Introduction

In recent years, there has been growing interest in the potential health benefits of selenium nanoparticles (SeNPs) due to their unique properties and biological activities. Selenium, an essential trace element, plays a crucial role in various physiological processes, including antioxidant defense and immune regulation1,2. The emergence of nanotechnology has allowed for the development of selenium in Nano particulate form, opening avenues for novel therapeutic applications3,4.

The intestinal microstructure is a critical determinant of overall health, particularly in avian species such as chickens5. The intricate balance between gut morphology and functionality significantly influences nutrient absorption, immune responses, and overall well-being. Disruptions in intestinal health can lead to various disorders, affecting growth, development, and disease susceptibility6,7,8.

After hatching, chicks are exposed to various harmful bacteria, and the susceptibility of the intestine to oxidative stress is heightened, resulting in increased inflammation9. IL-1β and IL-8, important inflammatory cytokines where IL-1β promotes inflammation and tissue injury, while IL-8 contributes to mucosal healing and immune cell recruitment10,11. The health of the broiler intestine is intricately linked to the functionality of the intestinal barrier proteins (claudin-5 and zonula occluden-1 (ZO-1)12,13. Serving as the initial defense line for the intestine, the intestinal mucus layer plays a pivotal role. The inner mucus layer functions as a protective shield against harmful bacteria, while the outer mucus layer provides nourishment for symbiotic microorganisms. Mucin2 (Muc2), the primary component of the intestinal mucus layer, is produced by goblet cells14. A recent study suggests that the release of mucin from intestinal goblet cells is contingent upon various biological processes. These processes encompass endocytosis, autophagy, reactive oxygen species (ROS) production, and the formation and activation of inflammasomes15. Pathogenic bacteria infiltrate the intestine, prompting goblet cells to endocytose Toll-like receptor (TLR) ligands. This initiation triggers the NLR family pyrin ___domain-containing 6 (NLRP6) inflammasome, culminating in Muc2 exocytosis facilitated by TLR- and MyD88-dependent ROS production16. Elevated levels of inflammatory substances such as IL-9, IL-13, and IL-25 have been shown to promote goblet cell differentiation and proliferation via the STAT6 signaling pathway17. Goblet cells play a crucial role in producing a protective mucus layer for the intestinal tissue.

Selenium (Se) is an essential trace element that plays a vital role in maintaining the health and productivity of poultry by participating in antioxidant defense, immune regulation, and thyroid hormone metabolism. In chickens, adequate selenium intake is crucial for growth performance, reproductive capacity, and disease resistance18,19. Selenium exerts its biological effects primarily through its incorporation into selenoproteins, such as glutathione peroxidases and thioredoxin reductases, which are integral to redox balance and cellular protection against oxidative stress. Recent studies have demonstrated that SeNPs may offer superior bioavailability and lower toxicity compared to inorganic and organic selenium sources, thereby enhancing their efficacy in modulating immune responses and gut health in poultry20,21. One notable aspect of intestinal health is the NLRP3 (NOD-like receptor family, pyrin ___domain-containing 3) signaling pathway, a key component of the innate immune system22,23. The NLRP3 inflammasome is implicated in the regulation of inflammatory responses and has been associated with various gastrointestinal conditions24,25. Understanding the interplay between SeNPs and the NLRP3 signaling pathway in the context of intestinal health is of paramount importance for advancing our knowledge of dietary interventions and potential therapeutic strategies. This research aims to investigate the impact of SeNPs on intestinal health in a chicken model and elucidate the underlying mechanisms involving the NLRP3 signaling pathway. By examining the morphological changes, immune responses, and molecular interactions within the intestinal environment, we seek to provide valuable insights into the potential of SeNPs as a modulator of intestinal health and its implications for avian well-being. This study contributes to the broader field of Nano medicine and holds promise for the development of targeted interventions to enhance intestinal resilience and overall health in poultry and potentially other species.

Materials and methods

Ethics statement

The current study was conducted according to the ARRIVE guidelines. All experiments were conducted according to The Institutional Animal Care and Use Committee (IACUC) approved and reviewed each of the experimental methods used in this research in line with the guidelines for Animal Care of the College of Biological Resource and Food Engineering, Qujing Normal University, 655011 Yunnan, China (Approval no.: QNU 2024-445).

