Ramipril ameliorates endometriosis by inducing oxidative stress-mediated apoptosis in the wistar rat

Endometriosis is illustrated by the presence of ectopic endometrial cells capable of evading apoptosis outside the uterus. Apoptotic and anti-apoptotic factors in the extra uterine microenvironment can be compromised by the impairment in oxidative status. Angiotensin Converting Enzyme (ACE) Inhibitors and Nitric Oxide (NO) modulators play pivotal role in inflammation, angiogenesis, apoptosis and in abrogating oxidative imbalance. Therefore, in the current study we investigate the role of ACE inhibitor and or NO modulators in mitigating the proliferation of ectopic endometrial lesions in rat model. Sixty adult female virgin wistar rats were utilized; out of which fifteen were used as donor rats and rest forty-two were randomly divided into seven groups after surgical implantation of endometrial explants into rats (group II–VII). Histomorphometric assessment of uteri and ectopic lesions was performed by Hematoxylin and eosin (H-E) staining, followed by immunohistochemical study for Proliferating cell nuclear antigen (PCNA), Bax and Bcl-2. Oxidative stress parameters were evaluated by biochemical estimations, succeeded by immunoblotting of Poly [ADP-ribose] polymerase 1 (PARP1). Additionally, immunoblotting of Vascular endothelial growth factor (VEGF), Bax, Bcl-2 and caspase-3 was also performed. Significant decrease in the diameter of lesions with diffused staining at the extracellular spaces of stromal cells for PCNA accompanied by significant decrease in the expression of VEGF (p < 0.00001) was observed in group III. Furthermore, increased expression of Bax:Bcl-2 ratio (p < 0.001) and cleaved caspase-3 (p ≤ 0.0001) in ectopic lesions of group III was also observed. Administration of ramipril alone results in triggering oxidative stress mediated cleavage of PARP1, augmenting apoptosis in the ectopic lesions. Similar content being viewed by others Data availability No datasets were generated or analysed during the current study. Abbreviations - OVX: - Ovariectomy - ACE: - Angiotensin converting enzyme - NO: - Nitric oxide - SNP: - Sodium nitroprusside - L-NAME: - Nω-Nitro-L-arginine methyl ester hydrochloride - PCNA: - Proliferating cell nuclear antigen - PARP1: - Poly (ADP-ribose) polymerase 1 - ROS: - Reactive oxygen species - SOD: - Superoxide dismutase - CAT: - Catalase - MDA: - Malondialdehyde - PBS: - Phosphate buffer saline - TBA-TCA: - 2-Thiobarbituric acid-trichloroacetic acid - DCFH-DA: - 2′,7′-Dichlorodihydrofuorescein diacetate - VCl3 : - Vanadium (III) chloride - NEDD: - N-(1-Naphthyl) ethylenediamine dihydrochloride - SULF: - Sulfanilamide - BSA: - Bovine serum albumin - RIPA: - Radioimmunoprecipitation assay buffer - NBT: - Nitro blue tetrazolium - BCIP: - 5-Bromo-4-chloro-3-indoyl-phosphate - AP: - Alkaline phosphatase - HRP: - Horseradish peroxidase - BCA: - Bicinchoninic acid method - HE: - Hematoxylin-Eosin - EDTA: - Ethylenediamine tetraacetic acid

Agarwal A, Gupta S, Sharma RK (2005) Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 3:28. https://doi.org/10.1186/1477-7827-3-28 Amoghimath S, Suresha RN, Jayanthi MK (2017) To evaluate the anti-inflammatory activity of ramipril in albino rats. Int J Basic Clin Pharmacol 7(1):16. https://doi.org/10.18203/2319-2003.ijbcp20175485 Arumugam K, Yip YC (1995) De novo formation of adhesions in endometriosis: the role of iron and free radical reactions. Fertil Steril 64(1):62–64. https://doi.org/10.1016/S0015-0282(16)57655-9 Augoulea A et al (2009) The role of the oxidative-stress