Introduction

Se is a trace element that plays an important role in animal and human immune function. Long-term Se deficiency decreases animal serum antibody concentrations, reduces disease resistance, and significantly suppresses the immune response [1]. The content of Se in the diet regulates the activity of phagocytic cells to kill microorganisms [2]. Animals fed with Se-deficient diets have weakened organismal resistance, a reduced B lymphocyte population, and a decreased response of lymphocyte blastogenesis to mitogens [3, 4]. Fat is a necessary substance to maintain human and animal health, and it plays a role in maintaining cell structure and function, protecting the body’s tissues, providing energy, and other functions. However, studies indicate that excessive intake of fat not only causes obesity but also promotes the production of inflammatory factors [5, 6]. High-fat diet can cause changes in the expression of systemic inflammatory factors, and inflammatory factors such as IFN-γ can regulate adaptive immunity in obesity [7, 8]. Long-term high-fat diet also interferes with insulin signaling pathways, leading to peripheral insulin resistance and immune disorders [9]. Blastogenesis of spleen lymphocytes was markedly inhibited when rats were fed with a high-fat diet [10]. A high-fat diet also significantly decreased the number of regulatory T cells in obese adipose tissue and led to an inflammatory state with insulin resistance [11].

HSPs and cytokines are reliable indicators of changes in immune status [12]. It has been demonstrated that Se deficiency causes higher expression of HSPs and leads to oxidative stress [13]. Immune function damage also significantly changes HSP expression [14, 15]. Also, lack of Se induced the production of immune-suppressive factors and promoted the release of oxygen metabolites [16]. Se deficiency can cause inflammation, thus increasing the expression of pro-inflammatory factors (IL-1γ, IL-6, and IL-7) and damaging the immune system [17]. The same phenomenon occurred in rats, for example, levels of IL-6, IL-1β, and TNF-α increased when rats were fed with a Se-deficient diet [18]. Studies have shown that selenium can regulate the changes in animals [19]. Selw served as an antioxidant in chicken myoblasts [20]. Selk knockout exhibited specific immune cell defects in mice [21]. In terms of immune cell function, selenoprotein deficiency leads to oxidant hypertonicity in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation [22]. Thus, Se and the regulation of selenoproteins by Se play important roles in immune function. The levels of inflammation-related cytokines (IL-1, IL-6, and IFN-γ), which are synthesized and secreted by immune cells in obese individuals, were boosted tremendously [8]. Stephen E et al. have reported that a high-fat diet increases tissue expression of TNF-α [23]. HSPs have been identified as adipokines; among them, HSP60 has been well described as the most closely related to metabolic and immune disorders caused by obesity [24].

The above studies suggested that low-Se and high-fat diet could cause immune disorders. When tissue or cell functions are disrupted, the expression of cytokines and HSPs shows abnormalities. Selenoproteins, as the principal bearers of the biological function of Se, also play an important role in the immune system. To our knowledge, the effects of low Se and high fat on the expression of cytokines, HSPs, and selenoproteins in pig peripheral blood lymphocytes are not reported. In order to explore the effects of low Se and high fat on the pig’s immune cells, we established low Se, high fat, and low-Se/high-fat pig models and detected the expression of selenoproteins, HSPs, and cytokine genes in pig peripheral blood lymphocytes, with further analysis of the effects of low Se and high fat on the expression of selenoprotein, HSP, and cytokine mRNA.

Materials and Methods

Materials or Chemicals

Lymphoprep was purchased from Tianjin Haoyang Biological Products Co., Ltd. TRIzol reagent was purchased from Invitrogen, America. The mRNA reverse transcription kit was provided by Thermo Fisher Scientific Inc. Chloroform, isopropanol, and ethanol were purchased from Tianjin Yong-largest chemical reagents Limited. All other chemicals were obtained from commercial sources at the highest quality available.

