Introduction

Selenium (Se) is an essential trace element to maintain homeostasis in humans and animals and has functions of regulating endocrine and immunity [1,2,3]. White muscle disease, mulberry heart, pancreatic atrophy, and exudative diathesis can be caused by Se deficiency in animals [4,5,6,7]. Brain tissue is one of the target organs for Se deficiency [8, 9], and previous studies had found that Se levels in the brain of Alzheimer’s patients were reduced [10, 11]. Nutritional brain softening could be induced by Se deficiency in chicken brain [12], and Se deficiency could increase the expression of inflammatory cytokines to induce inflammation [13, 14]. Inflammatory cytokines play a key role in most inflammation [15, 16]. For example, the expression of prostaglandin E synthase (PTGEs), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB), and inducible nitric oxide synthase (iNOS) could be increased by Se deficiency in the gastrointestinal tract of chickens, leading to inflammatory lesions of the digestive tract [17]. Interestingly, diet with Se deficiency could also increase the expression of inflammatory cytokines in peripheral blood lymphocyte in pigs and induce inflammation [18]. Additionally, previous study had shown that supplementation with Se significantly reduced the expression of NF-κB and COX-2 induced by bacterial endotoxin lipopolysaccharides [19]. It has also been reported that HSP60 and HSP90 had protective effects during erythrocyte injury induced by Se deficiency [20], and Se deficiency activated the expression of HSPs in the spleen and thymus [21, 22].

Nitric oxide (NO) is an autocrine and paracrine signaling pathway molecule that can diffuse freely in biofilms. In normal physiological state, the main physiological function of NO is to maintain blood vessel homeostasis [23, 24]. NO plays an important role in host defense, including regulation of inflammation [25]. NO is a molecular aggressor in inflammation, especially chronic inflammation [26]. NF-κB pathway is an important way to regulate the expression of inflammatory cytokines [27], and COX-2 and iNOS are two important target genes of NF-κB [28]. For example, small heterodimer partners inhibited the expression of COX-2 and iNOS by inhibiting the activity of NF-κB promoter in renal tubular injury [29], and iNOS expression and inflammation were inhibited via the NF-κB signaling pathway in acute renal ischemia reperfusion injury rats [30]. INOS could be activated via the NF-κB signal pathway following inflammatory stimulation such as meningitis in mouse choroid plexus cells [31], and inflammation in mucosa of rats could be ameliorated by NF-kB signaling pathway inhibition [32]. The activation of NF-κB pathway could be suppressed by heat shock treatment in heart inflammation of rats [33], and HSP70 could afford protection by the inhibition of NF-κB-mediated inflammation in ischemic acute renal failure of rats [34]. And HSP70 also could protect the brain from a variety of damage by the inhibition of iNOS and NO in the ischemia-reperfusion model [35].

The above studies suggested that Se deficiency could induce inflammation, which was closely related to iNOS/NF-κB signaling pathway, but the mechanism was still unclear. The aim of our experiment was to clarify the mechanism of Se deficiency-induced inflammation in the brain of pigs via the iNOS/NF-κB signaling pathway.

Materials and Methods

Establishment of Se Deficiency Animal Model and Grouping

All procedures used in this experiment were administered in accordance with the animal welfare guidelines and the Institutional Animal Care and Use Committee of Northeast Agricultural University. Twenty-four healthy 42-day-old pigs were randomly divided into two groups of 12 animals each. Group C was fed with 0.3 mg/kg inorganic Se, and group L was fed with 0.007 mg/kg inorganic Se. The diet was prepared according to NRC 2012 pig nutrition requirements, and the content of Se in control diet was 0.3 mg/kg by adding SeMet. The basic composition of the diet was shown in Table 1. At the 90th day of the experiment, the pigs were euthanized, brain tissue was extracted and partially fixated in 10% formalin, and left part was in liquid nitrogen for further use.

Table 1 The basic composition of the diet used in the present study
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Histological Observation of Pig Brain Tissue

After being fixed in 10% neutral formalin solution for fixation for at least 24 h, the tissues were embedded in paraffin, and sectioned and stained with hematoxylin and eosin (HE). The section was dehydrated in ethanol, and dried and sealed with neutral resin; it could be for the light microscopic observation when dried.

