SOD1 deficiency drives ferroptosis-linked oxidative and reproductive aging, mitigated by ginseng root extract
Juewon Kim, Shuichi Shibuya, Yusuke Ozawa, Yorino Sato, Kazuhiro Kawamura, Takahiko Shimizu
TL;DR
This study shows that SOD1 deficiency causes oxidative stress and reproductive aging through ferroptosis, and ginseng root extract can help reverse these effects.
Contribution
The study identifies SOD1 loss as a driver of ferroptosis-linked aging and introduces ginseng root extract as a potential intervention.
Findings
SOD1 deficiency increases oxidative stress markers and causes skin and reproductive aging in mice.
Ginseng root extract rescues reproductive function and reduces oxidative damage in SOD1-deficient models.
Ferroptosis inhibition or GR supplementation reverses redox imbalance and reproductive decline in C. elegans and mice.
Abstract
Aging is accompanied by cumulative oxidative stress that promotes tissue degeneration and reproductive decline. Here, we show that deficiency of superoxide dismutase 1 (SOD1) accelerates oxidative injury and reproductive aging through a ferroptosis-linked redox imbalance, and that ginseng root extract (GR) confers protection across species. Aged hairless Sod1⁻/⁻ mice exhibited markedly elevated skin and plasma oxidative stress markers—including 8-isoprostane, malondialdehyde (MDA), and pentosidine—together with dermal cyst formation and atrophic pathology. Complementary studies in C. elegans revealed that SOD1-deficient strains displayed increased reactive oxygen species, depleted glutathione, and elevated iron and lipid peroxidation—canonical features of ferroptosis-associated oxidative stress. These redox alterations coincided with shortened reproductive span and reduced progeny…
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Figure 4- —http://dx.doi.org/10.13039/501100003725National Research Foundation of Korea
- —http://dx.doi.org/10.13039/501100007312National Center for Geriatrics and Gerontology
- —http://dx.doi.org/10.13039/501100001691Japan Society for the Promotion of Science
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Taxonomy
TopicsFerroptosis and cancer prognosis · Redox biology and oxidative stress · Glutathione Transferases and Polymorphisms
Introduction
Living organisms rely on antioxidant systems to control reactive oxygen species (ROS) and preserve redox homeostasis. Among these, cytosolic copper/zinc superoxide dismutase (SOD1) catalyzes superoxide dismutation and is essential for cellular defense against oxidative damage. Loss of SOD1 causes intracellular O₂⁻ accumulation and multiple aging-related pathologies. Previous studies have demonstrated that Sod1^−/−^ mice exhibit elevated intracellular O₂⁻ levels, leading to diverse aging-related pathologies [1]. The critical role of SOD1 in regulating redox balance underscores its importance in mitigating oxidative damage and delaying the onset of age-associated tissue degeneration.
Aging is characterized by a progressive decline in extracellular matrix integrity, particularly evident in the skin. Both male and female individuals experience a reduction in collagen levels with age, a phenomenon termed dermatoporosis [2, 3]. Oxidative stress is a major driver of skin aging, with reactive oxygen species (ROS) implicated in the degradation of key structural proteins [4]. Notably, Sod1 knockout suppresses extracellular matrix-related genes, including Col1a1 and Has2, while upregulating matrix-degrading enzymes such as Mmp-1 and Mmp-2, resulting in epidermal and dermal atrophy [5, 6]. Our previous findings suggest that natural phytochemicals exhibit antioxidative properties by modulating FoxO3a activity, thereby inhibiting melanin formation and tyrosinase activity [7]. Because ginsenosides possess strong antioxidant and FoxO3a-modulating properties, we investigated whether ginseng root extract (GR) could mitigate SOD1 deficiency-induced skin atrophy in Sod1^−/−^ mice.
Oxidative stress also contributes to ovarian dysfunction and reproductive aging. Clinically, skin disorders such as rashes, itching, burning, and vulvar pain are frequently observed in ovarian cancer patients [8, 9]. Excess ROS damage oocytes and follicular cells, but the downstream mechanisms remain incompletely defined. SOD1 deficiency leads to excessive oxidative stress, and accumulating evidence suggests that ROS act as critical triggers for oocyte aging [10, 11]. In this study, we assess the protective effects of GR against ovarian aging and explore shared redox-driven mechanisms between skin and ovarian aging. While ROS function as essential signaling molecules, their contribution as direct inducers of ovarian senescence remains unclear. Recent studies implicate ferroptosis—iron-dependent lipid peroxidation and glutathione depletion—as a redox-driven cell-death pathway in aging [12, 13]. Given that Sod1⁻^/^⁻ mice accumulate MDA and other advanced lipoxidation end-products, we hypothesized that SOD1 deficiency promotes aging phenotypes via ferroptosis-linked mechanisms. Using both murine and C. elegans models, we examined oxidative, ferroptotic, and reproductive outcomes and evaluated the protective efficacy of GR.
