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Curcumin attenuates ischemia–reperfusion injury in rat testis
Curcumin attenuates ischemia–reperfusion injury in rat testis Si-Ming Wei, M.D., Ph.D.,a,d Zhi-Zhong Yan, M.S.,b and Jian Zhou, M.D.c a Department of Urology, Third Affiliated Hospital of Hangzhou City, Zhejiang Chinese Medical University, b Department of Reproductive Medicine, Red Cross Hospital of Hangzhou City, and c Department of Surgery, First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou City, Zhejiang Province, China; and d Department of Urology and Laboratory of Experimental Immunology, Campus Gasthuisberg (O & N), Catholic University of Leuven, Leuven, Belgium
Objective: To evaluate the effect of curcumin, a potent antioxidant, on testicular ischemia–reperfusion injury caused by overgeneration of reactive oxygen species (ROS) after testicular torsion–detorsion. Design: Controlled experimental study using rats. Setting: Research laboratory. Animal(s): Sixty adult male Sprague-Dawley rats. Intervention(s): Rats in the control group underwent a sham operation of the left testis. In the torsion–detorsion group, the left testis was rotated 720 for 2 hours. Rats in treatment group received the same surgical procedure as the torsion–detorsion group, but curcumin was administered IV at repair of testicular torsion. Main Outcome Measure(s): Testicular activity of xanthine oxidase, which catalyzes production of ROS; malondialdehyde level (an indicator of ROS content); protein expression level of heme oxygenase-1, which catalyzes antioxidant generation; and spermatogenesis. Result(s): Unilateral testicular torsion–detorsion caused significant increases in xanthine oxidase activity, malondialdehyde level, and heme oxygenase-1 protein expression level and caused a significant decrease in testicular spermatogenesis in ipsilateral testes. The rats treated with curcumin had significant decreases in xanthine oxidase activity and malondialdehyde level and had significant increases in heme oxygenase-1 protein expression level and testicular spermatogenesis in ipsilateral testes, compared with the torsion–detorsion group. Conclusion(s): The curcumin exerts a protective effect on testicular ischemia–reperfusion injury. (Fertil Steril 2009;91:271–7. 2009 by American Society for Reproductive Medicine.) Key Words: Curcumin, ischemia–reperfusion injury, testicular torsion
Testicular torsion or, more properly, torsion of the spermatic cord, is a urologic emergency. Its incidence has been estimated to be 1 in 4,000 in males by the age of 25 years (1). Testicular salvage rates with surgical detorsion have been reported to range from 42% to 88% (2, 3), but whether these testes truly were saved with respect to testicular spermatogenic function remains in question. Studies in human beings have shown an ipsilateral testicular atrophy rate of 33%–68% after testicular rescue (4, 5). Animal studies have found a significant decrease in ipsilateral testicular spermatogenesis after testicular salvage (6, 7). It appears that the main pathophysiology of testicular torsion–detorsion is ischemia–reperfusion injury of testis that is caused by the twisted spermatic cord and its release. In the ischemia-reperfusion injury, overgeneration of reactive oxygen species (ROS) is thought to play a critical role in the loss of ipsilateral testicular spermatogenesis (8). Reactive oxygen species can cause tissue damage through cell membrane lipid peroxidation, protein denaturation, and DNA impairment (9, 10).
It shows a wide range of pharmacological activities, including antioxidant and anti-inflammatory effects (11). Curcumin long has been used in Asian medicine for its medical properties (12). It has been reported that administration of curcumin can ameliorate ischemia–reperfusion injury in myocardium (13), liver (14), kidney (15), brain (16), and other organ systems. To our knowledge, no study has investigated the role of curcumin in testicular ischemia–reperfusion injury. In the present study, we evaluated the effect of curcumin on testicular spermatogenesis in a rat testicular ischemia–reperfusion injury model.
Curcumin is the major constituent of turmeric powder, which is extracted from rhizomes of plant Curcuma longa.
MATERIALS AND METHODS Animals and Surgical Procedure The 60 adult male Sprague-Dawley rats (250–300 g) were maintained on a 12:12-hour light–dark cycle with food and water ad libitum. All animal experiments followed a protocol that was approved by the ethics committee on animal research at our university. We followed appropriate care and the guidelines for experimental animals, as published by the US National Institutes of Health Board of Registry.
Received October 5, 2007; revised and accepted October 30, 2007. Reprint requests: Si-Ming Wei, M.D., Ph.D., Department of Urology and Laboratory of Experimental Immunology, Campus Gasthuisberg (O & N), Catholic University of Leuven, Herestraat 49, B-3000, Leuven, Belgium (E-mail: [email protected]).
Rats were randomly divided into three groups, each containing 20 rats. They were anesthetized with ketamine injection (50 mg/kg, IP). All operations were performed under sterile conditions. In the control group, the left testis was
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.10.082
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brought out through a left-sided ilioinguinal incision. An 11– 0 atraumatic silk suture was placed through the tunica albuginea, the left testis was replaced into the scrotum, and the incision was closed. In the torsion–detorsion group, the left testis was exposed through the same incision. The left testis was rotated 720 in a counterclockwise direction and was maintained in this torsion position by being fixed to the scrotum with 11–0 silk suture. The incision was sutured. After 2 hours of torsion, the incision was reopened, and the testis was counter-rotated to the natural position. The testis still was viable for restoration of blood flow and was reinserted into the scrotum. In the treatment group, the same surgical procedure was performed as in the torsion–detorsion group, but curcumin (200 mg/kg; Sigma Chemical Company, St. Louis, MO) was injected IV into the rat via the tail vein at the time of detorsion. Bilateral orchiectomy was performed on half of the rats in each experimental group at 4 hours after the repair of torsion for measurements of malondialdehyde level, xanthine oxidase activity, and heme oxygenase-1 protein expression level. Orchiectomy was performed on the remaining rats at 3 months after the repair of torsion, to allow analysis of testicular spermatogenesis.
Evaluation of Testicular Spermatogenesis Testicular spermatogenesis was evaluated by measuring testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score. Testis was excised and weighed. The testicular tissue was fixed in Bouin’s solution, postfixed in 70% alcohol, and embedded in paraffin block. A 5-mm section was obtained, deparaffinized, and stained with hematoxylin and eosin. The light-microscope histological evaluation was performed by an observer in a blind, randomly numbered fashion, without the observer having any knowledge of which testis was experimentally torsional or nontorsional. Mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score were used to evaluate the 20 roundest seminiferous tubules of each sample. Mean seminiferous tubular diameter was measured by using a microscope-adaptable micrometer. Number of germ cell layers in each tubule was determined by counting the numbers of germ cell layers from the basement membrane to the lumen at 90 , 180 , 270 , and 360 and averaging these numbers. The mean testicular biopsy score was graded by using Johnsen’s score (20). A score of 1 to 10 was given to each tubule, according to the maturity of the germ cells.
