SELENOF Mitigates Bovine Mastitis by Preserving Mitochondrial Homeostasis and Suppressing NLRP3-Mediated Pyroptosis

TL;DR

This study shows that Selenoprotein F protects cow mammary cells from mastitis by maintaining mitochondria health and reducing inflammation.

Contribution

The study identifies Selenoprotein F as a novel target for mitigating bovine mastitis through mitochondrial preservation and suppression of pyroptosis.

Findings

SELENOF overexpression restores mitochondrial membrane potential and reduces inflammation in mastitis-affected cells.

SELENOF suppresses NLRP3-mediated pyroptosis by inhibiting caspase-1/GSDMD-N pathway activation.

SELENOF is significantly downregulated in mastitic tissue, linking its deficiency to disease progression.

Abstract

Bovine mastitis is a common and costly inflammatory disease in dairy cows that damages mammary tissue and reduces milk production. This study investigated how a protein called Selenoprotein F helps protect cow mammary cells from this disease. The researchers examined tissue samples from cows with mastitis and found severe cell damage, inflammation, and a specific type of cell death called pyroptosis. They also discovered that Selenoprotein F levels were significantly lower in diseased tissue. Using laboratory cell experiments, the team showed that increasing Selenoprotein F levels could repair damaged mitochondria (the cell’s energy centers) and reduce harmful inflammation. These results point to Selenoprotein F as an important defender against mastitis, highlighting its potential to shield dairy cows from this damaging inflammatory condition. This discovery opens the door to a novel nutritional approach for safeguarding cattle health, which could in turn help curb the heavy reliance on antibiotics within the dairy sector.

Bovine mastitis threatens the dairy industry with limited effective therapies. The selenoprotein family offers potential anti-inflammatory interventions, yet the role of Selenoprotein F (SELENOF) remains unclear. This study investigated SELENOF in mitochondrial damage and pyroptosis using clinical mammary biopsies and a Staphylococcus aureus-induced Mammary alveolar cell-type T (MAC-T) cell model. Histology, TEM, immunofluorescence, Western blot, qPCR, RNA-seq, and mitochondrial staining (MitoTracker Red and JC-1) were employed. Mastitic mammary tissue exhibited severe architectural disruption, including focal necrosis with coalescing vacuoles of variable size, extensive epithelial denudation, and interstitial thickening with dense inflammatory infiltrates. At the ultrastructural level, mitochondrial swelling, cristae loss, and plasma membrane rupture were evident. Additionally, these tissue specimens exhibited marked upregulation of inflammatory mediator transcripts, notably IL-1β, IL-6, and TNF-α, alongside heightened abundance of pyroptosis-associated proteins including NOD-like receptor family pyrin domain containing 3 (NLRP3), cleaved caspase-1, and GSDMD-N (Gasdermin D N-terminal domain). RNA-seq identified SELENOF as significantly downregulated. The MAC-T model recapitulated the mitochondrial dysfunction, inflammatory response, and pyroptosis observed in mastitic tissue. SELENOF overexpression restored mitochondrial membrane potential, dampened the output of inflammatory signaling molecules, and suppressed NLRP3-mediated pyroptosis via attenuation of caspase-1/GSDMD-N pathway activation. These findings establish SELENOF as a novel target that mitigates bovine mastitis by preserving mitochondrial homeostasis and suppressing NLRP3-mediated pyroptosis.

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Full text

1. Introduction

Bovine mastitis, an inflammatory condition of mammary tissue, occurs ubiquitously across global dairy operations. Annual economic losses attributable to reduced milk yield, discarded milk, premature culling, and veterinary expenditures exceed billions of dollars [1]. Pathological damage associated with mastitis manifests as interstitial thickening and dense inflammatory infiltration within mammary tissue. Cytokines released by infiltrating immune cells amplify inflammatory signaling cascades, thereby promoting programmed cell death of mammary epithelial cells and exacerbating tissue injury [2]. Consequently, elucidation of the intrinsic molecular regulatory mechanisms governing host cellular responses to inflammatory damage, and identification of molecular targets capable of suppressing regulated demise of bovine mammary epithelial cells (BMECs), constitute critical priorities for mastitis prevention and therapeutic intervention.