Chemicals and antibodies

SeNPs used in the study were provided by Beijing Wahmix Biotechnology Co. Ltd. (Tangshan, China). The SeNPs were obtained by converting saline selenite through fermentation with the unaffected Bacillus subtilis S12. This process resulted in the production of crimson SeNPs with a diameter of approximately 170 nm from sodium selenite. Total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), MDA and CAT commercial kits were obtained from Jiancheng BioEngineering Institute (Nanjing, Jiangsu, China). ZO-1 and claudin-5 antibodies were obtained from Thermo Fisher Scientific (Madison, WI, USA). Bax, Caspase-3 and Bcl-2 antibodies were received from ABclonal (Wuhan, Hubei, China). IL-1, IL-8, TLR-2 and NLRP-3 antibodies were purchased from Wanlei (Wanleibio, Shenyang, China). HE and AB-PAS kits were purchased from YEASEM (Shanghai, China).

Birds and experimental design

A total of 150 Ross 308 1-day-old chicks were randomly selected from a pool of 1000 chicks and utilized in the experiment. The chicks were sourced from PIAST PASZE, a commercial hatchery. The experimental groups consisted of 50 chickens in each group. The control group received a standard commercial pelleted diet. The basal diet used in the study met or exceeded the dietary requirements for chicken broilers, as per the guidelines provided by the National Research Council (1994). Detailed formulations for each stage, including ingredient composition and calculated nutrient levels, have now been added to the revised manuscript as (Supplementary Table 1). The experimental groups were administered 0.20 mg/kg (low dose) and 0.40 mg/kg (high dose) of SeNPs. The experimental phase extended over 30 days, and all diets were formulated in pelleted and crushed forms. Feed intake and body weight of ducks per pen were recorded weekly, whereas feed intake and mortality per cage were recorded daily.

Detection of oxidative stress-related indexes

A 50 mg amount of tissue is weighed to make a 2% tissue suspension. Centrifuge 1000*g for 5 min and take the supernatant to be measured. The cellular activity of MDA, SOD and CAT was measured using specific commercial kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing. China) by manufacturer’s instructions.

Hematoxylin–eosin staining

Each chicken underwent the surgical removal of an identical segment of the small intestine, which was then trimmed and immersed in a fixative solution containing 4% paraformaldehyde for 24 h. After removal of the fixative, the tissue was subjected to a washing process, dehydrated using an ethanol concentration gradient, treated with a xylene solution for transparency, and finally impregnated with wax. Subsequently, the tissue was sectioned using a microtome and affixed to a slide. The sections underwent staining with hematoxylin for 10 min, followed by two rinses under running water. Subsequently, eosin staining solution was applied for 1 min, followed by two additional rinses under running water. The sections were then left to air dry and photographed under a microscope.

Immunohistochemical analysis

For the histological analysis, all samples underwent dewaxing in xylene and rehydration through immersion in concentrated ethanol. Antigen retrieval was performed by boiling the sections in citrate buffer. To block endogenous peroxidase, the sections were treated with 3% hydrogen peroxide. Subsequently, the sections were incubated with primary antibodies. Imaging and scanning of the slides were conducted using an SCN400 slide scanner (Leica). Image-Pro plus 6.0 software was employed for quantitative analysis of the images.

Quantitative real-time PCR (qRT-PCR)

Total RNA from the small intestine tissue was extracted with TRIzol reagent (Life Technologies, CA, USA) following the manufacturer’s protocol. The concentration of the extracted RNA was detected by using Nanodrop 2000C spectrophotometer (Thermo Fisher Scientific, USA) and then they were reverse transcribed to cDNA by HiScript II Reverse Transcriptase Kit (YEASEM, Shanghai, China). qRT-PCR was performed using Applied Biosystems 7500 (Life Technologies, CA, USA) and the experiment was carried out using the SYBR Green Master Mix Kit (YEASEM, Shanghai, China) following the manufacturer’s instruction. The relative mRNA transcriptional levels were calculated by normalizing to β-actin using the 2−∆∆CT method. The sequences of gene-specific primer were shown in Table S2.