Measurement of Malondialdehyde Level Malondialdehyde in testis was measured by using the thiobarbituric acid–reactive substance assay, as described by Ohkawa et al. (17). The principle of the method is based on the measurement of the concentration of pink chromogen compound that forms when malondialdehyde reacts with thiobarbituric acid. Malondialdehyde levels were expressed as nanomoles per milligram of protein.
Statistical Analysis Data were expressed as mean SD. Data among groups were compared by one-way analysis of variance, followed by the Student-Newman-Keuls test. Within groups, ipsilateral and contralateral data were compared by using Student’s t-test. P<.05 was considered statistically significant. GraphPad Prism software (GraphPad, San Diego, CA) was used for data analysis.
Xanthine Oxidase Activity Determination Xanthine oxidase activity was assayed spectrophotometrically at 293 nm, with xanthine as substrate (18). The formation of uric acid from xanthine results in an increase in absorbency. One unit of activity was defined as 1 mmol of uric acid formed per minute, at pH 7.5. Xanthine oxidase activity was expressed as international units per gram of protein. Detection of Heme Oxygenase-1 Protein Expression Level by Western Blot Protein extraction and concentration measurement were performed as described elsewhere (19). Proteins were separated by electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked with 5% nonfat milk and subsequently was incubated with anti–heme oxygenase-1 antibody (Stressgen) or anti–b-actin antibody (Sigma) overnight. Then, membrane was incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. Protein bands were visualized by using enhanced chemiluminescence detecting reagent. The density of each band was quantified. The ratio of density of the heme oxygenase-1 band to that of the internal control b-actin band from the same sample represented the relative expression level of heme oxygenase-1 protein. 272
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RESULTS Malondialdehyde Level and Xanthine Oxidase Activity Figure 1A and B show malondialdehyde level and xanthine oxidase activity in both ipsilateral and contralateral testes in control, torsion–detorsion, and treatment groups at 4 hours after detorsion. Malondialdehyde level and xanthine oxidase activity in ipsilateral testes were significantly increased in the torsion–detorsion group, as compared with in the control group (P<.05). The two parameters in ipsilateral testes in treatment group were significantly lower than those in the torsion–detorsion group (P<.05). These parameters of contralateral testes did not reveal any statistically significant difference among the three groups (P>.05). Protein Expression Level of Heme Oxygenase-1 Figure 2A and B display heme oxygenase-1 protein expression level in testes in the three groups at 4 hours after detorsion. Heme oxygenase-1 expression levels in ipsilateral testes of the torsion–detorsion group were significantly higher than those in the control group (P<.05). The parameter levels in ipsilateral testes of treatment group were significantly higher than those in the torsion–detorsion group (P<.05). No significant difference was detected in the heme oxygenase-1 expression level of the contralateral testes among the three groups (P>.05).
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FIGURE 1 Malondialdehyde level (A) and xanthine oxidase activity (B) in rat testicular tissue in control, torsion–detorsion (T/ D), and treatment groups 4 hours after detorsion. Hatched bars show the ipsilateral testes; open bars show the contralateral testes. *Value significantly different from that of control group (P< .05). #Value significantly different from that of contralateral testis in the same group (P< .05). xValue significantly different from that of ipsilateral testis in the T/ D group (P< .05).
Wei. Effect of curcumin on testicular torsion injury. Fertil Steril 2009.
Testicular Spermatogenesis Figures 3 and 4 show testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score in the control, torsion–detorsion, and treatment groups 3 months after detorsion. Compared with the control group, testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score in the ipsilateral testes that was obtained from the torsion–detorsion group were significantly lower (P<.05). The four parameters of ipsilateral testes in the treatment group were significantly higher than those in the torsion–detorsion group (P<.05). None of these parameters of contralateral testes revealed any significant difference among the three groups (P>.05). DISCUSSION Testicular torsion–detorsion is an ischemia–reperfusion injury for the testis. The ischemia–reperfusion injury is associated with overgeneration of ROS, such as superoxide anions, hydrogen peroxide, hydroxyl radicals, and nitric oxide. One important source of ROS generation is the hypoxanthine–xanthine oxidase reaction in parenchymal cells of the postischemic tissue (21). Xanthine oxidase is an important Fertility and Sterility
prerequisite factor in the process of superoxide anion and hydrogen peroxide production. Intracellular calcium concentration rises during ischemia because of calcium influx, leading to proteolytic conversion of xanthine dehydrogenase to xanthine oxidase (7). Furthermore, ischemia also causes an increase in intracellular hypoxanthine as a result of adenosine triphosphate breakdown (10). When oxygen is supplied during reperfusion, xanthine oxidase converts hypoxanthine to superoxide anion and hydrogen peroxide (22). Superoxide anions may react with hydrogen peroxide to form hydroxyl radicals (22). The ROS are difficult to quantify directly in tissue because of their high reactivity and short half-life. Malondialdehyde, a stable end product of lipid peroxidation generated by ROS, usually is used as an indirect indicator of ROS (8). In our study, an elevated level of malondialdehyde in ipsilateral testes of the torsion–detorsion group indicates increased oxidative stress. In addition, xanthine oxidase activity in ipsilateral testes also was enhanced in the torsion–detorsion group. The rise in enzyme activity provides an explanation for ROS overgeneration in testicular tissue. During ischemia–reperfusion, increased production of ROS inflicts significant injury on ischemic tissue through oxidization of cell membrane lipids, proteins, and DNA (9, 273
FIGURE 2 The protein expression level of heme oxygenase-1 in rat testes in control, torsion–detorsion, and treatment groups 4 hours after detorsion. (A) Representative Western blot result of heme oxygenase-1. Lanes 1L and 1R ¼ left (i.e., ipsilateral) and right (i.e., contralateral) testes in the control group; lanes 2L and 2R ¼ ipsilateral and contralateral testes in the torsion–detorsion group; lanes 3L and 3R ¼ ipsilateral and contralateral testes in the treatment group. (B) Quantitative analysis of heme oxygenase-1 Western blot data. Hatched bars show the ipsilateral testes; open bars show the contralateral testes. *Value significantly different from that of control group (P< .05). #Value significantly different from that of contralateral testis in the same group (P< .05). xValue significantly different from that of ipsilateral testis in torsion– detorsion (T / D) group (P< .05).
