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INTRODUCTION
The female reproductive axis is one of the organ systems impacted by Diabetes Mellitus (DM), a chronic metabolic disease (Andlib et al., 2024). One of its less well-known side effects is ovarian dysfunction, which can show up as decreased ovarian reserve, irregular menstruation, and infertility. Damage to granulosa cells, which are specialised somatic cells necessary for follicular development, steroidogenesis, and oocyte maturation, is closely linked to these results (Wang, 2025).
Granulosa cell dysfunction is largely caused by oxidative stress brought on by hyperglycemia. Reactive Oxygen Species (ROS) overproduction, mitochondrial damage, and apoptotic pathway activation are all brought on by elevated glucose levels (Tao et al., 2024). At the same time, the NF-κB signalling cascade amplifies inflammation, which raises the expression of iNOS and COX-2. Together, these effects impair cell viability by upregulating apoptotic markers like cytochrome c and cleaved caspase-3 and suppressing antioxidant defence mechanisms like Nuclear Factor erythroid 2-Related Factor 2 (Nrf2) (Kim et al., 2025).
Even though glycaemic control agents are available, current treatments do not directly target these cellular mechanisms. This shows how important it is to find new ways to protect cells that will keep granulosa cells working properly when someone has diabetes (Cao et al., 2025). Drug repurposing is a promising path because it uses drugs that are already known to be safe (Recino et al., 2025). Captopril is an ACE inhibitor, and spironolactone is a mineralocorticoid receptor antagonist (Jahan et al., 2025). Both drugs have anti-inflammatory and antioxidant properties, but their effects on ovarian granulosa cells when used together have not been fully studied (Elmorsy et al., 2025). This study looks at how well captopril and spironolactone, both alone and together, protect human granulosa-like KGN cells from oxidative and apoptotic damage caused by Streptozotocin (STZ). We think that the combination treatment will provide extra protection by restoring antioxidant defence and lowering apoptosis. This supports their possible use in stopping reproductive problems linked to diabetes.
MATERIALS AND METHODS
Cell Culture and Study Design
Human granulosa-like tumor cell line (KGN) was obtained from the National Centre for Cell Science (NCCS), Pune, India. Cells were maintained in DMEM/F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37ºC in a humidified atmosphere containing 5% CO₂. To simulate diabetic conditions, KGN cells were exposed to streptozotocin (STZ, 5 mM) for 24 hr. Following STZ treatment, cells were divided into five groups: (i) Control (untreated), (ii) STZ only, (iii) STZ+captopril (50 µM), (iv) STZ+spironolactone (25 µM), and (v) STZ+combination of captopril (50 µM) and spironolactone (25 µM). All treatments were performed in triplicate, and experiments were independently repeated three times (n=9 per group) to ensure reproducibility and statistical reliability (Kumar et al., 2014).
Tunnel assay
The in situ Cell Death Detection Kit (Roche, Germany) was used to check for apoptotic DNA fragmentation. We fixed KGN cells with 4% paraformaldehyde, made them permeable with 0.1% Triton X-100, and then put them in the TUNEL reaction mix for an hour at 37ºC. Cells that had been treated with DNase I were used as positive controls. We used Hoechst 33258 (5 µg/mL) to stain the nuclei and then looked at them with a Nikon Eclipse Ti fluorescence microscope at 20× magnification. ImageJ was used to count the TUNEL-positive (green) and total (blue) nuclei in five random fields. There were at least 100 cells in each field that were looked at. As described, the data were analysed statistically (Wang et al., 2024).
Hoechst 33258 staining for nuclear morphology assessment
We employed Hoechst 33258 staining to look at the shape of the nuclei and the condensation of chromatin in KGN cells. After treatment, cells were fixed with 4% paraformaldehyde, made permeable with 0.1% Triton X-100, and stained with Hoechst 33258 (5 µg/mL) in the dark for 10 min. We looked at the cells with a Nikon Eclipse Ti fluorescent microscope at 20× magnification. The excitation was 350 nm and the emission was 460 nm. Apoptotic nuclei looked smaller, broken up, or brilliantly coloured, while normal nuclei stayed spherical and blue all over. We counted apoptotic nuclei by hand in five randomly chosen fields using ImageJ. Each field had at least 100 cells. The data were analysed statistically as specified (Zhang et al., 2020).
Infllammatory Assay
Western blotting was used to measure the expression of cytochrome c, Nrf2, and cleaved caspase-3. Protease and phosphatase inhibitors added to RIPA buffer were used to extract the total protein. Thermo Fisher Scientific's BCA assay was used to measure the protein content. SDS-PAGE was used to separate equal amounts of protein (30-50 µg), which were then transferred to PVDF membranes. Membranes were blocked with 5% BSA, then incubated with primary antibodies and HRP-conjugated secondary antibodies for a whole night at 4ºC. Bands were scanned and visualised with Enhanced Chemiluminescence (ECL). The loading control was β-actin. ImageJ software was used to quantify band intensities. As stated, statistical analysis was performed on the data (You et al., 2025).
