Preventing Cell Damage to Restore Saliva Gland Balance in Obesity

Body weight was consistently elevated in all obese groups, including untreated ob/ob mice, compared to that in C57BL/6 control mice (Figure 1A). Moreover, the daily food intake significantly increased across these groups and reflected the characteristic leptin deficiency-associated hyperphagic behavior (Figure 1B). In parallel, fasting glucose levels were markedly elevated in the untreated ob/ob group, and remained similarly high in the FER and DFO groups throughout the experimental period (Figure 1C). These systemic metabolic alterations established a physiologically unfavorable environment that potentially fosters subsequent histological and functional deterioration of the salivary glands.

Histological analysis revealed pronounced acinar atrophy, expanded interstitial spaces, and extensive cytoplasmic vacuolization that was consistent with lipid-droplet accumulation in the salivary glands of untreated ob/ob mice (Figure 2A). Transmission electron microscopy (TEM) further confirmed the presence of abundant cytoplasmic lipid droplets within acinar cells. Notably, this accumulation was markedly reduced in both DFO- and FER-treated groups (Figure 2B). At the transcriptional level, ob/ob mice exhibited significantly elevated expression of key lipogenesis-related genes, including acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein-1c (SREBP-1c), and carbohydrate-responsive element-binding protein (ChREBP). Treatment with either ferrostatin-1 or deferoxamine substantially suppressed the expression of these lipogenic genes (Figure 2C). Collectively, these findings demonstrate that obesity promotes profound lipid accumulation in the salivary glands, as evident at the histological, ultrastructural, and molecular levels, which can be effectively mitigated by ferroptosis-targeted interventions. Additional representative histological and immunohistochemical images from independent sections are provided in Supplementary Figure S1.

To investigate whether obesity-induced salivary gland injury involves ferroptosis, we measured the key indices of oxidative damage and iron accumulation. ROS levels were significantly elevated in the ob/ob group, as determined by DCFH-DA fluorescence assay, and were effectively reduced by either ferrostatin-1 or deferoxamine (p < 0.001) (Figure 3A). Moreover, the MDA content—a marker of lipid peroxidation—increased in the ob/ob group. Ferrostatin-1 treatment induced a greater reduction in MDA levels than the deferoxamine treatment (p < 0.01 and p < 0.05, respectively) (Figure 3B). Similarly, the primary ferroptosis defense mechanism, indicated by GPX4 enzymatic activity, was markedly impaired in ob/ob mice. This activity was more robustly restored by ferrostatin-1 than by deferoxamine (p < 0.05) (Figure 3C). In contrast, cytosolic Fe²⁺ levels, which were significantly elevated in the ob/ob group, were comparably reduced by either treatment (p < 0.001) (Figure 3D). These results confirmed that obesity promotes ferroptosis-associated oxidative stress and iron overload in salivary glands. Furthermore, they suggest that, compared with deferoxamine, ferrostatin-1 exerts superior efficacy in mitigating lipid peroxidation and restoring antioxidant capacity in this model.

To evaluate whether obesity-induced ferroptosis is accompanied by fibrotic remodeling, we analyzed the histological and transcriptional markers in the salivary glands. In the ob/ob group, immunohistochemical staining revealed the increased expression of the profibrotic mediators TGF-β and Collagen I. These were visualized as blue-stained structures using a blue chromogen, indicating enhanced fibrotic activity. This increase was attenuated in both treatment groups. The qPCR analysis corroborated these histological findings. TGF-β mRNA was significantly upregulated in the ob/ob group and was more robustly suppressed by ferrostatin-1 than by deferoxamine (p < 0.01 and p < 0.05, respectively). In contrast, Collagen I mRNA expression showed a stronger suppressive response to deferoxamine (p < 0.01; Figure 4A,B). Masson's trichrome (MT) staining was performed to visualize collagen deposition. The ob/ob group exhibited extensive interstitial fibrosis, characterized by diffuse blue-stained collagen fibers, which were markedly reduced following either ferrostatin-1 or deferoxamine treatment (Figure 4C). These findings indicate that ferroptosis is closely linked to fibrotic remodeling in salivary glands under obese conditions, with ferrostatin-1 and deferoxamine exhibiting differential regulatory effects on specific fibrosis-related pathways. Additional representative histological and immunohistochemical images from independent sections are provided in Supplementary Figure S1.

