Selenium Deficiency via the ROS/NLRP3/IL-1β Signaling Pathway Leads to Pyroptosis Injury in Pig Spleen

The aim of the present study was to investigate the effect of selenium (Se) deficiency on the relationship between the pyroptosis and MAPK signaling pathway in spleen injury. A total of 10 two-month-old Sus scrofa domesticus specimens were allocated to two groups. The control group was fed a basal diet (0.15-mg/kg Se), and the experimental group was fed a 0.03-mg/kg Se-deficient diet for 2 months. The pig-spleen histopathological changes were observed with hematoxylin–eosin staining. Frozen sections were prepared to detect the content of ROS in pig-spleen cells. The oxidation stress related indexes were determined using a spectrophotometer. The levels of pyroptosis- and MAPK signaling pathway-related factors were detected via quantitative real-time polymerase chain reaction (qPCR) and western blotting (WB). The results of sections showed that the lymphocytes decreased in number, the spacing of cells widened, and some cells were necrotic in the spleen tissue of pigs fed a low-selenium diet. The content of ROS, malondialdehyde, nitric oxide, H2O2, and catalase activity in the low-selenium group was significantly higher than that in the control group, and SOD activity was decreased. The protein-ratio-levels of p-Nrf2 to Nrf2 were decreased. The expression levels of nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3), IL-1β, IL-18, ASC, gasdermin D, and caspase-1, the protein-ratio-levels of p-AKT serine/threonine kinase (p-AKT) to AKT, p-extracellular regulated protein kinases (ERK) to ERK, p-P38 MAPK to p-P38, and p–c-Jun N-terminal kinase (p-JNK) to JNK were significantly increased in the Se-deficient group compared with the control group. These results suggested that Se deficiency can induce oxidant stress, which increases pyroptosis- and inflammation-related factors of pig-spleen injury.


Introduction
An essential micronutrient in organisms, selenium (Se) is widely distributed in various tissues.Excessive or insufficient Se content leads to serious disease [1,2].It is related to the occurrence of complications such as arteriosclerosis, hyperlipidemia, and hyperglycemia [3].In organisms, Se functions mainly in the form of selenoproteins and some antioxidant enzymes.Selenoprotein P is involved in protecting organisms from damaging effects of free radicals and is a good indicator of selenium content in organisms [4].Selenoprotein W prevents organisms from being overoxidized and participates in muscle metabolism [5].Selenoprotein S controls the redox balance in cells [6].Selenium deficiency affects the expression of selenoproteins, and associated with splenic dysfunction and induced oxidative stress, excessive inflammation, abnormal autophagy, and endoplasmic reticulum stress [7].In summary, the function of Se is to regulate the immune balance of the organism through the antioxidant function of selenium protein.Se deficiency greatly reduces the body's antioxidant capacity and induces oxidative stress.
Oxidative stress is a pathological condition caused by an imbalance between the body's oxidant and antioxidant systems.The content of both H 2 O 2 and MDA was changed, as was the related CAT and SOD activity.When ROS increases to a certain point, there will be some damage to an organism's tissues or cells, including adipose-related MDA and nitric oxide (NO)-centered derivatives of RNS.Nrf2/ARE signaling is considered a promising target for the treatment of oxidative-stress-mediated diseases such as diabetes, fibrosis, neurotoxicity, and cancer [8,9].A key factor of oxidative-stress production is ROS content, and ROS production is closely related to mitochondria [10].
Oxidative stress can induce cell pyroptosis.Mitochondria ROS are closely related to the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome.NLRP3 is an important PRR in cytoplasm, which has a three-part carboxy-terminal leucine-rich repeat domain.NLRP3inflammasome activation is mediated by the related effects of mitochondria and lysosomes, and this process is related mainly to ROS production [11,12].A central nucleotide binding domain, which has ATPase activity and mediates self-oligomerization, and an amino-terminal pyrin domain recruit apoptosis-associated speck-like proteins containing CARD (ASC) [13].Subsequently, the assembled ASC recruits prucaspase-1 through homotypic CARD-CARD domain interaction to form the NLRP3-ASC-caspase-1 protein complex [14].The NLRP3 inflammasome is activated and induces the self-cleavage and activation of procaspase-1, which leads to the maturation of the proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18).In addition, activated caspase-1 cleaved gas protein D (GSDMD) releases its N-terminal domain, transfers to the cell membrane and forms pores, mediates the release of cell contents, including inflammatory cytokines IL-1β and IL-18, and induces inflammatory cell death, known as pyroptosis [15][16][17].NLRP3-inflammasome pyroptosis is a form of inflammatory PCD triggered by pathogen invasion and dependent on caspase activation.GSDMD is a coexecutor of pyroptosis, and there is growing evidence that other members of the gas protein family, including GSDMA3, GSDMB, and GSDME, can be cleaved and induce pyroptosis [16,[18][19][20][21].
MAPK/MAK/MRK overlapping kinase (MOK) belongs to the MAP-kinase superfamily, which plays an important role in regulating cell growth, division, and differentiation [22].As a key factor of pyroptosis, caspase-1 plays an important role in caspase-mediated inflammatory injury [23].Five distinct MAPK signal transduction pathways have been identified in mammalian organisms.Among them, the ERK1/2 signal transduction pathway regulates cell growth and differentiation, and the JNK and P38 signal transduction pathways play important roles in stress responses such as inflammation and apoptosis.Greeberg et al. demonstrated that cell survival or apoptosis is related to different degrees of ERK, JNK, and P38 activation [24].Cell survival or apoptosis depends on the ERK pathway's activation by growth factors and JNK-P38 pathway's activation by stress homeostasis.Various cytokines promote cell survival and inhibit programmed cell death through the ERK survival pathway, and the JNK and P38 protein kinase pathways are involved in mediating inflammation and apoptosis in response to stress [24,25].Moreover, studies have found that under the action of ROS and cytotoxins, JNK kinase is activated and transferred to the nucleus, promoting the expression of pyroptosis-related genes, initiating the formation of pyroptosis, and controlling the development of tumors [26].Activated JNK can be involved in the release of apoptotic factors (such as cytochrome C from mitochondria), leading to caspase activation and apoptosis [27].The MAPK pathway is related to the AKT pathway [28].Spleen the largest immune organ in the human body involved activation and induction of oxidant stress in the Se deficiency have been deeply studied and attracted great attention.In this study, we illuminate the relationships among Se, oxidant stress, and cell pyroptosis in pig spleen.