Statistical analysis

Statistical analysis was performed on obtained data and the results are presented as mean ± SEM. GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA) was employed for data analysis using one-way analysis of variance (ANOVA) and Scheffe’s SF test. P-values less than 0.05 were considered indicative of a significant difference.

Results

Effect of selenium nanoparticles on small intestine of chicken

To assess the impact of SeNPs on the chicken’s small intestine, various parameters were evaluated. Morphological observations indicated that the group treated with SeNPs exhibited improved small intestinal development and increased small intestine weight compared to the control group. Histological analysis using HE and AB-PAS staining revealed that villi height significantly increased in the 0.2 mg/kg SeNPs group compared to the 0.4 mg/kg SeNPs group. Additionally, there was a significant increase in the number of goblet cells in the 0.2 mg/kg selenium nanoparticles group compared to the 0.4 mg/kg SeNPs group (Fig. 1A–C). Moreover, mRNA expression of Muc2 was significantly higher in the 0.2 mg/kg SeNPs group compared to the 0.4 mg/kg SeNPs group (Fig. 1D).

Fig. 1
figure 1

HE staining was used to detect morphology of the small intestine of broilers of each group (A). AB-PAS staining was used to detect morphology of goblet cells the small intestine of broilers of each group (B). (C) The quantification of villi height, crypt depth and number of goblet cell number were measured by 10 villi/section/bird and 05 birds/group; Data was expressed as the mean ± standard deviation (SD), and different alphabets (a, b and c) indicate significant difference between the two groups, arrow shows (goblet cells). Scale bars 100 μm.

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Effect of selenium nanoparticles on oxidative stress and inflammation of the small intestine

In order to verify the effect of SeNPs on oxidative stress and inflammation in the small intestine, we first tested the activity of antioxidant enzymes in the small intestine, and the results were shown in (Fig. 2A), compared with the control group, the SeNPs groups decreased the serum MDA level, but increased enzyme activities of CAT and SOD. However, these changes were more improved by the 0.4 mg/kg SeNPs group compared with 0.2 mg/kg SeNPs group. We further examined indicators of inflammation, the results were shown in (Figs. 2B–D and 3), compared with the control group, the SeNPs groups decreased the inflammation in the small intestine. However, protein and gene expression of IL-1 were significantly down-regulated and IL-8 significantly up-regulated in 0.4 mg/kg SeNPs group compared with 0.2 mg/kg SeNPs group (Figs. 2 and 3A–D).

Fig. 2
figure 2

Antioxidant enzymes in the small intestine (A), Immunofluorescence and Immunohistochemical staining (B–C), and mRNA level of IL-1 in the small intestine of broiler (D), arrow shows (indicators of inflammation). Scale bars 50 and 100 μm

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Fig. 3
figure 3

Immunofluorescence and Immunohistochemical staining (A–B), and mRNA level of IL-8 in the small intestine of broiler (C), arrow shows (indicators of inflammation). Scale bars 50 and 100 μm.

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Effect of selenium nanoparticles on apoptosis and pyroptosis of the small intestine

As shown in (Figs. 4 and 5), compared with the control group, the SeNPs groups significantly decreased the apoptosis and pyroptosis of the small intestine. However, treatment with 0.4 mg/kg SeNPs group, apoptosis related protein and gene expression level of Bax, Caspase-3 and Bcl-2 were significantly decreased compared with 0.2 mg/kg SeNPs group (Fig. 4A–E). Furthermore, pyroptosis related protein and gene expression level of TLR2 and NLRP-3 significantly decreased in 0.4 mg/kg SeNPs group compared with 0.2 mg/kg SeNPs group (Fig. 5A–F).

Fig. 4
figure 4

Immunohistochemical staining (A–D), and mRNA level of Bax, Caspase-1 and Bcl-2 in the small intestine of broiler (E), arrow shows (immunoreactivity). Scale bars 50 and 100 μm.

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Fig. 5
figure 5

Immunofluorescence and Immunohistochemical staining (A-B & D-E), and mRNA level of TLR-2 and NLRP-3 in the small intestine of broiler (C & F), arrow shows (immunoreactivity). Scale bars 50 and 100 μm.