The pathogenesis of mastitis is intimately linked to damage and programmed death of mammary epithelial cells. This inflammatory mode of regulated cell demise, driven by caspase-1 activation downstream of inflammasome assembly, manifests through plasma membrane pore formation and subsequent osmotic lysis [3]. Inflammasomes represent critical intracellular multiprotein complexes responsible for initiating immune responses and regulating inflammatory processes. Among these, the nucleotide-binding oligomerization domain, leucine-rich repeat, and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a particularly pivotal role in pyroptotic cell death [4]. As a cytosolic pattern recognition receptor (PRR), NLRP3 serves as a sentinel of the innate immune system, detecting diverse pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to initiate host defense responses [5]. NLRP3 activation promotes the proteolytic activation of inflammatory caspases and subsequent cleavage of gasdermin D (GSDMD), resulting in membrane pore formation, cellular swelling, and release of pro-inflammatory cytokines (IL-1β and IL-18) [6], thus orchestrating immune reactions and exacerbating tissue injury. In a murine model of mammary infection, Hanna et al. documented abundant GSDMD-N-terminal-positive membrane pores in mammary alveolar epithelium, with significant positive correlations observed between milk levels of IL-1β and lactate dehydrogenase (LDH) and expression of the pyroptosis marker GSDMD-N (Gasdermin D N-terminal domain) [7], indicating a strong association between mastitis pathogenesis and pyroptosis. Given that the NLRP3 inflammasome serves as the critical nodal point for caspase-1 activation [8], and NLRP3 assembly is highly dependent upon mitochondrial reactive oxygen species (mtROS) and oxidized mitochondrial DNA (mtDNA) signaling [9], mitochondrial damage likely triggers NLRP3 activation, with subsequent GSDMD-mediated pyroptosis representing a central mechanism underlying persistent inflammation in mastitis.

Mitochondria function as intracellular dual sensors that activate innate immune signaling directly through mtROS and mtDNA leakage [10], while simultaneously serving as critical checkpoints governing cell fate through their structural and functional integrity. Multiple in vitro studies have demonstrated that exposure to PAMPs triggers rapid dissipation of mitochondrial membrane potential (ΔΨm) and diminished activity of Electron Transport Chain (ETC) Complexes I and III, resulting in mtROS burst [11]. Released oxidized mtDNA is subsequently recognized by the NLRP3 inflammasome [9], thereby catalyzing caspase-1 activation [12] and driving the processing and release of bioactive IL-1β and IL-18, establishing a positive feedback loop between mitochondria, reactive oxygen species (ROS), and NLRP3. Yan et al. demonstrated that free fatty acid-induced mitochondrial oxidative damage in diabetic cardiomyopathy facilitates mtDNA release into the cytoplasm, triggering NLRP3 inflammasome-dependent cardiomyocyte pyroptosis and cardiac hypertrophy [13]. An et al. reported that mitochondrial injury induces endothelial cell pyroptosis, thereby promoting the initiation and progression of atherosclerosis [14]. Qi et al. further established that mitochondrial injury triggered by kaempferol engages the NF-κB-NLRP3-caspase-1 circuitry, culminating in inflammatory programmed death of gastric carcinoma cells [15]. Whether mitochondrial structural damage and NLRP3-mediated pyroptosis occur in bovine mammary biopsy tissues, however, remains to be elucidated.

Given the escalating prevalence of antimicrobial resistance, nutritional interventions have emerged as critical alternative strategies for mastitis mitigation, with selenium (Se) garnering substantial attention due to its tripartite effects on antioxidant defense, immune modulation, and inflammatory regulation. Selenium, an essential micronutrient in mammals [16], participates in redox homeostasis, inflammatory modulation, and immune responses. Studies by Jing Zhang and colleagues have demonstrated that selenium and selenoproteins inhibit oxidative stress-induced mitochondrial damage and pyroptosis through maintenance of mitochondrial homeostasis, enhancement of antioxidant capacity, and modulation of the NLRP3 inflammasome pathway, consequently attenuating tissue injury [17,18,19,20]. Research conducted over the past half-century has consistently established that dietary selenium levels correlate closely with mammary gland health in dairy cattle, wherein selenium is co-translationally incorporated into selenoproteins as the 21st amino acid, selenocysteine (Sec) [21]. Dairy cattle with elevated blood selenium concentrations and glutathione peroxidase (GPX) activity exhibit significantly reduced incidences of clinical and subclinical mastitis, decreased somatic cell count (SCC), and lower detection rates of major mastitis pathogens [22]. However, existing investigations have predominantly focused on classical selenoproteins, including GPX1 and SELENOP (Selenoprotein P), leaving the role of SELENOF (Selenoprotein F) [23] in bovine mastitis poorly understood. Research indicates that SELENOF mRNA expression is markedly reduced in advanced-stage breast tumors compared to normal tissues, with low expression levels closely associated with unfavorable patient prognosis [24]; functional studies further confirm that SELENOF deficiency promotes aberrant proliferation and apoptosis resistance within the non-malignant mammary epithelial line Michigan Cancer Foundation-10A (MCF-10A), resulting in malignant phenotypes characterized by lumen filling in three-dimensional culture [25], whereas SELENOF overexpression triggers apoptosis and autophagic cell death in breast cancer cells through induction of mitochondrial swelling, outer membrane rupture, and DNA fragmentation [26]. As an endoplasmic reticulum-resident protein, SELENOF maintains mammary epithelial cell homeostasis through regulation of the p21/p27 cell cycle pathway [27]; nevertheless, its expression patterns in infectious inflammation-induced mammary injury, together with its modulatory actions over organelle dysfunction and inflammatory cell demise in BMECs, remain to be elucidated.

