Vortioxetine protects against methotrexate-induced ovarian toxicity through anti-inflammatory, antioxidant and antiapoptotic pathways: a multi-marker immunohistochemical study

Aims Methotrexate (MTX), a commonly used chemotherapeutic and immunosuppressive agent, is known to induce significant ovarian toxicity through mechanisms involving oxidative stress, inflammation, and apoptosis. Vortioxetine (VTX), a novel antidepressant with proven neuroprotective and anti-inflammatory properties, has not yet been evaluated in the context of chemotherapy-induced gonadotoxicity. This study aimed to investigate the protective effects of VTX against MTX-induced ovarian injury in a rat model by employing comprehensive histopathological and immunohistochemical evaluations.

and Results Thirty-two adult female Wistar Albino rats (300–350 g) were randomly divided into four equal groups (n = 8): Control, MTX, MTX + VTX, and VTX. Ovarian damage was induced with a single intraperitoneal injection of MTX (20 mg/kg), while VTX was administered daily (10 mg/kg) by oral gavage for five days. Rats were sacrificed on day 5, and bilateral ovaries were collected. Histopathological evaluation included follicular degeneration, vascular congestion, hemorrhage, and inflammatory cell infiltration. Immunohistochemical analyses were performed for 8-Hydroxy-2'-deoxyguanosine (8-OHdG), nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), caspase 3 (Cas-3), and Anti-Müllerian hormone (AMH) to assess oxidative stress, inflammation, apoptosis, and ovarian reserve. MTX administration caused severe follicular atresia, hemorrhage, and dense neutrophil infiltration. Immunohistochemically, 8-OHdG, NF-κB, TNF-α, and Cas-3 expressions were significantly elevated, while AMH was markedly reduced. VTX co-treatment significantly attenuated histological damage and modulated the expression of all biomarkers, indicating potent protective effects. VTX alone did not induce deleterious changes.

VTX exhibits a robust protective effect against MTX-induced ovarian injury via suppression of oxidative stress, inflammatory response and apoptotic pathways, while simultaneously preserving ovarian reserve. These findings highlight a novel application for VTX in fertility preservation strategies during chemotherapeutic interventions. Similar content being viewed by others Data Availability The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request. We have not used any AI tools or technologies to prepare this manuscript.

Aguilera-Aguirre L, Bacsi A, Radak Z, Hazra TK, Mitra S, Sur S, Brasier AR, Ba X, Boldogh I (2014) Innate inflammation induced by the 8-oxoguanine DNA glycosylase-1-KRAS-NF-κB pathway. J Immunol. https://doi.org/10.4049/jimmunol.1401625 Al Maruf A, O’Brien PJ, Naserzadeh P, Fathian R, Salimi A, Pourahmad J (2018) Methotrexate induced mitochondrial injury and cytochrome c release in rat liver hepatocytes. Drug Chem Toxicol 41(1):51–61. https://doi.org/10.1080/01480545.2017.1289221 Alboni S, Benatti C, Colliva C, Radighieri G, Blom JMC, Brunello N, Tascedda F (2021) Vortioxetine prevents Lipopolysaccharide-induced memory impairment without inhibiting the initial inflammatory cascade. Front Pharmacol 4(11):603979 Barbosa-Méndez S, Perez-Sánchez G, Salazar-Juárez A (2022) Vortioxetine treatment decreases cocaine-induced locomotor sensitization in rats. Physiol Behav 257:113989. https://doi.org/10.1016/j.physbeh.2022.113989 Canevarolo RR, Cury NM, Yunes JA (2023) The expression and activation of the NF-κB pathway correlate with methotrexate resistance and cell proliferation