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Article

Conjugated Linoleic Acid Ameliorates Staphylococcus aureus-Induced Inflammation, Oxidative Stress, and Mitophagy via the PPARG-UCP2 Pathway in Hu Sheep Mastitis

by
Yuzhi Jin
,
Hui Zhang
,
Xiaochang Xie
,
Nana Ma
* and
Xiangzhen Shen
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Agriculture 2026, 16(1), 99; https://doi.org/10.3390/agriculture16010099
Submission received: 17 November 2025 / Revised: 10 December 2025 / Accepted: 26 December 2025 / Published: 31 December 2025
(This article belongs to the Section Farm Animal Production)
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Abstract

Staphylococcus aureus (S. aureus)-induced mastitis poses a significant threat to animal husbandry. This condition triggers sustained mammary inflammation, oxidative stress, and disrupts mitochondrial homeostasis, ultimately impairing mammary gland function and milk yield. Conjugated linoleic acid (CLA) is a long-chain fatty acid found in meat and dairy products derived from ruminants. It exhibits multiple biological activities, including anti-cancer, anti-inflammatory, and antioxidative stress-alleviating effects. Thus, this study sought to determine whether CLA alleviates S. aureus-induced mastitis in Hu sheep through the PPARG-UCP2 axis. Fifteen lactating Hu sheep were randomly allocated into three groups (n = 5): control group, model group, and CLA group. The CLA group received 1 mg/mammary gland of CLA via intramammary infusion for seven days, followed by S. aureus challenge (5 × 107 cells/mL, 2 mL/mammary gland) in the model and CLA groups, while the control group received saline. Venous blood and mammary tissue samples were collected at two days post-infection. The results demonstrated that S. aureus infection significantly upregulated the expression of inflammatory factors (IL-1β, IL-6, and NF-κB) in the mammary tissue of Hu sheep, p < 0.01. Relative to the control, the model group showed increased ROS and MDA levels, a diminished NAD+/NADH ratio, and downregulated expression of the antioxidant factors SOD, Nrf2, HO-1, and SIRT3, p < 0.01. Furthermore, the expression of p-AMPK and mitophagy-related factors (PARKIN, PINK1, and LC3b) showed a statistically significant increase in the model group than in the control group, p < 0.01. S. aureus infection also suppressed the expression of PPARG and UCP2, p < 0.01. In contrast, the CLA group showed lower levels of inflammatory factors (IL-1β, IL-6, and NF-κB), ROS and MDA, while the NAD+/NADH ratio and the expression of antioxidant factors (SOD, p-Nrf2, HO-1, and SIRT3) were elevated compared with the model group, p < 0.01. Moreover, the expression of p-AMPK and mitophagy-related factors (PARKIN, PINK1, and LC3b) was reduced in the CLA group relative to the model group, p < 0.05. Concurrently, the expression of PPARG and UCP2 was higher in the CLA group than in the model group, p < 0.001. These findings demonstrated that S. aureus infection induced mastitis in Hu sheep mammary tissue, whereas CLA alleviated the infection by upregulating the PPARG-UCP2 pathway, thereby reducing inflammation, oxidative stress, and mitophagy levels. This study offers a novel perspective on mammary tissue repair during mastitis and expands the understanding of UCP2’s biological role.

