +Cd group. These results suggested that Se could antagonize Cd toxicity by reducing Cd accumulation in the chicken pancreas. Se is a necessary component of GSH-Px. Se deficiency may trigger a decrease in GSH-Px activity. Excess Se intake has been reported to inhibit GSH-Px activity and cause damage to multiple aspects of the body [30, 31]. B.I. Ognjanović notes that chronic Cd poisoning decreases SOD and GSH-Px activities in rat livers and kidneys, but the activity of CAT decreases in the liver and shows no remarkable change in the kidney. Se reduces the content of Cd in tissues by forming compounds and antagonizing Cd-induced oxidative stress [14]. In piglet Sertoli cells, a significant decrease in SOD, T-AOC, and GSH-Px activities and a significant increase in MDA levels were observed when the Cd content in the cells was 10 mg/kg [32]. Consistent with this research, our study revealed decreases in the +Cd group in antioxidant activity markers (CAT, SOD, GSH-Px, and T-AOC) and an increase in MDA levels. However, these Cd-induced changes were relieved in the Se+Cd group. Our study revealed that Se could antagonize Cd toxicity by reducing oxidative stress in the chicken pancreas.
The Protective Effect of Selenium on the Chicken Pancreas against Cadmium Toxicity via Alleviating... Table 5 Reactive oxygen indexes T-SOD (U/mg prot) CAT (U/mg prot) MDA (nmol/mg prot) GSH-Px (U/mg prot) T-AOC (U/mg prot)
Control
Se
Cd
Se+Cd
126.34 ± 2.69 17.59 ± 3.71 2.42 ± 1.30 18.03 ± 0.66 6.63 ± 2.59
133.78 ± 4.77 18.40 ± 2.35 2.59 ± 1.14 19.83 ± 0.98 7.00 ± 0.99
41.71 ± 1.48*a 11.26 ± 3.43*a 3.40 ± 0.75*a 9.51 ± 0.96*a 4.61 ± 1.64*a
48.279 ± 3.10*b 14.20 ± 3.33*b 2.63 ± 1.84* b 15.04 ± 2.56*b 5.30 ± 3.44*b
*Means a significant different from the control group (P < 0.05), while the different capital letter means a significant different from the adjacent data in same row
In recent years, more scholars have focused on autophagy [33]. The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, which plays a central role in the negative regulation of autophagy. Moreover, mTOR regulates autophagy under oxidative stress via the activation of Akt [34]. Several researchers have found that during starvation, mTOR, a nutrient-responsive kinase, was inhibited and that this process induced autophagy [35]. Wilson found that mTOR signal transduction pathway has a significant effect
on regulating autophagy in type II diabetes mellitus [36]. Beclin-1, the earliest known tumor suppressor gene associated with autophagy, participates in the regulation of autophagy and has an important role in development [37]. LC3-I, LC3-II, and Atg5 participate in autophagosome formation. LC3-I can convert into LC3-II during autophagy [38]. Atg5 forms a complex with the multimeric protein and promotes LC3 recruitment to autophagosomes by association with autophagosome membranes [39]. The effect of Se
Fig. 2 The effects of Se and Cd on mRNA and protein expression of autophagy-related genes (dynein, Beclin 1, LC3-1, LC3-2, TOR, and Atg5) in the chicken pancreas. Each value represents the mean±SD
(n = 5/group). Means with different letters represent statistically significant values (P < 0.05); the bars with a common letter are not significantly different (P > 0.05)
Liu et al.
deficiency in rats has been reported by Liu who indicated that Se deficiency induced an increase in the expression of LC3-I, LC3-II, and Atg5, resulting in the promotion of autophagy [40]. YJ Wang reported that autophagy and the expression levels of Beclin1 and LC3-II increased with Cd exposure [41]. Dynein is a family of cytoskeletal motor proteins. Several researchers have indicated that dynein plays an important role in transporting autophagosomes to lysosomes in the process of autophagosome-lysosome fusion [42, 43]. Ravikumar showed an increase in autophagosome marker levels and impaired autophagosome-lysosome fusion in both cell culture and mouse models [44]. In this study, the expression of Beclin1, LC3-I, LC3-II, Atg5, and dynein genes were increased and mTOR was decreased. Furthermore, the changes in autophagy-related genes in the +Cd group were reduced in the Se+Cd group, suggesting that Se reduced Cd-induced autophagy. The histopathology of the pancreas was also examined and revealed that Cd increased autophagy in the chicken pancreas and that Se co-treatment exerted a protective effect against Cd. In conclusion, Cd could accumulate in the chicken pancreas and lead to oxidative stress and autophagy. Se could antagonize Cd toxicity in the chicken pancreas by reducing Cd accumulation, alleviating oxidative stress, and inhibiting autophagy. This study showed a concrete mechanism by which Se antagonizes Cd and might provide a new clue to the detoxification of Cd poisoning.
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Acknowledgements and Grant Support The authors extend their sincere thanks to the Student Innovation Practical Training (No.201610224044) for supporting this research.
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