Treatment of Experimental Animals

All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. All experimental piglets were provided by normal pig farms and bred in the Northeast Agricultural University facility. Forty crossbred boar (Duroc × Landrace × Yorkshire, without castration) piglets with healthy lean body weights of 10 kg were randomly divided into four treatment groups of 10 piglets per treatment group. The study used a 2 × 2 factorial design, where the control group is group C (DE > 3290 kcal/kg, Se = 0.3 mg/kg), and the experimental groups are group L-Se (DE > 3290 kcal/kg, Se = 0 mg/kg), group H-fat (DE > 3490 kcal/kg, Se = 0.3 mg/kg), and group L-Se-H-fat (DE > 3490 kcal/kg, Se = 0 mg/kg). The diets were fed according to the experimental design and were prepared based on the normal fat diet with addition of 0 or 0.03 g/kg sodium selenite (1%, Se = 10,000 mg/kg). A complementary granulated diet including corn, soybean meal, and wheat bran, was obtained from Longjiang County, a typical Se-deficient region of Heilongjiang Province in China, which blends material without supplemental Se. Experimental animals were fed for 16 weeks.

Preparation of the Pig Peripheral Blood Lymphocytes for Analysis

Fresh porcine venous blood (30 mL) was mixed with sodium citrate tribasic in a 7:1 ratio. The lymphocyte preparations were enriched by centrifugation (2000 rpm) for 20 min. The upper layer was removed after centrifugation, and 18 ml of PBS was added to the remaining part, followed by mixing. The mixture was added to the cell separation medium at a ratio of 2:1. Then, the mixture was centrifuged (2000 rpm) for 15 min. The second layer was collected in tubes containing 30 mL of PBS, mixed well, and then centrifuged (1800 rpm) for 20 min. The cells were recovered from the tube, resuspended, and washed two times in 10 mL of PBS. Pig peripheral blood lymphocytes were prepared for further experiments.

Design of Primers and Quantitative PCR

The total RNA was isolated from pig peripheral blood lymphocyte samples using Trizol reagent according to the manufacturer’s instructions (Invitrogen, China). The concentration and purity of the total RNA were determined at 260 nm/280 nm using a spectrophotometer (Gene Quant 1300/100, General Electric Company, USA). First-strand cDNA was synthesized from 5 μg of total RNA using oligo dT primers and SuperScript II reverse transcriptase according to the manufacturer’s instructions (Roche, USA). The synthesized cDNA was diluted five times with sterile water and stored at − 80 °C before using.

The primers used for selenoprotein, HSP, cytokine, and inflammatory cytokine assays were designed by Premier Software (PREMIER Biosoft International, USA) and are summarized in Table 1 for quantitative PCR (qPCR). The β-actin gene was used as an internal reference. Gene expression levels were detected via qPCR using a Light Cycler® 480 System (Roche, Basel, Switzerland) and fast Universal SYBR Green Master Mix (Roche, Basel, Switzerland). The reactions were performed in a 20 μL reaction mixture containing 10 μL of 2 × SYBR Green I PCR Master Mix (TaKaRa, China), 2 μL of diluted cDNA, 0.4 μL of each primer (10 μM), 0.4 μL of 50 × ROX reference Dye II, and 6.8 μL of PCR-grade water. The PCR procedure for all of them was 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 60 °C for 30 s. The melting curve analysis showed only one peak for each PCR product. Electrophoresis was performed with the PCR products to verify primer specificity and product purity. The relative mRNA abundance was calculated according to the method of 2−ΔΔCT, which accounts for gene-specific efficiencies and is normalized to the mean expression of the above-mentioned index.

Table 1 Gene-specific primers for qPCR
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Statistical Analysis

Statistical analyses of all data were performed using GraphPad Prism (version 5.0, GraphPad Software Inc., San Diego, CA, USA). All datasets showed normal distributions and passed equal variance testing. Quantitative data are shown as the mean ± SD, and P < 0.05 was considered statistically significant. Bars with different superscript letters represent statistically significant differences (P < 0.05). We used two-way ANOVA to investigate the relationship between low Se and high fat.