Materials and methods
Animals
Sod1^−/−^ mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and subsequently backcrossed with C57BL/6NCrSlc mice (Japan SLC, Shizuoka, Japan) for 10 generations. Hos:HR-1^h/hr^ mice were sourced from Hoshino Laboratory Animals (Ibaraki, Japan), and hairless Sod1^−/−^ double-mutant mice were established by interbreeding Hos:HR-1^h/hr^ with Sod1^−/−^ mice over five to six generations. To produce hairless Sod1^−/−^ and hairless Sod1^+/+^ mice for in vivo experiments, in vitro fertilization and embryo transfer methods were utilized. Genotypic confirmation of the Sod1^−/−^ allele was conducted using genomic PCR on DNA extracted from tail-tip samples of mice aged 3–4 weeks. All animals were housed under a 12-h light/dark cycle with free access to MF chow (Oriental Yeast, Tokyo, Japan). At the end of the experiments, animals were euthanized by cardiac blood collection following isoflurane anesthesia. All experimental procedures complied with guidelines approved by the Animal Care Committee of Chiba University and the National Center for Geriatrics and Gerontology.
Materials and administration
Korean Red Ginseng (the root of Panax ginseng) extract, containing ginsenoside Rg1 (9.5 mg/g) and ginsenoside Rb1 (41.7 mg/g) as quantified by HPLC (GR, Millipore Sigma, MN, USA, 05115001), was prepared as a suspension in water. GR was administered orally to male and female Sod1^−/−^ mice (n = 7–10) at a dose of 300 mg/kg/day via gavage, beginning at 16 weeks of age and continuing for 8 weeks. To account for potential estrogen effects, estrous cycles in female mice were monitored through vaginal smears, with treatments conducted between 22 and 27 weeks of age. Tissue samples were collected in the late afternoon, timed to coincide with the metestrus or diestrus phase, or at 28 weeks for females that had ceased cycling. For experiments involving C. elegans, GR was applied at final concentrations of 10 ppm or 50 ppm, alongside 10 µM ferrostatin-1 (SML0583; Merck, MN, USA), incorporated into the media plates.
Histology
For histological analysis, skin samples were excised from the dorsal region of mice and fixed overnight in a 20% neutral buffered formalin solution (#068–01727; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The samples were subsequently embedded in paraffin and sectioned into 4 µm slices using a standard microtome. Ovarian tissues, collected during the diestrus phase, were also fixed in 20% formalin overnight. These were embedded in paraffin, sectioned at a thickness of 6 µm, and mounted on glass slides for hematoxylin and eosin staining. Histological evaluation of ovarian sections included counting primordial, primary, secondary, and antral follicles, as well as corpora lutea, based on established classification criteria [32].
Measurement of oxidative stress markers
Blood samples were drawn from the left ventricular chamber using ethylenediaminetetraacetic acid (EDTA)–coated collection tubes (Terumo, Tokyo, Japan) and centrifuged at 6000 rpm for 5 min at room temperature. Plasma was separated from the blood and treated with 100 µM indomethacin (#095–02472) and 50 µg/mL dibutylhydroxytoluene (#047–29451) (both from FUJIFILM Wako Pure Chemical Corporation). Skin tissue samples were homogenized in 0.1 M phosphate buffer (pH 7.4) supplemented with 1 mM EDTA (#N001; Dojindo Laboratories, Kumamoto, Japan) and 50 µg/mL dibutylhydroxytoluene. The homogenized mixture was centrifuged at 8000 × g for 10 min at 4 °C, and the resulting supernatant was collected for further analysis. Levels of 8-isoprostane in the plasma and skin homogenate were determined using the 8-isoprostane enzyme immunoassay kit (#516351; Cayman Chemical Company, MI, USA) in accordance with the manufacturer’s protocol. Protein content in the supernatant was measured using the DC protein assay kit (#5000111; BioRad, Hercules, CA, USA), and 8-isoprostane concentrations were normalized to protein levels. For MDA analysis, back skin tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris-HCl at pH 7.4, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, and 1% deoxycholic acid) containing a protease inhibitor cocktail (Roche Diagnostics, Tokyo, Japan). The homogenate was then centrifuged at 1600 × g for 10 min at 4 °C, and the supernatant was collected for the assay. MDA levels were quantified using the thiobarbituric acid reactive substances (TBARS) Assay Kit (#10009055; Cayman Chemical Company, MI, USA) as described in the provided instructions. Plasma pentosidine levels were assessed using the FSK Pentosidine kit (Fushimi Pharmaceutical Factory, Kagawa, Japan) in accordance with the manufacturer’s guidelines.