Accordingly, this investigation employed histopathological evaluation of clinical mammary biopsies, ultrastructural examination, and transcriptomic screening to characterize the occurrence of mitochondrial damage and pyroptosis and to determine SELENOF expression profiles within mastitic lesions. Integrated with in vitro functional experiments, the regulatory mechanism whereby SELENOF suppresses pyroptosis through maintenance of mitochondrial homeostasis was elucidated. These findings provide mechanistic insights into the target function of SELENOF in selenium-mediated antagonism of bovine mastitis, and establish a theoretical foundation for precision selenium supplementation strategies and novel nutritional interventions targeting mitochondrial damage and pyroptosis.

2. Materials and Methods

2.1. Mammary Tissue Collection

Tissue specimens were harvested from a commercial dairy operation located in Wuhan, Hubei Province, China. Parenchymal samples were obtained from both healthy animals and those diagnosed with clinical mastitis through percutaneous biopsy, immediately flash-frozen in liquid nitrogen, and maintained at −80 °C pending subsequent analyses. Selection criteria relied upon California Mastitis Test (CMT) scoring, with three CMT-negative animals designated as controls and three CMT-strongly positive individuals assigned to the disease group.

2.2. Cell Culture, Bacterial Strain, and Plasmid Constructs

The bovine mammary epithelial cell line Mammary alveolar cell-type (TMAC-T) was obtained as a gift from Prof. Mark Hanigan (Virginia Polytechnic Institute and State University, Blacksburg, VA, USA). Routine maintenance employed DMEM/F12 medium (1:1 mixture; Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China). Staphylococcus aureus (S. aureus) strain ATCC 29213 was provided by Prof. Xiangru Wang (Huazhong Agricultural University, Wuhan, China). For SELENOF overexpression studies, the full-length bovine SELENOF coding sequence (GenBank accession: NM_001034759.2) was inserted into pcDNA3.1 (+) vector, sequence-validated, and designated pcDNA3.1-SELENOF; empty pcDNA3.1 (+) functioned as the control (OE-NC). Plasmid DNA was purified using commercial endotoxin-free mini-prep kits (Omega Bio-tek, Norcross, GA, USA) and stored at −63 °C pending transfection experiments. Primer sequences appear in Table 1.

2.3. CMT

Milk specimens were combined with an equivalent volume of commercial CMT reagent, agitated for 10 s, and visually inspected for gel precipitation intensity. Interpretation followed standard criteria: absence of precipitate indicated negative status (−), whereas immediate solidification denoted strongly positive reaction (+++). Animals exhibiting negative CMT scores were classified as healthy, while those with strongly positive scores were categorized as mastitic for inclusion in downstream experiments.

2.4. Establishment of In Vitro Mastitis Model

Bacterial suspensions were rendered non-viable through heat inactivation (63 °C, 30 min) prior to cellular challenge. MAC-T monolayers were exposed to heat-inactivated S. aureus at MOI 10 for 12 h to simulate inflammatory conditions. Experimental arms comprised unstimulated controls and bacteria-treated groups.

2.5. Transfection Procedures

MAC-T cultures were plated in 12-well format and allowed to reach 70–80% confluency prior to transfection. Complexes were prepared by diluting 1 µg plasmid DNA in 100 µL jetPRIME buffer (Polyplus-transfection, Illkirch, France), combining with 1 µL transfection reagent, and incubating at ambient temperature for 10 min. The mixture was then applied directly to cultures. After 24 h, medium was exchanged for fresh complete growth medium, and incubation continued for 5 h before harvest. Study groups included OE-NC (vector control) and OE-SELENOF (experimental).

2.6. Quantitative Reverse Transcription PCR

RNA Isolation: Total RNA was extracted from snap-frozen tissue samples and cultured cell pellets using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. Solid tissue portions (not exceeding 20 mg) were homogenized in 1 mL TRIzol on ice using mechanical disruption; for adherent cultures, lysis was performed directly in the culture dish. Aqueous and organic phases were separated through chloroform addition, with subsequent RNA recovery via isopropanol precipitation. The resulting pellets underwent sequential washing with 75% ethanol, brief air-drying, and final dissolution in nuclease-free water.

cDNA Synthesis and Amplification: One microgram total RNA was reverse-transcribed using HiScript III RT SuperMix (Vazyme Biotech, Nanjing, China). Quantitative PCR was conducted with AceQ SYBR Green Master Mix (Vazyme Biotech) on a ViiA 7 platform (Applied Biosystems, Foster City, CA, USA). Relative quantification employed the 2^(−ΔΔCt)^ methodology normalized to ACTB. Primer details appear in Table 1.