1. Introduction

Mastitis constitutes a major animal health challenge within the dairy industry, being both widespread and serious. Bacterial infection is the most common cause of mastitis, with Staphylococcus aureus (S. aureus) being recognized as one of the primary causative pathogens [1,2]. S. aureus infection induces persistent mammary inflammation, oxidative stress, and disruption of mitophagy homeostasis, thereby impairing mammary gland function and leading to reduced or ceased milk production, which results in substantial economic losses [3,4]. Currently, the administration of antibiotics to eliminate pathogens is the primary therapeutic approach. However, issues concerning antibiotic residues and the emergence of resistance have raised increasing concerns [5]. Although bacterial elimination is a primary step, it does not address the subsequent requirement for mammary tissue repair [6]. As a result, mammary tissue repair is crucial for both preventing and treating mastitis.
Oxidative stress is a state of cellular damage resulting from an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defense mechanisms, where ROS accumulation exceeds the system’s capacity for detoxification. Oxidative stress and inflammation exhibit a strong interrelationship. Inflammation triggers increased ROS production, resulting in elevated oxidative stress and decreased superoxide dismutase (SOD) activity [7]. ROS directly activated the NF-κB transcription factor pathway, resulting in the upregulation of pro-inflammatory cytokines, which further exacerbated the inflammatory process [8,9]. Consequently, a reduction in oxidative stress may promote the resolution of inflammation. SIRT3, a key mitochondrial NAD+-dependent deacetylase, regulates ROS homeostasis through the deacetylation of mitochondrial enzymes, including superoxide dismutase 2 (SOD2) [10]. Mitophagy is a selective autophagy process by which cells degrade impaired or dysfunctional mitochondria. Excessive ROS accumulation induces mitochondrial depolarization and damage, triggering an upregulation of mitophagy to protect the mitochondrial network and maintain cellular homeostasis [11]. Despite the beneficial role of mitophagy in maintaining mitochondrial homeostasis, abnormally induced mitophagy can be detrimental, possibly culminating in cell death [12]. Following infection, S. aureus prolongs its intracellular survival by inducing host mitophagy [4,13,14]. Therefore, re-establishing balanced mitophagy is advantageous for tissue regeneration and bacterial clearance.
As a key transcription factor, peroxisome proliferator-activated receptor gamma (PPARG) is a ligand-activated nuclear receptor that significantly regulates processes including inflammation, oxidative stress, and immune responses [15,16,17]. PPARG reduces oxidative stress in human trophoblast cells through upregulation of uncoupling protein 2 (UCP2) [18,19]. Furthermore, PPARG agonists can increase UCP2 mRNA levels in adipocytes and muscle cells [20]. UCP2, an uncoupling protein, is situated within the inner membrane of mitochondria. It functions by dissipating the proton gradient, thereby reducing the electrochemical potential across the membrane. In an in vitro system modeling diabetic retinopathy, UCP2 modulates mitochondrial respiratory chain function and regulates ATP and ROS production by sensing NAD+ levels and regulating SIRT3 activity [21]. UCP2 participates in cardioprotection by mediating mitophagy in a myocardial ischemia/reperfusion model [22]. In a mouse model of meibomian gland dysfunction, modulation of the PPARG-mediated UCP2 signaling pathway improved meibomian gland function [23]. Based on these findings, we speculate that PPARG-UCP2 may play a similar role in mammary tissue infected with S. aureus.
Conjugated linoleic acid (CLA) is a long-chain fatty acid characteristic of ruminant-derived foods, including meat and dairy [24]. CLA has demonstrated anti-cancer, anti-inflammatory, and antioxidant effects in various studies [24,25]. CLA shares structural and functional similarities with endogenous ligands of PPARG [26]. Studies have demonstrated that CLA can enhance PPARG expression and activity in adipocytes, myocytes, rumen epithelial cells, and intestinal cells [27,28,29,30].
Based on these observations, we propose that CLA protects against S. aureus-induced mastitis by modulating the PPARG-UCP2 signaling pathway, thereby reducing inflammation, oxidative stress, and mitophagy. This study sought to determine whether CLA administration could alleviate S. aureus-induced mastitis in Hu sheep via the PPARG-UCP2 axis.

2. Materials and Methods

2.1. Ethics Statement

All animal procedures were approved by the Animal Care and Use Committee of Nanjing Agricultural University (Approval No.: NJAULLSC2024040, date of approval: 1 March 2024).

2.2. Animal Experiment

Fifteen healthy, multiparous (parity 2–3), one-month-postpartum, lactating Hu ewes were randomly allocated into three groups (n = 5 per group): a control group, a model group (infected with S. aureus), and a CLA group. Animals were allowed 7 days of acclimation to the feeding regimen. The CLA group received intramammary infusions of 1 mg of CLA (Macklin, Shanghai, China) per mammary quarter daily for seven consecutive days. Following CLA treatment, the model and CLA groups received intramammary infusions of 2 mL of S. aureus suspension per mammary quarter, while the control group received an equal volume of sterile saline. S. aureus mastitis was induced in Hu sheep by following a standardized intramammary challenge protocol, administering 2 mL of S. aureus (5 × 107 cells/mL, CVCC2086, Beijing, China) suspension per mammary quarter. Control sheep received sterile saline via the same route.

2.3. Sample Collection

Following intramammary infusion of S. aureus suspension, the physical condition of Hu sheep was monitored. The ewes exhibited systemic symptoms including fever, anorexia, and lethargy, along with apparent redness, swelling, and localized heat in the mammary glands. Hu sheep were scheduled for slaughter and sample collection 48 h after intramammary infusion of S. aureus. Prior to euthanasia, the average body weights of the three groups were recorded (Control group: 52.0 kg; Model group: 46.0 kg; CLA group: 50.1 kg). Milk samples were individually collected from each ewe. Subsequently, 10 mL of blood was drawn from the jugular vein. The blood samples were centrifuged at 3000× g for 10 min at room temperature to obtain plasma. Plasma and milk samples were stored at −80 °C for subsequent analysis. Following euthanasia, mammary tissue samples were collected and divided into two portions for preservation. One aliquot was snap-frozen in liquid nitrogen for subsequent protein and RNA extraction, while the other was fixed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining.

2.4. H&E Staining

Breast tissues fixed in 4% paraformaldehyde were processed into paraffin sections. Following deparaffinization, the sections were stained with hematoxylin and eosin (H&E), mounted with resin, and coverslipped. Images were acquired using a microscope and Slide Viewer 2.5 software.

2.5. Plasma Samples ELISA

IL-6, IL-8, and TNF-α concentrations were determined using commercially available ELISA kits (YH-091206S, YH-091208S and YH-201407S, Yihe Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.

2.6. Determination of ROS

A ROS staining working solution was prepared according to the manufacturer’s instructions. Fixed samples were then immersed in the working solution and permeabilized. Samples were stained with the fluorescent dye DHE (Beyotime S0063, Beyotime biotechnology Co., Ltd., Shanghai, China). After sufficient incubation, samples were thoroughly washed with PBS or other appropriate buffer to remove unbound dye. Finally, the samples were visualized, and images were acquired using a laser scanning confocal microscope (LSCM).