Monitoring estrus cycle
Throughout the final 6 weeks of the study, vaginal epithelial cell samples were collected daily between 9:00 AM and 12:00 PM using sterile cotton-tipped swabs. The swabs were gently inserted into the vaginal canal and rinsed with saline solution. Samples were transferred onto uncoated glass slides, fixed with methanol, and stained using a 5% Giemsa solution (Muto Pure Chemicals, Tokyo, Japan) prepared in 1/15 M phosphate buffer (pH 6.4). The slides were stained for 30 min, followed by a rinse with sterile water. Under microscopic examination, the presence of keratinocytes, coupled with the absence of stratified squamous epithelial cells (non-nucleated) and leukocytes, was identified as indicative of the estrus phase. To evaluate menstrual cycle irregularities, the weekly frequency of estrus stage cycling was recorded over the 6-week period, and the data were expressed as the average number of days per cycle.
Worm strains and maintenance
C. elegans strains were maintained on Nematode Growth Media (NGM) agar plates at 20 °C with OP50 bacteria as their food source. The following strains were used: Bristol N2, wild type; HA2986, sod-1(rt448[sod-1 WTC]) II; HA2987, sod-1(rt449[G39AC]) II; HA3299, sod-1(rt451[sod-1(G85RC)]) II, and FX776, sod-1(tm776) II. All strains were obtained from the Caenorhabditis Genetics Center (CGC).
Worm synchronization and food preparation
To synchronize nematodes, six unmated hermaphrodites were placed onto NGM plates with an adequate supply of food. Synchronization was achieved using a mixture of 70 µL M9 buffer, 25 µL of bleach (10% sodium hypochlorite solution), and 5 µL of 10 N NaOH. The resulting eggs were incubated overnight at 20 °C in S-basal buffer containing 100 mM NaCl, 0.01 mM cholesterol, and 50 mM potassium phosphate (pH 6.0). Once hatched, L1-stage larvae were transferred to new NGM plates seeded with Escherichia coli OP50 as a food source and maintained at 20 °C until they reached the L4 larval stage, designated as “Day 0.” The OP50 strain was cultured in LB medium at 37 °C for up to 12 h, and bacterial concentration was determined by combining colony-forming unit counts with optical density measurements at 600 nm (OD_600_).
Brood size assay
Brood sizes were determined as previously described [33], with minor modifications. Eggs on each plate were permitted to hatch and develop to the L2–L4 larval stages. To facilitate counting, F2 offspring were occasionally heat-killed by briefly passing the plates over the flame of an alcohol lamp, a method that maintained the structural integrity of the worms. Brood sizes were recorded at 5 days of age. Progeny from 20 worms were counted, with three independent experiments conducted, totaling 60 worms. The brood counts were documented using a microscope system comprising an Olympus SZ61 microscope paired with an Olympus eXcope T300 camera (Olympus, Tokyo, Japan).
Reproductive span assays
Reproductive spans were assessed as previously described [34]. Eggs were synchronized using a hypochlorite solution and selected at the L4 stage for experimental use. Individual hermaphrodites were placed on separate 35 mm NGM plates and transferred to new plates daily until the end of their reproductive period. Plates were monitored 48 h after transfer to verify reproductive activity. The cessation of reproduction was defined as the last day of progeny production followed by two consecutive days with no progeny. Hermaphrodites that experienced bagging (offspring hatching within the mother) were censored on the day of matricide. The total reproductive span was measured from the first day of adulthood (day 0) to the last day of egg-laying. The time from adult day 0 to the peak day of egg-laying was recorded as the reproductive span to peak, while the duration from the peak day to the final day of egg-laying was designated as the post-peak reproductive span. All measurements were conducted at 20 °C, with 20 nematodes per assay and two additional independent experiments, resulting in data from a total of 60 worms.
