Eucalyptol attenuates testicular ischemia-reperfusion injury in rats

Document Type : Original Article

Authors

1 Department of Surgery, Faculty of Veterinary Sciences, Hatay Mustafa Kemal University, Hatay, Türkiye

2 Department of Biochemistry, Faculty of Veterinary Sciences, Hatay Mustafa Kemal University, Hatay, Türkiye

3 Department of Pathology, Faculty of Veterinary Sciences, Aksaray University, Aksaray, Türkiye

4 Department of Pathology, Faculty of Veterinary Sciences, Sivas Cumhuriyet University, Sivas, Türkiye

Abstract
The efficacy of eucalyptol on testicular ischemia-reperfusion (I/R) injury remains uncertain. This study aimed to investigate the protective effects of eucalyptol on I/R injury induced by testicular torsion/detorsion (T/D) in rats. A total of 32 rats were divided into four groups, including control, T/D, E100 (T/D + 100 mg kg-1 eucalyptol administered 30 min prior to torsion), and E200 (T/D + 200 mg kg-1 eucalyptol administered 30 min prior to torsion). Testicular T/D was induced in the left testis via a 720° clockwise torsion for 3 hr, followed by 3 hr of detorsion. Testicular tissues were harvested for histopathological, immunohistochemical including cleaved caspase-3, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and proliferating cell nuclear antigen (PCNA), and biochemical parameter as glutathione peroxidase (GPx), reduced glutathione (rGSH), glucose-6-phosphate dehydrogenase (G6PD), vitamin C, and malondialdehyde (MDA) analyses. In the T/D group, significant reductions in GPx, rGSH, G6PD, and vitamin C levels were observed, alongside increased MDA levels compared to the control group. Immuno-histochemically, the T/D group exhibited increased expression of cleaved caspase-3 and Bax, along with decreased expression of Bcl-2 and PCNA, compared to the control group. Histologically, Johnsen and Cosentino scores were irregular in the T/D group. Pre-treatment with eucalyptol resulted in reduced MDA, cleaved caspase-3, and Bax levels, while GPx, rGSH, vitamin C, G6PD, PCNA, and Bcl-2 levels increased. Additionally, improvements in Cosentino and Johnsen scores and histopathological damage were observed. These findings suggest that eucalyptol may exert a protective effect against I/R injury caused by testicular T/D, likely due to its anti-oxidant and anti-apoptotic properties.

