Ameliorative Effects of Quercetin and Metformin and Their Combination Against Experimental Endometriosis in Rats

Endometriosis, as the leading cause of infertility, is attributed to oxidative stress, inflammation, and autophagy dysregulation. This study was conducted to evaluate the effect of quercetin and metformin, alone or in combination, on the ectopic and eutopic endometrial tissues in a rat model of endometriosis. We divided 60 female rats into 6 groups, including SH, Endo, Endo + Oil, Endo + Q, Endo + M, and Endo + Q + M. The last five groups underwent a surgery, so that we could induce endometriosis, and after 4 weeks, daily treatment began, lasting for a month. Subsequently, the size and histoarchitecture of the endometrial implants, serum levels of 17β-estradiol, progesterone and tumor necrosis factor (TNF)-α, and markers of oxidative stress and autophagy were assessed utilizing ELISA and gene expression analysis. Our results shed light to the fact that serum TNF-α and 17β-estradiol levels significantly increased in endometriosis rats. Moreover, NADPH: quinone oxidoreductase (NQO1) enzyme activity and gene expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and autophagy markers significantly decreased; meanwhile, mammalian target of rapamycin (mTOR) gene expression increased in the ectopic endometrial tissues, as compared with eutopic ones. Surprisingly, our results demonstrated that the treatment in which we applied the combination of quercetin and metformin significantly reversed these changes and had a pronounced effect on the endometrial implant size and gene expression levels of mTOR and autophagy markers in ectopic endometrium. The findings of the present study suggest that quercetin, metformin, and their combination were of potential therapeutic effects on the rat model of endometriosis. Similar content being viewed by others

Zhang B, et al. The ginsenoside PPD exerts anti-endometriosis effects by suppressing estrogen receptor-mediated inhibition of endometrial stromal cell autophagy and NK cell cytotoxicity. Cell Death Dis. 2018;9(5):1–13. Miller JE, Ahn SH, Monsanto SP, Khalaj K, Koti M, Tayade C. Implications of immune dysfunction on endometriosis associated infertility. Oncotarget. 2017;8(4):7138–47. Perrotta AR, et al. The vaginal microbiome as a tool to predict rASRM stage of disease in endometriosis: a pilot study. Reprod Sci. 2020:1–10. Agarwal A, et al. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol. 2012;10(1):1–31. Jamali N, Mostafavi-Pour Z, Zal F, Kasraeian M, Poordast T, Ramezani F, et al. Combination effect of caffeine and caffeic acid treatment on the oxidant status of ectopic endometrial cells separated from patients with endometriosis. Iran J Med Sci. 2019;44(4):315–24. Szczepańska M, Koźlik J, Skrzypczak J, Mikołajczyk M. Oxidative stress may be a piece in the endometriosis puzzle. Fertil Steril. 2003;79(6):1288–93. Wei S, Pei Y, Wang Y, Guan H, Huang Y, Xing T, et al. Role of human Keap1 S53 and S293 residues in modulating the binding of Keap1 to Nrf2. Biochimie. 2019;158:73–81. Marcellin L, et al. Alteration of Nrf2 and glutamate cysteine ligase expression contribute to lesions growth and fibrogenesis in ectopic endometriosis. Free Radic. Biol Med. 2017;110:1–10. Choi J, Jo M, Lee E, Kim HJ, Choi D. Differential induction of autophagy by mTOR is associated with abnormal apoptosis in ovarian endometriotic cysts. Mol Hum Reprod. 2014;20(4):309–17. Ruiz A, Rockfield S, Taran N, Haller E, Engelman RW, Flores I, et al. Effect of hydroxychloroquine and characterization of autophagy in a mouse model of endometriosis. Cell Death Dis. 2016;7(1):e2059–9. Sui X, Li Y, Sun Y, Li C, Li X, Zhang G. Expression and significance of autophagy genes LC3, Beclin1 and MMP-2 in endometriosis. Exp Ther Med. 2018;16(3):1958–62. Honda H, Barrueto FF, Gogusev J, Im DD, Morin PJ. Serial analysis of gene expression reveals differential expression between endometriosis and normal endometrium. Possible roles for AXL and SHC1 in the pathogenesis of endometriosis. Reprod Biol Endocrinol. 