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The generally undesired effects of exocannabinoids on male reproduction include alterations in testicular cell proliferation and function, as well as apoptosis induction. However, this paradigm has been challenged by the ability of endocannabinoids to regulate reproductive function. The present study addresses these paradoxical facts by investigating the effects of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on mouse Sertoli cells' survival and apoptosis, with a mechanistic insight into Sertoli cell-based growth factors' production. The Mus musculus Sertoli cell line (TM4) was exposed to different concentrations of 2-AG, and cell viability was evaluated using MTT assay. Growth factors' gene and protein expressions were analyzed through RT-PCR and western blotting. 2-AG concentration dependently increased TM4 viability, with a slight increase starting at 0.0001 µM, a peak of 190% of the control level at 1 µM, and a decrease at 3 µM. Moreover, 2-AG paradoxically altered mRNA expression of caspase-3 and growth factors. Caspase-3 mRNA expression was down-regulated, and growth factors mRNA and protein expression were up-regulated when using a low concentration of 2-AG (1 µM). Opposite effects were observed by a higher concentration of 2-AG (3 µM). These paradoxical effects of 2-AG can be explained through the concept of hormesis. The results indicate the pivotal role of 2-AG in mediating Sertoli cell viability and apoptosis, at least in part, through altering growth factors secretion. Furthermore, they suggest the involvement of endocannabinoids in Sertoli cell-based physiological and pathological conditions and reflect the ability of abnormally elevated 2-AG to mimic the actions of exocannabinoids in reproductive dysfunction.

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BACKGROUND: Global rise in cannabis abuse during reproductive years has placed a large number of men at risk for the adverse consequences of δ-9-tetrahydrocannabinol (THC), the primary active component of cannabis. It has been reported that THC affects male fertility and causes testicular cell dysfunction and apoptosis. This study aimed to investigate the possible protective role of zinc pretreatment against the toxic effects of THC in cultured mouse Sertoli cells and the underlying mechanism. METHODS: The Mus Musculus Sertoli cell line (TM4) was cultured, exposed to THC alone (470 µM, 24 h), co-administered with zinc (8 µM, 48 h), and investigated in three groups: control, THC, and THC + zinc. The MTT was performed to evaluate cell viability. TUNEL assay was also applied for the detection of cell apoptosis and a western blot was performed for measuring protein expression levels of Caspase3, Pro-caspase3, SOD, and PDGF-A. RESULTS: THC significantly decreased cell viability (p < 0.001) and expression levels of SOD, PDGF-A, and pro-caspase3 proteins (p < 0.05 for all), whereas increased Sertoli cells apoptosis (p < 0.001) and expression level of cleaved caspase3 protein (p < 0.001). Pretreatment with zinc reversed THC-induced apoptotic and oxidative effects and reduced cleaved caspase3/pro-caspase3 ratio but could not reverse THC-induced reduction of PDGF-A expression level in TM4 cells. CONCLUSION: The present data suggest that THC induces Sertoli cell damage through a multitarget mechanism. Zinc was reported to protect against THC-induced Sertoli cell damage due to its antiapoptotic and antioxidant activities, indicating its clinical importance against THC-induced testicular toxicity among addicted men.

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BACKGROUND: Chronic use of morphine is associated with reproductive complications, such as hypogonadism and infertility. While the side effects of morphine have been extensively studied in the testis, much less is known regarding the effects of morphine on Sertoli cells and the effects of zinc on morphine-induced testicular injury as well as their underlying mechanisms. Therefore, the purpose of this study was to investigate the effect of morphine (alone and co-administered with zinc) on cell viability and apoptosis of the testicular (Sertoli) cells as well as the tumor suppressor p53 and phosphorylated-protein kinase B (p-Akt) protein levels in both in vitro and in vivo models. METHODS: Cultured Sertoli cells were exposed to morphine (23 µM), zinc (8 µM), and zinc prior to morphine and their effects on Sertoli cell viability and apoptosis were investigated. Morphine (3 mg/kg) and zinc (5 mg/kg, 1 h before morphine) were also injected intraperitoneally to rats and then the apoptotic changes in the testis were evaluated. RESULTS: Cell viability and p-Akt protein levels decreased in morphine-treated cells, while apoptosis and p53 protein expression increased in these cells. Pretreatment with zinc recovered morphine-induced apoptotic effects, as well as over-expression of p53 and down-regulation of p-Akt. These findings were supported by a subsequent animal study. CONCLUSION: The present data indicated the protective effect of zinc against morphine-induced testicular (Sertoli) cell toxicity via p53/Akt pathways in both in vivo and in vitro models and suggested the clinical importance of zinc on infertility among chronic opioid users and addicted men.