To assess the secretory function of salivary glands, we performed immunohistochemical staining for functional markers. In the ob/ob group, the expression of α-amylase—a key digestive enzyme—was markedly reduced compared to that in C57BL/6 controls. This reduction was partially reversed in both treatment groups; however, a more pronounced recovery was observed in the FER group (Figure 5A). Similarly, the expression of aquaporin-5 (AQP5)—a water-channel protein that is essential for saliva secretion—decreased in the ob/ob group. Compared to deferoxamine treatment, ferrostatin-1 treatment more effectively restored AQP5 expression (Figure 5B). These findings suggest that obesity-induced ferroptosis significantly impairs salivary gland secretory function and that pharmacological inhibition of this pathway, particularly with ferrostatin-1, can effectively facilitate functional recovery. Additional representative histological and immunohistochemical images from independent sections are provided in Supplementary Figure S1.

Considering the observed phenotypic changes, we examined the mitochondrial ultrastructure and regulation of selective autophagy pathways. In the ob/ob group, TEM revealed severe mitochondrial ultrastructural damage, which was characterized by swelling, loss of cristae, and outer membrane rupture. These morphological changes were partially mitigated by ferrostatin-1 or deferoxamine treatment (Figure 6A). Western blotting revealed distinct alterations in key autophagy- and mitophagy-regulatory proteins. The expression of LC3B-II (the lipidated form, indicated at 14 kDa) was most prominently induced in the ob/ob group. Concurrently, the levels of p62 (SQSTM1)—an autophagy substrate—were markedly decreased in ob/ob mice compared with controls. This pattern (high LC3B-II and low p62 levels) suggested enhanced autophagic degradation (increased flux) in the obese state. Both treatments reduced LC3B-II levels and partially restored p62 levels, which suggested the normalization of autophagic flux. The mitophagy regulators PINK1 and PRKN (parkin) were upregulated in the ob/ob group. Either treatment decreased the expression of these proteins. Notably, ferrostatin-1 maintained PINK1 expression at levels that were similar to those in the control whereas deferoxamine resulted in a near-complete loss of the PINK1 signal. NCOA4—a cargo receptor for ferritinophagy—is selectively elevated in ob/ob mice, which suggests increased ferritin turnover. NCOA4 levels were suppressed in both the control and treatment groups (Figure 6B). These findings indicate that obesity-induced salivary gland pathology involves significant mitochondrial damage and the dysregulation of selective autophagy pathways, including mitophagy and ferritinophagy. The modulation of autophagic flux and mitophagy markers by ferroptosis inhibitors, particularly the preservation of PINK1 by ferrostatin-1, likely contributed to the observed tissue-protective effects.

Leptin-deficient (ob/ob) mice, characterized by excessive food intake and pronounced obesity, are widely used as robust models of metabolic disorders. In this study, we used ob/ob mice to investigate the contribution of ferroptosis to obesity-induced alterations in the salivary glands. Six-week-old female C57BL/6-Jms Slc mice (n = 8) and age-matched C57BL/6J ob/ob mice (n = 24) were obtained from Japan SLC (Shizuoka, Japan). Following a 2-week acclimation period, the animals were randomly assigned to four groups (n = 8 per group) as follows: wild-type C57BL/6 (control), untreated ob/ob, ferrostatin-1-treated ob/ob (FER), and deferoxamine-treated ob/ob (DFO). All animals were fed standard chow ad libitum throughout the study period. Beginning at 8 weeks of age, mice in the FER and DFO groups received intraperitoneal (IP) injections of ferrostatin-1 (5 µM/kg) or deferoxamine (100 mg/kg), respectively, three times weekly for 8 weeks. The control and untreated ob/ob groups received vehicle (sterile saline) injections according to the same schedule. To monitor pathological progression, baseline and endpoint assessments were conducted at 8 and 16 weeks of age. At the study endpoint, the animals were anesthetized via isoflurane inhalation and blood samples were collected from the inferior vena cava. Subsequently, the mice were euthanized and salivary gland tissues were harvested for subsequent analysis. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Pusan National University Hospital (IACUC approval no. PNUH-2023-223).

The level of malondialdehyde (MDA), a biomarker of lipid peroxidation, was quantified using a commercial detection kit (Abcam, Cambridge, UK). Tissue homogenates and standards were incubated with thiobarbituric acid (TBA) at 95 °C for 1 h. Following rapid cooling on ice, the absorbance of the MDA-TBA adduct was spectrophotometrically measured at 532 nm. The MDA concentrations were determined by interpolating a standard curve.