2008;6:59. Yagyu T, et al. Activation of mammalian target of rapamycin in postmenopausal ovarian endometriosis. Int J Gynecol Cancer. 2006;16(4):1545–51. PARK YB, HEO GM, MOON HK, CHO SJ, SHIN YC, EOM KS, et al. Pulmonary endometriosis resected by video-assisted thoracoscopic surgery. Respirology. 2006;11(2):221–3. Kiani, K., et al., Medicinal plants and natural compounds in the treatment of experimental endometriosis: a systematic review protocol. Evid Based Preclin Med, 2016. 3(2): p. 1–6. Ashrafizadeh M, Ahmadi Z, Farkhondeh T, Samarghandian S. Autophagy as a molecular target of quercetin underlying its protective effects in human diseases. Arch Physiol Biochem. 2019:1–9. Samare-Najaf M, et al. Stereological and histopathological evaluation of doxorubicin-induced toxicity in female rats’ ovary and uterus and palliative effects of quercetin and vitamin E. Hum Exp Toxicol. 2020:0960327120937329. Wang K, Liu R, Li J, Mao J, Lei Y, Wu J, et al. Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR-and hypoxia-induced factor 1α-mediated signaling. Autophagy. 2011;7(9):966–78. Rotermund C, Machetanz G, Fitzgerald JC. The therapeutic potential of metformin in neurodegenerative diseases. Front Endocrinol. 2018;9:400. Hardie, D.G., Fiona A. Ross, and Simon A. Hawley, AMP-activated protein kinase: a target for drugs both ancient and modern. Chem Biol, 2012. 19(10): p. 1222–1236. Kim YC, Guan K-L. mTOR: a pharmacologic target for autophagy regulation. J Clin Invest. 2015;125(1):25–32. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril. 1985;44(5):684–94. Neisy A, Zal F, Seghatoleslam A, Alaee S. Amelioration by quercetin of insulin resistance and uterine GLUT4 and ERα gene expression in rats with polycystic ovary syndrome (PCOS). Reprod Fertil Dev. 2019;31(2):315–23. Oner G, Ozcelik B, Ozgun MT, Serin IS, Ozturk F, Basbug M. The effects of metformin and letrozole on endometriosis and comparison of the two treatment agents in a rat model. Hum Reprod. 2010;25(4):932–7. Arinç E, et al. Stimulatory effects of benzene on rabbit liver and kidney microsomal cytochrome P-450 dependent drug metabolizing enzymes. Arch. Toxicol. 1991;65(3):186–90. Lowry OH, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–75. Zhan, L., J. Li, and B. Wei, Autophagy in endometriosis: friend or foe? Biochem Biophys Res Commun, 2018. 495(1): p. 60–63. Patel BG, Lenk EE, Lebovic DI, Shu Y, Yu J, Taylor RN. Pathogenesis of endometriosis: interaction between endocrine and inflammatory pathways. Best Pract Res Clin Obstet Gynaecol. 2018;50:50–60. Baboo KD, Chen Z-Y, Zhang X-M. Role of oxidative stress and antioxidant therapies in endometriosis. Reprod Dev Med. 2019;3(3):170. Bina F, Soleymani S, Toliat T, Hajimahmoodi M, Tabarrai M, Abdollahi M, et al. Plant-derived medicines for treatment of endometriosis: a comprehensive review of molecular mechanisms. Pharmacol Res. 2019;139:76–90. Chantalat E, Valera MC, Vaysse C, Noirrit E, Rusidze M, Weyl A, et al. Estrogen receptors and endometriosis. Int J Mol Sci. 2020;21(8):2815. Foda AA, Aal IAA. Metformin as a new therapy for endometriosis, its effects on both clinical picture and cytokines profile. Middle East Fertil Soc J. 2012;17(4):262–7. Hong Y, Yin Y, Tan Y, Hong K, Jiang F, Wang Y. Effect of quercetin on biochemical parameters in letrozoleinduced polycystic ovary syndrome in rats. Trop J Pharm Res. 2018;17(9):1783–8. Yang H, Liu J, Wang M, Wang L, Zhang L, Zhang F. Effect of quercetin on bone metabolism and serum osteocalcin in osteoporotic rats. Trop J Pharm Res. 2020;19(2):277–81. Shahzad H, Giribabu N, Sekaran M, Salleh N. Quercetin induces dose-dependent differential morphological and proliferative changes in rat uteri in the presence and in the absence of estrogen. J Med Food. 2015;18(12):1307–16. Mansfield R, Galea R, Brincat M, Hole D, Mason H. Metformin has direct effects on human ovarian steroidogenesis. Fertil Steril. 