2.7. Hematoxylin–Eosin (H&E) and Masson’s Trichrome Staining

Tissue fragments were immersion-fixed in 10% neutral-buffered formalin, progressively dehydrated through ethanol gradients, and paraffin-embedded. Five-micrometer serial sections were prepared for H&E staining (morphological assessment) and Masson’s trichrome staining (collagen visualization). Micrographs were captured at 20× magnification. Injury severity was evaluated via a modified semi-quantitative scoring system adapted from Lai et al. [28] for bovine applications. Two independent evaluators, blinded to group allocation, assessed three parameters (0–3 scale each): (i) inflammatory infiltrate density; (ii) acinar architectural integrity/necrotic changes; and (iii) collagen deposition intensity. Aggregate scores ranged 0–9, with higher values indicating more severe damage. Final scores averaged both observers’ ratings.

2.8. Ultrastructural Analysis by Transmission Electron Microscopy (TEM)

For the ultrastructural examination, samples underwent initial rinsing with phosphate-buffered saline followed by primary fixation in 2.5% glutaraldehyde at 4 °C. After thorough buffer washing, tissues were post-fixed using 1% osmium tetroxide. A graded ethanol series served for dehydration before infiltration and embedding in epoxy resin. Ultrathin sections (70 nm) were prepared via ultramicrotomy, subsequently stained with uranyl acetate and lead citrate for contrast enhancement. Observations were conducted on a Hitachi H7650 TEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV.

2.9. Western Blotting

Protein extraction was performed by homogenizing frozen tissue specimens and cell pellets in pre-chilled Radioimmunoprecipitation Assay (RIPA) lysis buffer containing 1 mM Phenylmethylsulfonyl fluoride (PMSF) alongside protease and phosphatase inhibitors, with agitation for 30 min. Lysates were then cleared through high-speed centrifugation (12,000× g, 15 min at 4 °C). Total protein concentrations were quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) method. Western blotting was performed according to standard protocols [29,30]. Briefly, for each sample, 30 µg of protein was separated by electrophoresis on 10% polyacrylamide gels under denaturing conditions and subsequently transferred onto 0.22 µm Polyvinylidene Difluoride (PVDF) membranes. Membrane blocking involved 5% skim milk for 4 h at room temperature. Primary antibodies were applied for overnight incubation at 4 °C, followed by exposure to Horseradish Peroxidase -linked secondary antibodies for 1 h at room temperature. Specific protein bands were visualized through Enhanced Chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) and documented using a Tanon gel documentation system (Tanon Science & Technology, Shanghai, China). Band intensities were quantified with ImageJ software version 1.54i (NIH, Bethesda, MD, USA), normalizing against β-actin signal. Detailed antibody information appears in Table 2.

2.10. Tissue Immunofluorescence

For immunofluorescence staining of tissue sections, paraffin-embedded samples were first subjected to antigen unmasking via heat-induced epitope retrieval using sodium citrate buffer (pH 6.0). Nonspecific binding sites were subsequently blocked with 5% normal goat serum (Beyotime Biotechnology, Shanghai, China). Sections were then probed overnight at 4 °C with primary antibodies directed against NLRP3 and GSDMD. Following extensive PBS washes, appropriate fluorophore-labeled secondary antibodies were applied at ambient temperature under light-protected conditions. Nuclear visualization was achieved through 4′,6-Diamidino-2-Phenylindole (DAPI) incubation (5 min), after which specimens were preserved using antifade mounting medium. Microscopic evaluation was carried out on an Olympus IX73 system (Olympus, Tokyo, Japan). Specific antibody information is listed in Table 2.

2.11. Mitochondrial Visualization

For mitochondrial imaging, MAC-T cells cultured on glass coverslips were incubated with 100 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, Shanghai, China) diluted in serum-free medium for 30 min at 37 °C in the dark. After two consecutive PBS rinses, specimens were fixed using 4% paraformaldehyde for 15 min, followed by nuclear counterstaining with DAPI for 5 min. Fluoromount-G served as the mounting medium before confocal acquisition on a Zeiss LSM 980 microscope (Zeiss, Oberkochen, Germany). Subsequent image analysis and fluorescence intensity measurements were conducted using ImageJ software (NIH).