in the endometriosis-related infertility. Gynecol Endocrinol 25(2):75–81. https://doi.org/10.1080/09513590802485012 Aydin Y et al (2011) Remission of endometriosis by hyperbaric oxygen treatment in rats. Reprod Sci 18(10):941–947. https://doi.org/10.1177/1933719111400635 Barbe AM et al (2020) Expression and significance of matrix metalloproteinase-2 and matrix metalloproteinas-9 in endometriosis. J Med Life 13(3):314. https://doi.org/10.25122/JML-2020-0117 Barthelmebs M, Grima M, Imbs J-L (1995) Ramipril-induced decrease in renal lithium excretion in the rat. Br J Pharmacol 116(4):2161–2165. https://doi.org/10.1111/J.1476-5381.1995.TB15048.X Becker JB et al (2005) Strategies and methods for research on sex differences in brain and behavior. Endocrinology 146(4):1650–1673. https://doi.org/10.1210/EN.2004-1142 Benedetti S et al (2022) A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen. Heliyon. https://doi.org/10.1016/J.HELIYON.2022.E10642 Biswas P et al (2019) High-Protein diet ameliorates arsenic-induced oxidative stress and antagonizes uterine apoptosis in rats. Biol Trace Elem Res 192(2):222–233. https://doi.org/10.1007/S12011-019-1657-2 Bravo R, Macdonald-Bravo H (1987) Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 105(4):1549–1554. https://doi.org/10.1083/JCB.105.4.1549 Cenksoy PO et al (2015) A potential novel treatment strategy: inhibition of angiogenesis and inflammation by resveratrol for regression of endometriosis in an experimental rat model. Gynecol Endocrinol 31(3):219–224. https://doi.org/10.3109/09513590.2014.976197 Chakraborty TR, Gore AC (2004) Aging-related changes in ovarian hormones, their receptors, and neuroendocrine function. Exp Biol Med 229(10):977–987. https://doi.org/10.1177/153537020422901001 Chand P et al (2018) Evaluation of immunohistochemical profile of breast cancer for prognostics and therapeutic use. Niger J Surg 24(2):100. https://doi.org/10.4103/NJS.NJS_2_18 Folkman J, Klagsbrun M (1987) Angiogenic factors. Science 235(4787):442–447. https://doi.org/10.1126/SCIENCE.2432664 Foyouzi N et al (2004) Effects of oxidants and antioxidants on proliferation of endometrial stromal cells. Fertil Steril 82(SUPPL. 3):1019–1022. https://doi.org/10.1016/j.fertnstert.2004.02.133 Giudice LC, Kao LC (2004) Endometriosis. The Lancet 364(9447):1789–1799. https://doi.org/10.1016/S0140-6736(04)17403-5 Greenwald RA (2018) Handbook methods for oxygen radical research. Handbook methods for oxygen radical research, pp. 1–447. https://doi.org/10.1201/9781351072922/HANDBOOK-METHODS-OXYGEN-RADICAL-RESEARCH-ROBERT-GREENWALD/ACCESSIBILITY-INFORMATION. Howell RJ, Dowsett M, Edmonds DK (1994) Endometriosis: oestrogen and progesterone receptors in endometriosis: heterogeneity of different sites. Hum Reprod 9(9):1752–1758. https://doi.org/10.1093/oxfordjournals.humrep.a138788 ‘IHC staining protocol Paraffin, frozen and free-floating sections "Materials and methods" IHC staining protocol Contents’ (no date) Van Langendonckt A, Casanas-Roux F, Donnez J (2002) Oxidative stress and peritoneal endometriosis. Fertil Steril 77(5):861–870. https://doi.org/10.1016/S0015-0282(02)02959-X Li H, Horke S, Förstermann U (2014) Vascular oxidative stress, nitric oxide and atherosclerosis. Atherosclerosis 237(1):208–219. https://doi.org/10.1016/J.ATHEROSCLEROSIS.2014.09.001 Liang W et al (2008) Ramipril improves oxidative stress-related vascular endothelial dysfunction in db/db mice. J Physiol Sci 58(6):405–411. https://doi.org/10.2170/PHYSIOLSCI.RP012808 Liao JK et al (1995) Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Investig 