Keywords

Subjects


1.     Abdel-Gaber SA, Mohammed RK, Refaie MMM. Mechanism mediating the protective effect of diacerein in ischemia-reperfusion-induced testicular injury in rats. Life Sci 2018; 209: 57-62.
2.     Mohamed MZ, Morsy MA, Mohamed HH, et al. Paeonol protects against testicular ischaemia‐reperfusion injury in rats through inhibition of oxidative stress and inflammation. Andrologia 2020; 52(6): e13599. doi: 10.1111/and.13599.
3.     Wei SM, Yan ZZ, Zhou J. Protective effect of rutin on testicular ischemia-reperfusion injury. J Pediat Surg 2011; 46(7): 1419-1424.
4.     Arena S, Iacona R, Antonuccio P, et al. Medical perspective in testicular ischemiareperfusion injury. Exp Ther Med 2017; 13(5): 2115-2122.
5.     Akhigbe RE, Odetayo AF, Akhigbe TM, et al. Patho-physiology and management of testicular ischemia/ reperfusion injury: lessons from animal models. Heliyon 2024; 10(9): e27760. doi: 10.1016/j.heliyon. 2024.e27760.
6.     Akhigbe RE, Hamed MA, Odetayo AF, et al. Omega-3 fatty acid rescues ischaemia/perfusion-induced testicular and sperm damage via modulation of lactate transport and xanthine oxidase/uric acid signaling. Biomed Pharmacother 2021; 142: 111975. doi: 10.1016/j.biopha.2021.111975. 
7.     Lian BS, Ong CC, Chiang LW, et al. Factors predicting testicular atrophy after testicular salvage following torsion. Eur J Pediatr Sur 2016; 26(1): 17-21.
8.     Abdel-Kader MS, Abdel-Rahman RF, Althurwi HN, et al. Samarcandin protects against testicular ischemia/ reperfusion injury in rats via activation of Nrf2/HO-1-mediated antioxidant responses. Saudi Pharm J 2023; 31(7): 1186-1196.
9.     Kazak F, Akcakavak G, Alakus I, et al. Proanthocyanidin alleviates testicular torsion/detorsion-induced ischemia/reperfusion injury in rats. Tissue Cell 2024; 89: 102459. doi: 10.1016/j.tice.2024.102459.
10. Oyedokun PA, Akhigbe RE, Ajayi LO, et al. Impact of hypoxia on male reproductive functions. Mol cell Biochem 2023; 478(4): 875-885.
11. Abbaszadeh A, Assadollahi V, Alasvand M, et al. Protective effects of royal jelly on testicular torsion induced ischaemia reperfusion injury in rats. Andrologia 2020; 52(9): e13716. doi: 10.1111/ and.13716.
12. Hoch CC, Petry J, Griesbaum L, et al. 1, 8-cineole (eucalyptol): a versatile phytochemical with therapeutic applications across multiple diseases. Biomed Pharmacother 2023; 167: 115467. doi: 10.1016/j.biopha.2023.115467.
13. Cai ZM, Peng JQ, Chen Y, et al. 1, 8-Cineole: a review of source, biological activities, and application. J Asian Nat Prod Res 2021; 23(10): 938-954.
14. Akcakavak G, Kazak F, Karatas O, et al. Eucalyptol regulates Nrf2 and NF-kB signaling and alleviates gentamicin-induced kidney injury in rats by downregulating oxidative stress, oxidative DNA damage, inflammation, and apoptosis. Toxicol Mech Methods 2024; 34(4): 413-422.
15. Akcakavak G, Kazak F, Yilmaz Deveci MZ. Eucalyptol protects against cisplatin-induced liver injury in rats. Biol Bull Russ Acad Sci 2023; 50(5): 987-994.
16. Kazak F, Deveci MZY, Akçakavak G. Eucalyptol alleviates cisplatin-induced kidney damage in rats. Drug Chem Toxicol 2024; 47(2): 172-179.
17. Bezerra DS, de Araujo Delmondes GA, Lopes MJP, et al. Eucalyptol prevents pilocarpine-induced seizure and neuronal damage in mice, through the cholinergic, monoaminergic and antioxidant pathways. Food Biosci 2023; 53: 102824. doi: 10.1016/j.fbio.2023.102824.
18. Ganjiani V, Ahmadi N, Raayat Jahromi A. Protective effects of Stevia rebaudiana aqueous extract on experimental unilateral testicular ischaemia/ reperfusion injury in rats. Andrologia 2020; 52(2): e13469. doi: 10.1111/and.13469. 
19. Unsal V, Kolukcu E, Gevrek F, et al. Sinapic acid reduces ischemia/reperfusion injury due to testicular torsion/detorsion in rats. Andrologia 2021; 53(8): e14117. doi: 10.1111/and.14117. 
20. Akcakavak G, Karataş Ö, Tuzcu N, et al. Determination of apoptosis, necroptosis and autophagy markers by real-time PCR in naturally infected pneumonic pasteurellosis caused by pasteurella multocida and mannheimia haemolytica in cattle. Pak Vet J 2024; 44(2): 483-489.
21. Johnsen SG. Testicular biopsy score count - - a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1970; 1(1): 2-25.
22. Cosentino MJ, Nishida M, Rabinowitz R, et al. Histopathology of prepubertal rat testes subjected to various durations of spermatic cord torsion. J Androl 1986; 7(1): 23-31.
23. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95(2): 351-358.
24. Haag W. Methodology and practical importance of the determination of ascorbic acid in cattle in the past and present, Giessen, Germany: Justus Liebig University 1985; 196.
25. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82(1): 70-77.
26. Beutler E. A manual of biochemical methods. 2nd ed. New York, USA: Grune & Stratton 1975; 112-114.