Results

MRNA Levels of Selenoprotein in Pig Peripheral Blood Lymphocytes

To determine the effects of low Se, high fat, and their combination on the mRNA level of selenoprotein in pig peripheral blood lymphocytes, we used qPCR to examine 23 selenoproteins (Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio2, Dio3, TrxR1, TrxR2, TrxR3, Sephs, Sep, Selh, Seli, Selm, Sepp, Selt, Selx, SelK, Sepn, Sels, Selo, and Sepw). However, we did not detect certain transcripts, such as Dio1, Dio2, Dio3, TrxR2, TrxR3, Sels, and Selo; this result might be explained by tissue differences, with several selenoproteins failing to express significant levels in the pig peripheral blood lymphocytes.

Consequently, we present the 16 selenoprotein genes that showed significant changes in expression levels. Data analysis was performed from three perspectives. First, Fig. 1 shows the effects of four diets on selenoprotein mRNA levels. Group C was the control group and was compared to the normal Se and normal fat content diet group. Group L-Se was the normal fat/low-Se group, group H-fat was fed a diet with normal Se/high-fat content, and group L-Se-H-fat was the high-fat content/low-Se content group. Compared with group C, the mRNA level of selenoprotein was lowest in the pig peripheral blood lymphocytes from group L-Se. Among these selenoprotein genes, we noted that Se deficiency had the most pronounced effects on the mRNA expression of Gpx2, Selx, and Sep in the pig peripheral blood lymphocytes. Compared with group C, the mRNA levels of Gpx2, Selx, and Sep in group L-Se were reduced by 73–80%. In addition, it was noted that in group L-Se-H-fat, the mRNA level of selenoprotein was lower than in group C. The effect on the mRNA expression of Selk, Sepn, and Sep in the pig peripheral blood lymphocytes in group L-Se-H-fat was 82–84% lower compared with group C. Second, the levels of selenoprotein for further study are shown in Fig. 2. The upper part of the chart shows Se = 0.3 mg/kg, and the lower part of the chart shows Se < 0.03 mg/kg. Our results show that the effect of fat content on selenoprotein mRNA levels follows a similar trend. For most selenoprotein genes, the changes for each selenoprotein gene follow a similar pattern across the groups. Third, to summarize the patterns of Se content and effects of fat content on selenoprotein in pig peripheral blood lymphocytes, a two-way ANOVA was conducted. The results are presented in Table 2. The primary source of variation in parameters of selenoprotein in pig peripheral blood lymphocytes was the concentration of dietary Se. The concentration of dietary fat was a minor factor in most cases. Among these selenoprotein genes, Selx is the exception in that the minor factor is the interaction between the concentration of Se and fat.

Fig. 1
figure 1

The selenoproteins mRNA levels. Analysis of the mRNA levels of selenoproteins from pig peripheral blood lymphocytes. The results are from at least four independent experiments. Data are represented as the mean ± SD (n = 4). Bars with different superscript letters represent statistically significant differences (P < 0.05) among group C, group L-Se, group H-fat, and group L-Se-H-fat

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Fig. 2
figure 2

The mRNA levels of selenoproteins. Effects of selenoproteins in the different dietary content groups in pig peripheral blood lymphocytes

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Table 2 Results of a repeated measure two-way ANOVA performed to test the effects of selenium content and fat content on selenoprotein levels in pigs
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MRNA Levels of HSPs in Pig Peripheral Blood Lymphocytes