2.12. Assessment of Mitochondrial Membrane Potential

Mitochondrial membrane potential was evaluated with JC-1 staining (Beyotime Biotechnology, Shanghai, China). Following experimental treatments, cells were incubated with 5 µg/mL JC-1 working solution prepared in serum-free medium for 20 min at 37 °C under light-shielded conditions. Post-staining washes preceded microscopic examination using an Olympus IX73 inverted microscope (Olympus, Tokyo, Japan). Healthy mitochondria with intact membrane potential display red-orange fluorescence corresponding to J-aggregate formation (excitation 560 nm, emission 595 nm), whereas compromised mitochondria manifest green fluorescence indicative of monomeric JC-1 (excitation 485 nm, emission 535 nm). Each experimental condition was assessed using three independent replicates.

2.13. ROS Detection by Flow Cytometry

Cellular ROS levels were measured using a DCFH-DA-based detection kit (S0033, Beyotime Biotechnology, Shanghai, China). The fluorescent probe was diluted to 10 µM in serum-free culture medium and loaded into cells for 20 min at 37 °C in darkness. Following incubation, cells underwent extensive PBS rinsing to eliminate unbound probe. Fluorescence detection was performed on a Beckman CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA), with excitation at 488 nm and emission collection at 525 nm. Elevated DCF fluorescence indicates increased intracellular oxidative stress.

2.14. Transcriptome Sequencing

Total RNA from flash-frozen mammary specimens was extracted with TRIzol, quantified by Qubit 4.0, and quality-verified using Qsep400 bioanalyzer (BiOptic Inc., New Taipei City, Taiwan, China) (RNA Integrity Number ≥ 7). Polyadenylated transcripts were captured with oligo (dT) magnetic beads, fragmented, and converted to double-stranded cDNA. Library construction included end repair, A-tailing, adapter ligation, and size selection (300–350 bp) using AMPure XP beads, followed by PCR enrichment. Sequencing was performed on Illumina Nova Seq 6000 (Illumina, San Diego, CA, USA) generating paired-end 150 bp reads with ≥8 Gb clean data per sample (Q30 ≥ 96%). Reads were mapped to Bos taurus ARS-UCD1.2 reference using HISAT2 version 2.2.1, with feature Counts generating gene-level quantifications. Differential expression analysis employed DESeq2 version 1.22.1 (mastitis versus normal, |log_2_ FC| ≥ 1, FDR < 0.05).

2.15. Statistical Analyses

Statistical evaluation relied on GraphPad Prism 9.0. Intergroup comparisons applied Student’s t-test (two groups) or one-way ANOVA (three or more groups). Data represent mean ± SD, with p < 0.05 considered significant.

3. Results

3.1. Pathological Damage and Inflammatory Response in Mastitic Bovine Mammary Tissue

Experimental animals were selected based on CMT scores, comprising healthy cows with CMT-negative (-) status and cows with clinical mastitis exhibiting CMT-strong positive (+++) reactions. Mammary parenchymal tissue was collected via biopsy gun (Figure 1A). Expression levels of IL-1β, IL-18, and NLRP3 were markedly elevated in mastitic mammary tissue compared to normal tissue (p < 0.05) (Figure 1B). Gross examination revealed distinct phenotypic differences between groups. Mammary glands from healthy cows appeared normal with soft consistency, whereas glands from mastitic cows displayed characteristic inflammatory manifestations including erythema, edematous enlargement, and induration. Histological analysis demonstrated well-defined architectural organization in normal mammary tissue. In contrast, mastitic tissue exhibited disrupted lobular architecture, focal necrosis with coalescing vacuoles of variable dimensions, extensive epithelial denudation, and dense inflammatory cell infiltration. Acinar structures were compromised, with increased collagen deposition (blue staining) and inflammatory infiltrates (red staining) evident, indicating substantial tissue damage and cellular disorganization. Histopathological scores were significantly higher in mastitic tissue relative to normal controls (p < 0.01) (Figure 1C). Collectively, these data indicate that mastitis induces pronounced inflammatory responses and pathological alterations in mammary tissue architecture.

3.2. Mitochondrial Damage and Pyroptosis in Mastitic Bovine Mammary Tissue

TEM revealed well-preserved cellular architecture with morphologically intact mitochondria in normal mammary tissue, whereas mastitic tissue exhibited characteristic ultrastructural damage including mitochondrial swelling and cristae dissolution (Figure 2A). Immunofluorescence analysis demonstrated substantially enhanced fluorescence intensity for GSDMD (green) and NLRP3 (red) in mastitic tissue, with markedly increased co-localization (yellow fluorescence in merged images) (Figure 2B). RT-qPCR analysis showed significant upregulation of GSDMD, caspase-1, and Apoptosis-associated Speck-like Protein with a CARD (ASC) mRNA expression in mastitic tissue compared to normal controls (p < 0.05) (Figure 2C). Furthermore, Western blot and densitometric quantification indicated pronounced elevation of cleaved caspase-1, IL-18, GSDMD-N, and NLRP3 protein levels in mastitic tissue (p < 0.001) (Figure 2D,E). These findings demonstrate that mastitic mammary tissue undergoes substantial mitochondrial damage and activation of NLRP3-mediated pyroptosis.