Animals and Diets
Two-month-old inbred male landrace pigs (Sus scrofa domesticus) were provided by the Anyang Fuwang pig farm (Anyang, China) and kept in an environmentally controlled cages (randomly assigned to three pigs in each pen) in 10 pens that were assigned to the control group and Se-deficient group based on their initial weight, which was analyzed with Statistical Product and Service Solutions (SPSS) soft.The experiment used commercial feeds supplied by COFCO Corporation.Diets were formulated to meet the nutrient requirements of swine (NRC, 2012), and the nutritional values are shown in Supplement Table 1.The Se-deficient group was fed an Se-deficient diet containing 0.03 mg/kg Se; the corn in the feed was purchased from Anda, China, which is selenium deficiency area.And no Na 2 SeO 3 was added into the Sedeficient diet.The control group was fed a basal diet (adding 0.24 mg/kg Na 2 SeO 3 to an Se-deficient diet mixture feed; the Se content in the feed is 0.15 mg/kg; the powder was supply by Sigma-Aldrich, America).The content of Se in the feed was tested by FUDA testing group using cyanide atomic fluorescence spectrometry.The feed and distilled water were supplied ad libitum.After two months of treatment, 1 pig from each pen was anesthetized (sodium pentobarbital, i.v, 30 mg/ kg) and slaughtered to collect blood and spleen samples.

Histopathology
The spleen-tissue sample was cut into pieces and fixed with a 4% formaldehyde solution for at least 24 h.Dehydration was performed by 70%, 80%, and 90% ethanol solution for 30 min each, and then 95% and 100% ethanol solution twice for 20 min each.The 100% ethanol solution and dimethylbenzene equal mixture for 15 min, dimethylbenzene I for 15 min, and dimethylbenzene II for 15 min until the tissue transparent.For paraffin penetration, the tissues were putted in the mixture of xylene and paraffin for 15 min, and putted in paraffin I and paraffin II for 60 min each.Then, the tissues were putted in the liquid paraffin.Pathological paraffin Sections (5 μm) were produced using a conventional method and stained with hematoxylin and eosin.The pathological changes were observed under an optical microscope (Nikon, Instruments Inc., Japan).

Immunofluorescence of Frozen Sections
The frozen spleen tissues were cut by freezing microtome.The fluorescence quencher, ROS dye solution (SIGMA D7008 1:500) were added on to the prepared frozen spleen tissues section for 5 min.The slides were placed in PBS (PH7.4) and washed three times by shaking on a decolorization shaker, 5 min each time.After the sections had dried slightly, DAPI dye solution (Servicebio G1012) was added to the rings, and the slices were incubated for 10 min at room temperature in the dark.Sealing: The slides were placed in PBS (PH7.4) and washed three times by shaking on a decolorization shaker, 5 min each time.The slices were sealed with an antifluorescence quenching tablet.Microscopic examination and photography: The sections were observed, and images were collected under a fluorescence microscope (DAPI ultraviolet excitation wavelength: 330-380 nm, emission wavelength: 420 nm, blue light; CY3 excitation wavelength: 510-560 nm, emission wavelength: 590 nm, red light).

