ارزیابی نقش تیمار همزمان لتروزول و کروسین بر آسیبهای اکسیداتیو و هورمون تستوسترون در بافت بیضه موشهای صحرایی آزواسپرمی شده با داروی بوسولفان.

Background & Aims: Azoospermia (lack of sperm in ejaculation) is one of the most severe types of infertility in men, due to various defects in the sperm production process or the testicular system . Azoospermia disease is caused by either obstructive mechanism (Obstructive azoospermia-OA) due to blockages in the natural sperm production system, or non-obstructive mechanism (NOA- Non obstructive azoospermia) due to defects in testicular sperm production, with over 60% of infertile men being afflicted with non-obstructive azoospermia (NOA). Treatment with busulfan may have adverse effects on various organs such as reproductive organs. Based on the understanding of hormonal control of sperm production, several drugs such as gonadotropins, androgens, estrogen receptor blockers, and aromatase inhibitors are used as experimental treatments for idiopathic male infertility. Letrozole, as an aromatase inhibitor, has been widely used in the treatment of female infertility, because the chemical structure of letrozole includes a triazole group that selectively interacts with the heme group of the enzyme P450arom, thereby reversibly inhibiting its biological activity Limited studies have evaluated the therapeutic potential of Letrozole in male infertility. In addition, excessive consumption of chemical drugs causes changes in the activity of antioxidant enzymes (TOS) and the stability of oxidative status (TAC) in the ROS pathway, leading to an imbalance between antioxidants and oxidants, resulting in oxidative stress. This phenomenon has led researchers towards the use of herbal medicines, which naturally have fewer side effects. Crocin has been identified as the main active biological component in saffron. Crocins are a type of water-soluble carotenoids that are either mono- or diglycosyl polyene esters, where D-gentiobiose and/or D-glucose appear as carbohydrate residues. A carotenoid with a 20-carbon dicarboxylic structure. Saffron contains various carotenoid chemicals that include small amounts of crocin, alpha and beta carotene, and lycopene. Six different forms of glycosylated esters from the crocin family have been found in saffron. Trans-crocins 3 and 4 are the most common analogs of crocins, which include crocins 1-4, and are essentially the glycosides of trans-crocin in saffron. Crocin can act as an antioxidant and improve the quality of sperm through increasing the expression of antioxidant genes. Therefore, phytoestrogens such as crocin bind to testicular estrogen receptors and stimulate spermatogenesis by strategies like increasing epithelial layers and the diameter of seminiferous tubules and lumen. The aim of this study was to investigate the synergistic effect of letrozole and crocin on the changes of antioxidant enzymes and testosterone hormone synthesis in the induction of spermatogenesis in azoospermia model mice with busulfan using ELISA technique. Methods: In this clinical trial, crocin and letrozole drugs were purchased from Sigma Aldrich. 30 male Wistar mice weighing 180-220 grams and aged approximately 8-10 weeks were purchased from the Pasteur Institute of Iran and transferred to the laboratory. They were kept under new conditions (at a temperature of 22-24 degrees Celsius, relative humidity of 55-60%, with a 12- hour light-dark cycle). To halt spermatogenesis and induce azoospermia, a dose of 10 mg/kg of busulfan was injected intraperitoneally for 10 days. Finally, the rats were divided into 5 groups of 6: the control group was injected with normal saline and citrate buffer inside the hemocoel; the treatment group received a single dose of 10 mg busulfan intra protanealat at a volume of 1.0 mL for 10 days; the treatment group was administered with a single dose of 10 mg busulfan intra protanealat at a volume of 1.0 mL, along with 5 mg letrozole with Black Soldier Fly (BSF) as the solvent; the treatment group received 15 mg crocin extract with normal saline, along with a single dose of 10 mg busulfan intra protanealat a volume of 1.0 mL; and the treatment group was given a single dose of 10 mg busulfan intra protanealat at a volume of 1.0 mL, along with 5 mg letrozole with BSF as the solvent, and 15 mg crocin extract with normal saline and underwent histopathological, histochemical immunological, and hormonal analyses. The data were presented as mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey's post hoc test was used to compare the means in the study groups (P <0.05), and statistically significant differences were considered. Graphs were also plotted using Graph Prism software. Results: The results demonstrated that the simultaneous treatment of letrozole and crocin increased spermatogonia, spermatocytes, and spermatids; Sertoli and Leydig cells were also enhanced. Additionally, letrozole and crocin treatment led to the preservation of the thickness of the basement membrane and the germinal epithelium of testicular tissue; the inner and outer diameters of the tubules were increased. While in the azoospermia group treated with letrozole or crocin, this level of population destruction was somewhat reduced; however, the use of letrozole or crocin in the study groups did not show a significant difference in the inner and outer diameter of the tubules. Biochemical analysis showed a significant increase in the activity of the Total Antioxidant Capacity (TAC) enzyme activity and testosterone hormone levels in the group treated with letrozole + crosine compared to the azoospermia group; however, a significant decrease was found in the letrozole or crosine treatment group compared to the control group, while the Total Oxidant Status (TOS) enzyme activity in the letrozol+crosine treatment group showed a significant decrease compared to the azoospermia group(P<0.05). Conclusion: Some of the improvements in sperm parameters can be attributed to the antioxidant properties of crocin. It seems that sperm lose a large volume of their cytoplasm due to lack of antioxidants during spermatogenesis and become more sensitive to increases in ROS. These results indicate that crocin can act as an antioxidant and enhance sperm quality by increasing the expression of antioxidant genes. Therefore, the use of a plant-derived antioxidant like crocin appears necessary without any side effects and with essential strengthening effects. Crocin can likely act as an antioxidant and potentially improve sperm quality due to its antioxidant activity. Additionally, it has been found that letrozole improves spermatogenesis without causing any observable pathological changes in testicular tissue. According to the studies of Ribeiro et al, Uzun et al, and Shoshany and colleagues, letrozole reversibly inhibits the aromatase enzyme and prevents the conversion of androgen precursors to estradiol in adipose tissue. Moreover, crocin can neutralize the negative effects of oxidative stress and increase cell ability to overcome oxidative stress conditions by preventing glutathione reduction and increasing antioxidant capacity. One of the limitations of the research can be attributed to the lack of studies and sample restrictions, as well as the death of mice during the experimental process. Further investigations and focusing on the precise dosage of crocin, along with attention to the relevant mechanisms of action, seem essential. Overall, crocin has antioxidant and defensive effects, improves the quality of some sperms, and increases morphology, viability, motility, and natural count. In fact, testosterone regulates the secretion of LH hormone from the anterior pituitary gland through a negative feedback mechanism. Busulfan induced apoptosis in germinal epithelial cells and co-administration of letrozole and crocin reduced busulfan-induced apoptosis leading to infertility; therefore, a rational relationship between apoptosis of spermatogonia in response to busulfan exposure can be obtained. It can be suggested that crocin, one of the key components of saffron, acts as a potent antioxidant that can prevent the toxic effects of busulfan and DNA damage that may occur due to oxidative stress. [ABSTRACT FROM AUTHOR]