3.3. Screening and Identification of SELENOF in Mammary Gland Tissues from Cows with Mastitis

Transcriptomic profiling of bovine mammary tissue revealed distinct gene expression patterns between mastitic and healthy groups, with high intra-group consistency observed among biological replicates (Figure 3A). A total of 5082 differentially expressed genes (DEGs) were identified, comprising 2621 upregulated and 2461 downregulated genes in mastitic tissue relative to controls (Figure 3B). Volcano plot visualization indicated SELENOF as one of the significantly downregulated DEGs (Figure 3C). Gene Ontology (GO) enrichment analysis demonstrated significant enrichment of DEGs in multiple mitochondria-linked functional categories, encompassing protein assemblies housing mitochondrial components, complexes residing within the organelle’s inner membrane, and the construction of respiratory chain machinery (Figure 3D). Heatmap analysis further revealed marked downregulation of selenoprotein family members, including SELENOF and Selenoprotein M (SELENOM), in mastitic tissue (Figure 3E). Validation of sequencing data was performed using RT-qPCR and Western blot. SELENOF mRNA (Figure 3F) and protein (Figure 3G) levels were significantly reduced in mastitic tissue compared to normal controls (p < 0.01), corroborating the transcriptomic findings. These transcriptomic analyses demonstrate substantial gene expression reprogramming in mastitic mammary tissue and implicate mitochondrial dysfunction and the associated regulatory factor SELENOF in the pathogenesis of bovine mastitis.

3.4. S. aureus Induces Mitochondrial Damage and Pyroptosis in MAC-T Cells

A cell-based experimental system was constructed through exposure of MAC-T cultures to thermally attenuated S. aureus challenge (Figure 4A). TEM revealed intact cellular architecture and well-preserved mitochondrial morphology in control cells, whereas S. aureus-treated cells exhibited characteristic ultrastructural damage including mitochondrial matrix expansion, disorganized and fragmented cristae, and outer membrane rupture (Figure 4B). S. aureus stimulation significantly upregulated mRNA expression of IL-6, IL-1β, and NLRP3 (p < 0.001) (Figure 4C). MitoTracker Red staining demonstrated elongated, tubular, and orderly arranged mitochondria in control cells, while S. aureus-treated cells displayed disrupted mitochondrial networks with punctate fragmentation indicative of morphological damage (Figure 4D). JC-1 probe analysis showed intense red fluorescence (JC-1 aggregates) with minimal green fluorescence in control cells; conversely, S. aureus-treated cells exhibited markedly diminished red fluorescence and enhanced green fluorescence (JC-1 monomers), indicating reduced mitochondrial membrane potential (Figure 4E). Flow cytometric analysis confirmed significantly elevated intracellular ROS levels in S. aureus-treated cells compared to controls (Figure 4F). Western blot and densitometric quantification demonstrated significantly higher protein levels of NLRP3, cleaved caspase-1, and GSDMD-N in S. aureus-treated cells relative to controls (p < 0.05) (Figure 4G,H). These data demonstrate that S. aureus induces mitochondrial damage and activates NLRP3-mediated pyroptosis in MAC-T cells.

3.5. SELENOF Alleviates Mitochondrial Damage and Pyroptosis

In vitro, ectopic expression of SELENOF was verified by markedly elevated protein levels relative to both control and empty vector groups (p < 0.001) (Figure 5A). The S. aureus + OE-SELENOF group exhibited reduced mRNA levels of TNF-α, IL-18, and IL-1β relative to the S. aureus + OE-NC group (Figure 5E). Protein expression of NLRP3, cleaved caspase-1, and GSDMD was significantly lower in the OE-SELENOF group compared to the S. aureus + OE-NC group (p < 0.001) (Figure 5B,D). Following S. aureus stimulation, the OE-NC group displayed intense green fluorescence with weak red fluorescence; conversely, the OE-SELENOF group showed attenuated green fluorescence intensity and enhanced red fluorescence intensity relative to the OE-NC group (Figure 5C). These findings indicate that elevated SELENOF levels mitigate bacteria-induced mitochondrial dysfunction and caspase-1-dependent pyroptosis in bovine mammary epithelial cells.