Wei. Effect of curcumin on testicular torsion injury. Fertil Steril 2009.
10). The present study showed that unilateral testicular torsion–detorsion caused a significant increase in testicular malondialdehyde level (an indicator of ROS content) and caused a significant decrease in spermatogenesis in the ipsilateral testes, suggesting that testicular spermatogenesis was severely injured by ROS. Testicular lesions were characterized by decreases in testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score. The elimination of ROS has been shown to be beneficial in treating several diseases relevant to ischemia–reperfusion injury, such as myocardial infarction (13), renal transplantation (15), and cerebral stroke (16). Curcumin is a powerful scavenger of ROS such as superoxide anions, hydroxyl radicals, and nitric oxide (23, 24). Curcumin treatment successfully has been used to decrease ischemia–reperfusion injury in multiple organ systems, including heart (13), liver (14), kidney (15), and brain (16). This success led us to attempt such treatment in a model of testicular torsion–detorsion. In the present study, we found 274
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that rats treated with curcumin had a significant reduction in malondialdehyde level and had a significant increase in spermatogenesis in ipsilateral testes, as compared with the torsion–detorsion group. In addition, curcumin has achieved beneficial effects on renal ischemia–reperfusion injury in a clinical trial (15). It did not have side effects in those patients who were receiving renal transplantation (15). Therefore, it is reasonable to suggest that clinical application of curcumin may be a useful new approach for therapy of testicular torsion, as an addition to conventional detorsion. Testicular torsion is an ischemic process for testis. Blood cannot flow into testis during torsion. If curcumin is injected into rats through their tail vein at the time of testicular torsion, that is, before detorsion, it will not flow into the testis to exert its beneficial effect. Testicular detorsion is a reperfusion process of blood. Consequently, we injected curcumin at the time of detorsion. Some other reports have indicated that curcumin at the dosage of 200 mg/kg is effective in treating oxidative stress–mediated injury in rat forebrain (16), heart (13), liver (25), and so on. Therefore, we chose this dosage in our rat experiment. One strategy to reduce the effect of ROS involves their elimination through the use of ROS scavengers. Reactive oxygen species scavengers, such as superoxide dismutase and catalase, can convert toxic ROS into water and oxygen (10). Although the treatment with superoxide dismutase and catalase had a protective effect in 1 hour of testicular torsion, no significant rescue was observed in 2 hours of testicular torsion (10). In contrast, we found that administration of curcumin provided a significant rescue of testicular spermatogenesis after 2 hours of testicular torsion. The observations could not be explained by a simple scavenging ROS effect of curcumin. Our study showed yet that curcumin may significantly reduce xanthine oxidase activity and malondialdehyde level in ipsilateral testes. These results suggest that curcumin may reduce ROS production by inhibiting xanthine oxidase. As a result, we believe that curcumin may provide an additional protective effect by diminishing ROS generation. Heme oxygenase-1 is a widely distributed enzyme in mammalian tissues and plays an important cytoprotective role against oxidative stress (26). Its main function is associated with the degradation of heme to iron, carbon monoxide, and biliverdin, the latter being converted to bilirubin by the cytosolic enzyme biliverdin reductase (27). Both biliverdin and bilirubin possess antioxidant properties (28). Ample evidence supports the notion that up-regulation of heme oxygenase-1 expression provides potent cytoprotective effects in many in vitro and in vivo models of oxidative stress–induced cellular and tissue injury (15, 19, 28–30). Many recent studies have revealed that curcumin potently induces heme oxygenase-1 expression, such as in cardiac myoblasts (29), hepatocytes (30), and monocytes (28), leading to increased resistance to oxidative stress–mediated damage. However, its effect on testicular germ cells has not been examined previously. In this study, we explored whether curcumin treatment could increase protein
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FIGURE 3 Rat testicular weight (A), mean seminiferous tubular diameter (B), number of germ cell layers (C), and mean testicular biopsy score (D) in control, torsion–detorsion (T/D), and treatment groups, 3 months after detorsion. Hatched bars show the ipsilateral testes; open bars show the contralateral testes. *Value significantly different from that of the control group (P< .05). #Value significantly different from that of contralateral testis in the same group (P< .05). xValue significantly different from that of ipsilateral testis in the T/D group (P< .05).
Wei. Effect of curcumin on testicular torsion injury. Fertil Steril 2009.
expression of heme oxygenase-1 in rat testes after ischemia– reperfusion. The present study showed that heme oxygenase1 expression levels in ipsilateral testes harvested from the torsion–detorsion group were significantly higher than those in the control group. It is suggested that oxidative stress–induced increase in heme oxygenase-1 expression has only a regulatory rather than a therapeutic role in attenuating the deleterious effects of oxidative stress (31). The heme oxygenase-1 expression levels in ipsilateral testes of the curcumin treatment group increased significantly, as compared with the torsion–detorsion group. In addition, curcumin treatment also attenuated testicular injury and reduced malondialdehyde level in ipsilateral testes in comparison with the torsion–detorsion group. These results suggest that induction of heme oxygenase-1 expression after curcumin treatment may protect testes against ischemia–reperfusion injury by antioxidation. Therefore, induction of heme oxygenase-1 expression is an important mechanism of curcumin against testicular ischemia–reperfusion injury. Treatment with curcumin (200 mg/kg) significantly rescued ipsilateral testicular spermatogenesis in our experiment, but the saved spermatogenesis was not restored to normal value. In the current study, we did not investigate the effect Fertility and Sterility
of curcumin on testicular ischemia–reperfusion injury at different doses, or at different administration times. Because these factors may affect the effect of curcumin, further studies are required to elucidate answers to these questions so that curcumin can be used to optimal effect. There still is controversy about whether unilateral testicular torsion affects the contralateral testis. Some investigators have found that unilateral testicular torsion causes contralateral testicular damage (32, 33), whereas other investigators have not (34, 35). In our study, although unilateral testicular torsion resulted in significant changes in ipsilateral testicular malondialdehyde level, xanthine oxidase activity, heme oxygenase-1 expression level, and spermatogenesis, our results did not show any change in the contralateral testes. As a result, we believe that unilateral testicular torsion does not lead to contralateral injury. In conclusion, we have demonstrated the protective effect of curcumin on testicular ischemia–reperfusion injury for the first time. We propose that curcumin may be a novel approach for therapy of testicular ischemia–reperfusion injury. The protective mechanism of curcumin may be explained as follows. Curcumin can scavenge ROS. In addition, it reduces 275
FIGURE 4 Testicular histology 3 months after detorsion. (A) The bilateral testes in control group and the contralateral testes in torsion–detorsion and treatment groups all indicated normal seminiferous tubular diameter, number of germ cell layers, and spermatogenesis from spermatogonium in the basement membrane to spermatozoon in the center of tubule. (B) The ipsilateral testes in torsion–detorsion group showed seminiferous tubular atrophy, reduction of germ cell layer number, and spermatogenous arrest. (C) The ipsilateral testes in the treatment group displayed normality of most seminiferous tubules and abnormality of a few tubules (arrows). All panels, hematoxylin and eosin staining (original magnification, 100 in panels A and B and 50 in panel C).
