IntroductionCisplatin (CIS) is a widely used chemotherapeutic agent for various solid tumors; however, its clinical application is often limited by serious off-target toxicities, notably ovarian damage, leading to infertility and premature ovarian insufficiency in young female patients1,2,3. Ovarian toxicity following cisplatin administration involves a complex interplay of oxidative stress, inflammation, apoptosis, and disruption of local proliferative and stress responses within the ovarian microenvironment4.Proliferating cell nuclear antigen (PCNA) serves as a marker of DNA replication and repair, playing a critical role in the cellular response to injury. Elevated PCNA expression in ovarian follicles has been associated with reparative proliferation attempts following toxic insults, yet excessive or dysregulated proliferation may exacerbate follicular depletion5,6. Simultaneously, heat shock protein-70 (HSP-70) is a highly inducible molecular chaperone that is upregulated under conditions of cellular stress, including chemotherapeutic exposure. HSP-70 mediates protein stabilization and anti-apoptotic signaling but can also reflect the severity of tissue injury when overly expressed7,8.Kisspeptin-1 (KISS-1), initially identified as a metastasis suppressor, is now recognized as a critical regulator of reproductive axis function, including gonadotropin-releasing hormone (GnRH) secretion and ovarian folliculogenesis9,10. Disruption in ovarian KISS-1 expression following toxic injury suggests an alteration in local endocrine signaling and impaired follicular dynamics. However, studies investigating KISS-1 alterations under CIS-induced ovarian damage remain scarce.Dapagliflozin (DAPA), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, has recently emerged as a promising agent beyond its glucose-lowering effects, demonstrating potent antioxidant, anti-inflammatory, and cytoprotective properties through mechanisms involving nuclear factor erythroid 2-related factor 2 (NRF2)/ heme oxygenase-1 (HO-1) activation and suppression of pro-inflammatory cascades11,12,13.Therefore, the present study aimed to evaluate the immunohistochemical expressions of PCNA, HSP-70, and KISS-1 in ovarian tissues subjected to CIS-induced toxicity and to investigate the protective effects of DAPA. By focusing on these molecular markers, we sought to elucidate whether DAPA can attenuate CIS-induced ovarian injury via modulation of proliferative, stress-related, and reproductive signaling pathways, thereby offering a novel therapeutic strategy for preserving female reproductive health during chemotherapy.Although several studies have investigated the protective effects of dapagliflozin against cisplatin-induced nephrotoxicity and cardiotoxicity, to our knowledge, no study has examined its impact on ovarian tissue or on the expression of PCNA, HSP-70, and KISS-1. This study is the first to investigate the potential protective effects of dapagliflozin against cisplatin-induced ovarian injury in rats, thereby contributing novel insights into female reproductive protection mechanisms.Materials and methodsEthical approvalAll experimental procedures were approved by the Animal Experiments Local Ethics Committee of Suleyman Demirel University (Approval No: 11.07.2024-08/311) All methods were carried out in accordance with relevant guidelines and regulations. The study was conducted in compliance with the ARRIVE 2.0 guidelines.AnimalsThirty-two healthy female Wistar albino rats (250–300 g) were obtained from the Experimental Animal Research Laboratory of Suleyman Demirel University, Türkiye. Animals were housed under controlled conditions (22–25 °C, 12-h light/dark cycle, 55% humidity) with free access to standard chow and water. After a one-week acclimatization period, the rats were randomly divided into four experimental groups (n = 8 per group) as follows:1.Control group: Received 1 mL of saline orally once daily for 7 consecutive days and a single intraperitoneal (i.p.) injection of 0.1 mL saline on day 4.2.CIS group: Received 1 mL of saline orally once daily for 7 days and a single i.p. injection of CIS (7.5 mg/kg, Koçak Farma, Türkiye) on day 4 (Bilgic et al., 2020).3.CIS + DAPA group: Administered DAPA (10 mg/kg, oral, Forziga, AstraZeneca, Türkiye) once daily for 7 days, with a single i.p. injection of CIS (7.5 mg/kg) on day 4 (Abd El-Fattah et al., 2022).4.DAPA group: Received DAPA (10 mg/kg, oral) once daily for 7 days and a single i.p. injection of 0.1 mL saline on day 4.At 24 h after the final treatment, animals were anesthetized using ketamine (90 mg/kg, i.p., Keta-Control, Doğa