27. Beutler E. Red cell metabolism manual of biochemical methods. 12th ed. London, UK: Academic Press 1971; 68-70.
28. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193(1): 265-275.
29. Meng C, Zeng W, Lv J, et al. 1,8-cineole ameliorates ischaemic brain damage via TRPC6/CREB pathways in rats. J Pharm Pharmacol 2021; 73(7): 979-985.
30. Elshaari FA, Elfagih RI, Sheriff DS, et al. Oxidative and antioxidative defense system in testicular torsion/detorsion. Indian J Urol 2011; 27(4): 479-484.
31. Lee JW, Kim JI, Lee YA, et al. Inhaled hydrogen gas therapy for prevention of testicular ischemia/ reperfusion injury in rats. J Pediatr Surg 2012; 47(4): 736-742.
32. Aboubakr M, Elbadawy M, Ibrahim SS, et al. Allicin and lycopene possesses a protective effect against methotrexate-induced testicular toxicity in rats. Pak Vet J 2023; 43(3): 559-566.
33. Sangodele JO, Inuwa Z, Lawal B, et al. Proxeed plus salvage rat testis from ischemia-reperfused injury by enhancing antioxidant’s activities and inhibition of iNOS expression. Biomed Pharmacother 2021; 133: 111086. doi: 10.1016/j.biopha.2020.111086. 
34. Davoodi F, Taheri S, Raisi A, et al. Leech therapy (Hirudo medicinalis) attenuates testicular damages induced by testicular ischemia/reperfusion in an animal model. BMC Vet Res 2021; 17: 256. Doi: 10.1186/s12917-021-02951-5.
35. Karaguzel E, Kadihasanoglu M, Kutlu O. Mechanisms of testicular torsion and potential protective agents. Nat Rev Urol 2014; 11(7): 391-399.
36. Jafari A, Ghasemnejad-Berenji H, Nemati M, et al. Beneficial effects of memantine on ischemia/re-perfusion injury following torsion/detorsion induced testicular damage in rats: improvement in histological and biochemical parameters. J Pediatr Urol 2021; 17 (4): 441. e1-441-e7. doi: 10.1016/j.jpurol.2021.04.014.
37. Taheri S, Davoodi F, Raisi A, et al. Co‐administration of Salvia miltiorrhiza and verapamil inhibits detrimental effects of torsion/detorsion on testicular tissue in rats. Andrologia 2021; 53(6): e14049. doi: 10.1111/ and.14049.
38. Abu-Baih RH, Abu-Baih DH, Abdel-Hafez SMN, et al. Activation of SIRT1/Nrf2/HO-1 and Beclin-1/AMPK/mTOR autophagy pathways by eprosartan ameliorates testicular dysfunction induced by testicular torsion in rats. Sci Rep 2024; 14(1): 12566. doi: 10.1038/s41598-024-62740-6.
39. Rotimi DE, Ojo OA, Olaolu TD, et al. Exploring Nrf2 as a therapeutic target in testicular dysfunction. Cell Tissue Res 2022; 390(1): 23-33.
40. Xu G, Guo J, Sun C. Eucalyptol ameliorates early brain injury after subarachnoid haemorrhage via antioxidant and anti-inflammatory effects in a rat model. Pharm Biol 2021; 59(1): 114-120.
41. Shokoohi M, Shoorei H, Soltani M, et al. Protective effects of the hydroalcoholic extract of Fumaria parviflora on testicular injury induced by torsion/ detorsion in adult rats. Andrologia 2018; 50(7): e13047. doi: 10.1111/and.13047.
42. Sahoo G, Samal D, Khandayataray P, et al. A review on caspases: key regulators of biological activities and apoptosis. Mol Neurobiol 2023; 60(10): 5805-5837.
43. Azarabadi M, Heidari F, Khaki AA, et al. Minocycline attenuates testicular damages in a rat model of ischaemia/reperfusion (I/R) injury. Andrologia 2020; 52(9): e13704. doi: 10.1111/and.13704.
44. Al-Maghrebi M, Renno WM, Al-Ajmi N. Epigallocatechin-3-gallate inhibits apoptosis and protects testicular seminiferous tubules from ischemia/reperfusion-induced inflammation. Biochem Biophys Res Commun 2012; 420(2): 434-439.
45. Kim DY, Kang MK, Lee EJ, et al. Eucalyptol inhibits amyloid-β-induced barrier dysfunction in glucose-exposed retinal pigment epithelial cells and diabetic eyes. Antioxidants (Basel) 2020; 9(10): 1000. doi: 10.3390/antiox9101000.
46. Wang Y, Zhang X, Fu Y, et al. 1, 8-cineole protects against ISO-induced heart failure by inhibiting oxidative stress and ER stress in vitro and in vivo. Eur J Pharmacol 2021; 910: 174472. doi: 10.1016/j.ejphar. 2021.174472.
47. Sun W, Liu H, Zhu H, et al. Eucalyptol antagonized the apoptosis and immune dysfunction of grass carp hepatocytes induced by tetrabromobisphenol A by regulating ROS/ASK1/JNK pathway. Environ Toxicol 2023; 38(4): 820-832.
48. Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 2003; 116(Pt 15): 3051-3060.
49. Chandorkar N, Tambe S, Amin P, et al. A systematic and comprehensive review on current understanding of the pharmacological actions, molecular mechanisms, and clinical implications of the genus Eucalyptus. Phytomed Plus 2021; 1(4): 100089. doi: 10.1016/ j.phyplu.2021.100089.
50. Thaker H, Nelson CP. Adjuvant pharmacological and surgical therapy for testicular torsion: current state of the art. J Pediatr Urol 2020; 16(6): 807-814.
Volume 16, Issue 11
November 2025
Pages 611-620

  • Receive Date 30 December 2024
  • Revise Date 17 February 2025
  • Accept Date 22 April 2025