2.7. Measurement of NAD+/NADH, ATP

NAD+, NADH, and ATP levels were determined using commercial assay kits (YH-140104S, YH-140112S, and YH-012016S, Yihe Biotechnology Co., Ltd., Shanghai, China, respectively), according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader.

2.8. Measurement of SOD, MDA

SOD activity and MDA levels were determined using commercially available assay kits (A001-3-2 and A003-1-2, Jiancheng Bioengineering Research Institute Co., Ltd., Nanjing, China, respectively), according to the manufacturer’s protocol. Absorbance was measured at 450 nm (SOD) and 532 nm (MDA) using a microplate reader.

2.9. Immunofluorescence Staining of Tissues

The paraffin-embedded mammary tissue sections were deparaffinized and rehydrated by sequential immersion in xylene, absolute ethanol, 85% ethanol, 75% ethanol, and finally distilled water. Antigen retrieval was performed on tissue sections using an antigen retrieval buffer (pH 6.0). To quench endogenous peroxidase activity, tissue sections were treated with 3% hydrogen peroxide and incubated at room temperature under dark conditions. After blocking with serum, the sections were incubated with primary antibody at 4 °C overnight. Primary antibody details are listed in Supplementary Table S1. After washing with PBS, the sections were incubated with HRP-conjugated secondary antibody at room temperature for 50 min. The sections were incubated with freshly prepared 3,3′-diaminobenzidine (DAB) chromogenic solution. Color development was monitored under a microscope, with positive signals appearing as a brown-yellow color. The reaction was stopped by rinsing the sections with tap water. Subsequently, the nuclei were counterstained with Harris hematoxylin for 3 min. Finally, the sections were dehydrated, cleared, and mounted. Staining results were examined under a microscope, and images were captured for analysis.

2.10. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)

RNA was extracted from mammary gland tissue using the Trizol method. Briefly, 0.1 g of tissue was homogenized in 600 µL of Trizol reagent (R1100; Solarbio Science & Technology Co., Ltd., Beijing, China). The concentration and purity of the RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Samples with an A260/A280 ratio between 1.9 and 2.1 were considered of acceptable quality and were used for subsequent experiments. RNA integrity was checked using agarose gel electrophoresis. Subsequently, Total RNA (1000 ng) was reverse-transcribed into complementary DNA (cDNA) using the Evo M-MLV Reverse Transcription Premix Kit (AG11728; Accurate Biotechnology, Hunan, China) according to the manufacturer’s instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a premixed qPCR kit (AG11756; Accurate Biotechnology, Shanghai, China) on a Bio-Rad real-time PCR system (Bio-Rad Laboratories Co., Ltd., Hercules, CA, USA). Relative gene expression levels were calculated using the 2−ΔΔCt method with β-actin as the internal reference gene. Primers used in this study were designed using Oligo 7 software (Ruiqi Technology Co., Ltd., Beijing, China). Primer details are listed in Supplementary Table S2.

2.11. Western Blot

Approximately 0.1 g of mammary gland tissue, stored in liquid nitrogen, was pulverized into a fine powder under liquid nitrogen. The powder was then transferred to a 1.5 mL microcentrifuge tube and homogenized in 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (PC101; Epizyme Biotech, Shanghai, China) supplemented with 10 μL of phenylmethylsulfonyl fluoride (PMSF) (GRF101; Epizyme Biotech). After incubation for 20 min, the lysate was centrifuged at 12,000× g for 5 min at 4 °C. The supernatant was collected, and the total protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (ZJ101; Epizyme Biotech) according to the manufacturer’s instructions. All samples were diluted to a concentration of 5 μg/mL. Subsequently, 5× SDS-PAGE loading buffer (Catalog No. BL502A; Shanghai, China) equal to one-fifth of the total sample volume was added, thoroughly mixed, and then denatured at 99 °C for 10 min.
Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a precast gel (PG112; Epizyme Biotech, Shanghai, China), with 10 μL of sample loaded per lane. Following electrophoresis, the separated proteins were transferred onto a methanol-activated polyvinylidene fluoride (PVDF) membrane (1620177; Bio-Rad Laboratories, Inc.). The membrane was then blocked with 5% skimmed milk powder for 2 h at room temperature. Subsequently, it was incubated overnight at 4 °C with the appropriately diluted primary antibody. After incubation, the membrane was washed with Tris-buffered saline containing 0.1% Tween-20 (1× TBST) and then incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody corresponding to the species of the primary antibody. Following a final wash with 1× TBST, protein bands were visualized using enhanced chemiluminescence (ECL) substrate and captured with a ChemiDoc MP imaging system (Bio-Rad). Band intensities were quantified using Image Lab software (version 5.2.1.62311; Bio-Rad).

2.12. Statistical Analysis

Statistical analysis was performed using GraphPad Prism (version 8.4.0). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was applied to determine whether significant differences existed between group means. Student’s t-test was used to assess the significance of differences between two groups. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.