To examine whether low Se, high fat, and their combination could cause the synthesis of stress proteins, we detected the mRNA expression of HSP40, HSP60, HSP70, and HSP90. Up-regulation of the mRNA for HSP40, HSP60, HSP70, and HSP90 in pig peripheral blood lymphocytes was observed after pigs were fed with the low-Se/high-fat diet (Fig. 3). Compared with group C, the mRNA expression of HSP40, HSP60, HSP70, and HSP90 significantly increased due to Se deficiency and high fat (P < 0.05). Se deficiency and high fat induced the sharpest up-regulation of HSP90. Compared with group C, group H-fat, group L-Se, and group L-Se-H-fat were 5.28-, 52.08-, and 62.5-fold higher, respectively. Also, two-way ANOVA was conducted to summarize the patterns of Se content and fat content effects on HSP40, HSP60, HSP70, and HSP90. The results are shown in Table 3. The primary source of variation in the parameters in pig peripheral blood lymphocytes was the concentration of fat. A secondary source of variation in the parameters was the concentration of Se. In addition, our results in Table 3 show a 13.16–16.31% total variation caused by the interaction of low Se and high fat for HSP40, HSP60, and HSP70. Among them, the most obvious effects of fat content are on HSP90, which showed 97.84% of the total variation.

Fig. 3
figure 3

The mRNA levels of HSPs. Analysis of the mRNA levels of HSPs in pig peripheral blood lymphocytes. The results are taken from at least four independent experiments. Data are presented as the mean ± SD (n = 4). Bars with different superscript letters represent statistically significant differences (P < 0.05) among group C, group L-Se, group H-fat, and group L-Se-H-fat

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Table 3 Results of repeated measures two-way ANOVA performed to test the effects of selenium content and fat content on the levels of HSPs, cytokines, and inflammatory cytokines in pigs
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MRNA Levels of Cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-9, TGF-β1, IFN-γ, iNOS, COX-2, NF-κB, and TNF-α) in Pig Peripheral Blood Lymphocytes

Figure 4 shows the mRNA levels of cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-9, TGF-β1, and IFN-γ). Compared with group C, the mRNA levels of the cytokines IL-1α, IL-1β, IL-6, IL-8, and IL-9 are higher, and the others are lower. Group L-Se, group H-fat, and group L-Se-H-fat were 1.26- to 49.44-fold higher than group C. In this dataset, IL-1α and IL-6 in group L-Se-H-fat showed obvious changes. IL-1α was 49.44 times higher and IL-6 48 times higher than the control group. Figure 5 shows the mRNA levels of inflammatory cytokines (iNOS, COX-2, NF-κB, and TNF-α). Compared with group C, the mRNA expression of inflammatory factors in group L-Se, group H-fat, and group L-Se-H-fat all increased. In addition, the increased expression of TNF-α was the most dramatic; group L-Se-H-fat was 39.44 times higher than group C.

Fig. 4
figure 4

The cytokines mRNA levels. Analysis of the mRNA levels of cytokines in pig peripheral blood lymphocytes. The results are taken from at least four independent experiments. Data are presented as the mean ± SD (n = 4). Bars with different superscript letters represent statistically significant differences (P < 0.05) among group C, group L-Se, group H-fat, and group L-Se-H-fat

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Fig. 5
figure 5

The mRNA levels of inflammatory cytokines. Analysis of the mRNA levels of inflammatory cytokines in the pig peripheral blood lymphocytes. The results are from at least four independent experiments. Data are presented as the mean ± SD (n = 4). Bars with different superscript letters represent statistically significant differences (P < 0.05) among group C, group L-Se, group H-fat, and group L-Se-H-fat

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To examine the patterns of Se content and fat content effects on cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-9, TGF-β1, and IFN-γ) and inflammatory cytokines (iNOS, COX-2, NF-κB, and TNF-α), we conducted a two-way ANOVA. The results are shown in Table 3. The primary source of variation in the parameters (including cytokines and inflammatory cytokines) in pig peripheral blood lymphocytes was the concentration of fat. The exceptions were COX-2 and IL-1α in which the primary source of variation was the concentration of Se. Secondary sources of variation in the parameters included the concentration of Se. Also, the interaction between Se content and fat content on NF-κB p50 and IL-6 was similar to the effect of only Se. The interaction between Se content and fat content on IL-6 is 29.01% of the total variation, while the Se content is 30.53% of the total variation.