2003;79(4):956–62. Takemura Y, Osuga Y, Yoshino O, Hasegawa A, Hirata T, Hirota Y, et al. Metformin suppresses interleukin (IL)-1β-induced IL-8 production, aromatase activation, and proliferation of endometriotic stromal cells. J Clin Endocrinol Metab. 2007;92(8):3213–8. Zheng W, Wu J, Gu J, Weng H, Wang J, Wang T, et al. Modular characteristics and mechanism of action of herbs for endometriosis treatment in Chinese medicine: a data mining and network pharmacology–based identification. Front Pharmacol. 2020;11:147. Karslıoglu T, Karasu AFG, Yildiz P. The effects of micronized progesterone and cabergoline on a rat autotransplantation endometriosis model: a placebo controlled randomized trial. J Investig Surg. 2020:1–5. Abdelkader NF, Eitah HE, Maklad YA, Gamaleldin AA, Badawi MA, Kenawy SA. New combination therapy of gliclazide and quercetin for protection against STZ-induced diabetic rats. Life Sci. 2020;247:117458. Omer, N.A., M.A. Taher, and H.D.S. Aljebory, Effect of metformin treatment on some blood biomarkers in women with endometriosis. Iraq J Pharm Sci (P-ISSN: 1683–3597, E-ISSN: 2521–3512), 2016. 25(1): p. 28–36. Sobočanec S, Šarić A, Mačak Šafranko Ž, Popović Hadžija M, Abramić M, Balog T. The role of 17β-estradiol in the regulation of antioxidant enzymes via the Nrf2–Keap1 pathway in the livers of CBA/H mice. Life Sci. 2015;130:57–65. Darband SG, Sadighparvar S, Yousefi B, Kaviani M, Ghaderi-Pakdel F, Mihanfar A, et al. Quercetin attenuated oxidative DNA damage through NRF2 signaling pathway in rats with DMH induced colon carcinogenesis. Life Sci. 2020;253:117584. Luo X, Cheng W, Wang S, Chen Z, Tan J. Autophagy suppresses invasiveness of endometrial cells through reduction of fascin-1. Biomed Res Int. 2018;2018:1–9. Chen, N. and V. Karantza, Autophagy as a therapeutic target in cancer. Cancer Biol Ther, 2011. 11(2): p. 157–168. Honda H, et al. Serial analysis of gene expression reveals differential expression between endometriosis and normal endometrium. Possible roles for AXL and SHC1 in the pathogenesis of endometriosis. Reprod Biol Endocrinol. 2008;6(1):59. Wang Qq, et al. Treatment with metformin and sorafenib alleviates endometrial hyperplasia in polycystic ovary syndrome by promoting apoptosis via synergically regulating autophagy. Journal of cellular physiology. 2020;235(2):1339–48. Klappan AK, Hones S, Mylonas I, Brüning A. Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity. Histochem Cell Biol. 2012;137(1):25–36. Jia L, Huang S, Yin X, Zan Y, Guo Y, Han L. Quercetin suppresses the mobility of breast cancer by suppressing glycolysis through Akt-mTOR pathway mediated autophagy induction. Life Sci. 2018;208:123–30. Laganà AS, et al. Translational animal models for endometriosis research: a long and windy road. Ann Transl Med. 2018:6(22). Funding The present study was performed as a part of a PhD student thesis by Navid Jamali (Biochemistry Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran), and supported by Grant Number 20441 from the office of Vice Chancellor for Research and the Committee for Advanced Biomedical Sciences, Shiraz University of Medical Sciences. Author information Authors and Affiliations Corresponding authors Ethics declarations All the animal surgeries and procedures were approved by the Institutional Animal Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1398.977) and conducted in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Conflict of Interest The authors declare that they have no conflict of interest. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions About this article Cite this article Jamali, N., Zal, F., Mostafavi-Pour, Z. et al. Ameliorative Effects of Quercetin and Metformin and Their Combination Against Experimental Endometriosis in Rats. Reprod. Sci. 28, 683–692 (2021). https://doi.org/10.1007/s43032-020-00377-2 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-020-00377-2