3. Results

3.1. S. aureus Infection Elicits Inflammation in the Mammary Glands of Hu Sheep

Hematoxylin and eosin staining of mammary tissues revealed significant inflammatory changes in the model group relative to the control group, characterized by disrupted alveolar architecture and extensive inflammatory cell infiltration (Figure 1A). Serum concentrations of the pro-inflammatory cytokines IL-8, IL-6, and TNFα were elevated in the model group relative to the control (Figure 1B–D). Collectively, these data strongly suggest the successful induction of a S. aureus mastitis model in Hu sheep. Furthermore, compared with the control group, the model group exhibited significantly increased relative mRNA expression of IL-1β, IL-6, and NF-Kb (Figure 1E), as well as elevated protein levels of IL-1β and phosphorylated p65 (p-p65) in mammary tissue (Figure 1F–I). These findings demonstrated that S. aureus successfully induced an inflammatory response in the mammary tissue of Hu sheep.

3.2. S. aureus-Induced Mastitis Induces Oxidative Stress

The pathogenesis of inflammation is driven not only by pro-inflammatory mediators but also by oxidative stress, which plays a pivotal role throughout its development. To assess oxidative stress, we measured key markers of oxidative damage, including ROS, SOD, and MDA. An impaired antioxidant status was observed in the model group, as indicated by the upregulation of Nrf2, HO-1, SIRT3 concurrent with elevated MDA levels and suppressed SOD activity compared to the control group. (Figure 2A,B). Tissue sections were stained with DHE to visualize ROS production. Compared to the control, a marked increase in DHE fluorescence intensity was observed in the model group by confocal microscopy (Figure 2C). Quantitative real-time PCR analysis revealed a significant reduction in the relative mRNA expression of NFE2L2 and HMOX1 in the breast tissue of the model group (Figure 2D). Western blot analysis demonstrated a corresponding decrease in phosphorylated Nrf2 (p-Nrf2), HO-1, and SIRT3 (Figure 2E–I). Given the critical role of the NAD+/NADH ratio in maintaining redox homeostasis [31], we quantified this ratio using an NAD+/NADH assay kit. The results showed that the NAD+/NADH ratio was markedly reduced in the model group relative to the control group (Figure 2J). SIRT3, an NAD+-dependent deacetylase known for its role in mitigating oxidative stress, was assessed using immunohistochemistry. Consistent with decreased NAD+/NADH ratio, the model group exhibited weaker SIRT3 staining intensity in breast tissue compared to the control group (Figure 2K), suggesting reduced SIRT3 expression. These results demonstrated that S. aureus infection significantly elevated oxidative stress in the mammary tissue.

3.3. S. aureus-Induced Mastitis Induces Elevated Levels of Mitophagy

Given the importance of PINK1, PARKIN, and LC3B in the classical mitophagy pathway, we assessed their expression in mammary tissue. Compared to the control group, the model group exhibited significantly increased relative mRNA (Figure 3A) and protein expressions (Figure 3B–D) of PARKIN and PINK1 in mammary tissue. Observation by immunohistochemistry indicated a discernible increase in LC3b-associated DAB staining in the mammary tissue of the model group relative to controls. The protein expression of phosphorylated AMPK (p-AMPK) was significantly increased in the mammary tissue of the model group compared to the control group (Figure 3F–H). A significant decrease in ATP content was detected in the mammary tissue of the model group relative to the control group (Figure 3I). These results indicated that S. aureus infection significantly enhanced mitophagy in the mammary tissue.

3.4. S. aureus Stimulation Suppressed PPARG and UCP2 Expression in Mammary Tissue

Figure 4A–D showed a marked downregulation in both the relative mRNA and protein expression levels of PPARG and UCP2 in the mammary tissue of the model group compared to the control (p < 0.05). Immunohistochemical analysis revealed that the DAB staining intensity of UCP2 was significantly weaker in the model group relative to the control group. (Figure 4E). These findings demonstrated that S. aureus infection significantly downregulated the expression of both PPARG and UCP2.

3.5. CLA Attenuated S. aureus-Induced Inflammation in Ovine Mammary Tissue

The CLA group received 1 mg/mammary quarter of CLA via intramammary infusion for seven days, followed by S. aureus challenge (2 mL/mammary quarter) in the model and CLA group. The CLA group preserved mammary alveolar structure and reduced inflammatory cell infiltration compared to the model group (Figure 5A). This was accompanied by decreased serum IL-8, IL-6, and TNF-α levels (Figure 5B–D), as well as reduced mRNA expression of IL-1β, IL-6, and NF-κB (Figure 5E) and protein expression of IL-1β and p-p65 in mammary tissue (Figure 5F–I). These results indicated that CLA alleviated S. aureus-induced inflammation in ovine mammary tissue.