Wei. Effect of curcumin on testicular torsion injury. Fertil Steril 2009.
ROS formation by inhibiting xanthine oxidase. Curcumin is a potent inducer of heme oxygenase-1 in testes. The increased heme oxygenase-1 expression is an important element in curcumin-mediated cytoprotection against oxidative stress. All these together lead to spermatogenous protection in ischemia–reperfusion for testis that is treated with curcumin. REFERENCES 1. Barada JH, Weingarten JL, Cromie WJ. Testicular salvage and age-related delay in the presentation of testicular torsion. J Urol 1989;142: 746–8. 2. Parker RM, Robison JR. Anatomy and diagnosis of torsion of the testicle. J Urol 1971;106:243–7. 3. Cattolica EV, Karol JB, Rankin KN, Klein RS. High testicular salvage rate in torsion of the spermatic cord. J Urol 1982;128:66–8. 4. Macnicol MF. Torsion of the testis in childhood. Br J Surg 1974;61: 905–8. 5. Krarup T. The testes after torsion. Br J Urol 1978;50:43–6.
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6. Ozturk H, Ozturk H, Yagmur Y. PAF antagonist BN-52021 reduces intercellular adhesion molecule-1 expression and oxidative stress in rats with reperfusion damage due to unilateral testicular torsion. Pediatr Surg Int 2006;22:191–6. 7. Cay A, Alver A, Kucuk M, Isik O, Eminagaoglu MS, Karahan SC, et al. The effects of N-acetylcysteine on antioxidant enzyme activities in experimental testicular torsion. J Surg Res 2006;131:199–203. 8. Turner TT, Tung KS, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997;57:1267–74. 9. Filho DW, Torres MA, Bordin AL, Crezcynski-Pasa TB, Boveris A. Spermatic cord torsion, reactive oxygen and nitrogen species and ischemia-reperfusion injury. Mol Aspects Med 2004;25:199–210. 10. Prillaman HM, Turner TT. Rescue of testicular function after acute experimental torsion. J Urol 1997;157:340–5. 11. Jagetia GC. Radioprotection and radiosensitization by curcumin. Adv Exp Med Biol 2007;595:301–20. 12. Ammon HP, Wahl MA. Pharmacology of Curcuma longa. Planta Med 1991;57:1–7. 13. Nirmala C, Puvanakrishnan R. Protective role of curcumin against isoproterenol induced myocardial infarction in rats. Mol Cell Biochem 1996;159:85–93.
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14. Shen SQ, Zhang Y, Xiang JJ, Xiong CL. Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes. World J Gastroenterol 2007;13:1953–61. 15. Shoskes D, Lapierre C, Cruz-Correa M, Muruve N, Rosario R, Fromkin B, et al. Beneficial effects of the bioflavonoids curcumin and quercetin on early function in cadaveric renal transplantation: a randomized placebo controlled trial. Transplantation 2005;80:1556–9. 16. Ghoneim AI, Abdel-Naim AB, Khalifa AE, El-Denshary ES. Protective effects of curcumin against ischaemia/reperfusion insult in rat forebrain. Pharmacol Res 2002;46:273–9. 17. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. 18. Prajda N, Weber G. Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas. FEBS Lett 1975;59: 245–9. 19. Yang S, Shih HJ, Chow YC, Tsai PS, Wang TY, Wang PS, et al. The protective role of heme oxygenase-1 induction on testicular tissues after testicular torsion and detorsion. J Urol 2007;177:1928–33. 20. 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;1:2–25. 21. Mccord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159–63. 22. Gilgun-Sherki Y, Melamed E, Offen D. The role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy. J Neurol 2004;251:261–8. 23. Zhao BL, Li XJ, He RG, Cheng SJ, Xin WJ. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell Biophys 1989;14:175–85. 24. Li JK, Lin-Shia SY. Mechanisms of cancer chemoprevention by curcumin. Proc Natl Sci Counc Repub China B 2001;25:59–66. 25. Reyes-Gordillo K, Segovia J, Shibayama M, Vergara P, Moreno MG, Muriel P. Curcumin protects against acute liver damage in the rat by in-
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26. 27. 28.
29.
30.
31.
32.
33.
34.
35.
hibiting NF-kappaB, proinflammatory cytokines production and oxidative stress. Biochim Biophys Acta 2007;1770:989–96. Shiraishi K, Naito K. Increased expression of Leydig cell haem oxygenase1 preserves spermatogenesis in varicocele. Hum Reprod 2005;20:2608–13. Kikuchi G, Yoshida T, Noguchi M. Heme oxygenase and heme degradation. Biochem Biophys Res Commun 2005;338:558–67. Rushworth SA, Ogborne RM, Charalambos CA, O’Connell MA. Role of protein kinase C delta in curcumin-induced antioxidant response element-mediated gene expression in human monocytes. Biochem Biophys Res Commun 2006;341:1007–16. Abuarqoub H, Green CJ, Foresti R, Motterlini R. Curcumin reduces cold storage-induced damage in human cardiac myoblasts. Exp Mol Med 2007;39:139–48. Mcnally SJ, Harrison EM, Ross JA, Garden OJ, Wigmore SJ. Curcumin induces heme oxygenase-1 in hepatocytes and is protective in simulated cold preservation and warm reperfusion injury. Transplantation 2006;81: 623–6. Srisook K, Cha YN. Super-induction of HO-1 in macrophages stimulated with lipopolysaccharide by prior depletion of glutathione decreases iNOS expression and NO production. Nitric Oxide 2005;12:70–9. Uguralp S, Bay Karabulut A, Mizrak B, Kaymaz F, Kiziltay A, Hasirci N. The effect of sustained and local administration of epidermal growth factor on improving bilateral testicular tissue after torsion. Urol Res 2004;32:323–31. Rodriguez MG, Rival C, Theas MS, Lustig L. Immunohistopathology of the contralateral testis of rats undergoing experimental torsion of the spermatic cord. Asian J Androl 2006;8:576–83. Tunckiran A, Cayan S, Bozlu M, Yilmaz N, Acar D, Akbay E. Protective effect of vascular endothelial growth factor on histologic changes in testicular ischemia-reperfusion injury. Fertil Steril 2005;84:468–73. Salmasi AH, Beheshtian A, Payabvash S, Demehri S, Ebrahimkhani MR, Karimzadegan M, et al. Effect of morphine on ischemia-reperfusion injury: experimental study in testicular torsion rat model. Urology 2005;66:1338–42.