Assay of NO Levels and iNOS Activity

NO levels and iNOS activity were assayed using NO and iNOS assay kits (Nanjing Institute of Bioengineering, Nanjing, China).

Real-Time Quantitative PCR Analysis

Total RNA was extracted from brain tissues by using Trizol reagent according to the manufacturer’s instructions, and the reverse transcription steps of cDNA were also based on the manufacturer’s instructions (Roche, Shanghai, China). The primers used in our experiment were shown in Table 2. qRT-PCR was performed using the Light Cycler® 480 System (Roche, Basel, Switzerland) and Fast Universal SYBR Green Master Mix (Roche, Basel, Switzerland). Only one peak for each PCR product was shown in the melting curve analysis. The genes’ relative abundance of mRNA was calculated by using the 2-ΔΔCT method, accounting for gene-specific efficiencies and was normalized to the mean expression of the above mentioned indexes.

Table 2 The primers used in the present study
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Western Blot Analysis

After total protein being subjected to 12% SDS-polyacrylamide gel electrophoresis, separated proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol at 4 °C. Membranes were blocked with 5% skim milk at 37 °C for 2 h and then incubated overnight with diluted primary antibodies against rabbit at 4 °C, and the diluted concentration was shown in Table 3. And then, it was incubated with peroxidase-conjugated secondary antibodies against rabbit IgG (1:5000, Santa Cruz, USA) for 50 min at 37 °C. Finally, the signal was detected with X-ray films (TransGen Biotech Co., Beijing, China).

Table 3 The primary antibodies used in present study
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Statistical Analysis

The data were analyzed with t tests, using GraphPad Prism software (version 7.0, GraphPad Software Inc., San Diego, CA, USA). The data for each group are reported as mean ± standard (SD) deviation. The difference is considered statistically significant at P < 0.05.

Results

Detection Results of Histological Changes in Brain Tissue

As shown in Fig. 1, compared with group C, the number of nerve cells was decreased in group L, the cell morphology was irregular, the structure was incomplete, the nucleus was dissolved and fragmented, and the cytoplasm staining deepened; the number of glial cells was increased irregularly, and the nucleus condensed and darkened. Moreover, there were small vessel congestion and inflammatory cell infiltration in group L. It indicated that inflammation occurred in the brain tissue of pigs due to Se deficiency.

Fig. 1
figure 1

Effects of Se deficiency on histological in the brain of pigs. a 20× group C. The yellow arrow represents normal nerve cells, and the blue arrow represents glial cells in the stroma. (b) 40× group C. The yellow arrow represents normal nerve cells, and the blue arrow represents glial cells in the stroma. (c) 20× group L. Red arrows denote damage of nerve cells—neurotropic, blue arrows denote damage of glial cells, and yellow arrow denotes the small vessel congestion. (d) 40× group L. The red arrow denotes damage to nerve cells—neurotropic, the yellow arrow denotes infiltration of inflammatory cell, the blue arrow denotes damage of nerve cell, and the black arrow denotes damage of glial cells

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Detection Results of NO Levels and iNOS Activity in Brain Tissue

As shown in Fig. 2, compared with group C, iNOS activity and NO levels were increased by 20% and 97% in group L, with significant differences (P < 0.05). It indicated that Se deficiency decreased the ability of brain tissue to clear RNS, induced inflammatory lesions.

Fig. 2
figure 2

The activity of iNOS and the level of NO in the brain tissue of pig. Each value represents the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the L and the C groups

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Detection Results of HSP Expression in Brain Tissue

The mRNA and protein expression levels of HSPs were shown in Fig. 3. Compared with group C, the mRNA expression levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were increased by 133%, 51%, 17%, 266%, and 147% in group L, and the protein expression levels of HSP70 and HSP90 were increased by 750% and 235%, with significant differences (P < 0.05). These results provided evidence that HSPs were involved in the compensatory regulation of inflammation induced by Se deficiency.

Fig. 3
figure 3

The mRNA and protein levels of HSP27, HSP40, HSP60, HSP70, and HSP90. a The mRNA levels of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) in the brain of pigs. Each value represents the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the L and the C groups. b The protein levels of HSP70 and HSP90. Each value represents the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the L and the C groups

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Detection Results of Inflammatory Cytokine Expression in Brain Tissue

The mRNA and protein expression levels of inflammatory cytokines were shown in Fig. 4. Compared with group C, the mRNA expression levels of PTGEs, COX-2, NF-κB, and iNOS were increased by 144%, 252%, 62%, and 280% in group L, and the protein expression levels of NF-κB and iNOS were increased by 500% and 22%, with significant differences (P < 0.05). These results indicated that Se deficiency upregulated the expression of inflammatory cytokines in pig brain tissue.