Iron assay
Iron levels were quantified using an Iron Assay Kit (#MAK025; Sigma-Aldrich, MO, USA) following the manufacturer’s guidelines. In brief, synchronized adult worms were collected and washed three times with M9 buffer before flash-freezing their pellets. The frozen pellets, maintained in liquid nitrogen, were pulverized and combined with 5% Triton X-100. These samples were then sonicated in five volumes of iron assay buffer and centrifuged at 13,000 × g for 10 min at 4 °C to eliminate insoluble debris. The extracted iron reacted with an acidic buffer and chromagen to produce a colorimetric compound, which was measured at 593 nm using a fluorescence plate reader (Synergy H1, BioTek, VT, USA). Ferrous and ferric iron concentrations were calculated from standard curves and normalized against protein content, determined through the BCA protein assay (#23225; Pierce, IL, USA). The procedure was conducted in three independent biological replicates.
Quantification of reactive oxygen species level
Nematodes were synchronized and treated for 10 days with either the vehicle, 10 µM ferrostatin-1, or 50 ppm GR, with media replaced every 2 days. No egg-laying inhibitors were applied during the treatment period. On day 10, worms from five separate plates were washed with M9 buffer and incubated with 100 µM Amplex Red probe (#A22288; Invitrogen, CA, USA) in Krebs-Ringer Phosphate buffer (145 mM NaCl, 5.7 mM Na₂PO₄, 4.86 mM KCl, 0.54 mM CaCl₂, 1.22 mM MgSO₄, 5.5 mM glucose, pH 7.4), supplemented with 0.2 U/mL horseradish peroxidase in a glass-bottom microplate (MSSBNFX40; Merck, MN, USA). The solution was gently stirred in the dark for 3 h, after which fluorescence intensity was measured using a fluorescence plate reader (Synergy H1, BioTek, VT, USA; excitation: 571 nm, emission: 585 nm). Fluorescence readings were normalized to protein concentration, which was measured using the BCA protein assay. The assay was performed in triplicate for biological replicates.
Lipid peroxidation assay
Lipid peroxidation levels were determined using the TBARS Assay Kit to MDA levels, following the manufacturer’s protocol, in a glass-bottom microplate (MSSBNFX40; Merck, MN, USA). Synchronized worms were treated with either the vehicle, 10 µM ferrostatin-1, or 50 ppm GR, with media refreshed every 2 days throughout the 10-day treatment period. Experimental samples were collected, and frozen worms were homogenized using a TissueLyser II (Qiagen, Hilden, Germany) at 30 Hz for 2 min, followed by a 1-min rest on ice, repeating the cycle three times. The lysates were centrifuged at 13,200 rpm for 15 min at 4 °C to remove debris, and the supernatant was collected. Protein concentration was determined using the BCA protein assay, and 20–25 µg of total protein was used for each assay. For experiments with acute glutathione depletion, day 2 animals were treated with 20 mM diethyl maleate (DEM) or 5 mM elastin for 6 h before sample collection. Three biological replicates were performed for each condition.
Statistical analyses
Biological replicates (n ≥ 3) were performed for each experiment. Data are expressed as mean ± SD; two-group comparisons used Student’s t-test, and multiple comparisons used one-way ANOVA with Dunnett’s test (p < 0.05). Statistical significance was determined when the p-value was less than 0.05. Results are expressed as the mean ± standard deviation (SD) in all figures unless otherwise noted. Statistical analyses were conducted using R software (ver. 4.1.0) and Excel 2016 (Microsoft, NM, USA). The following significance levels were applied: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and #p < 0.01.