Discussion

Lymphocytes are important components of the immune response, which is produced by lymphoid organs and mediated by the organism’s cellular immunity, humoral immunity, and other immunological functions. Cytokine is a general term that refers to biologically active small proteins secreted by cells, which mediate inflammation and the immune response, regulating cell physiology and immune system function, and are important cellular signal molecules. The changes in cytokine expression in lymphocytes represent immunological damage of low Se and high fat. In order to further detect Se-deficient and high-fat-induced lymphocyte damage and host defenses, such as inflammatory reactions and immune functions, the mRNA expression levels of IL-1α, IL-1β, IL-6, IL-8, IL-9, TGF-β1, IFN-γ, iNOS, COX-2, NF-κB, and TNF-α were examined by qPCR. IL-1α, IL-1β, IL-6, IL-8, and IL-9 are pro-inflammatory factors that are important indicators of inflammation. Increases in their levels represent the generation of inflammation in the organism and can cause other organism-wide pathological damage. IL-8 is produced by various types of inflammatory stimuli and then exerts a variety of functions on leukocytes. IL-9 is a key molecule that affects differentiation of T(reg) function and T(H)17 cells, promoting the differentiation of IL-6 [25]. TNF-α and IL-1β can be used as indirect indexes for detecting immune function because they inhibit activated immune cells in inflammation [26]. TGF-β1 plays a predominant role in immunological activation and suppression [27]. The NF-κB pathway is an important pathway for the regulation of inflammatory factors, and Se can regulate IL-1, IL-6, and TNF-a through the NF-κB pathway [28]. Two target genes of NF-κB are the inflammatory enzymes COX-2 and iNOS [29, 30]. IFN-γ can rapidly activate host macrophages and enhance the immune responses of organisms [31]. TGF-β1 and IFN-γ are anti-inflammatory factors, and their expression decreases in inflammation. In our experiments, the mRNA levels of the cytokines IL-1α, IL-1β, IL-6, IL-8, IL-9, iNOS, COX-2, NF-κB, and TNF-α in group L-Se, group H-fat, and group L-Se-H-fat all increased, and the other cytokines are lower than group C. Also, group L-Se-H-fat underwent a remarkable change. This is consistent with previous studies showing that high fat and low Se induced inflammatory responses and changes in immune function in lymphocytes [10, 32]. Therefore, we conclude that low Se or high fat could induce inflammation and decrease immune function. We also conclude that low Se might cause more serious injury that is induced by high-fat diet in the lymphocyte.

The major biological form of Se is the amino acid selenocysteine, which is present in the active sites of selenoproteins. Se plays a role through selenoproteins. A low-Se diet can cause a variety of selenoprotein expression changes in animals such as the pig, rat, and chicken [17, 33, 34]. At the same time, animals that lack Se show immune function decline and immune disorders of the organism [1]. Selenoproteins in the organism play a number of roles, such as regulating antioxidant capacity, which is closely related to the organism’s immune function [35]. Dietary Se deficiency and SelW knockdown increased mitochondrial Ca2+ levels and oxidative stress [36, 37]. Immune cell function is obviously changed in Selk knockout mice [21]. Long-term high-fat diets lead to chronic inflammation, which can increase the expression of pro-inflammatory cytokines, leading to immune function disorders [38]. Existing research shows that Selw regulates the expression of other selenoproteins (Gpx3, Gpx4, Txnrd1, Selt, Selh, Sepp1, Sels, and Sep15) and also regulates inflammation-related cytokines (IL-1, IL-6, IL-8, IL-17, IL-4, IL-10, and IFN-γ) [39, 40]. Yu Dong et al. also proved that silencing of Selw significantly up-regulates inflammation-related genes (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in lymphocytes [32]. High-fat diet-induced inflammation in pigs is associated with thioredoxin and oxidoreductase systems and related selenoproteins [41]. Lymphocytes are important immune cells to test the immune injury induced by low Se and high fat. Experiments showed that low Se and high fat can induce an inflammatory response and change lymphocyte function [4, 42]. Thus, expression of 16 selenoprotein genes was determined in pig peripheral blood lymphocytes in 4 types of responses to dietary Se deficiency: normal, high fat, and Se deficiency/high fat. Our results showed that the mRNA level of selenoproteins was lower in group L-Se and group L-Se-H-fat than in group C in pig peripheral blood lymphocytes. This is consistent with previous research. Also, a high-fat diet changes the selenoprotein mRNA levels in pig peripheral blood lymphocytes. Our result showed that four selenoproteins (Gpx1, Gpx2, Selt, and Selm) examined in obese pigs were elevated. This is different from a previous report where 12 selenoproteins (Gpx3, Gpx4, Gpx6, Dio1, Dio2, Txnrd1, Selv, Seli, Sels, Selm, Selo, and Sep15) examined in high-fat pigs were elevated in 6 tissues. This may be related to the differences among the tissues. Existing studies have shown that Se deficiency and high-fat diet may change the mRNA level of selenoprotein genes in pig immune cells, and our results also verify this model [43]. Our two-way ANOVA showed that the primary source of variation in selenoprotein parameters in pig peripheral blood lymphocytes was the concentration of dietary Se, and the concentration of dietary fat content was a minor factor in most cases. These results suggested that dietary Se content and fat content can regulate the levels of selenoprotein genes in peripheral blood lymphocytes. The precise mechanism needs further research.