4. Discussion

Inflammatory disease of the bovine udder imposes considerable financial burdens upon worldwide milk production operations, and the escalating prevalence of antibiotic resistance has rendered conventional therapeutic strategies increasingly problematic. Consequently, elucidation of programmed cell death mechanisms in mammary epithelial cells during inflammatory injury and identification of their regulatory targets are critical for the development of novel preventive and therapeutic approaches for mastitis. This study systematically delineates the central roles of mitochondrial damage and pyroptosis in the pathogenesis of bovine mastitis and provides the first demonstration of selenoprotein F regulatory mechanisms in this disease. Significant mitochondrial damage and pyroptosis were observed in bovine mastitic tissue. RNA-seq analysis of 2461 downregulated genes in mastitic tissue identified SELENOF as significantly suppressed. In vitro overexpression of SELENOF reversed S. aureus-induced mitochondrial damage and NLRP3-mediated pyroptosis in MAC-T cells, thereby attenuating inflammation. In summary, these findings establish the molecular mechanism by which SELENOF inhibits NLRP3-mediated pyroptosis through maintenance of mitochondrial homeostasis, laying the groundwork for evidence-based, customized dietary interventions that address mastitis pathogenesis in milk-producing cattle.

Mammary parenchymal tissue was collected from clinically naturally occurring mastitis cases using a biopsy gun, rather than the postmortem tissues from slaughterhouses or experimentally induced models traditionally employed in mastitis research. Compared with postmortem tissues, which are frequently compromised by autolysis, ischemia–reperfusion injury, and unclear medical histories, the biopsy technique utilized in this study—conducted according to standardized protocols established by Farr et al. and Daley et al. [27,31]—enabled acquisition of high-quality viable tissue with preserved RNA and protein integrity. Furthermore, rigorous differentiation of healthy versus clinical mastitis cases based on the CMT [32] provided a reliable histological foundation for accurate pathological mechanism analysis. Transcriptome sequencing for differential gene screening was performed on these biopsy tissues, with subsequent functional validation conducted in S. aureus-induced MAC-T cell models. This integrated approach effectively addresses limitations inherent to single-model systems, such as exclusive reliance on cell lines or murine models. Most notably, this study represents the first identification of significantly downregulated SELENOF in bovine mastitis and demonstrates its capacity to suppress NLRP3-mediated pyroptosis through maintenance of mitochondrial homeostasis. These findings not only fill a critical knowledge gap regarding SELENOF in mammary inflammation but also reveal a novel anti-inflammatory mechanism for the selenoprotein family that extends beyond classical antioxidant pathways to encompass regulation of mitochondrial damage and pyroptosis, thereby providing a new molecular target for the development of precision nutritional intervention strategies targeting SELENOF.

In dairy herds, mastitis emerges as an immune-mediated tissue response predominantly elicited by infectious agents breaching mammary defenses [1], characterized by histopathological alterations including inflammatory cell infiltration, epithelial cell detachment, and fibrosis. However, whether pathogenic infection induces mitochondrial damage and pyroptosis in mammary cells remains incompletely understood. Accumulating evidence indicates that mitochondria function not merely as cellular powerhouses [33] but also serve as critical hubs for sensing infection and initiating inflammatory responses. This study integrated histopathological assessment with transmission electron microscopy-based ultrastructural observation to demonstrate mitochondrial damage and NLRP3-mediated pyroptosis through multiple methodological dimensions both in vivo and in vitro. Clinical mastitis biopsy tissues exhibited mitochondrial ultrastructural disruption alongside upregulated expression of NLRP3, caspase-1, and GSDMD-N. In vitro, S. aureus stimulation of MAC-T cells induced mitochondrial damage (manifested as reduced membrane potential and ROS accumulation) and pyroptosis. These findings align with previous reports: Shimada et al. [34] confirmed that upon mitochondrial injury, oxidized DNA released into the cytosol acts as a crucial self-derived signal instigating NLRP3 inflammasome response, while Wang et al. [35] confirmed S. aureus-induced NLRP3 activation and pyroptosis in MAC-T cells through a potassium efflux-dependent mechanism. Additionally, prior studies have established that inhibition of mitochondrial ROS production significantly attenuates NLRP3-mediated pyroptosis and tissue damage in mastitis models both in vivo and in vitro [17]. Collectively, these results confirm the presence of a cascading response involving mitochondrial damage-triggered, NLRP3-mediated pyroptosis in bovine mastitis, providing a theoretical foundation for targeting mitochondrial homeostasis in mastitis prevention and control strategies.