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Exocannabinoids such as tetrahydrocannabinol (THC) may alter the physiological function of endocannabinoids in male reproduction and thus affect male fertility. This study aimed to investigate the apoptotic effects of THC via mechanisms related to p53 and AKT signaling pathways on Sertoli cells and seminiferous germinal cells, as well as the possible protective role of selenium pretreatment in both in vitro and in vivo models. The Mus musculus Sertoli cell line, TM4, was used for in vitro experiments. The TM4 cells were cultured and exposed to selenium (2 µM, 48 h) and THC (470 µM, 24 h). The MTT test was performed to evaluate cell viability. Fifteen male Wistar rats (220 ± 20 g) were used for in vivo experiments and divided into three groups: (1) control, (2) tetrahydrocannabinol (THC, 5 mg/kg, dissolved in DMSO 5%, i.p., for 21 consecutive days), and (3) THC + selenium (selenium, 0.5 mg/kg per day, i.p.). At the end of the experiments, Sertoli cells and testis tissue samples were collected for biochemical (AKT, P53), cell apoptosis, and histological analyses. The results of the in vitro study revealed that THC significantly decreases the cell viability (p < 0.001) and expression of the p-AKt protein (p < 0.05) and increases Sertoli cells' apoptosis (p < 0.001) and p53 protein expression (p < 0.001). The in vivo effects of THC were in line with the in vitro results. Pretreatment with selenium (as sodium selenite) significantly decreased the THC-induced Sertoli cell and testicular tissue damages in the rats. Pathological changes were significantly alleviated in the selenium-pretreated rats. Collectively, these data suggest that pretreatment with selenium is able to protect against THC-induced testicular cell damage. The attenuating effect of selenium may be due to its anti-apoptotic activity through the p53 and AKT modulation.

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Abstract Introduction: It is hypothesized that increased macrophage migration inhibitory factor (MIF) expression may contribute to diabetic nephropathy (DN) pathogenesis. The aim of the present study was to investigate the renal effects of MIF inhibition in a diabetic experimental model. Methods: Eighteen male Wistar rats (230 ± 20 g) were divided into three groups: 1) control, 2) diabetic (STZ, 50 mg/kg, dissolved in saline, ip), 3) diabetic + MIF antagonist (p425, 1 mg/kg per day, ip, on the 21th day, for 21 consecutive days). The treatment started since we founwd a significant increase in urine albumin excretion (UAE) rate in the diabetic rats in comparison with the control rats. The rats were kept individually in metabolic cages (8 AM-2 PM) and urine samples were collected in the 21 and 42th day. At the end, blood and tissue samples were collected for biochemical (BS, UPE, urine GAG, BUN, Cr, Na, and K) and histological analyses. Results: The results of this study showed that MIF antagonist (p425) significantly decreased urine protein and GAG excretion, urine protein/creatinine ratio, and serum BUN and Cr in the streptozotocin-induced DN in the rats. Pathological changes were significantly alleviated in the MIF antagonist (p425)-administered DN rats. Conclusion: Collectively, these data suggested that MIF antagonist (p425) was able to protect against functional and histopathological injury in the DN.