in acute lymphoblastic leukemia. Genes (Basel) 27(10):1880. https://doi.org/10.3390/genes14101880 Caruso G, Grasso M, Fidilio A, Torrisi SA, Musso N, Geraci F, Tropea MR, Privitera A, Tascedda F, Puzzo D, Salomone S, Drago F, Leggio GM, Caraci F (2021) Antioxidant activity of Fluoxetine and Vortioxetine in a non-transgenic animal model of Alzheimer’s disease. Front Pharmacol 12:809541. https://doi.org/10.3389/fphar.2021.809541 Çelik H, Atılgan R, Akpolat N, Gürateş B et al (2005) Tek ve Multi Doz Methotrexate Uygulamalarına Bağlı Overyan Histopatolojik Değişimler ve Folikül Gelişimi Üzerine Etkileri. Türk Jinekolojik Onkoloji Dergisi 8(1):14–18 Chen Y, Yang J, Zhang L (2023) The impact of follicular fluid oxidative stress levels on the outcomes of assisted reproductive therapy. Antioxidants (Basel). https://doi.org/10.3390/antiox12122117 Demir S, Türkmen Alemdar N, Ayazoglu Demir E, Mentese A, Aliyazıcıoğlu Y (2024) Gallic acid alleviates methotrexate-induced oxidative ovarian damage in rats. Farabi Med J 3(4):119–125. https://doi.org/10.59518/farabimedj.1544287 Di Renzo L, De Lorenzo A, Fontanari M et al (2022) Immunonutrients involved in the regulation of the inflammatory and oxidative processes: implication for gamete competence. J Assist Reprod Genet 39:817–846. https://doi.org/10.1007/s10815-022-02472-6 Dong Z, Dong Z, Xu L, Zhang J, Li L, Wang R, Huang X, Zou Z (2025) Vortioxetine alleviates motor, cognitive and emotional disorders in post-stroke rats by regulating the TLR-2/NF-κB pathway. Front Pharmacol 12(16):1555079. https://doi.org/10.3389/fphar.2025.1555079 Feng J, Ma WW, Li HX, Pei XY, Deng SL, Jia H, Ma WZ (2022) Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression. Front Endocrinol (Lausanne) 3(13):895095. https://doi.org/10.3389/fendo.2022.895095 Gökçek I, Gözer A, Kutlu T, Güvenç M, Aydın L, Uyanık G (2024). Protective effect of tyrosol on cisplatin–ınduced ovarian ınflammation and oxidative stress in rats. Revista Científica, FCV-LUZ / 34, rcfcv-e34444. https://doi.org/10.52973/rcfcv-e34444. Gowkielewicz M, Lipka A, Zdanowski W, Waśniewski T, Majewska M, Carlberg C (2024) Anti-Müllerian hormone: biology and role in endocrinology and cancers. Front Endocrinol (Lausanne) 16(15):1468364. https://doi.org/10.3389/fendo.2024.1468364 Gunyeli I, Saygin M, Ozmen O (2021) Methotrexate-induced toxic effects and the ameliorating effects of astaxanthin on genitourinary tissues in a female rat model. Arch Gynecol Obstet 304(4):985–997. https://doi.org/10.1007/s00404-021-06000-2 Hanoodi M, Mittal M (2024). Methotrexate. In StatPearls. StatPearls Publishing. Keçeci M, Karaoluk N (2025) Effect of curcumin on methotrexate-induced ovarian damage and follicle reserve in rats: the role of PARP-1 and P53. Ann Med 57(1):2446688. https://doi.org/10.1080/07853890.2024.2446688 Kiremitli T, Kiremitli S, Akselim B, Yilmaz B, Mammadov R, Tor IH, Yazici GN, Gulaboglu M (2021) Protective effect of coenzyme Q10 on oxidative ovarian and uterine damage induced by methotrexate in rats. Hum Exp Toxicol 40(9):1537–1544. https://doi.org/10.1177/09603271211002891 Kyaw MT, Sakthiswary R, Ani Amelia Z, Rahana AR, Munirah MM (2020) Effects of methotrexate therapy on the levels of gonadotropic hormones in rheumatoid arthritis patients of reproductive age. Cureus. https://doi.org/10.7759/cureus.7632 Li L, Wang S, Zhou W (2022) Balance cell apoptosis and pyroptosis of Caspase-3-activating chemotherapy for better antitumor therapy. Cancers (Basel). https://doi.org/10.3390/cancers15010026 Liu T, Zhang L, Joo D, Sun SC (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2:17023. https://doi.org/10.1038/sigtrans.2017.23 Liu D, Zhong Z, Karin M (2022) NF-κB: a double-edged sword controlling inflammation. Biomedicines 27(6):1250. https://doi.org/10.3390/biomedicines10061250 Lo SZ, Steer JH, Joyce DA (2011) Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-kappaB-dependent pathway. Arthritis Res Ther. https://doi.org/10.1186/ar3248 Melekoglu R, Ciftci O, Eraslan S, Cetin A, Basak N (2018) Beneficial effects of curcumin and capsaicin on cyclophosphamide-induced premature ovarian failure in a rat model. J Ovarian Res 11:33. https://doi.org/10.1186/s13048-018-0409-9 Rosen MP, Johnstone E, McCulloch CE, Schuh-Huerta SM, Sternfeld B, Reijo-Pera RA, Cedars MI (2012) A characterization of the relationship of ovarian reserve markers with age. Fertil Steril 97(1):238–243. https://doi.org/10.1016/j.fertnstert.2011.10.031 Samdanci ET et al (2019) Cytoprotective effects of molsidomine against methotrexate-induced hepatotoxicity: an experimental rat study. Drug des Dev Ther 13:13–21 Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi H (2002) Eight-hydroxy-2’-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program. Fertil Steril 77(6):1184–1190. https://doi.org/10.1016/s0015-0282(02)03103-5 Shafiek MS, Mekky RY, Nassar NN, El-Yamany MF, Rabie MA (2024) Vortioxetine ameliorates experimental autoimmune encephalomyelitis model of multiple sclerosis in mice via activation of PI3K/Akt/CREB/BDNF cascade and modulation of serotonergic pathway signaling. Eur J Pharmacol 5(982):176929. https://doi.org/10.1016/j.ejphar.2024.176929 Silva MSB, Giacobini P (2021) New insights into anti-Müllerian hormone role in the hypothalamic-pituitary-gonadal axis and neuroendocrine development. Cell Mol Life Sci 78(1):1–16. https://doi.org/10.1007/s00018-020-03576-x Soylu Karapinar O, Pinar N, Özcan O, Özgür T, Dolapçıoğlu K (2017) Protective effect of alpha-lipoic acid in methotrexate-induced ovarian oxidative injury and decreased ovarian reserve in rats. Gynecol Endocrinol 33(8):653–659. https://doi.org/10.1080/09513590.2017.1306847 Stika CS (2012) Methotrexate: the pharmacology behind medical treatment for ectopic pregnancy. Clin Obstet Gynecol 55(2):433–439. https://doi.org/10.1097/GRF.0b013e3182510a35 Sun Y, Lin Y, Li H et al (2012) 2,5-hexanedione induces human ovarian granulosa cell apoptosis through BCL-2, BAX, and CASPASE-3 signaling pathways. Arch Toxicol 86:205–215. https://doi.org/10.1007/s00204-011-0745-7 Sze SCW, Zhang L, Zhang S, Lin K, Ng TB, Ng ML, Lee KF, Lam JKW, Zhang Z, Yung KKL (2022) Aberrant transferrin and ferritin upregulation elicits iron accumulation and oxidative inflammaging causing ferroptosis and undermines estradiol biosynthesis in aging rat ovaries by upregulating NF-Κb-activated inducible nitric oxide synthase: first demonstration of an intricate mechanism. Int J Mol Sci 23(20):12689. https://doi.org/10.3390/ijms232012689 Talmon M, Rossi S, Pastore A, Cattaneo CI, Brunelleschi S, Fresu LG (2018) Vortioxetine exerts anti-inflammatory and immunomodulatory effects on human monocytes/macrophages. Br J Pharmacol 175(1):113–124. https://doi.org/10.1111/bph.14074 Topsakal S, Ozmen O, Asci H, Gulal A, Ozcan KN, Aydin B. Dapagliflozin prevents reproductive damage caused by acute systemic inflammation through antioxidant, anti-inflammatory, and antiapoptotic mechanisms. Basic Clin Pharmacol Toxicol. 