Selenium deficiency induces spleen pathological changes in pigs by decreasing selenoprotein expression, evoking oxidative stress, and activating inflammation and apoptosis

Abstract

Background: The immune system is one aspect of health that is affected by dietary selenium (Se) levels and selenoprotein expression. Spleen is an important immune organ of the body, which is directly involved in cellular immunity. However, there are limited reports on Se levels and spleen health. Therefore, this study established a Se-deficient pig model to investigate the mechanism of Se deficiency-induced splenic pathogenesis.

Methods: Twenty-four pure line castrated male Yorkshire pigs (45 days old, 12.50 ± 1.32 kg, 12 full-sibling pairs) were divided into two equal groups and fed Se-deficient diet (0.007 mg Se/kg) or Se-adequate diet (0.3 mg Se/kg) for 16 weeks. At the end of the trial, blood and spleen were collected to assay for erythroid parameters, the osmotic fragility of erythrocytes, the spleen index, histology, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining, Se concentrations, the selenogenome, redox status, and signaling related inflammation and apoptosis.

Results: Dietary Se deficiency decreased the erythroid parameters and increased the number of osmotically fragile erythrocytes (P < 0.05). The spleen index did not change, but hematoxylin and eosin and TUNEL staining indicated that the white pulp decreased, the red pulp increased, and splenocyte apoptosis occurred in the Se deficient group. Se deficiency decreased the Se concentration and selenoprotein expression in the spleen (P < 0.05), blocked the glutathione and thioredoxin antioxidant systems, and led to redox imbalance. Se deficiency activated the NF-κB and HIF-1α transcription factors, thus increasing pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, and TNF-α), decreasing anti-inflammatory cytokines (IL-10, IL-13, and TGF-β) and increasing expression of the downstream genes COX-2 and iNOS (P < 0.05), which in turn induced inflammation. In addition, Se-deficiency induced apoptosis through the mitochondrial pathway, upregulated apoptotic genes (Caspase3, Caspase8, and Bak), and downregulated antiapoptotic genes (Bcl-2) (P < 0.05) at the mRNA level, thus verifying the results of TUNEL staining.

Conclusions: These results indicated that Se deficiency induces spleen injury through the regulation of selenoproteins, oxidative stress, inflammation and apoptosis.

Keywords: Apoptosis; Inflammation; Oxidative stress; Pigs; Selenium deficiency; Spleen.

Fig. 1

The erythroid parameters of RBC, MCV, HCT, HGB, MCH, and MCHC, and osmotically fragile erythrocytes in the Se-adequate group and Se-deficient group. Values are means ± SEM, n = 12. * Different from Se-A, P < 0.05; ** different from Se-A, P < 0.01

Fig. 2

Hematoxylin and eosin staining, Se concentrations, and spleen index in the Se-adequate group and Se-deficient group spleens. Se deficiency caused blurring of the boundary between red and white pulp, and decreased the white pulp volume and cell density (yellow arrow) in the spleen, particularly narrowing of the periarterial lymphatic sheath (blue arrow). In addition, Se deficiency significantly decreased the density and number of splenic cord cells (red arrow), thereby increasing the splenic sinusoid space and the splenic red pulp volume. Photomicrographs are shown at 100× and 200× magnification

Fig. 3

Relative mRNA and protein levels of selenoproteins in the spleen in the Se-adequate group and Se-deficient group. Values are means ± SEM, n = 12. * Different from Se-A, P < 0.05; ** different from Se-A, P < 0.01

Fig. 4

The activity of GPX, TXNRD, CAT, SOD, and TNOS; the hydroxyl radical inhibition capacity; and the content of MDA and H₂O₂ in the spleen in the Se-adequate group and Se-deficient group. Values are means ± SEM, n = 12. * Different from Se-A, P < 0.05; ** different from Se-A, P < 0.01

Fig. 5

The content of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, IL-17, and TNF-α; anti-inflammatory cytokines IL-10, IL-13, and TGF-β; transcription factors NF-κB and HIF-1α; NF-κB p65 and COX-2; and the relative mRNA levels of inflammatory genes in the spleen in the Se-adequate group and Se-deficient group. Values are means ± SEM, n = 12. * Different from Se-A, P < 0.05; ** different from Se-A, P < 0.01

Fig. 6

TUNEL staining of spleen tissue and relative mRNA levels of apoptosis genes in the spleen in the Se-adequate group and Se-deficient group. Values are means ± SEM, n = 12. * Different from Se-A, P < 0.05; ** different from Se-A, P < 0.01