İlaç, Türkiye) and xylazine (8–10 mg/kg, i.p., XylazineBio, Bioveta, Czech Republic), followed by euthanasia via exsanguination through the inferior vena cava.The right ovaries were harvested and stored at − 80 °C for molecular analysis, whereas the left ovaries were fixed in 10% formalin for histopathological and immunohistochemical evaluations.Histopathological analysisOvarian tissues collected from each group were fixed in 10% formaldehyde solution for 72 h, followed by overnight washing under running water. The tissues were then processed through routine histological series (%50–%60–%70–%80–%96–%100), cleared in xylene, and embedded in paraffin blocks. From these paraffin blocks, 5 µm thick sections were obtained. The sections were stained with Hematoxylin–Eosin (HE) and Martius Scarlet Blue (MSB) and evaluated under a light microscope.HE stainingOvarian tissues fixed in 10% formalin were processed via standard paraffin embedding protocols. Sections of 5 µm thickness were cut and deparaffinized, rehydrated and stained with HE. They were evaluated histological parameters, including vascular congestion, hemorrhage, follicle count reduction, follicular cell degeneration, follicular invagination, vacuolization, and collagenous material accumulation, were scored using a semi-quantitative scale ranging from 0 to 3 (0: no histopathological finding, 1 (mild): histopathology in 66% of the section area (Table 1)14.Table 1 Histopathological scores ov ovarian damage.Full size tableMSB stainingTo evaluate increased fibrotic areas in the stroma, MSB staining was performed. Tissue Sects. (5 µm) were deparaffinized by incubation at 60 °C for 60 min. After deparaffinization, sections were passed through xylene and graded alcohol series to rehydrate and prepare them for staining. Staining was carried out according to the manufacturer’s instructions (RRSK2-100, Atom Scientific)15. Fibrotic areas were graded on a 0–3 scale based on the extent of collagen deposition14. For each ovarian section, 10 non-overlapping fields were randomly selected and examined at 20 × magnification to ensure representative assessment of fibrosis.Immunohistochemical analysisDeparaffinized Sects. (5 µm) were subjected to antigen retrieval and blocked with hydrogen peroxide and Ultra V Block (Thermo Scientific). Slides were incubated with primary antibodies against:PCNA (1:200, ab220208, Abcam)HSP-70 (1:200, ab79852, Abcam)KISS-1 (1:200, PA5-106920, Thermo Scientific)for 60 min at room temperature. Subsequently, sections were treated with a biotinylated secondary antibody (TP-125-BN, Thermo Scientific) and streptavidin-HRP complex (TS-125-HR, Thermo Scientific), developed with 3,3′-diaminobenzidine (DAB) substrate, counterstained with Mayer’s hematoxylin, and mounted. Staining intensity was evaluated semi-quantitatively by two independent observers blinded to the experimental groups. The intensity was scored on a 0–3 scale as follows: 0, no staining; 1, weak/focal ( +); 2, moderate/diffuse (+ +); and 3, strong/intense (+ + +). For each ovary, ten randomly selected microscopic fields were evaluated at × 400 magnification. All preparations were examined, evaluated, and photographed using an Eclipse E-600 Nikon photomicroscope (Japan) equipped with an image analysis system (NIS Elements, Nikon, Japan).Gene expression analysis by RT-qPCRTotal RNA was extracted from frozen ovarian tissues using the RiboEx™ RNA Isolation Kit (GeneAll, Korea) following the manufacturer’s protocol. RNA purity and concentration were determined spectrophotometrically (BioSpec-Nano, Shimadzu, Japan). Complementary DNA (cDNA) was synthesized using 1 µg RNA with a commercial cDNA synthesis kit (A.B.T., Atlas Biotechnology, Türkiye).Quantitative real-time PCR (qPCR) was conducted using SYBR Green Master Mix (Nepenthe, Türkiye) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). GAPDH was used as a housekeeping gene. Primer sequences are provided in Table 2. The relative mRNA expression levels were calculated using the 2−ΔΔCt method. PCR cycling conditions were:Table 2 Adjusted P values for pairwise comparisons of experimental groups.Full size tableInitial denaturation: 94 °C for 10 min (1 cycle).Denaturation: 95 °C for 15 s.Annealing/extension: 55 °C for 30 s (40 cycles total).ResultsHistopathological resultsHE stainingHE staining demonstrated severe histopathological alterations in the CIS group, including vascular congestion, hemorrhage, follicular degeneration, follicular invagination, decrease in follicle number, and vacuolization. All parameters were significantly worsened in the CIS group compared to control (p