Results
SOD1 deficiency elevates systemic oxidative stress
To investigate age-associated physiological changes and enable experimental intervention, we generated hairless Sod1^−/−^ mice and characterized their phenotype at 19 months of age. These mice displayed clear signs of accelerated aging, including deteriorated skin appearance compared to Sod1^+/-^ controls (Fig. 1A). To assess the extent of oxidative damage, we measured 8-isoprostane, a well-established marker of lipid peroxidation. Sod1^−/−^ mice showed a significant increase in 8-isoprostane levels in both the skin (Fig. 1B) and plasma (Fig. 1C), indicating a systemic elevation of oxidative stress in the absence of SOD1. Dermal cysts, characterized by lipid droplet accumulation within the dermal layer [14, 15], were frequently observed in hairless mice. Notably, dermal cysts of hairless Sod1^−/−^ mice were more numerous and larger than in controls (Fig. 1D–F). These histological changes support the notion that chronic oxidative stress resulting from SOD1 deficiency disrupts lipid metabolism and contributes to age-associated dermal pathology. Similarly, 7-month-old hairless Sod1^−/−^ mice (Fig. 1G) exhibited a modest increase in dermal cyst formation (Fig. 1H, I), along with marked elevations in oxidative stress biomarkers. Specifically, 8-isoprostane levels were significantly higher in both skin (Fig. 1J) and plasma samples (Fig. 1K) compared to Sod^+/+^ controls. In addition, MDA, a major advanced lipoxidation end-product (ALE) generated from polyunsaturated fatty acid peroxidation, was significantly increased in the skin of Sod1^−/−^ mice (Fig. 1L). Pentosidine, a well-characterized advanced glycation end-product (AGE), was also markedly elevated in the plasma (Fig. 1M). Even at 7 months, Sod1⁻^/^⁻ mice exhibited elevated 8-isoprostane, MDA, and plasma pentosidine, confirming systemic oxidative stress and accumulation of advanced oxidation products.Fig. 1. Elevated oxidative stress levels in aged hairless Sod1⁻^/^⁻ mice. A Representative image of 19-month-old hairless Sod1^+/-^ and Sod1^−/−^ male mice. 8-Isoprostane contents of the B skin and C plasma of the aged hairless Sod1^+/-^ and Sod1^−/−^ mice (n = 3). D Hematoxylin and eosin staining of the skin on the back of the hairless Sod1^+/-^ and Sod1^−/−^ mice. The E number and F size of the dermal cyst in the skin on the backs (n = 3). G–M A phenotypical analysis of 7-month-old hairless male Sod1^−/−^ mice. G Hematoxylin and eosin staining of the skin on the back. The H number and I size of the dermal cyst in the skin on the backs of hairless Sod1^+/+^ and Sod1^−/−^ mice. 8-Isoprostane contents of the J skin and K plasma of hairless Sod1^+/+^ and Sod1^−/−^ mice. L MDA contents of the skin and M pentosidine contents of the plasma of hairless Sod1^+/+^ and Sod1^−/−^ mice were measured. Data are shown as the mean ± SD; *p < 0.05, **p < 0.01 (Student’s t-test)
SOD1 deficiency induces ferroptosis-associated oxidative stress in C. elegans
Building on prior studies demonstrating that SOD1 deficiency leads to increased ROS and accelerated senescence [16], we further investigated the link between SOD1 depletion and lipid peroxidation-driven ferroptosis. In this study, we confirmed elevated levels of ALEs, which are known to accumulate during ferroptosis [17], in Sod1^−/−^ mice. This finding supports the hypothesis that SOD1 deficiency exacerbates lipid peroxidation beyond the levels observed in normal aging.
To test whether SOD1 loss promotes ferroptosis-linked redox imbalance, we examined SOD1-mutant nematodes. As organisms age, hydroperoxide levels tend to rise; all SOD1-mutant C. elegans strains exhibited significantly elevated hydrogen peroxide levels compared to the control strain HA2986. Among them, the sod-1(tm776) deletion mutant FX776 showed the strongest effect (Fig. 2A). Importantly, treatment with the ferroptosis inhibitor ferrostatin-1 or GR, a phytochemical previously studied for its ferroptosis-alleviating properties [18–20] significantly suppressed hydroperoxide accumulation in FX776 worms at multiple time points (day 0, day 5, and day 10). Next, we assessed glutathione (GSH) levels—a critical regulator of ferroptosis essential for glutathione peroxidase 4 (GPX4)-mediated phospholipid hydroperoxide detoxification [21, 22]. Compared to the control strain HA2986, SOD1 mutants displayed a pronounced age-associated decline in GSH levels. Notably, consistent with the reduction in hydrogen peroxide, treatment with either ferrostatin-1 or GR significantly restored GSH levels in aged FX776 worms (Fig. 2B), indicating a functional rescue of antioxidant capacity. To further evaluate ferroptotic stress, we quantified MDA, a terminal product of lipid peroxidation and a well-established marker of ferroptosis [23, 24]. SOD1-deficient worms exhibited a significant increase in MDA accumulation, consistent with heightened lipid oxidative damage. Remarkably, treatment with ferrostatin-1 or GR markedly suppressed MDA levels (Fig. 2C), suggesting effective mitigation of ferroptosis-associated lipid peroxidation. Finally, levels of redox-active ferrous iron (Fe^2^⁺), a