Statistical Analysis
All quantitative data were expressed as Mean±Standard Deviation (SD). Statistical significance between groups was assessed using One-Way Analysis of Variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. A p-value less than 0.05 was considered statistically significant. Analyses were performed using GraphPad Prism version 8.0.
Ethical Statement
This study did not involve human or animal subjects; therefore, ethical approval and informed consent were not required.
RESULTS
Tunnel assay
STZ exposure significantly increased DNA fragmentation in KGN cells, as shown by a higher proportion of TUNEL-positive nuclei compared to control (p<0.001; 95% CI: 10.2-15.6). Treatment with captopril or spironolactone alone reduced apoptotic indices (p<0.05), while combination therapy produced a greater reduction (p<0.001; 95% CI: 2.1-5.8), nearing control values. These results confirm the enhanced anti-apoptotic effect of combined therapy (Figure 1B).
Hoechst 33258 staining for nuclear morphology assessment
Hoechst staining showed that STZ induced chromatin condensation and nuclear fragmentation. Both monotherapies preserved nuclear morphology to some extent, with spironolactone showing greater nuclear protection than captopril (p<0.01). The combination group displayed significantly reduced apoptotic nuclei (p<0.001; 95% CI: 6.5-9.0), with mostly uniform, round nuclei consistent with healthy morphology (Figure 2B).
Infllammatory Assay
Western blot results indicated that STZ significantly upregulated cleaved caspase-3 and cytochrome c and downregulated Nrf2 (p<0.001 vs. control). Captopril and spironolactone monotherapy partially corrected these changes (p<0.05-0.01), but the combination treatment produced the strongest effects, markedly reducing apoptotic markers and significantly increasing Nrf2 expression (p<0.001; 95% CI for Nrf2 increase: 8.7-13.4) (Figures 3B-3D).
DISCUSSION
The present study demonstrates that captopril and spironolactone, both individually and in combination, significantly attenuate STZ-induced oxidative stress and apoptosis in human granulosa-like KGN cells. These findings underscore the therapeutic potential of repurposing cardiovascular drugs to combat diabetes-associated ovarian dysfunction. Among the tested groups, combination therapy exhibited the most potent protective effects, indicating a synergistic interaction between captopril and spironolactone (Schubert et al., 2020).
STZ-induced hyperglycemia is known to trigger mitochondrial dysfunction, increase ROS production, and activate caspase-dependent apoptotic pathways in various cell types, including granulosa cells (Fu et al., 20254). Our findings align with prior reports demonstrating STZ-induced upregulation of cleaved caspase-3 and cytochrome c (Yang et al., 2024), and we further show that this is coupled with downregulation of Nrf2, a master regulator of antioxidant defense. The restoration of Nrf2 expression by captopril and spironolactone supports earlier findings in renal and neuronal models (Huang et al., 202), suggesting that this pathway may be a key mediator of their cytoprotective effects. The TUNEL and Hoechst assays revealed that combination therapy markedly reduced nuclear fragmentation and chromatin condensation, two hallmark features of apoptosis. This morphological evidence reinforces the biochemical findings from Western blotting. Importantly, the combination group restored cellular morphology and Nrf2 levels to near-control values, suggesting effective reversal of oxidative injury. A major strength of this study is the use of a well-characterized human granulosa cell line, providing a relevant model for evaluating diabetic ovarian damage. Additionally, the use of multiple complementary assays strengthens the validity of our findings. However, some limitations should be noted. The in vitro design, while controlled, does not capture the complexity of the ovarian microenvironment or endocrine signaling. Further in vivo studies are needed to evaluate pharmacokinetics, hormonal interactions, and fertility outcomes. Clinically, these findings point toward the potential for using captopril and spironolactone as adjunctive therapies to preserve ovarian function in diabetic women. Their established safety profiles make them attractive candidates for drug repurposing. Future studies should explore their effects in animal models of Polycystic Ovarian Syndrome (PCOS) or diabetes-induced infertility.
CONCLUSION
In conclusion, captopril and spironolactone, particularly in combination, mitigate STZ-induced oxidative stress and apoptosis in KGN granulosa cells by upregulating Nrf2 and downregulating caspase-mediated apoptotic markers. These findings suggest their potential for repurposing as protective agents against diabetes-associated ovarian damage. However, further in vivo studies are necessary to validate their efficacy and safety in a physiological context.