Intracellular ferrous iron (Fe²⁺) levels were assessed using a colorimetric assay kit (Abcam, ab83366). Approximately 30 mg of salivary gland tissue was homogenized in Iron Assay Buffer and centrifuged at 16,000× g for 10 min at 4 °C. The supernatant was reacted with an iron probe under acidic conditions to produce a colored complex, which was detected spectrophotometrically at 593 nm. Data were normalized to the total protein content that was determined using the bicinchoninic acid (BCA) assay.

ROS generation was measured using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO, USA). Homogenized tissue samples were incubated with 10 µM DCFH-DA at 37 °C for 30 min in the dark. Oxidation of the probe yielded fluorescent dichlorofluorescein (DCF), which was measured fluorometrically (e.g., using a fluorescence microplate reader) at excitation/emission wavelengths of 485/535 nm. The fluorescence intensity was normalized to the protein concentration.

Glutathione peroxidase 4 (GPX4) activity was evaluated using a commercial assay kit (Abcam). Enzymatic activity was quantified by measuring the rate of NADPH oxidation. Protein concentrations were determined using the BCA method, and activity was calculated according to the manufacturer's protocol.

Salivary glands were excised immediately after euthanasia, fixed overnight in 4% formalin, and paraffin-embedded. Because mouse salivary glands are very small, multiple specimens from animals within the same experimental group were embedded together and stained on the same slide to ensure uniform staining conditions and to allow direct comparison across samples. Serial paraffin sections were prepared so that histologic and immunohistochemical analyses could be performed at anatomically comparable levels among animals.

Sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO, USA) for general morphology or Masson's trichrome (MT; Sigma-Aldrich, St. Louis, MO, USA) for fibrosis assessment. For immunohistochemistry (IHC), deparaffinized sections underwent antigen retrieval by heating in 10 mM sodium citrate buffer (pH 6.0; sodium citrate buffer (pH 6.0)) for 15 min using a microwave oven, followed by cooling at room temperature (23 °C). Sections were then incubated with horseradish peroxidase-conjugated secondary antibodies (ENZO Biochem Inc., New York, NY, USA) for 1 h at room temperature (23 °C), followed by visualization using the chromogen 3,3′-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA, USA) and counterstaining with hematoxylin. Negative controls were processed in parallel by substituting PBS containing 1% BSA for the primary antibody.

Representative images were acquired from the central region of each gland using a Leica light microscope at 200× magnification. For each animal, 3–4 serial sections were evaluated, and 5–7 microscopic fields per section were analyzed in a fully blinded manner to ensure methodological rigor.

For ultrastructural analysis, salivary gland tissues were pre-fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) in phosphate buffer and post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA). After dehydration through a graded ethanol series, samples were embedded in Epon 812 resin. Ultrathin sections (50–60 nm) were stained with uranyl acetate and lead citrate and examined using a JEOL JEM-1200EXII (JEOL Ltd., Tokyo, Japan) transmission electron microscope.

To ensure adequate quantitative assessment of mitochondrial morphology, three animals per group were examined. For each animal, 2–3 ultrathin sections were prepared, and 8–10 TEM fields per section were analyzed. Mitochondrial integrity was assessed in a blinded manner, focusing on cristae structure, membrane continuity, and the degree of swelling.

Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) synthesis was synthesized using a reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR (qPCR) was conducted on an ABI PRISM 7900 HT system using SYBR Green Chemistry. The relative gene expression levels were calculated using the 2^−ΔΔCt method, with GAPDH normalized as the endogenous reference gene. The primer sequences are listed in Table 1.

Tissue lysates were prepared in a radioimmunoprecipitation assay (RIPA) buffer that was supplemented with protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Lysates were cleared by centrifugation at 12,000× g for 15 min at 4 °C. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. These membranes were blocked (e.g., with 5% non-fat milk) and incubated overnight at 4 °C with the following primary antibodies: anti-LC3B, anti-p62, anti-PRKN, anti-PINK1, and anti-NCOA4 (all from Abcam, Cambridge, UK; 1:1000 dilution), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated secondary antibodies (1:5000) were applied for 2 h at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) reagent and visualized using an imaging system.

All data are presented as mean ± standard deviation (SD). Intergroup comparisons were performed using one-way analysis of variance (ANOVA) followed by Scheffé's post hoc test using SPSS (v19.0; IBM Corp., Armonk, NY, USA). A p-value < 0.05 was considered as the cutoff for statistical significance.