96(6):2661–2666. https://doi.org/10.1172/JCI118332 Martin JP, Dailey M, Sugarman E (1987) Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation. Arch Biochem Biophys 255(2):329–336. https://doi.org/10.1016/0003-9861(87)90400-0 Mashimo M et al (2021) The 89-kDa PARP1 cleavage fragment serves as a cytoplasmic PAR carrier to induce AIF-mediated apoptosis. J Biol Chem. https://doi.org/10.1074/JBC.RA120.014479 Miranda KM, Espey MG, Wink DA (2001) A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5(1):62–71. https://doi.org/10.1006/NIOX.2000.0319 Murphy AA et al (1998) Evidence for oxidatively modified lipid-protein complexes in endometrium and endometriosis. Fertil Steril 69(6):1092–1094. https://doi.org/10.1016/S0015-0282(98)00087-9 Nakao T et al (2017) Expression of angiotensin ii types 1 and 2 receptors in endometriotic lesions. Gynecol Obstet Invest 82(3):294–302. https://doi.org/10.1159/000447591 Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358. https://doi.org/10.1016/0003-2697(79)90738-3 Ota H et al (1999) Immunohistochemical assessment of superoxide dismutase expression in the endometrium in endometriosis and adenomyosis. Fertil Steril 72(1):129–134. https://doi.org/10.1016/S0015-0282(99)00152-1 Ota H et al (2000) Aberrant expression of glutathione peroxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis. Fertil Steril 74(2):313–318. https://doi.org/10.1016/S0015-0282(00)00638-5 Overton C et al (1996) Peritoneal fluid cytokines and the relationship with endometriosis and pain. Hum Reprod 11(2):380–386. https://doi.org/10.1093/HUMREP/11.2.380 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159 Parasar P, Ozcan P, Terry KL (2017) Endometriosis: epidemiology, diagnosis and clinical management. Curr Obstet Gynecol Rep 6(1):34–41. https://doi.org/10.1007/s13669-017-0187-1 Polak G et al (2001) Total antioxidant status of peritoneal fluid in infertile women. Eur J Obstet Gynecol Reprod Biol 94(2):261–263. https://doi.org/10.1016/S0301-2115(00)00352-3 Roshanaee MK et al (2022) Protective effect of minocycline on Bax and Bcl-2 gene expression, histological damages and oxidative stress induced by ovarian torsion in adult rats. Int J Fertil Steril 16(1):30. https://doi.org/10.22074/IJFS.2021.522550.1069 Sampson JA (1927) Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 14(4):422–469. https://doi.org/10.1016/s0002-9378(15)30003-x Snyder CM et al (2009) Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members. PLoS ONE. https://doi.org/10.1371/JOURNAL.PONE.0007059 Szczepańska M et al (2003) Oxidative stress may be a piece in the endometriosis puzzle. Fertil Steril 79(6):1288–1293. https://doi.org/10.1016/S0015-0282(03)00266-8 Tait SWG, Green DR (2010) Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 11(9):621–632. https://doi.org/10.1038/nrm2952 Tzifi F et al (2012) The role of BCL2 family of apoptosis regulator proteins in acute and chronic leukemias. Adv Hematol. https://doi.org/10.1155/2012/524308 Ulas M, Cay M (2011) Effects of 17β-estradiol and vitamin E treatments on blood trace element and antioxidant enzyme levels in ovariectomized rats. Biol Trace Elem Res 139(3):347–355. https://doi.org/10.1007/S12011-010-8669-2/FIGURES/4 Vernon MW, Wilson EA (1985) Studies on the surgical induction of endometriosis in the rat. Fertil Steril. https://doi.org/10.1016/S0015-0282(16)48988-0 Wan H et al (2024) PARP1 inhibition prevents oxidative stress in age-related hearing loss via PAR-Ca2+-AIF axis in cochlear strial marginal cells. Free Radical Biol Med 220:222–235. https://doi.org/10.1016/J.FREERADBIOMED.2024.05.020 Yerlikaya G et al (2016) Comprehensive study of angiogenic factors in women with endometriosis compared to women without endometriosis. Eur J Obstet Gynecol Reprod Biol 204:88–98. https://doi.org/10.1016/j.ejogrb.2016.07.500 Zhang L et al (2023) Network pharmacology analysis of the mechanisms underlying the therapeutic effects of Yangjing Zhongyu Tang on thin endometrium. Drug des Dev Ther 17:1805–1818. https://doi.org/10.2147/DDDT.S409659 Zhang Z et al (2020) Involvement of angiotensin II receptor type 1/NF-κB signaling in the development of endometriosis. Exp Ther Med 20(4):3269. https://doi.org/10.3892/ETM.2020.9071

Not applicable. Funding This work was supported by a grant from the scientific project {1198(Sanc.)/STBT-11012(15)},) funded by the Department of Science and Technology and Biotechnology, West Bengal, India. We are appreciative towards FRPDF grant of Presidency University, Kolkata. We are also grateful to DST-FIST ((DLS[SR/FST/LSI-560/2013(C)]), Government of India and DBT-BUILDER (BT/INF/22/SP45088/2022). Author information Authors and Affiliations Contributions PM has conceptualized, investigated, designed & performed the experiments, analyzed the results and written the original draft. SSB has conceptualized, supervised, acquired fund and laboratory resources, and revised the original draft. Both the authors reviewed the manuscript before the final submission. Corresponding author Ethics declarations Conflict of interest The authors declare no competing interests. Ethics approval and consent to participate All animal procedures were performed in accordance with the Committee for Control and Supervision of Experiments on Animals (CCSEA) guidelines for the Humane Care of Laboratory Animals. All procedures were approved by the Presidency University, Kolkata, India University Institutional Animals Care and Use Committee. Consent for publication Not applicable. 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. 10735_2025_10397_MOESM2_ESM.tif (download TIF ) Fig. S2 Immunohistochemical localization pattern of PCNA, Bax and Bcl-2 in the eutopic uterine section under 100X oil immersion. Representative photomicrographs of uterine sections stained for PCNA in Group I (a), Group II (b), Group III (c), Group IV (d), Group V (e), Group VI (f) and Group VII (g); for Bax in Group I (h), Group II (i), Group III (j), Group IV (k), Group V (l), Group VI (m) and Group VII (n); for Bcl-2 in Group I (o), Group II (p), Group III (q), Group IV (r), Group V (s), Group VI (t) and Group VII (u). Black thick arrows represent the distinctive pattern of PCNA, Bax and Bcl-2 in the endometrium lining 10735_2025_10397_MOESM3_ESM.tif (download TIF ) Fig. S3 Immunohistochemical localization pattern of PCNA, Bax and Bcl-2 in the ectopic lesion under 100X oil immersion. Pictorial representation of ectopic endometrial sections stained for PCNA in Group II (a), Group III (b), Group IV (c), Group V (d), Group VI (e) and group VII (f); for Bax in Group II (g), Group III (h), Group IV (i'), Group V (j), Group VI (k) and Group VII (l); for Bcl-2 in Group II (m), Group III (n), Group IV (o), Group V (p), Group VI (q) and group VII (r). Blue thick arrows represent variable staining pattern of Bax and Bcl-2 in the ectopic endometrial glands and stroma 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 Mazumdar, P., Biswas, S.S. Ramipril ameliorates endometriosis by inducing oxidative stress-mediated apoptosis in the wistar rat. J Mol Histol 56, 117 (2025). https://doi.org/10.1007/s10735-025-10397-4 Received: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s10735-025-10397-4