Introduction

Aflatoxins are secondary toxic metabolites produced by the genus Aspergillus-coumarin derivatives containing a dihydrofurofuran moiety [1]. Aflatoxin B1 (AFB1), the most toxic form of aflatoxins, is frequently encountered in cereal crops and peanut meal [2, 3]. This toxin can disturb protein synthesis, and impede metabolic systems, resulting in hepatotoxic, mutagenic, and immunosuppressive effects on animals and humans [4,5,6,7,8].

The spleen, the most important periphery lymphoid organ, contains a large number of lymphocytes and macrophages, playing a crucial role in the cellular and humoral immunity [9]. Documents have revealed that AFB1 could impede the development of spleen [10, 11], inhibit the expression of cytokines [12], as well as led oxidative stress [13] and excessive apoptosis of splenocyte [14].

Selenium (Se), an essential element for the animal body, participates in the normal function of immune system [15,16,17]. Se could amend the disease process [18] and ameliorate the AFB1-induced negative effects [19, 20] including the hindered development and injured histological structure of immune organs, lowered T cell subset proportion, as well as declined antibody levels [17, 21,22,23].

However, no information on the ameliorative aspects of Se on AFB1-caused excess apoptosis in chicken splenocytes through endoplasmic reticulum and death receptor and pathways in vivo was available. This research aimed to explore these unknown areas by flow cytometry and TUNEL assays as well as qRT-PCR method. The outcomes from the present study may rich the knowledge of the detoxification mechanism of Se.

Materials and Methods

Animals and Diets

Two hundred sixteen healthy neonatal Cobb chickens (male) were purchased from a commercial rearing farm (Wenjiang poultry farm, Sichuan province, China) and were randomized into four dietary treatments: control (0 mg/kg AFB1), +Se (0.4 mg/kg supplement Se), AFB1 (0.6 mg/kg AFB1), and AFB1 + Se (0.6 mg/kg AFB1 + 0.4 mg/kg supplement Se), respectively. There were three replicates/treatment and 24 birds/replicate. The control diet was the corn-soybean basal diet and provided by the Animal Nutrition Institute of Sichuan Agricultural University (Chengdu, Sichuan, China). Nutritional requirements were sufficient in accordance with National Research Council (1994) (National Research Council, 1994) [24] and Chinese Feeding Standard of Chicken (NY/T33–2004) (Table 1). The 0.6 mg/kg AFB1 (Sigma-Aldrich, USA, A6636)-contaminated diets were produced based on the Kaoud’s report [25]. Furthermore, 1% sodium selenite (feed-grade) was mingled into the control diet by a stepwise dilution method to make +Se and AFB1 + Se diets with 0.4 mg/kg Se supplement. The Se content of control treatment diet was 0.332 mg/kg by the test of hydride-generation atomic absorption spectroscopy. The doses of AFB1 and Se supplement were determined on the basis of our early studies [26, 27]. Chickens in cages with electric heating installation were given water and the above described diets ad libitum throughout 21 days of experiment. Sichuan Agricultural University Animal Care and Use Committee approved this operation in this research involving animals.

Table 1 Composition of the basal diet
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Apoptosis Analysis by Flow Cytometry

The spleen tissues from six chickens in each treatment were obtained for determination of the apoptotic percentages by flow cytometry (BD FACSCalibur) on day 7, 14, and 21 of the experiment. Immediately, the sample splenocyte suspensions that could pass through the nylon screen with a 300-mesh were made by scissors. After being rinsed with ice-cold PBS, the suspensions were adjusted to be 1 × 106 cells/mL. One hundred microliters of cell suspensions with 5 μL Annexin V-Fluorescein isothiocyanate (V-FITC) and 5 μL propidium iodide (PI) were incubated for 15 min at 25 °C in the dark. After 400 μL 1 × Annexin-binding buffer was put into the mixture; the apoptotic percentages were immediately tested by flow cytometer. The annexin V-FITC Kit was obtained from BD Pharmingen (USA, 556547).

TUNEL Assay

On day 7, 14, and 21, six spleen tissues from each treatment were obtained, immediately fixed in 4% paraformaldehyde (PFA), and processed for the routine paraffin embedded section with 5 μm for TUNEL assay as reported by Zheng [28]. The numbers of TUNEL-positive cells in spleens were conducted by a digital microscope camera system (Nikon DS-Ri1, Japan) and Image-Pro Plus5.1 (USA) image analysis software. Five fields at × 400 magnification (corresponding to 0.064 mm2 per field) were randomly chosen in each section, and all TUNEL-positive cells showing yellow or brown nuclear were counted in each field of vision. The TUNEL cell apoptosis detection kit was obtained from QIA33, Merck, Germany.