3.6. CLA Protected Mammary Tissue from Oxidative Damage Associated with Mastitis

CLA significantly decreased MDA levels and increased SOD activity in mammary tissue compared to the model group (Figure 6A,B). ROS levels were also lower in the CLA group (Figure 6C). These changes were associated with increased mRNA expression of NFE2L2 and HMOX1 (Figure 6D) and increased p-Nrf2, HO-1, and SIRT3 (Figure 6E–I), suggesting activation of the Nrf2/HO-1 pathway. CLA also increased the NAD+/NADH ratio (Figure 6J) and decreased SIRT3 staining intensity (Figure 6K). These results demonstrated that CLA augmented the systemic antioxidant defense.

3.7. CLA Reduced Mitophagy in Mammary Tissue During Mastitis

Compared to the model group, CLA significantly reduced PARKIN and PINK1 mRNA and protein expression in mammary tissue (Figure 7A–D), indicating decreased mitophagy. Relative to the model group, the CLA group exhibited weaker LC3b immunostaining, which is indicative of reduced mitophagic activity (Figure 7E). CLA also significantly reduced p-AMPK protein expression (Figure 7F–H) and ATP content (Figure 7I) relative to the model group.

3.8. CLA Upregulated PPARG and UCP2 Expression in Mammary Tissue

Administration of CLA significantly upregulated the expression of both PPARG and UCP2 in mammary tissue at the mRNA (Figure 8A) and protein (Figure 8B–D) levels compared to the model group. Consistently, immunohistochemical analysis demonstrated enhanced UCP2 protein expression, as evidenced by stronger DAB staining intensity in the CLA group (Figure 8E).