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Objective: To evaluate the effect of curcumin, a potent antioxidant, on testicular ischemia–reperfusion injury caused by overgeneration of reactive oxygen species (ROS) after testicular torsion–detorsion. Design: Controlled experimental study using rats. Setting: Research laboratory. Animal(s): Sixty adult male Sprague-Dawley rats. Intervention(s): Rats in the control group underwent a sham operation of the left testis. In the torsion–detorsion group, the left testis was rotated 720 for 2 hours. Rats in treatment group received the same surgical procedure as the torsion–detorsion group, but curcumin was administered IV at repair of testicular torsion. Main Outcome Measure(s): Testicular activity of xanthine oxidase, which catalyzes production of ROS; malondialdehyde level (an indicator of ROS content); protein expression level of heme oxygenase-1, which catalyzes antioxidant generation; and spermatogenesis. Result(s): Unilateral testicular torsion–detorsion caused significant increases in xanthine oxidase activity, malondialdehyde level, and heme oxygenase-1 protein expression level and caused a significant decrease in testicular spermatogenesis in ipsilateral testes. The rats treated with curcumin had significant decreases in xanthine oxidase activity and malondialdehyde level and had significant increases in heme oxygenase-1 protein expression level and testicular spermatogenesis in ipsilateral testes, compared with the torsion–detorsion group. Conclusion(s): The curcumin exerts a protective effect on testicular ischemia–reperfusion injury. (Fertil Steril 2009;91:271–7. 2009 by American Society for Reproductive Medicine.) Key Words: Curcumin, ischemia–reperfusion injury, testicular torsion
Testicular torsion or, more properly, torsion of the spermatic cord, is a urologic emergency. Its incidence has been estimated to be 1 in 4,000 in males by the age of 25 years (1). Testicular salvage rates with surgical detorsion have been reported to range from 42% to 88% (2, 3), but whether these testes truly were saved with respect to testicular spermatogenic function remains in question. Studies in human beings have shown an ipsilateral testicular atrophy rate of 33%–68% after testicular rescue (4, 5). Animal studies have found a significant decrease in ipsilateral testicular spermatogenesis after testicular salvage (6, 7). It appears that the main pathophysiology of testicular torsion–detorsion is ischemia–reperfusion injury of testis that is caused by the twisted spermatic cord and its release. In the ischemia-reperfusion injury, overgeneration of reactive oxygen species (ROS) is thought to play a critical role in the loss of ipsilateral testicular spermatogenesis (8). Reactive oxygen species can cause tissue damage through cell membrane lipid peroxidation, protein denaturation, and DNA impairment (9, 10).
It shows a wide range of pharmacological activities, including antioxidant and anti-inflammatory effects (11). Curcumin long has been used in Asian medicine for its medical properties (12). It has been reported that administration of curcumin can ameliorate ischemia–reperfusion injury in myocardium (13), liver (14), kidney (15), brain (16), and other organ systems. To our knowledge, no study has investigated the role of curcumin in testicular ischemia–reperfusion injury. In the present study, we evaluated the effect of curcumin on testicular spermatogenesis in a rat testicular ischemia–reperfusion injury model.
Curcumin is the major constituent of turmeric powder, which is extracted from rhizomes of plant Curcuma longa.
MATERIALS AND METHODS Animals and Surgical Procedure The 60 adult male Sprague-Dawley rats (250–300 g) were maintained on a 12:12-hour light–dark cycle with food and water ad libitum. All animal experiments followed a protocol that was approved by the ethics committee on animal research at our university. We followed appropriate care and the guidelines for experimental animals, as published by the US National Institutes of Health Board of Registry.
Received October 5, 2007; revised and accepted October 30, 2007. Reprint requests: Si-Ming Wei, M.D., Ph.D., Department of Urology and Laboratory of Experimental Immunology, Campus Gasthuisberg (O & N), Catholic University of Leuven, Herestraat 49, B-3000, Leuven, Belgium (E-mail: [email protected]).
Rats were randomly divided into three groups, each containing 20 rats. They were anesthetized with ketamine injection (50 mg/kg, IP). All operations were performed under sterile conditions. In the control group, the left testis was
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2007.10.082
Fertility and Sterility Vol. 91, No. 1, January 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
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brought out through a left-sided ilioinguinal incision. An 11– 0 atraumatic silk suture was placed through the tunica albuginea, the left testis was replaced into the scrotum, and the incision was closed. In the torsion–detorsion group, the left testis was exposed through the same incision. The left testis was rotated 720 in a counterclockwise direction and was maintained in this torsion position by being fixed to the scrotum with 11–0 silk suture. The incision was sutured. After 2 hours of torsion, the incision was reopened, and the testis was counter-rotated to the natural position. The testis still was viable for restoration of blood flow and was reinserted into the scrotum. In the treatment group, the same surgical procedure was performed as in the torsion–detorsion group, but curcumin (200 mg/kg; Sigma Chemical Company, St. Louis, MO) was injected IV into the rat via the tail vein at the time of detorsion. Bilateral orchiectomy was performed on half of the rats in each experimental group at 4 hours after the repair of torsion for measurements of malondialdehyde level, xanthine oxidase activity, and heme oxygenase-1 protein expression level. Orchiectomy was performed on the remaining rats at 3 months after the repair of torsion, to allow analysis of testicular spermatogenesis.
Evaluation of Testicular Spermatogenesis Testicular spermatogenesis was evaluated by measuring testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score. Testis was excised and weighed. The testicular tissue was fixed in Bouin’s solution, postfixed in 70% alcohol, and embedded in paraffin block. A 5-mm section was obtained, deparaffinized, and stained with hematoxylin and eosin. The light-microscope histological evaluation was performed by an observer in a blind, randomly numbered fashion, without the observer having any knowledge of which testis was experimentally torsional or nontorsional. Mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score were used to evaluate the 20 roundest seminiferous tubules of each sample. Mean seminiferous tubular diameter was measured by using a microscope-adaptable micrometer. Number of germ cell layers in each tubule was determined by counting the numbers of germ cell layers from the basement membrane to the lumen at 90 , 180 , 270 , and 360 and averaging these numbers. The mean testicular biopsy score was graded by using Johnsen’s score (20). A score of 1 to 10 was given to each tubule, according to the maturity of the germ cells.