Quantitative Real-Time PCR

On day 7, 14, and 21 of the experiment, six spleens from four treatments were taken and stored in liquid nitrogen. Total RNA of spleen was extracted by RNAiso Plus (9108/9109, Takara, Otsu, Japan) based on the manufacturer’s guide. The mRNA was reversely transcribed, and cDNA was formed by Prim-Script™ RT reagent Kit (RR047A, Takara, Japan). The cDNA as a template was used for PCR analysis. The β- actin gene expression value was chosen as calibration of gene expression tool in the control group. The results were analyzed using the 2-ΔΔCT method [29]. Sangon Biotech (Shanghai, China) provided the genes primers designed with Primer 5 (Table 2).

Table 2 Primer sequence for apoptotic genes
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Statistical Analysis

The statistical analyses were done by one-way analysis of variance and Dunnett T3 in SPSS 20.0 software (IBM Corp, Armonk, NY, USA) for windows. The data of this experiment were shown as mean ± standard deviation (X ± SD). Statistical significant differences were considered at p < 0.05 and markedly significant differences were considered at p < 0.01.

Results

Apoptosis Percentage of Splenocytes by Flow Cytometry

The apoptotic percentages of splenocytes were demonstrated in Fig. 1. The percentages of apoptosis in the AFB1 treatment were evidently raised during the experiment compared with the control treatment (p < 0.01). In comparison to the AFB1 treatment, the apoptotic percentages in the AFB1 + Se treatment were significantly dropped (p < 0.05) although they still kept obviously higher level compared with +Se or control treatments (p < 0.05 or 0.01). There was on evident difference in these parameters between the control and + Se treatments (p > 0.05).

Fig. 1
figure 1

The splenic cell apoptosis by flow cytometry analysis. a–d Representative scattergram of apoptotic splenocytes by flow cytometry assay in the control (a), +Se (b), AFB1 (c), and AFB1 + Se (d) treatments on 21 days of the experiment. e Apoptotic percentages of splenocytes by flow cytometry assay. Uppercase letters A, B, C, and D represent the significant difference (p < 0.01) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively. Lowercase letters a, b, c, and d represent difference (p < 0.05) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively.

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The Apoptosis of Splenocytes by TUNEL Assay

The apoptosis of splenocytes using TUNEL test was presented in Fig. 2. TUNEL-positive cells which showed yellow or brown nuclear were mainly distributed in the white pulp. The TUNEL-positive cell numbers in the AFB1 and AFB1 + Se treatments were significantly increased (p < 0.01) compared with the +Se and control treatments. However, in comparison to the AFB1 treatment, the TUNEL-positive cell numbers in the AFB1 + Se treatment were significantly declined (p < 0.05). These parameters had no obvious difference between the +Se and control treatments (p > 0.05).

Fig. 2
figure 2

The apoptosis splenocytes by TUNEL assay. a–d TUNEL-positive cells (TUNEL assay, Scale bar 50 μm) in the control (a), +Se (b), AFB1(c), and AFB1 + Se treatments (d) on day 21. e The numbers of TUNEL-positive cells (microscopic quantitative analysis). Uppercase letters A, B, C, and D represent the significant difference (p < 0.01) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively. Lowercase letters a, b, c, and d represent difference (p < 0.05) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively.

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The mRNA Expression of Apoptosis Regulatory Molecules by RT-qPCR

The mRNA expressions of CASP-3, CASP-8, CASP-10, TNF-α, TNF-R1, FAS, and FASL in spleen involved in death receptor pathway were exhibited in Fig. 3. When compared with the control and + Se treatments, the mRNA expressions of CASP-3, CASP-8, CASP-10, TNF-α, TNF-R1, FAS, and FASL in the AFB1 treatment were obviously increased during the experiment (p < 0.05 or p < 0.01). In comparison to the AFB1 treatment, these parameters in the AFB1 + Se treatment were obviously decreased (p < 0.05 or p < 0.01). No evident differences were observed in these gene expressions between the +Se and control treatments (p > 0.05).

Fig. 3
figure 3

The mRNA expression levels of FAS, FASL, TNF-a, TNF-R1, CASP-3, CASP-10, and CASP-8 of splenocytes (fold of control). Uppercase letters A, B, C, and D represent the significant difference (p < 0.01) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively. Lowercase letters a, b, c, and d represent difference (p < 0.05) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively.

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The mRNA expression levels of GRP78 and GRP94 related to endoplasmic reticulum pathway were demonstrated in Fig. 4. Compared with the control treatment, the gene expression levels of GRP78 and GRP94 in the AFB1 and AFB1+ Se treatments were evidently raised during the experiment (p < 0.05 or p < 0.01). However, the parameters in the AFB1 + Se treatment were evidently lower than those in the AFB1 treatment (p < 0.05 or p < 0.01). Furthermore, these parameters exhibited no obvious differences between the +Se and control treatments (p > 0.05).