Resumo Introdução: Supõe-se que elevações da expressão do fator de inibição da migração de macrófagos (MIF) possam contribuir para a patogênese da nefropatia diabética (ND). O objetivo do presente estudo foi investigar os efeitos renais da inibição do MIF em um modelo experimental diabético. Métodos: Dezoito ratos Wistar machos (230 ± 20g) foram divididos em três grupos: 1) controle, 2) diabético (STZ 50 mg/kg dissolvida em soro fisiológico, IP), 3) diabético + antagonista do MIF (p425 1 mg/kg por dia IP no 21o dia por 21 dias consecutivos). O tratamento começou após a identificação de aumento significativo na albuminúria nos ratos diabéticos em relação aos controles. Os ratos foram mantidos individualmente em gaiolas metabólicas (8h-14h) e amostras de urina foram colhidas no 21o e no 42o dia. Ao final do estudo, amostras de sangue e tecido foram colhidas para análises bioquímicas (BS, excreção urinária de proteína, excreção urinária de GAGs, BUN, Cr, Na e K) e histológicas. Resultados: O presente estudo demonstrou que o antagonista do MIF (p425) diminuiu significativamente proteinúria, excreção urinária de GAGs , relação proteína/creatinina na urina, BUN e Cr no grupo com ND induzida por estreptozotocina. As alterações patológicas foram significativamente abrandadas nos ratos com ND que receberam antagonista do MIF (p425). Conclusão: Coletivamente, os dados sugerem que o antagonista do MIF (p425) teve efeito protetor contra lesões funcionais e histopatológicas da ND.

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INTRODUCTION: It is hypothesized that increased macrophage migration inhibitory factor (MIF) expression may contribute to diabetic nephropathy (DN) pathogenesis. The aim of the present study was to investigate the renal effects of MIF inhibition in a diabetic experimental model. METHODS: Eighteen male Wistar rats (230 ± 20 g) were divided into three groups: 1) control, 2) diabetic (STZ, 50 mg/kg, dissolved in saline, ip), 3) diabetic + MIF antagonist (p425, 1 mg/kg per day, ip, on the 21th day, for 21 consecutive days). The treatment started since we founwd a significant increase in urine albumin excretion (UAE) rate in the diabetic rats in comparison with the control rats. The rats were kept individually in metabolic cages (8 AM-2 PM) and urine samples were collected in the 21 and 42th day. At the end, blood and tissue samples were collected for biochemical (BS, UPE, urine GAG, BUN, Cr, Na, and K) and histological analyses. RESULTS: The results of this study showed that MIF antagonist (p425) significantly decreased urine protein and GAG excretion, urine protein/creatinine ratio, and serum BUN and Cr in the streptozotocin-induced DN in the rats. Pathological changes were significantly alleviated in the MIF antagonist (p425)-administered DN rats. CONCLUSION: Collectively, these data suggested that MIF antagonist (p425) was able to protect against functional and histopathological injury in the DN.

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Chronic alcohol ingestion is associated with pronounced detrimental effects on the renal system. In the current study, the protective effect of ginger extract on ethanol-induced damage was evaluated through determining 8-OHdG, cystatin C, glomerular filtration rate, and pathological changes such as cell proliferation and fibrosis in rats' kidneys. Male wistar rats were randomly divided into three groups and were treated as follows: (1) control, (2) ethanol and (3) ginger extract treated ethanolic (GETE) groups. After a six weeks period of treatment, the results revealed proliferation of glomerular and tubular cells, fibrosis in glomerular and peritubular and a significant rise in the level of 8-OHdG, cystatin C, plasma urea and creatinine. Moreover, compared to the control group, the ethanol group showed a significant decrease in the urine creatinine and creatinine clearance. In addition, significant amelioration of changes in the structure of kidneys, along with restoration of the biochemical alterations were found in the ginger extract treated ethanolic group, compared to the ethanol group. These findings indicate that ethanol induces kidneys abnormality by oxidative DNA damage and oxidative stress, and that these effects can be alleviated using ginger as an antioxidant and anti-inflammatory agent.

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