HSPs are proteins that are abundantly expressed during stress. They protect cells against stimulation, improve tolerance, and deal with the adverse effects of stimulation. HSPs can directly stimulate cells and enhance the organism’s immune function. However, when cells undergo stress, such as oxidative stress, which is induced by Se deficiency and high fat, the expression quantity will increase. For example, a certain concentration of manganese can induce high expression of HSP40, HSP60, HSP70, and HSP90 in spleen lymphocytes in vitro [44]. That may be explained by the negative feedback regulation. Previous studies demonstrated that Se deficiency or high fat could induce the expression of HSPs. Khoso’s results showed that immunosuppression was accompanied by a downregulation of mRNA expression levels of selenoproteins and an upregulation of the HSP mRNA expression levels, and the expression levels of IL-2, IL-6, IL-8, IL-10, IL-17, IFN-α, IFN-β, and IFN-γ were lower [45]. Selenium can reduce oxidative stress and inflammatory response induced by lead [46, 47]. A Se-deficient diet induced oxidative stress and impaired immune function [48, 49]. HSP70 is one of the most strongly induced after stresses such as oxidative stress [50]. HSP can induce production of cytokines such as TNF-α and other cell types, and they can deliver maturation signals and peptides to antigen-presenting cells through receptor-mediated interactions [51]. HSP60 treatment of mouse and human macrophages increased the production of IL-12, IL-15, INF-γ, and TNF-α. Khoso et al. and Chen et al. have reported that Se deficiency activates HSP expression in splenic lymphocytes, the thymus and neutrophils [52, 53]. HSP60 and HSP90 may have protective effects during Se deficiency-induced erythrocyte injury [54]. Upon hyperlipidemia, HSP expression increased significantly in the intracranial cerebral arteries of rabbits [55]. In response to a high-fat diet, interactions of HSPs, which act as danger signals, influenced the signaling pathways and the inflammatory responses of cells [56]. A high-fat diet inhibits lymphocyte immune function in rats, and lipoprotein is one of the inhibiting factors [10]. Our result is consistent with the findings that HSPs are extensively elevated compared with the control group. Not only group L-Se but also group H-fat and group L-Se-H-fat have an observable change. Se deficiency and high-fat diet can cause injury to lymphocytes in the organism, and our results show that their interaction is not a simple superposition. Low Se will promote high-fat damage to lymphocytes, causing the organism to be in a more serious state of stress. This serves as a basis for the potential correlation of Se and lipids in the organism.

Conclusion

In summary, our results indicated that low-Se/high-fat diet could cause relevant selenoprotein expression changes and promote the expression of pro-inflammatory factors and HSPs. Also, low Se will promote changes associated with high-fat diet. This information is helpful in understanding pig peripheral blood lymphocytes with low-Se and high-fat diet.