Determination of Pig-Spleen Oxidant-Stress-Related Index
The frozen spleen tissues were homogenized in ice-cold saline.The supernatants were collected after the homogenate was centrifuged at 3000 g and 4 °C for 10 min.SOD, CAT, H 2 O 2 , MDA, and NO levels were measured with a spectrophotometer using commercially available assay kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).The protein concentrations in tissue homogenates were measured with the Bradford protein assay using bovine serum albumin as the standard (Jiancheng Bioengineering Institute, Nanjing, China).   1.The melting curve and dissociation curve were extrapolated to confirm primer specificity and product purity.The relative abundance of each mRNA was calculated with the formula 2 −(ΔΔCt) , where ΔΔCt = (Ct Target − Ct GAPDH ) treatment − (Ct Target − Ct GAPDH ) control.

Western-Blotting Analysis of Pig-Spleen Pyroptosis and MAPK-Signal-Pathway-Related Protein Expression
For protein-expression analysis, the extraction and isolation of proteins were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China) according to the manufacturer's instructions.protein concentration was determined using a BCA assay kit (Beyotime, Nanjing, China).Equal amounts of protein extracts were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions in concentrate protein gel 5% (pH = 6.8) and separating protein gel 12% (pH = 8.8).
The separated proteins were transferred to PVDF membranes using tank transfer for 2 h at 200 mA in tris-glycine buffer with 15% methanol.The membranes were blocked with 5% skim milk for 3 h and incubated for 12 h with corresponding antibodies for 2 h at 37 °C.The secondary antibodies (IgG/HRP) were incubated for 2 h at 37 °C.The antibody information is shown in Table 2.The images of the blots were visualized using ECL (Genshare, Xi'an, China).

Statistical Analysis
The results were presented in terms of five single observation replications (n = 5), and duplicated for each set and having coefficient of variation below 5%.Oneway ANOVA followed by Duncan's multiple range test (p < 0.05) with SPSS 20.0 (SPSS Inc, Chicago, IL, USA) was applied for the mean values compared.

Histopathological Observation of the Spleen
Spleen histopathology after L-Se treatment is illustrated in Fig. 1.In the control group, the amount of white pulp, the morphology of red pulp, and the number of lymphocytes were normal.The lymphocytes in the white pulp were closely arranged, and the connective tissue in the red pulp was closely arranged and exhibited normal cell spacing (Fig. 1, NC).In the L-Se treatment group, the amount of white pulp and number of lymphocytes in the spleen were lower, and the lymphocytes in the white pulp were loosely arranged or it was no white pulp structure in the spleen (blue arrow).The red pulp was extensively

Selenium-Deficiency-Induced Oxidative Stress in Pig Spleen
The content of MDA, H 2 O 2 , and NO; the activity of CAT and SOD; and the expression of Nrf2 were tested (see Fig. 2).In the Se-deficient group, the content of MDA, H 2 O 2 , and NO was significantly increased (Fig. 2A-C) and the activity of SOD was significantly decreased (Fig. 2E) compared with those in the control group (p < 0.05, n = 5).By contrast, the activity of CAT was significantly increased after Se-deficiency treatment (p < 0.05, n = 5; Fig. 2D).The fluorescence results for frozen sections showed that the ROS content in the L-Se group's spleen cells was significantly higher than that in the control group (p < 0.05, n = 5; Fig. 2F).The protein expression of phosphorylated Nrf2 was significantly increased in the Se-deficient group (p < 0.05, n = 5; Fig. 2G).The results indicated that Se deficiency induced oxidant stress in pig spleen.

Selenium-Deficiency-Induced Pyroptosis in Pig Spleen
To evaluate the effect of selenium deficiency on pyroptosis in pig-spleen cells, the NLRP3, ASC, caspase-1, GSDMD, IL-1β, and IL-18 were determined with quantitative realtime PCR (Fig. 3A) and WB (Fig. 3B).The mRNA-expression levels of NLRP3, ASC, caspase-1, GSDMD, IL-1β, and IL-18 genes were significantly increased in the Se-deficient group compared with those in the control group (p < 0.05, n = 5).The results of WB were similar to those of qPCR, and the relative average value of GSDMD expression in the low-selenium group was higher than that in control group (Fig. 3B; n = 5).The results indicated that Se deficiency induced pyroptosis in pig spleen.