4. Discussion

S. aureus mastitis impairs mammary gland function and impedes the restoration of milk yield. Consequently, therapeutic interventions should encompass not only antimicrobial strategies but also approaches to promote mammary tissue repair [6]. S. aureus infection induces inflammation and elevates oxidative stress levels in mammary tissues [32,33]. Furthermore, by inducing mitophagy, S. aureus employs a strategy to enhance its intracellular survival [4,13,14]. Sustained inflammation, oxidative stress, and mitophagy impede mammary tissue repair. In our study, infection of Hu sheep mammary tissues with S. aureus disrupted mammary gland architecture and exacerbated mammary tissue inflammation.
Oxidative stress typically arises from the excessive accumulation of intracellular ROS, surpassing the antioxidant defense capacity of the organism, thereby leading to cellular damage. Under normal conditions, the organism maintains redox homeostasis through antioxidant enzymes, such as SOD. Upon the onset of inflammation, pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) stimulate the production of reactive oxygen species (ROS) while reducing the levels of antioxidant enzymes [9]. ROS can directly activate the NF-κB transcription factor pathway, which in turn promotes the production of pro-inflammatory cytokines and exacerbates the inflammatory response [8,9]. The nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway is one of the crucial cellular pathways that mediates antioxidant stress responses [34,35]. In the LPS-induced murine mastitis model, ROS production is increased, Nrf2 and HO-1 expression is suppressed, and the level of oxidative stress is elevated [36]. In our study, S. aureus stimulation of mammary tissue resulted in decreased antioxidant factors, increased ROS levels, and elevated oxidative stress. Furthermore, sirtuin 3 (SIRT3) plays a crucial role in the antioxidant process. SIRT3 alleviates intracellular oxidative stress primarily by activating specific antioxidant enzymes [37]. A key mechanism involves the deacetylation and subsequent activation of enzymes such as superoxide dismutase (SOD) [38,39]. SIRT3 can also indirectly reduce ROS production by accelerating electron transport [40,41]. SIRT3 activity is contingent upon the availability of nicotinamide adenine dinucleotide (NAD+); increased NAD+ concentrations correlate with enhanced SIRT3 activity [42,43]. In a murine model of skin aging, by elevating NAD+ levels and SIRT3 expression, NMN-sEVs treatment attenuated both cellular oxidative stress and mitochondrial dysfunction, mediated through the NAD+/SIRT3 pathway [44]. In experimental myocardial ischemia/reperfusion (I/R) injury, SIRT3 knockout resulted in a larger infarct area in the myocardium after I/R. However, augmenting SIRT3 expression led to increased antioxidant enzyme activity, thereby exerting a protective effect against myocardial I/R injury [45]. Previous research has indicated that infection with S. aureus induced SIRT3 degradation [46]. Additional research suggests that inflammation promotes a decrease in NAD+ biosynthesis [47,48]. Corroborating these studies, we observed a reduction in NAD+ and SIRT3 synthesis in mammary tissue following S. aureus stimulation. These observations indicate that impaired SIRT3 synthesis may be an important contributor to oxidative stress.
Mitophagy, a highly conserved process, enables cells to selectively degrade damaged mitochondria through autophagy. PINK1 and Parkin are pivotal proteins within the classical mitophagy pathway. When cells experience excessive reactive oxygen species (ROS), mitochondrial DNA damage, altered membrane potential, and modified activity of oxidized proteins ensue, leading to mitochondrial impairment. Consequently, mitophagy is enhanced to preserve mitochondrial network integrity and cellular homeostasis [49,50]. Although mitophagy contributes to the maintenance of mitochondrial homeostasis, abnormally heightened mitophagy can have deleterious effects, potentially inducing cell death [12]. Prior research suggests that S. aureus enhances intracellular survival via mitophagy induction [4,51]. In bovine macrophages, S. aureus infection triggered mitophagy via the PINK1/Parkin pathway to facilitate its intracellular survival [52]. Our study corroborated these observations by demonstrating that S. aureus challenge induces PINK1/PARKIN-mediated mitophagy in Hu sheep mammary tissue. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial cellular energy sensor that plays an important role in mitochondrial function and mitophagy [53,54]. AMPK induces mitophagy in hydrogen peroxide-stimulated porcine epithelial cells [55]. ROS and ATP modulate AMPK signaling. Increased ROS levels or decreased ATP levels activate the AMPK pathway [56,57,58]. Increased AMPK phosphorylation is observed in host cells undergoing a starvation-like response following S. aureus infection, as reported in previous literature [59]. Mirroring these results, we observed decreased ATP levels and increased AMPK phosphorylation in mammary tissue following S. aureus infection of Hu sheep mammary glands.
UCP2, characterized by high proton transport activity, plays a crucial role in regulating mitochondrial energy metabolism and is closely involved in processes like mitochondrial ROS production and ATP synthesis [60]. Under physiological conditions, UCP2 mediates the direct influx of protons into the mitochondrial matrix, decreasing the electrochemical gradient across the inner mitochondrial membrane, thereby uncoupling electron transfer and leading to reduced ATP synthesis [61]. Concurrently, the reduced proton electrochemical gradient across the inner membrane decreases electron leak from the respiratory chain, leading to diminished ROS production [60]. Nevertheless, we found that, in the pathological setting of S. aureus infection, cellular ATP consumption is elevated. Given the disruption of the intracellular ATP production and consumption equilibrium, UCP2 favors the restoration of intracellular ATP balance under pathological conditions, leading to enhanced intracellular ATP production. Earlier studies suggest a mechanism whereby UCP2 regulates SIRT3 expression by altering the NAD+/NADH ratio, and this regulation subsequently leads to the amelioration of oxidative stress [62]. Overexpression of UCP2 in a diabetic retinopathy model reduced intracellular ROS levels and increased NAD+ levels in retinal endothelial cells, promoting SIRT3 expression and mitigating high glucose-induced oxidative stress and senescence in these cells [21]. Additionally, UCP2 is capable of regulating AMPK phosphorylation [63]. The UCP2/PINK1/PARKIN signaling pathway regulated mitophagy and reduced myocardial damage in a myocardial I/R injury model [22]. We, therefore, propose that mitophagy is modulated by the UCP2/AMPK/PINK1 signaling pathway in our study. S. aureus infection was observed to suppress UCP2 expression but stimulate both oxidative stress and mitophagy in this experiment.
CLA is a naturally occurring fatty acid with anti-inflammatory, anti-cancer, and antioxidant properties [64]. Studies have shown that CLA shares structural and physiological similarities with PPARG ligands and can promote PPARG expression in various tissues [26,27,28,29,30]. In experimental models of atherosclerosis, CLA ameliorated the disease by upregulating PPARG expression in the aorta, which subsequently decreased the expression of pro-inflammatory mediators [65]. Administration of a PPARG agonist ameliorated S. aureus-induced mastitis in mice, an effect accompanied by upregulated PPARG expression [66]. Evidence indicated that PPARG induces the expression of UCP2 [20,67,68]. The PPARG-UCP2 signaling pathway protects primary human trophoblast cells from mitochondrial dysfunction [18]. Reduced PPARG expression has been observed following S. aureus infection in earlier reports [69,70]. Consistent with these findings, we observed that S. aureus infection decreased PPARG and UCP2 expression in the mammary tissue of Hu sheep. In this study, CLA increased PPARG and UCP2 expression in the mammary tissue. Furthermore, CLA increased the NAD+/NADH ratio and ATP content, upregulated SIRT3 expression, decreased AMPK phosphorylation, and alleviated oxidative stress and mitophagy, ultimately restoring mammary tissue homeostasis. These results suggest that CLA exerts its protective effect against mastitis by modulating the PPARG-UCP2 signaling pathway.

5. Conclusions

In summary, our findings demonstrate that CLA promotes PPARG and UCP2 expression in Hu sheep mammary tissue. This is accompanied by the upregulation of antioxidant-related factors (SIRT3, SOD, Nrf2, HO-1) and the reduction in ROS and MDA levels, leading to the alleviation of oxidative stress associated with mastitis. Furthermore, CLA attenuated mitophagy by downregulating mitophagy-related proteins (PARKIN, PINK1, LC3b), thereby restoring mitophagy homeostasis. These findings indicate that CLA alleviates S. aureus-induced inflammation in the mammary tissue of Hu sheep by activating the PPARG-UCP2 signaling pathway. As illustrated in Figure 9. This study provides novel insights into mammary tissue repair in mastitis and expands our understanding of the biological functions of UCP2.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture16010099/s1, Table S1: Information of the antibodies used in Western blot and Immuno-histochemical assay. Table S2: The list of primers used in RT-qPCR. Table S3: Information of the feed formula.