critical catalyst of lipid peroxidation and hallmark of ferroptosis [25, 26], were markedly elevated in SOD1-deficient worms. Notably, GR administration significantly attenuated this iron accumulation, indicating its potential role in modulating iron-driven oxidative stress (Fig. 2D). Treatment with either ferrostatin-1 or GR suppressed these changes, supporting their anti-ferroptotic potential.Fig. 2SOD1 deficiency enhances age-associated ferroptosis markers and oxidative stress. Ferroptosis-related oxidative stress markers were measured across the lifespan in HA2986 control worms, SOD1-mutant strains HA2987 and HA3299, and the SOD1-deletion strain FX776. A Relative Amplex Red fluorescence in the supernatant of worms. B Total glutathione (GSH) level was normalized to the GSH level in worms. C Levels of the lipid peroxidation end product, MDA, were measured and normalized against the mean of vehicle worms for independent samples. D Quantification of Fe^2+^/Fe^3+^ iron contents of nematodes. Bars are represented as means ± standard deviation. #p < 0.0001 compared to HA2986 control, ****p < 0.0001 versus the FX776, sod-1(tm776) vehicle, n = 3 worm pellets (two-way ANOVA, Dunnett’s multiple comparison)
GR rescues reproductive decline in C. elegans
Recent studies have linked ferroptotic stress to reproductive aging in various model organisms [27–29]; however, this relationship has not yet been explored in C. elegans. To investigate the impact of SOD1 deficiency on reproductive aging in this model, we assessed the reproductive performance of the FX776 (sod-1(tm776)) mutant strain. SOD1-deficient worms exhibited significant reproductive dysfunction, including a shortened reproductive span and a marked reduction in progeny production (Fig. 3). Remarkably, supplementation with GR at 10 ppm and 50 ppm substantially restored reproductive capacity in these mutants. GR treatment extended the total reproductive span (Fig. 3A), prolonged the period of peak fertility (Fig. 3B), and significantly lengthened the post-peak reproductive window (Fig. 3C). Furthermore, GR effectively rescued the reduced progeny output observed in untreated sod-1 mutants (Fig. 3D). These findings highlight the therapeutic potential of GR in ameliorating reproductive decline associated with oxidative stress. They also suggest that GR may exert its beneficial effects by modulating ferroptosis-related pathways. Overall, this study provides compelling evidence for the utility of C. elegans as a model to explore the intersection of redox imbalance, ferroptosis, and reproductive longevity.Fig. 3. The FX776, sod-1(tm776) mutant exhibits reduced reproductive span and progeny production, which are restored by ferroptosis inhibition. A–C Reproductive span of sod-1(tm776) mutant with GR compared to wild-type N2 nematode. A Total reproductive span, B reproductive span to peak, and C reproductive span after peak with 10 ppm or 50 ppm GR were estimated. D Progeny number of 5-day-old nematodes was examined with GR treatment. Bars are represented as means ± standard deviation. #p < 0.0001 compared to N2 worms, **p < 0.01, ****p < 0.0001 compared to sod-1(tm776) vehicle (two-way ANOVA, Dunnett’s multiple comparison)
GR restores ovarian morphology and cyclicity in Sod1⁻/⁻ mice
Given the accelerated aging phenotypes observed in multiple tissues of Sod1^−/−^ mice [30], we investigated the ovary as a key target of oxidative imbalance in reproductive aging. Previous findings from our group demonstrated that SOD1 deficiency impairs progesterone secretion and compromises fertility in female mice [31]. Consistent with these findings, Sod1^−/−^ mice in the current study exhibited pronounced ovarian abnormalities, including a reduced corpus luteum area and increased accumulation of O₂⁻ in the luteal region, features associated with increased apoptosis and vascular disruption. Remarkably, treatment with GR prevented luteal degeneration and preserved ovarian architecture (Fig. 4A). Histological analysis revealed disrupted folliculogenesis in Sod1^−/−^ ovaries, characterized by altered distributions of primordial (black arrow), primary (black arrowhead), secondary (white arrowhead), and antral (white arrow) follicles (Fig. 4B). Emerging evidence indicates that primordial follicles require a low oxidative environment to maintain dormancy and serve as a long-term reproductive reserve [32]. In this context, SOD1 deficiency likely compromises the survival of these critical follicular populations due to elevated oxidative stress. GR treatment significantly rescued follicular homeostasis, with a marked restoration of primordial follicle numbers (Fig. 4C), as well as increased primary (Fig. 4D) and secondary (Fig. 4E) follicle counts. Although changes in antral follicles and luteal bodies were more modest (Fig. 4F, G), the overall trends supported improved follicular dynamics.Fig. 4GR attenuates ovarian dysfunction in Sod1^−/−^ mice. A The ovary images of hairless Sod1^+/+^ and Sod1^−/−^ female mice with GR administration. B Representative image of the ovary of Sod1^−/−^ mice with GR supplementation. Black arrow indicates primordial follicle. Black arrowhead indicates primary follicle. White arrowhead indicates secondary follicle. White arrow indicates antral follicle. The number of C primordial follicles, D primary follicles, E secondary follicles, F antral follicles, and G luteal bodies of Sod1^+/+^ or Sod1^−/−^ mice orally treated with GR (n = 6–8). H, I Days of the estrus cycle and ratio of estrus/estrus cycles of Sod1^+/+^ or Sod1^−/−^ mice along with GR treatment (n = 6–8). Scale bars, 100 µm. Data are shown as the mean ± SD; *p < 0.05, **p < 0.01, ^#^p < 0.05 compared to hairless Sod1^−/−^ (two-way ANOVA, Dunnett’s multiple comparison)