High-throughput RNA sequencing technology, as a transcriptomics approach, has been extensively applied in recent years to elucidate the molecular pathological mechanisms underlying bovine mastitis [36,37,38]. These analyses have revealed that S. aureus or Escherichia coli (E. coli) infection induces substantial gene expression reprogramming in mammary epithelial cells, identifying hundreds to thousands of differentially expressed genes enriched in pathways associated with inflammasome activation, immune responses, and metabolic disorders [39,40]. The present study employed RNA-seq analysis to screen and identify SELENOF as a significantly downregulated candidate gene. GO enrichment analysis demonstrated significant enrichment of differentially expressed genes in mitochondria-related categories, including mitochondrial protein complex and oxidative phosphorylation, implicating mitochondrial dysfunction and SELENOF deficiency in mastitis pathogenesis. Consistent with these findings, Gaspa et al. [40] identified significant enrichment of mitochondria-associated genes during inflammatory responses in milk somatic cell transcriptomes, accompanied by downregulation of oxidative phosphorylation pathways. Additionally, significant downregulation of selenoprotein family members SELENOF and SELENOM was observed in mastitic tissue. As important antioxidant and immune regulatory proteins, decreased selenoprotein expression may compromise cellular antioxidant capacity, thereby exacerbating mitochondrial damage and inflammatory responses [41]. These findings provide transcriptome-level evidence for investigations into the protective roles of selenoproteins in mastitis. RNA-seq analysis reveals the transcriptional characteristics of mammary tissue under mastitic conditions and provides a foundation for elucidating the mechanisms by which mitochondrial dysfunction and selenoprotein regulation contribute to mastitis pathogenesis.

SELENOF is a low molecular weight protein containing the essential trace element selenium in the form of selenocysteine [25]. As an endoplasmic reticulum-resident oxidoreductase, SELENOF regulates immune cell function through maintenance of redox homeostasis, with deficiency resulting in oxidative stress and protein misfolding [42,43]. Although selenoprotein family members possess anti-inflammatory activities, including reactive oxygen species scavenging, Nuclear Factor-kappa B (NF-κB) pathway inhibition [41], and suppression of NLRP3 inflammasome activation [44], the target specificity and regulatory mechanisms of SELENOF in inflammatory diseases remain incompletely characterized. Despite growing recognition of SELENOF function in oxidative stress and inflammatory regulation, its expression patterns and mechanistic roles in bovine mastitis have not been previously investigated. The present study demonstrates significantly reduced SELENOF expression in mastitic tissue and elucidates its intervention mechanisms through in vitro overexpression: SELENOF restoration significantly recovered mitochondrial membrane potential, attenuated NLRP3-mediated GSDMD-N activation, downregulated pro-inflammatory cytokine expression, and alleviated S. aureus-induced inflammatory injury in MAC-T cells. Ren et al. [43] demonstrated that SELENOF regulates cellular redox status through maintenance of endoplasmic reticulum protein folding homeostasis, with deficiency causing ROS accumulation and mitochondrial membrane potential collapse. The inhibitory effect of SELENOF overexpression on NLRP3 inflammasome activation supports the anti-inflammatory functions of the selenoproteins family; Skalny et al. [45] demonstrated that selenium blocks ROS-mediated NLRP3 activation through upregulation of selenocysteine-containing antioxidant enzymes, thereby suppressing pyroptosis and IL-1β maturation. Regarding mitochondrial protection, SELENOF indirectly preserves mitochondrial membrane integrity through maintenance of calcium homeostasis and inhibition of oxidative stress, despite its endoplasmic reticulum localization, thereby preventing mitochondrial ROS release and subsequent NLRP3 inflammasome activation. These findings delineate a previously unrecognized mechanism whereby SELENOF attenuates bovine mastitis pathogenesis, establishing a theoretical basis for targeted modulation of mitochondrial function and pyroptosis pathways in mastitis prevention and treatment strategies.

The present study is subject to several limitations that warrant addressing in future investigations. Given the extensive functional redundancy within the mammalian selenoprotein family, the analysis focused exclusively on SELENOF without systematic examination of compensatory or synergistic mechanisms with homologous members including SELENOM [46,47,48]. Subsequent studies should employ systematic siRNA screening, multiplex gene knockdown, or conditional knockout models to comprehensively elucidate the interaction networks among selenoproteins. Furthermore, although SELENOF overexpression was demonstrated to attenuate mitochondrial damage and pyroptosis, the specific molecular targets and downstream signaling mechanisms require further experimental validation.

5. Conclusions

The present study provides the first characterization of significant mitochondrial damage and NLRP3-mediated pyroptosis in bovine mastitic tissue and in vitro cell models. Through transcriptomic screening, SELENOF was identified as significantly downregulated in this pathological context. Mechanistic analysis demonstrated that SELENOF exerts protective effects in bovine mastitis through modulation of mitochondrial homeostasis and subsequent suppression of NLRP3-mediated pyroptosis. These findings establish SELENOF as a novel molecular target and provide a theoretical foundation for precision nutritional interventions utilizing selenium in the prevention and treatment of bovine mastitis.