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Effect of selenium nanoparticles on intestinal barrier of the small intestine

Finally, after SeNPs treatment, the immunohistochemistry and qPCR were performed to detect intestinal barrier related proteins and genes. Compared with the control group, SeNPs groups significantly decreased the tight and gape junction proteins and genes of the small intestine. However, treatment with 0.4 mg/kg SeNPs group, intestinal barrier related proteins and genes expression level of ZO-1 and claudin-5were significantly decreased compared with 0.2 mg/kg SeNPs group (Fig. 6A–C).

Fig. 6
figure 6

Immunohistochemical staining (A–B), and mRNA level of ZO-1 and Claudin-5 in the small intestine of broiler (C), arrow shows (immunoreactivity). Scale bars 50 and 100 μm.

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Discussion

Selenium nanoparticles (SeNPs) are very essential for chicken’s health development26,27. High quantity of selenium may decrease mortality rate. This study found that dietary SeNPs can lower broiler mortality28. In previous research also have discovered that dietary SeNPs can significantly decrease chicken mortality29. In commercial farming, chicks may face difficulties from oxidative stress and microbial pathogens. Adding SeNPs to the feed might reduce inflammation and increase antioxidant status, leading to improved broiler growth performance.

Intestinal morphological variation associated with nutrient absorption surface area and intestinal development30,31. In the present study, the villi height and crypt depth of the jejunum adding 0.2 mg/kg SeNPs resulted in the highest villi height and crypt depth value, followed by 0.4 mg/SeNPs in the small intestine. These findings are consistent with previous studies that have demonstrated the beneficial effects of SeNPs on intestinal morphology by promoting antioxidant status and cellular proliferation in the gut epithelium, thereby supporting better growth performance and gut health in poultry and other animal models32,33. Researchers discovered that dietary SeNPs supplementation improved villi height and crypt depth in broilers’ intestines. Dietary Selenium supplementation might boost gut health through modulating flora34. Tang et al.35 discovered that changing the gut flora led to increased nutrition and villi growth. Selenium promotes intestinal tissue development through regulating inflammatory cytokine activity and elevating antioxidant levels36. SeNPs properly reduce inflammation and oxidative stress in the gastrointestinal tract. Oxidative stress in the intestine is primarily driven by excessive generation of reactive oxygen species (ROS), which can damage lipids, proteins, and DNA, leading to compromised epithelial function and triggering inflammatory responses37. Thus, we propose that SeNPs improve gut morphology involves reducing harmful microorganism growth and inflammation. Intestinal epithelial cells create different kind of intestinal barriers that allow penetration, expression of claudin proteins produce tight junction barrier, and reduce solute permeability38,39,40. Improving tight junction proteins enhances intestinal integrity and reduces paracellular permeability, increasing the epithelial barrier and inhibiting microorganisms from entering the body. Tight junctions serve as receptors for pathogen toxins, specifically Clostridium perfringens enterotoxin41. Pathogenic bacteria use TJs as receptors to create pores in intestinal epithelial cells. Calcium infiltration through these pores causes cell destruction. Our study demonstrated that SeNPs reduced ZO-1 and claudin-5 expression in the small intestine compared to the control group. Supplementing diets with SeNPs significantly decreased the expression of intestinal claudin-1, ZO-1, and ZO-232. According to Ali et al.32 SeNPs can decrease the attachment sites of pathogen bacteria through decreasing claudin-1, ZO-1, and ZO-2 levels, including extra selenium in the diet enhanced gut barrier integrity by decreasing oxidative stress42.