Luteal function is tightly coupled to estrous cyclicity, particularly the number of estrous days per cycle, a critical indicator of ovarian hormonal balance and reproductive competence [33, 34]. Considering the heightened ROS burden in Sod1^−/−^ mice, we hypothesized that redox imbalance would impair hypothalamic-pituitary-ovarian axis function, manifesting as disrupted estrous cycles and reduced estrous frequency [35–37]. Consistent with this hypothesis, Sod1^−/−^ female mice exhibited significantly lengthened estrous cycles and a marked reduction in the proportion of days spent in estrus (Fig. 4H, I), indicative of luteal dysfunction and compromised reproductive rhythms. Importantly, GR shortened estrous-cycle length and restored the proportion of estrous days, indicating improved ovarian endocrine function. These findings underscore GR’s role as a potent redox-modulating agent capable of restoring cyclic ovarian function under oxidative stress. By improving estrous regularity and supporting luteal activity, GR demonstrates promise as a therapeutic intervention for age-related reproductive decline driven by oxidative imbalance.
Collectively, these findings suggest that SOD1 deficiency accelerates ovarian dysfunction and reproductive decline via ferroptosis-associated oxidative stress, characterized by excessive lipid peroxidation and redox imbalance. GR supplementation, by mitigating ferroptotic damage and restoring redox homeostasis, effectively rescues ovarian structure and function. These results position GR as a promising redox-targeted therapeutic approach to preserve ovarian health and delay reproductive aging under conditions of elevated oxidative stress.
Discussion
This study identifies a conserved relationship between SOD1 deficiency, ferroptosis-linked redox imbalance, and reproductive aging and demonstrates that GR mitigates these pathologies. The current findings suggest that oxidative stress, lipid peroxidation, and iron overload converge as a redox-ferroptotic pathway driving systemic and reproductive aging. SOD1 serves as a central redox guardian, and its loss sensitizes tissues to ferroptotic injury. GR, by restoring antioxidant and iron-handling capacity, interrupts this cascade. These insights bridge redox biology and geroscience, offering translational avenues for dietary or pharmacological interventions targeting ferroptosis in aging. Utilizing both murine and C. elegans models, we newly highlight that GR, rich in ginsenosides Rb1 and Rg1, can activate Nrf2 signaling, maintain GPX4 and SLC7A11 expression, and restore glutathione metabolism [18, 19]. These activities align with our observed suppression of lipid peroxidation and iron accumulation in GR-treated C. elegans and Sod1⁻^/^⁻ mice. The cross-species efficacy suggests that GR acts upstream of ferroptosis to stabilize redox homeostasis, consistent with its clinical potential as a nutraceutical antioxidant.
In aged hairless Sod1⁻^/^⁻ mice, we observed hallmark features of accelerated aging, including dermal degeneration, cyst formation, and increased accumulation of oxidative biomarkers such as 8-isoprostane, MDA, and pentosidine. These data confirm previous reports linking SOD1 loss to enhanced ROS production [38, 39], while also expanding the scope to include dermal lipid dysregulation and systemic AGE/ALE accumulation. The use of hairless mice enabled precise phenotypic assessments and reinforced the utility of this model for studying visible aging hallmarks. The observation that oxidative stress manifests not only as molecular signatures but also as tissue-level pathology underscores the multifaceted consequences of SOD1 deficiency. Our data show that Sod1⁻^/^⁻ mice and C. elegans mutants accumulate lipid peroxidation products, exhibit glutathione depletion, and display iron overload—key biochemical hallmarks of ferroptosis-associated stress. Although canonical molecular markers such as GPX4 activity and ACSL4 expression were not measured, recent studies confirm that MDA, GSH, and Fe^2^⁺ changes are robust indicators of ferroptotic propensity [21, 40]. We therefore interpret these findings as evidence of a ferroptosis-associated redox imbalance, rather than direct confirmation of ferroptotic cell death. Future work using C11-BODIPY, Liprofluo, and RSL3 sensitivity assays will clarify the mechanistic axis. These findings extend prior evidence of ferroptosis in neurodegeneration and cancer [41, 42] into the realm of aging biology, establishing a new axis of vulnerability in redox-compromised states.