Fig. 4
figure 4

The mRNA expression levels of GRP78 and GRP94 of splenocytes (fold of control). Uppercase letters A, B, C, and D represent the significant difference (p < 0.01) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively. Lowercase letters a, b, c, and d represent difference (p < 0.05) between the treatment and control, +Se, AFB1, and AFB1 + Se treatments, respectively.

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Discussions

As a crucial component in several vital metabolic pathways, Se displays an antioxidant effective oxygen-free radical scavenging, protects the organs and tissues from oxidative damage, and improves the immune function [30, 31]. Generally, Se is absorbed for animals by food as the main source. Increasing reports showed that Se can defend animal and humans from different toxic substances [30, 32].

Se can alleviate AFB1-induced damages in broilers’ immune system, including the growth retardation of immune organs [23], the declined percentages of T cell subsets in spleen and thymus [17, 21], the lowered contents of immunoglobulins in the small intestine [22], and the G2/M phase arrest of jejunum [26], along with the excess apoptosis in immune organs [17, 23, 33]. In this study, the flow cytometry results showed that apoptotic levels in the AFB1 treatment were obviously raised compared with the +Se and control treatments. And, these parameters in the AFB1+ Se treatment were significantly declined compared with the AFB1 treatment, although they were still evidently higher than those of the +Se and control treatments. In addition, the changing pattern of the apoptotic numbers using TUNEL staining was similar to that by flow cytometry assay. The above results indicated that dietary 0.4 mg/kg Se supplement partially alleviated excess apoptosis caused by 0.6 mg/kg AFB1, which was in accordance with previous researches in the chicken’s central immune organs [17, 23]. However, the alliterative mechanism of Se on the AFB1-induced excess apoptosis of splenocytes was required to be elucidated by further investigations. Meanwhile, the apoptosis rates in four groups by flow cytometry test showed an increasing trend with the age, while the TUNEL-positive cells’ numbers did not show such a tendency by age, which may be due to the fact that TUNEL staining mainly reveals the late apoptotic cells, whereas the flow cytometry detects both the early and late apoptotic cells.

Apoptosis is a modulated physiological process resulting in cell death in order to keep homeostasis. It is also an active course that contains a variety of actions such as activation, expression, and regulation of genes so as to accommodate to the living surroundings [34]. CASP-8, -9, -10, and -12 belong to pro-apoptotic molecules, while, CASP-3,-6, and -7 function as effector apoptotic molecules [35]. FAS and TNF-α binding with its ligand FASL and TNF-R1 respectively activate CASP- 8 and -10 and CASP-3, resulting in cell apoptosis [36, 37]. In this research, compared with the +Se and control treatments, the mRNA expressions of CASP-3, CASP-8, CASP-10, TNF-α, TNF-R1, FAS, and FASL in the AFB1 treatment were obviously increased. And, these gene expressions in the AFB1 + Se treatment were significantly decreased compared with the AFB1 treatment, although they were still evidently higher than those of the control treatment. Moreover, no obvious differences in these gene expressions between the +Se and control treatments were observed. These results implicated that supplement dietary Se with 0.4 mg/Kg could down regulate the gene expression of death receptor pathway molecules induced by AFB1.

The endoplasmic reticulum (ER) has many functions such as lipid biosynthesis and glycosylation, as well as protein folding and assembly [38]. Unfolded or misfolded proteins are accumulated in the ER lumen when the protein-folding capacity in the ER is overwhelmed, resulting in ER stress [39] and then the activation of CASP-12 mediated apoptosis [40]. These reactions will induce the expression of glucose 2-regulated protein 78kD (GRP78), GRP94, and other endoplasmic reticulum molecular chaperones which have protective roles and also lead to apoptosis independently [41,42,43]. In the present research, compared with the control treatment, the gene expressions of molecular chaperone GRP78 and GRP94 in the AFB1 treatment were evidently raised. However, these values in the AFB1 + Se treatment were significantly decreased in comparison to the AFB1 treatment. These results demonstrated that supplement dietary Se with 0.4 mg/kg could partially downregulate the gene expressions of GRP78 and GRP94 caused by AFB1.

In summary, supplement dietary Se with 0.4 mg/kg could partially alleviate AFB1-induced excess apoptosis in chicken splenocytes via downregulation of the gene expressions of endoplasmic reticulum and death receptor pathway molecules. The outcomes from the present study may enrich the knowledge of the detoxification mechanism of Se on AFB1-induced apoptosis.