Author Contributions

Methodology, Y.J., H.Z. and X.X.; formal analysis, Y.J. and H.Z.; data curation, Y.J.; writing—original draft preparation, Y.J.; writing—review and editing, Y.J. and N.M.; project administration, N.M. and X.S.; funding acquisition, N.M. and X.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (No. 32302945, No. 32373087, No. 32172933) and the China Postdoctroal Science Foundation (the 72nd batch of grants, 82202614).

Institutional Review Board Statement

All animal procedures were approved by the Animal Care and Use Committee of Nanjing Agricultural University (Approval No.: NJAULLSC2024040, date of approval: 1 March 2024).

Data Availability Statement

The data generated and analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. S. aureus infection elicits inflammation in the mammary glands of Hu sheep. (A) Hematoxylin and eosin (H&E) staining of mammary gland tissue (scale bar = 20 μm). (B–D) Concentrations of IL-8, IL-6, and TNF-α in plasma from Hu sheep. (E) mRNA expression levels of inflammation-related factors IL-1β, IL-6, and NF-κB in mammary gland tissue. (F–I) Protein expression levels of inflammation-related factors IL-1β, p65, and phospho-p65 (pp65) in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 1. S. aureus infection elicits inflammation in the mammary glands of Hu sheep. (A) Hematoxylin and eosin (H&E) staining of mammary gland tissue (scale bar = 20 μm). (B–D) Concentrations of IL-8, IL-6, and TNF-α in plasma from Hu sheep. (E) mRNA expression levels of inflammation-related factors IL-1β, IL-6, and NF-κB in mammary gland tissue. (F–I) Protein expression levels of inflammation-related factors IL-1β, p65, and phospho-p65 (pp65) in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 2. S. aureus-induced mastitis induces oxidative stress through depressing SIRT3. (A,B) SOD activity and MDA levels in mammary gland tissue. (C) Fluorescence detection of reactive oxygen species (ROS) expression in mammary gland tissue. (D) mRNA expression levels of oxidative stress-related factors NFE2L2 and HMOX1 in mammary gland tissue. (E–I) Protein expression levels of antioxidant-related factors Nrf2, p-Nrf2, HO-1, and SIRT3 in mammary gland tissue. (J) NAD+/NADH ratio in mammary gland tissue. (K) Immunohistochemical staining of SIRT3 in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2. S. aureus-induced mastitis induces oxidative stress through depressing SIRT3. (A,B) SOD activity and MDA levels in mammary gland tissue. (C) Fluorescence detection of reactive oxygen species (ROS) expression in mammary gland tissue. (D) mRNA expression levels of oxidative stress-related factors NFE2L2 and HMOX1 in mammary gland tissue. (E–I) Protein expression levels of antioxidant-related factors Nrf2, p-Nrf2, HO-1, and SIRT3 in mammary gland tissue. (J) NAD+/NADH ratio in mammary gland tissue. (K) Immunohistochemical staining of SIRT3 in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 3. S. aureus-induced mastitis induces elevated levels of mitophagy. (A) mRNA expression levels of mitophagy-related factors PARKIN and PINK1 in mammary gland tissue. (B–D) Protein expression levels of PARKIN and PINK1 in mammary gland tissue. (E) Immunohistochemical staining of LC3b in mammary gland tissue. (F–H) Protein expression levels of AMPK and phospho-AMPK (p-AMPK) in mammary gland tissue. (I) ATP content in mammary gland tissue. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001.
Figure 3. S. aureus-induced mastitis induces elevated levels of mitophagy. (A) mRNA expression levels of mitophagy-related factors PARKIN and PINK1 in mammary gland tissue. (B–D) Protein expression levels of PARKIN and PINK1 in mammary gland tissue. (E) Immunohistochemical staining of LC3b in mammary gland tissue. (F–H) Protein expression levels of AMPK and phospho-AMPK (p-AMPK) in mammary gland tissue. (I) ATP content in mammary gland tissue. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001.
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Figure 4. S. aureus stimulation suppressed PPARG and UCP2 expression in mammary tissue. (A) mRNA expression of PPARG and UCP2 in mammary gland tissue. (B–D) Protein expression levels of PPARG and UCP2 in mammary gland tissue. (E) Immunohistochemical staining of UCP2 in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4. S. aureus stimulation suppressed PPARG and UCP2 expression in mammary tissue. (A) mRNA expression of PPARG and UCP2 in mammary gland tissue. (B–D) Protein expression levels of PPARG and UCP2 in mammary gland tissue. (E) Immunohistochemical staining of UCP2 in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 5. CLA attenuated S. aureus-induced inflammation in ovine mammary tissue. (A) H&E staining of mammary gland tissue (scale bar = 20 μm). (B–D) Concentrations of IL-8, IL-6, and TNF-α in plasma from Hu sheep. (E) mRNA expression levels of inflammation-related factors IL-1β, IL-6, and NF-κB in mammary gland tissue. (F–I) Protein expression levels of inflammation-related factors IL-1β, p65, and phospho-p65 (pp65) in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5. CLA attenuated S. aureus-induced inflammation in ovine mammary tissue. (A) H&E staining of mammary gland tissue (scale bar = 20 μm). (B–D) Concentrations of IL-8, IL-6, and TNF-α in plasma from Hu sheep. (E) mRNA expression levels of inflammation-related factors IL-1β, IL-6, and NF-κB in mammary gland tissue. (F–I) Protein expression levels of inflammation-related factors IL-1β, p65, and phospho-p65 (pp65) in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 6. CLA protected mammary tissue from oxidative damage associated with mastitis. (A,B) SOD activity and MDA levels in mammary gland tissue. (C) Fluorescence detection of reactive oxygen species (ROS) expression in mammary gland tissue. (D) mRNA expression levels of oxidative stress-related factors NFE2L2 and HMOX1 in mammary gland tissue. (E–I) Protein expression levels of antioxidant-related factors Nrf2, p-Nrf2, HO-1, and SIRT3 in mammary gland tissue. (J) NAD+/NADH ratio in mammary gland tissue. (K) Immunohistochemical staining of SIRT3 in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6. CLA protected mammary tissue from oxidative damage associated with mastitis. (A,B) SOD activity and MDA levels in mammary gland tissue. (C) Fluorescence detection of reactive oxygen species (ROS) expression in mammary gland tissue. (D) mRNA expression levels of oxidative stress-related factors NFE2L2 and HMOX1 in mammary gland tissue. (E–I) Protein expression levels of antioxidant-related factors Nrf2, p-Nrf2, HO-1, and SIRT3 in mammary gland tissue. (J) NAD+/NADH ratio in mammary gland tissue. (K) Immunohistochemical staining of SIRT3 in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 7. CLA reduced mitophagy in mammary tissue during mastitis. (A) mRNA expression levels of mitophagy-related factors PARKIN and PINK1 in mammary gland tissue. (B–D) Protein expression levels of PARKIN and PINK1 in mammary gland tissue. (E) Immunohistochemical staining of LC3b in mammary gland tissue. (F–H) Protein expression levels of AMPK and phospho-AMPK (p-AMPK) in mammary gland tissue. (I) ATP content in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 7. CLA reduced mitophagy in mammary tissue during mastitis. (A) mRNA expression levels of mitophagy-related factors PARKIN and PINK1 in mammary gland tissue. (B–D) Protein expression levels of PARKIN and PINK1 in mammary gland tissue. (E) Immunohistochemical staining of LC3b in mammary gland tissue. (F–H) Protein expression levels of AMPK and phospho-AMPK (p-AMPK) in mammary gland tissue. (I) ATP content in mammary gland tissue. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 8. CLA upregulated PPARG and UCP2 expression in mammary tissue. (A) mRNA expression of PPARG and UCP2 in mammary gland tissue. (B–D) Protein expression levels of PPARG and UCP2 in mammary gland tissue. (E) Immunohistochemical staining of UCP2 in mammary gland tissue. Data are presented as mean ± standard deviation. *** p < 0.001.
Figure 8. CLA upregulated PPARG and UCP2 expression in mammary tissue. (A) mRNA expression of PPARG and UCP2 in mammary gland tissue. (B–D) Protein expression levels of PPARG and UCP2 in mammary gland tissue. (E) Immunohistochemical staining of UCP2 in mammary gland tissue. Data are presented as mean ± standard deviation. *** p < 0.001.
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Figure 9. Graphical Abstract. Red arrows denote the action of S. aureuson specific intracellular molecules; black arrows denote the action of CLA.
Figure 9. Graphical Abstract. Red arrows denote the action of S. aureuson specific intracellular molecules; black arrows denote the action of CLA.
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MDPI and ACS Style