The intestinal mucous layer comprises two distinct layers: a cohesive inner mucus and a detached, loosely structured outer mucus43. Mucus primarily consists of Muc2, which is produced and released by goblet cells44. Pathogenic bacteria can compromise the mucus barrier, triggering goblet cells to produce and release more Muc2 to prevent penetration45. Goblet cells play a crucial role in shielding intestinal crypts from germs that have breached the inner mucus layer16. Inadequate selenium during broiler growth may lead to increased cytoplasm vacuolization and goblet cell disintegration46. SeNPs have been shown to enhance the number of goblet cells and improve the structure of mucus in the small intestine47,48. Bami et al.33 observed that chickens fed 0.3 mg/kg SeNPs developed higher goblet cell density in the different parts of small intestine. Similarly, our findings showed that adding 0.2 mg/kg SeNPs dramatically enhanced the number of goblet cells and their RNA expression of Muc2 in the small intestine. The avian intestinal mucosa’s pro-inflammatory cytokines make a significant contribution to the host’s response to infectious pathogens49. IL-1b, a pro-inflammatory cytokine, affects both antibody and cell-mediated immune responses50. IL-8, a CXC chemokine, attracts leukocytes, specifically neutrophils, to regions of inflammation49. Selenium deprivation resulted in apoptosis of chicken intestinal cells through an inflammatory signaling-induced death receptor pathway and reduced immunity through activation of the NF-kB signaling pathway, which is controlled via redox activity46,51. Selenium injection in ovo has been shown to increase immune responses against poultry necrotic enteritis by increasing levels of IL-1b, IL-6, and IL-8 gene transcripts52,53. Although the oxidative damage model hadn’t been developed, this study demonstrated that increasing the concentration of selenium nanoparticles drastically reduced mRNA expression of IL-1β in the small intestine. Furthermore, higher doses of selenium nanoparticles did not increase IL-8 expression. The results showed that exogenous supplementation with SeNPs successfully reduced oxidative stress and inflammation in the small intestine.

Apoptosis plays a crucial role in the growth, homeostasis, and immunological control of multicellular organisms54. Cellular dysfunction, triggered by the overproduction of reactive oxygen species, pathogens, or host chemicals, can lead to inflammation and cell death55. This study utilized immunohistochemistry to illustrate that the 0.2 and 0.4 mg/kg SeNPs groups exhibited significantly reduced levels of apoptosis compared to the control group. Selenium nanoparticles substantially decreased the mRNA expression of Bax, caspase-1, and Bcl-2 in the small intestine. Selenium deprivation resulted in the death of intestinal villi cells and damage to the mucosal barrier of the small intestine46,51. The balance between Bcl-2 and Bax frequently reflected by the Bax/Bcl-2 ratio is critical for determining cell fate56. A reduction in both genes suggests that SeNPs may exert a regulatory effect by suppressing the overall activation of apoptotic pathways, contributing to enhanced intestinal cell survival57. The concomitant decrease in Caspase-3 expression further supports the anti-apoptotic potential of SeNPs, indicating that their protective effect on intestinal tissue may be mediated through downregulation of mitochondrial-dependent apoptosis58. The NLRP3/caspase-1/IL-1β pathway is a major active mechanism of inflammation, induced by reactive oxygen species and pathogens to prevent infections54. When the pathway is activated, NLRP3 triggers caspase-1, leading to IL-1β-induced inflammation54,59. Mucin secretion from intestinal goblet cells depends on the construction and activation of inflammasomes15. Among them, NLRP6 has been shown to regulate Muc2 secretion in goblet cells and enhance mucus layer formation60. Intestinal inflammation triggers NLRP6 in goblet cells, regulating Muc2 release, and promoting mucus layer development61. Goblet cells respond to Toll-like receptor (TLR) ligands by activating NLRP6, which is downstream of TLR and MyD88-dependent reactive oxygen species synthesis16. NLRP3 has been demonstrated to reduce oxidative stress and inflammation in chicken kidneys, acting as an antagonist to lead-induced inflammation62. Limited studies have explored how the inflammasome regulates intestinal cells in chickens. The expression levels of NLRP3, Caspase-1, IL-8, and TLR2 decreased linearly as selenium nanoparticles concentrations increased. Selenomethionine inhibited the NLRP3 signaling pathway, reducing lipopolysaccharide-induced inflammation in the chicken liver63. Collectively, these findings suggest that selenium nanoparticles reduce intestinal oxidative stress and inflammation through the NLRP3/caspase-1/IL-1β signaling pathway.

Conclusion

The treatment with 0.2 mg/kg SeNPs inhibits the oxidative stress and inflammation in the small intestine through modifying the activation of the NLRP3 signaling pathway in birds. High-dose SeNPs were effective in reducing mortality, apoptosis, oxidative stress, and inflammation, while also downregulating key markers associated with intestinal barrier function and immune response.