Fig. 4
figure 4

The mRNA and protein levels of PTGEs, COX-2, NF-κB, and iNOS. a The mRNA levels of inflammatory cytokines (PTGEs, COX-2, NF-κB, iNOS) in the brain of pigs. Each value represents the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the L and the C groups. b The protein levels of inflammatory cytokines iNOS and inflammatory cytokines NF-κB. Each value represents the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the L and the C groups

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Discussion

The trace element Se plays an important biological role in the body. Many studies had shown that Se regulated inflammatory response and maintained normal physiological function in humans and animals [36, 37]. A large number of data provided evidence that inflammatory cytokines and HSPs had cross talk in Se deficiency-induced inflammation in the brain. For example, the inflammatory lesions of chicken brain tissue could be induced by Se deficiency [12]. The results of our experiment proved that diet with Se deficiency activated the iNOS/NF-κB signaling pathway and induced inflammatory lesions in pig brain; the results of histological observation also displayed it. At the same time, HSPs were involved in the compensatory regulation of inflammation.

Lots of NO can be produced by the activation of iNOS, which in turn induces inflammation. An increase of NO levels and iNOS activity was observed in the intestinal tract of inflammatory injury in Se deficiency chickens [38]. Moreover, the intrinsic relationship between NO levels and iNOS activity in the chicken inflammatory lesion duodenum was also reported [39]. In the present study, the iNOS activity and the NO levels in group L were significantly upregulated compared with group C. It was consistent with the above; histological observation also displayed that Se deficiency could cause inflammatory lesions in pig brain. The activation of iNOS could increase NF-κB expression. Activation of NF-κB was closely related to inflammation [40] and could increase the expressions of iNOS, COX-2, and PTGEs. This mechanism was observed in chicken respiratory inflammation and pneumonia response [41, 42]. Many studies had shown that blocking the NF-κB signaling pathway in renal tubular and rat brains could inhibit inflammation by reducing the expression of inflammatory cytokines (PTGEs, COX-2, and iNOS) and NO levels [29, 43,44,45]. In our study, the mRNA levels of PTGEs, COX-2, NF-κB, and iNOS and the protein expression levels of NF-κB and iNOS in group L were upregulated, and it was consistent with the above studies.

HSPs are a series of highly conserved proteins that protect cells from stimuli, improve tolerance, and attenuate the adverse effects of stimuli. Many studies showed that large amounts of HSPs were induced to protect the cells and maintain normal metabolic function when a tissue was damaged [46,47,48], such as inflammation. Induction of HSP40, HSP60, and HSP70 could inhibit inflammatory response by inhibiting lymphocyte damage during Se deficiency-induced inflammation in peripheral blood lymphocytes of pigs [18]. Recent studies had found that Se deficiency could increase the expression of inflammatory cytokines to lead inflammation and was accompanied by high expression of HSP27, HSP40, HSP60, HSP70, and HSP90 in mouse skeletal muscle and chicken erythrocytes [20, 49]. Tao et al. showed that COX-2 and iNOS were approximate promoters of inflammatory bowel cancer, HSP70 could exert tumor suppressive effects by limiting inflammatory cytokines [50], and Helicobacter pylori promoted the apoptosis of gastric glandular epithelial cells by activating COX-2 and inhibiting HSP70 [51]. Our results showed that the mRNA expression of HSP27, HSP40, HSP60, and HSP70 and the protein expression of HSP70 were increased in group L, which was consistent with the above studies. Furthermore, previous study had shown that HSP90 was involved in the transcription of iNOS; inhibition of HSP90 expression could inhibit iNOS expression and indicated that HSP90 could promote inflammatory response [52]; the mRNA and protein expression levels of HSP90 were both increased in our experiment, and it was consistent with the above.

In summary, our present experiments provided evidence that Se deficiency can activate the iNOS/NF-κB pathway to upregulate the expression of inflammatory cytokines, thereby leading inflammatory lesions in the pig’s brain, and HSPs are involved in the compensatory regulation of inflammation.