Measurement of Malondialdehyde Level Malondialdehyde in testis was measured by using the thiobarbituric acid–reactive substance assay, as described by Ohkawa et al. (17). The principle of the method is based on the measurement of the concentration of pink chromogen compound that forms when malondialdehyde reacts with thiobarbituric acid. Malondialdehyde levels were expressed as nanomoles per milligram of protein.
Statistical Analysis Data were expressed as mean SD. Data among groups were compared by one-way analysis of variance, followed by the Student-Newman-Keuls test. Within groups, ipsilateral and contralateral data were compared by using Student’s t-test. P<.05 was considered statistically significant. GraphPad Prism software (GraphPad, San Diego, CA) was used for data analysis.
Xanthine Oxidase Activity Determination Xanthine oxidase activity was assayed spectrophotometrically at 293 nm, with xanthine as substrate (18). The formation of uric acid from xanthine results in an increase in absorbency. One unit of activity was defined as 1 mmol of uric acid formed per minute, at pH 7.5. Xanthine oxidase activity was expressed as international units per gram of protein. Detection of Heme Oxygenase-1 Protein Expression Level by Western Blot Protein extraction and concentration measurement were performed as described elsewhere (19). Proteins were separated by electrophoresis and transferred to nitrocellulose membrane. The membrane was blocked with 5% nonfat milk and subsequently was incubated with anti–heme oxygenase-1 antibody (Stressgen) or anti–b-actin antibody (Sigma) overnight. Then, membrane was incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. Protein bands were visualized by using enhanced chemiluminescence detecting reagent. The density of each band was quantified. The ratio of density of the heme oxygenase-1 band to that of the internal control b-actin band from the same sample represented the relative expression level of heme oxygenase-1 protein. 272
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RESULTS Malondialdehyde Level and Xanthine Oxidase Activity Figure 1A and B show malondialdehyde level and xanthine oxidase activity in both ipsilateral and contralateral testes in control, torsion–detorsion, and treatment groups at 4 hours after detorsion. Malondialdehyde level and xanthine oxidase activity in ipsilateral testes were significantly increased in the torsion–detorsion group, as compared with in the control group (P<.05). The two parameters in ipsilateral testes in treatment group were significantly lower than those in the torsion–detorsion group (P<.05). These parameters of contralateral testes did not reveal any statistically significant difference among the three groups (P>.05). Protein Expression Level of Heme Oxygenase-1 Figure 2A and B display heme oxygenase-1 protein expression level in testes in the three groups at 4 hours after detorsion. Heme oxygenase-1 expression levels in ipsilateral testes of the torsion–detorsion group were significantly higher than those in the control group (P<.05). The parameter levels in ipsilateral testes of treatment group were significantly higher than those in the torsion–detorsion group (P<.05). No significant difference was detected in the heme oxygenase-1 expression level of the contralateral testes among the three groups (P>.05).
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FIGURE 1 Malondialdehyde level (A) and xanthine oxidase activity (B) in rat testicular tissue in control, torsion–detorsion (T/ D), and treatment groups 4 hours after detorsion. Hatched bars show the ipsilateral testes; open bars show the contralateral testes. *Value significantly different from that of control group (P< .05). #Value significantly different from that of contralateral testis in the same group (P< .05). xValue significantly different from that of ipsilateral testis in the T/ D group (P< .05).
Wei. Effect of curcumin on testicular torsion injury. Fertil Steril 2009.
Testicular Spermatogenesis Figures 3 and 4 show testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score in the control, torsion–detorsion, and treatment groups 3 months after detorsion. Compared with the control group, testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score in the ipsilateral testes that was obtained from the torsion–detorsion group were significantly lower (P<.05). The four parameters of ipsilateral testes in the treatment group were significantly higher than those in the torsion–detorsion group (P<.05). None of these parameters of contralateral testes revealed any significant difference among the three groups (P>.05). DISCUSSION Testicular torsion–detorsion is an ischemia–reperfusion injury for the testis. The ischemia–reperfusion injury is associated with overgeneration of ROS, such as superoxide anions, hydrogen peroxide, hydroxyl radicals, and nitric oxide. One important source of ROS generation is the hypoxanthine–xanthine oxidase reaction in parenchymal cells of the postischemic tissue (21). Xanthine oxidase is an important Fertility and Sterility
prerequisite factor in the process of superoxide anion and hydrogen peroxide production. Intracellular calcium concentration rises during ischemia because of calcium influx, leading to proteolytic conversion of xanthine dehydrogenase to xanthine oxidase (7). Furthermore, ischemia also causes an increase in intracellular hypoxanthine as a result of adenosine triphosphate breakdown (10). When oxygen is supplied during reperfusion, xanthine oxidase converts hypoxanthine to superoxide anion and hydrogen peroxide (22). Superoxide anions may react with hydrogen peroxide to form hydroxyl radicals (22). The ROS are difficult to quantify directly in tissue because of their high reactivity and short half-life. Malondialdehyde, a stable end product of lipid peroxidation generated by ROS, usually is used as an indirect indicator of ROS (8). In our study, an elevated level of malondialdehyde in ipsilateral testes of the torsion–detorsion group indicates increased oxidative stress. In addition, xanthine oxidase activity in ipsilateral testes also was enhanced in the torsion–detorsion group. The rise in enzyme activity provides an explanation for ROS overgeneration in testicular tissue. During ischemia–reperfusion, increased production of ROS inflicts significant injury on ischemic tissue through oxidization of cell membrane lipids, proteins, and DNA (9, 273
FIGURE 2 The protein expression level of heme oxygenase-1 in rat testes in control, torsion–detorsion, and treatment groups 4 hours after detorsion. (A) Representative Western blot result of heme oxygenase-1. Lanes 1L and 1R ¼ left (i.e., ipsilateral) and right (i.e., contralateral) testes in the control group; lanes 2L and 2R ¼ ipsilateral and contralateral testes in the torsion–detorsion group; lanes 3L and 3R ¼ ipsilateral and contralateral testes in the treatment group. (B) Quantitative analysis of heme oxygenase-1 Western blot data. Hatched bars show the ipsilateral testes; open bars show the contralateral testes. *Value significantly different from that of control group (P< .05). #Value significantly different from that of contralateral testis in the same group (P< .05). xValue significantly different from that of ipsilateral testis in torsion– detorsion (T / D) group (P< .05).