Jin, Y.; Zhang, H.; Xie, X.; Ma, N.; Shen, X. Conjugated Linoleic Acid Ameliorates Staphylococcus aureus-Induced Inflammation, Oxidative Stress, and Mitophagy via the PPARG-UCP2 Pathway in Hu Sheep Mastitis. Agriculture 2026, 16, 99. https://doi.org/10.3390/agriculture16010099

AMA Style

Jin Y, Zhang H, Xie X, Ma N, Shen X. Conjugated Linoleic Acid Ameliorates Staphylococcus aureus-Induced Inflammation, Oxidative Stress, and Mitophagy via the PPARG-UCP2 Pathway in Hu Sheep Mastitis. Agriculture. 2026; 16(1):99. https://doi.org/10.3390/agriculture16010099

Chicago/Turabian Style

Jin, Yuzhi, Hui Zhang, Xiaochang Xie, Nana Ma, and Xiangzhen Shen. 2026. "Conjugated Linoleic Acid Ameliorates Staphylococcus aureus-Induced Inflammation, Oxidative Stress, and Mitophagy via the PPARG-UCP2 Pathway in Hu Sheep Mastitis" Agriculture 16, no. 1: 99. https://doi.org/10.3390/agriculture16010099

APA Style

Jin, Y., Zhang, H., Xie, X., Ma, N., & Shen, X. (2026). Conjugated Linoleic Acid Ameliorates Staphylococcus aureus-Induced Inflammation, Oxidative Stress, and Mitophagy via the PPARG-UCP2 Pathway in Hu Sheep Mastitis. Agriculture, 16(1), 99. https://doi.org/10.3390/agriculture16010099

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