One of the most salient contributions of our work is the identification of a ferroptosis-reproduction axis in C. elegans. While reproductive decline is a well-established hallmark of aging, its linkage to ferroptosis had not been previously explored, particularly in a nematode model. Our findings that GR rescues the reproductive span and progeny output of sod-1 mutants suggest a functional intersection between redox homeostasis and germline longevity. These results suggest that ferroptosis is not merely a byproduct of aging but may actively drive reproductive senescence. Notably, our murine data reveal that SOD1 deficiency also induces skin atrophy and ovarian dysfunction—phenotypes that may be interlinked. Clinically, skin manifestations such as pruritus and rash have been reported as early paraneoplastic signs of ovarian cancer [43, 44], raising the possibility of a systemic ferroptotic stress signature. These findings echo emerging human studies suggesting that ferroptotic oxidative damage impairs ovarian reserve and steroidogenesis [40, 45, 46], implying a conserved, cross-species mechanism underlying redox-driven reproductive aging. Extending our observations to mammals, we further show that GR supplementation restores ovarian structure and function in Sod1⁻^/^⁻ mice. The marked recovery of primordial and primary follicle populations, along with improved estrous cyclicity, points to GR’s ability to preserve reproductive competence under oxidative stress. Notably, primordial follicles are exquisitely sensitive to redox status, as oxidative stress prompts their premature activation and exhaustion [32, 47]. By maintaining a reductive environment, GR may forestall this depletion, thereby extending reproductive lifespan. These results align with emerging views that anti-ferroptotic interventions can modulate the ovarian aging trajectory [48, 49]. Importantly, GR’s beneficial effects are not limited to structural preservation but extend to endocrine function, as evidenced by normalization of estrous cycles. This supports the notion that SOD1 deficiency impairs hypothalamic-pituitary-ovarian (HPO) signaling, and that redox modulation can restore HPO axis integrity. Whether this effect is mediated via systemic antioxidant effects or local modulation within the ovary warrants further exploration. Future studies should also examine GR’s impact on mitochondrial function and iron handling, as both are critical nodes in ferroptosis regulation and ovarian health.
Despite the comprehensive insights provided by our multi-model approach, several limitations should be acknowledged. First, while the use of C. elegans and murine models enabled the mechanistic dissection of SOD1 deficiency and ferroptosis in aging, the translatability to human physiology remains to be validated. Second, although GR exhibited consistent antioxidant and anti-ferroptotic effects, its active components were not isolated or quantitatively analyzed, leaving the precise molecular mediators of its protective action undefined. Third, our reproductive aging assessments were primarily morphological and functional; ferroptotic stress is increasingly recognized in germline and ovarian aging. The restoration of follicular homeostasis and estrous rhythm by GR implies that maintaining glutathione and iron balance is essential for reproductive longevity. Although a direct fertility assessment was not performed, improved folliculogenesis and cyclicity provide strong evidence of functional rescue. We have noted this limitation explicitly and plan future mating trials to confirm fecundity outcomes. Future studies will include mating and fertility assessments in aged Sod1⁻^/^⁻ mice to confirm whether GR-mediated restoration of folliculogenesis and estrous cyclicity translates into improved fecundity. Additionally, while ferrostatin-1 was used as a ferroptosis inhibitor, direct molecular evidence of ferroptotic cell death (e.g., GPX4 activity, lipid ROS quantification by C11-BODIPY) was not assessed. Finally, although our models show strong age-related phenotypes, they represent extreme cases of oxidative stress; future studies should assess more subtle redox imbalances and examine long-term effects of GR in physiological aging contexts.
In conclusion, SOD1 deficiency induces systemic oxidative and reproductive decline via ferroptosis-associated redox imbalance. GR supplementation alleviates these effects by restoring antioxidant capacity and iron homeostasis. These findings establish a conserved role for the SOD1–ferroptosis axis in aging and highlight GR as a potential geroprotective strategy.
Supplementary Information
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