Selenium-Deficiency Activity MAPK Signaling Pathway in Pig Spleen
The effects of selenium deficiency on JNK, p-JNK, P38, p-P38, ERK1/2, p-ERK1/2 AKT, and p-AKT are shown in Fig. 4. Selenium deficiency significantly enhanced the phosphorylation proportion of related proteins in the treatment group compared with the control group (p < 0.05, n = 5).The protein-ratio levels of p-AKT1 to AKT, p-ERK

Discussion
Selenium-deficiency-induced spleen injury destructed spleen's antioxidant capacities.In the Se-deficient pathological tissue sections we observed, the number of cells in the white pulp and red pulp was significantly reduced, and the connective tissue was loosely arranged and exhibited decisively necrotic cells.Selenium is a component of GSH structure and has antioxidant capacities.Studies have found that oxidative stress caused by selenium deficiency can affect spleen function [22].The most intuitive manifestation of oxidative stress is the increase of ROS.To detect ROS levels in the spleen, this experiment tested typical nitrogencontaining compounds, ROS levels, and detected the loss caused by oxidative stress.The results showed that Se deficiency clearly increased the content of MDA and ROS.This indicates that in the presence of O 2 , biofilms are attacked and decomposed by free radicals and their active derivatives.
The increase of NO content also indicates the increase of free radicals in the body.When oxidative stress occurs, the oxidative system in the body is strong and activates the antioxidant system.The results also showed that Se deficiency increased H 2 O 2 content.CAT, responsible for catalyzing the decomposition of H 2 O 2 , was significantly increased, but Se deficiency induced a decrease in SOD, suggesting that Se deficiency disturbed the oxidative balance and induced oxidative stress.Nrf2 is a key factor in the regulation of oxidative stress and a defense mechanism of the body that is activated when oxidative stress occurs [29].It can regulate related inflammatory factors through the MAPK signaling pathway.The content of p-Nrf2 is significantly reduced after H 2 O 2 treatment [30].As Fig. 2 shows, under the seleniumdeficient condition, the expression of p-Nrf2 and the activity of SOD were significantly decreased; those induced the content of NO, H  and other oxidants increases, and the balance of oxidation and antioxidation in organisms shifts, thus causing damage to cells or tissues.In this state, the NLRP3 inflammasome was increased, caspase-1 underwent greater activation, and GSDMD was effectively cleaved [31].With increased H 2 O 2 content, caspase-1, which affects pyroptosis, is also significantly increased, which can be manifested as the release of IL-1β, IL-18, or other inflammatory factors [22].This molecular regulatory process is distinct from apoptosis.The previous results showed that Se deficiency can cause apoptosis; the ratios of Bax/Bcl2, caspase 3, and caspase 8 were significantly increased [32].Our study found that the content of related pyroptosis proteins ASC, NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 were increased significantly in the low-selenium treatment compared with the control group.The activation of the NLRP3 inflammasome is governed by GSTO1-promoted ASC deglutathionylation in macrophages [33].The expression of ASC, NLRP3, and caspase-1 increased in the low-selenium group.ASC is involved in NLRP3 activation.NLRP3 can bind to ASC protein, which in turn binds to pro-caspase-1 to form the NLRP3 inflammasome.Thus, when inflammation is severe, the ASC in neutrophils also increases.Caspase-1 regulates cell inflammation by promoting the processing and release of inflammatory factors.When activated by the inflammatory body, caspase-1 excises IL-1β precursors and IL-18 precursors to make them active, and caspase-1 promotes the release of these processed and mature inflammatory factors.The experimental results clearly showed an increased expression of IL-1β and IL-18 inflammatory factors.When cells are in an inflammatory environment, the feedback leads to cell death.
Oxidative stress is closely related to inflammation, and various signaling pathways and signaling factors are interlinked in the body.The process of pyroptosis is regulated by the MAPK pathway.MAKP-signal-pathway-related proteins were further identified using WB.The content of Nrf2, an antioxidant protein associated with the inflammatory response, was significantly reduced, and elevated AKT may participate in the inflammatory response via the PI3K/ AKT pathway.The levels of P38 and P-JNK proteins, which are associated with the inflammatory response, were significantly increased.H 2 O 2 can activate the p-ERK1/2 protein, and our results showed that its content was also significantly increased (Fig. 4).The expression level of the GSDMD gene, which is a key protein of cell pyroptosis, was significantly increased.
To summarize, we examined five indicators of oxidative stress in pig spleen and identified the expression of 11 inflammatory proteins and factors.We found that Se deficiency may increase the expression level of free radicals, pyroptosis, and inflammation.The results indicate that selenium deficiency may be involved in the pyroptosis of porcine spleen cells by regulating the ROS/NLRP3/IL-1β signaling pathway.

Fig. 1
Fig. 1 Pig-spleen histopathology.Lymphocytes in the white pulp were loosely arranged (blue arrow) and edematous, with loose connective tissue and wide cell spacing (black arrow) and necrotic cell

Fig. 2
Fig. 2 Selenium-deficiency-induced oxidative stress in pig spleen.A-C Content of MDA, H 2 O 2 , and NO, respectively; D-E activity of CAT and SOD, respectively; F ROS levels; G the ratio protein-level of p-Nrf2; *p < 0.05, n = 5

Table 2
Antibodies used in the present study