2024 Nov;135(5):561-574. https://doi.org/10.1111/bcpt.14077 Topsakal S, Ozmen O, Ozcan KN, Asci H, Imeci OB, Sevuk MA, Kosar PA (2025) Fluvoxamine inhibited NLRP3 and NF-κB inflammatory pathways and maintained genital functions by ameliorating CD-MPR, KISS-1, AQP4 and Claudin-1 expressions. Basic Clin Pharmacol Toxicol 136(3):e70000. https://doi.org/10.1111/bcpt.70000 Turkler C, Onat T, Yildirim E, Kaplan S, Yazici GN, Mammadov R, Sunar M (2020) An experimental study on the use of lycopene to prevent infertility due to acute oxidative ovarian damage caused by a single high dose of methotrexate. Adv Clin Exp Med 29(1):5–11. https://doi.org/10.17219/acem/111809 Ulug P, Oner G (2014) Evaluation of the effects of single or multiple dose methotrexate administration, salpingectomy on ovarian reserve of rat with the measurement of anti-Müllerian hormone (AMH) levels and histological analysis. Eur J Obstet Gynecol Reprod Biol 181:205–209. https://doi.org/10.1016/j.ejogrb.2014.07.011 Yalcin B, Kalkan KT, Koseoglu E, Karaman E & Yay A (2024). Assessment of the effects of two different doses of methotrexate in rat ovarian tissue: A histological study. Ann Med Res. https://www.annalsmedres.org/index.php/aomr/article/view/4720. Accessed 23 August 2025. Yamada K, Ito M, Nunomura H, Nishigori T, Furuta A, Yoshida M et al (2025) Interplay of oxidative stress, autophagy, and Rubicon in ovarian follicle dynamics: orchestrating ovarian aging. Antioxidants 14(8):919. https://doi.org/10.3390/antiox14080919 Yamamoto Y, Kuwahara A, Taniguchi Y, Yamasaki M, Tanaka Y, Mukai Y, Yamashita M, Matsuzaki T, Yasui T, Irahara M (2015) Tumor necrosis factor alpha inhibits ovulation and induces granulosa cell death in rat ovaries. Reprod Med Biol 14(3):107–115. https://doi.org/10.1007/s12522-014-0201-5 Zhang W, Wu F (2024) Linoleic acid induces human ovarian granulosa cell inflammation and apoptosis through the ER-FOXO1-ROS-NFκB pathway. Sci Rep. https://doi.org/10.1038/s41598-024-56970-x Zhao LL, Jayeoye TJ, Ashaolu TJ, Olatunji OJ (2023) Pinostrobin, a dietary bioflavonoid exerts antioxidant, anti-inflammatory, and anti-apoptotic protective effects against methotrexate-induced ovarian toxicity in rats. Tissue Cell 85:102254

The authors thank Suleyman Demirel University Scientific Research Project Unit (SDU-BAP) for providing financial support for the study (project no. TSG-2024-9556). Funding Suleyman Demirel University Scientific Research Project Unit (SDU-BAP) provided financial support for the study (project no. TSG-2024–9556). Author information Authors and Affiliations Contributions H.A., E.S. and K.N.O. designed the study. E.S. and I.N. performed the experimental procedures and histological evaluations. E.S. and I.N. carried out the immunohistochemical analysis and statistical evaluation. H.A. and E.S. wrote the initial draft of the manuscript. H.A. and O.K. prepared the figures and tables. All authors reviewed and approved the final version of the manuscript. Corresponding author Ethics declarations Conflicts of Interest The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request. We have not used any AI tools or technologies to prepare this manuscript. Sarman E. and the co-authors have no conflicts of interest to declare in association with this study. The authors have no potential conflicts of interest to disclose. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Sarman, E., Asci, H., Ozcan, K.N. et al. Vortioxetine protects against methotrexate-induced ovarian toxicity through anti-inflammatory, antioxidant and antiapoptotic pathways: a multi-marker immunohistochemical study. J Mol Histol 56, 356 (2025). https://doi.org/10.1007/s10735-025-10642-w Received: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s10735-025-10642-w