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10). The present study showed that unilateral testicular torsion–detorsion caused a significant increase in testicular malondialdehyde level (an indicator of ROS content) and caused a significant decrease in spermatogenesis in the ipsilateral testes, suggesting that testicular spermatogenesis was severely injured by ROS. Testicular lesions were characterized by decreases in testicular weight, mean seminiferous tubular diameter, number of germ cell layers, and mean testicular biopsy score. The elimination of ROS has been shown to be beneficial in treating several diseases relevant to ischemia–reperfusion injury, such as myocardial infarction (13), renal transplantation (15), and cerebral stroke (16). Curcumin is a powerful scavenger of ROS such as superoxide anions, hydroxyl radicals, and nitric oxide (23, 24). Curcumin treatment successfully has been used to decrease ischemia–reperfusion injury in multiple organ systems, including heart (13), liver (14), kidney (15), and brain (16). This success led us to attempt such treatment in a model of testicular torsion–detorsion. In the present study, we found 274
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that rats treated with curcumin had a significant reduction in malondialdehyde level and had a significant increase in spermatogenesis in ipsilateral testes, as compared with the torsion–detorsion group. In addition, curcumin has achieved beneficial effects on renal ischemia–reperfusion injury in a clinical trial (15). It did not have side effects in those patients who were receiving renal transplantation (15). Therefore, it is reasonable to suggest that clinical application of curcumin may be a useful new approach for therapy of testicular torsion, as an addition to conventional detorsion. Testicular torsion is an ischemic process for testis. Blood cannot flow into testis during torsion. If curcumin is injected into rats through their tail vein at the time of testicular torsion, that is, before detorsion, it will not flow into the testis to exert its beneficial effect. Testicular detorsion is a reperfusion process of blood. Consequently, we injected curcumin at the time of detorsion. Some other reports have indicated that curcumin at the dosage of 200 mg/kg is effective in treating oxidative stress–mediated injury in rat forebrain (16), heart (13), liver (25), and so on. Therefore, we chose this dosage in our rat experiment. One strategy to reduce the effect of ROS involves their elimination through the use of ROS scavengers. Reactive oxygen species scavengers, such as superoxide dismutase and catalase, can convert toxic ROS into water and oxygen (10). Although the treatment with superoxide dismutase and catalase had a protective effect in 1 hour of testicular torsion, no significant rescue was observed in 2 hours of testicular torsion (10). In contrast, we found that administration of curcumin provided a significant rescue of testicular spermatogenesis after 2 hours of testicular torsion. The observations could not be explained by a simple scavenging ROS effect of curcumin. Our study showed yet that curcumin may significantly reduce xanthine oxidase activity and malondialdehyde level in ipsilateral testes. These results suggest that curcumin may reduce ROS production by inhibiting xanthine oxidase. As a result, we believe that curcumin may provide an additional protective effect by diminishing ROS generation. Heme oxygenase-1 is a widely distributed enzyme in mammalian tissues and plays an important cytoprotective role against oxidative stress (26). Its main function is associated with the degradation of heme to iron, carbon monoxide, and biliverdin, the latter being converted to bilirubin by the cytosolic enzyme biliverdin reductase (27). Both biliverdin and bilirubin possess antioxidant properties (28). Ample evidence supports the notion that up-regulation of heme oxygenase-1 expression provides potent cytoprotective effects in many in vitro and in vivo models of oxidative stress–induced cellular and tissue injury (15, 19, 28–30). Many recent studies have revealed that curcumin potently induces heme oxygenase-1 expression, such as in cardiac myoblasts (29), hepatocytes (30), and monocytes (28), leading to increased resistance to oxidative stress–mediated damage. However, its effect on testicular germ cells has not been examined previously. In this study, we explored whether curcumin treatment could increase protein
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FIGURE 3 Rat testicular weight (A), mean seminiferous tubular diameter (B), number of germ cell layers (C), and mean testicular biopsy score (D) in control, torsion–detorsion (T/D), and treatment groups, 3 months after detorsion. Hatched bars show the ipsilateral testes; open bars show the contralateral testes. *Value significantly different from that of the control group (P< .05). #Value significantly different from that of contralateral testis in the same group (P< .05). xValue significantly different from that of ipsilateral testis in the T/D group (P< .05).
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expression of heme oxygenase-1 in rat testes after ischemia– reperfusion. The present study showed that heme oxygenase1 expression levels in ipsilateral testes harvested from the torsion–detorsion group were significantly higher than those in the control group. It is suggested that oxidative stress–induced increase in heme oxygenase-1 expression has only a regulatory rather than a therapeutic role in attenuating the deleterious effects of oxidative stress (31). The heme oxygenase-1 expression levels in ipsilateral testes of the curcumin treatment group increased significantly, as compared with the torsion–detorsion group. In addition, curcumin treatment also attenuated testicular injury and reduced malondialdehyde level in ipsilateral testes in comparison with the torsion–detorsion group. These results suggest that induction of heme oxygenase-1 expression after curcumin treatment may protect testes against ischemia–reperfusion injury by antioxidation. Therefore, induction of heme oxygenase-1 expression is an important mechanism of curcumin against testicular ischemia–reperfusion injury. Treatment with curcumin (200 mg/kg) significantly rescued ipsilateral testicular spermatogenesis in our experiment, but the saved spermatogenesis was not restored to normal value. In the current study, we did not investigate the effect Fertility and Sterility
of curcumin on testicular ischemia–reperfusion injury at different doses, or at different administration times. Because these factors may affect the effect of curcumin, further studies are required to elucidate answers to these questions so that curcumin can be used to optimal effect. There still is controversy about whether unilateral testicular torsion affects the contralateral testis. Some investigators have found that unilateral testicular torsion causes contralateral testicular damage (32, 33), whereas other investigators have not (34, 35). In our study, although unilateral testicular torsion resulted in significant changes in ipsilateral testicular malondialdehyde level, xanthine oxidase activity, heme oxygenase-1 expression level, and spermatogenesis, our results did not show any change in the contralateral testes. As a result, we believe that unilateral testicular torsion does not lead to contralateral injury. In conclusion, we have demonstrated the protective effect of curcumin on testicular ischemia–reperfusion injury for the first time. We propose that curcumin may be a novel approach for therapy of testicular ischemia–reperfusion injury. The protective mechanism of curcumin may be explained as follows. Curcumin can scavenge ROS. In addition, it reduces 275
FIGURE 4 Testicular histology 3 months after detorsion. (A) The bilateral testes in control group and the contralateral testes in torsion–detorsion and treatment groups all indicated normal seminiferous tubular diameter, number of germ cell layers, and spermatogenesis from spermatogonium in the basement membrane to spermatozoon in the center of tubule. (B) The ipsilateral testes in torsion–detorsion group showed seminiferous tubular atrophy, reduction of germ cell layer number, and spermatogenous arrest. (C) The ipsilateral testes in the treatment group displayed normality of most seminiferous tubules and abnormality of a few tubules (arrows). All panels, hematoxylin and eosin staining (original magnification, 100 in panels A and B and 50 in panel C).
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ROS formation by inhibiting xanthine oxidase. Curcumin is a potent inducer of heme oxygenase-1 in testes. The increased heme oxygenase-1 expression is an important element in curcumin-mediated cytoprotection against oxidative stress. All these together lead to spermatogenous protection in ischemia–reperfusion for testis that is treated with curcumin. REFERENCES 1. Barada JH, Weingarten JL, Cromie WJ. Testicular salvage and age-related delay in the presentation of testicular torsion. J Urol 1989;142: 746–8. 2. Parker RM, Robison JR. Anatomy and diagnosis of torsion of the testicle. J Urol 1971;106:243–7. 3. Cattolica EV, Karol JB, Rankin KN, Klein RS. High testicular salvage rate in torsion of the spermatic cord. J Urol 1982;128:66–8. 4. Macnicol MF. Torsion of the testis in childhood. Br J Surg 1974;61: 905–8. 5. Krarup T. The testes after torsion. Br J Urol 1978;50:43–6.
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6. Ozturk H, Ozturk H, Yagmur Y. PAF antagonist BN-52021 reduces intercellular adhesion molecule-1 expression and oxidative stress in rats with reperfusion damage due to unilateral testicular torsion. Pediatr Surg Int 2006;22:191–6. 7. Cay A, Alver A, Kucuk M, Isik O, Eminagaoglu MS, Karahan SC, et al. The effects of N-acetylcysteine on antioxidant enzyme activities in experimental testicular torsion. J Surg Res 2006;131:199–203. 8. Turner TT, Tung KS, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997;57:1267–74. 9. Filho DW, Torres MA, Bordin AL, Crezcynski-Pasa TB, Boveris A. Spermatic cord torsion, reactive oxygen and nitrogen species and ischemia-reperfusion injury. Mol Aspects Med 2004;25:199–210. 10. Prillaman HM, Turner TT. Rescue of testicular function after acute experimental torsion. J Urol 1997;157:340–5. 11. Jagetia GC. Radioprotection and radiosensitization by curcumin. Adv Exp Med Biol 2007;595:301–20. 12. Ammon HP, Wahl MA. Pharmacology of Curcuma longa. Planta Med 1991;57:1–7. 13. Nirmala C, Puvanakrishnan R. Protective role of curcumin against isoproterenol induced myocardial infarction in rats. Mol Cell Biochem 1996;159:85–93.
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14. Shen SQ, Zhang Y, Xiang JJ, Xiong CL. Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes. World J Gastroenterol 2007;13:1953–61. 15. Shoskes D, Lapierre C, Cruz-Correa M, Muruve N, Rosario R, Fromkin B, et al. Beneficial effects of the bioflavonoids curcumin and quercetin on early function in cadaveric renal transplantation: a randomized placebo controlled trial. Transplantation 2005;80:1556–9. 16. Ghoneim AI, Abdel-Naim AB, Khalifa AE, El-Denshary ES. Protective effects of curcumin against ischaemia/reperfusion insult in rat forebrain. Pharmacol Res 2002;46:273–9. 17. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. 18. Prajda N, Weber G. Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas. FEBS Lett 1975;59: 245–9. 19. Yang S, Shih HJ, Chow YC, Tsai PS, Wang TY, Wang PS, et al. The protective role of heme oxygenase-1 induction on testicular tissues after testicular torsion and detorsion. J Urol 2007;177:1928–33. 20. 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;1:2–25. 21. Mccord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159–63. 22. Gilgun-Sherki Y, Melamed E, Offen D. The role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy. J Neurol 2004;251:261–8. 23. Zhao BL, Li XJ, He RG, Cheng SJ, Xin WJ. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell Biophys 1989;14:175–85. 24. Li JK, Lin-Shia SY. Mechanisms of cancer chemoprevention by curcumin. Proc Natl Sci Counc Repub China B 2001;25:59–66. 25. Reyes-Gordillo K, Segovia J, Shibayama M, Vergara P, Moreno MG, Muriel P. Curcumin protects against acute liver damage in the rat by in-
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26. 27. 28.
29.
30.
31.
32.
33.
34.
35.
hibiting NF-kappaB, proinflammatory cytokines production and oxidative stress. Biochim Biophys Acta 2007;1770:989–96. Shiraishi K, Naito K. Increased expression of Leydig cell haem oxygenase1 preserves spermatogenesis in varicocele. Hum Reprod 2005;20:2608–13. Kikuchi G, Yoshida T, Noguchi M. Heme oxygenase and heme degradation. Biochem Biophys Res Commun 2005;338:558–67. Rushworth SA, Ogborne RM, Charalambos CA, O’Connell MA. Role of protein kinase C delta in curcumin-induced antioxidant response element-mediated gene expression in human monocytes. Biochem Biophys Res Commun 2006;341:1007–16. Abuarqoub H, Green CJ, Foresti R, Motterlini R. Curcumin reduces cold storage-induced damage in human cardiac myoblasts. Exp Mol Med 2007;39:139–48. Mcnally SJ, Harrison EM, Ross JA, Garden OJ, Wigmore SJ. Curcumin induces heme oxygenase-1 in hepatocytes and is protective in simulated cold preservation and warm reperfusion injury. Transplantation 2006;81: 623–6. Srisook K, Cha YN. Super-induction of HO-1 in macrophages stimulated with lipopolysaccharide by prior depletion of glutathione decreases iNOS expression and NO production. Nitric Oxide 2005;12:70–9. Uguralp S, Bay Karabulut A, Mizrak B, Kaymaz F, Kiziltay A, Hasirci N. The effect of sustained and local administration of epidermal growth factor on improving bilateral testicular tissue after torsion. Urol Res 2004;32:323–31. Rodriguez MG, Rival C, Theas MS, Lustig L. Immunohistopathology of the contralateral testis of rats undergoing experimental torsion of the spermatic cord. Asian J Androl 2006;8:576–83. Tunckiran A, Cayan S, Bozlu M, Yilmaz N, Acar D, Akbay E. Protective effect of vascular endothelial growth factor on histologic changes in testicular ischemia-reperfusion injury. Fertil Steril 2005;84:468–73. Salmasi AH, Beheshtian A, Payabvash S, Demehri S, Ebrahimkhani MR, Karimzadegan M, et al. Effect of morphine on ischemia-reperfusion injury: experimental study in testicular torsion rat model. Urology 2005;66:1338–42.
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