Efecto protector de naringina en un modelo experimental de hipogonadismo asociado a Diabetes mellitus tipo 1

Hypogonadism is a complication associated with type 1 Diabetes mellitus (DM1). Hyperglycemia and lack of insulin trigger oxidative and nitrosative stress weakening the Leydig cells, which produce testosterone. Our objective was to evaluate the effect of the flavonoid naringin (NAR) as a protector of Leydig cells in an experimental model of DM1. Control male Wistar rats (C), rats treated with 60 mg streptozotocin/kg b.w. (STZ) and STZ rats treated with NAR (20, 40 or 80 mg/kg b.w., STZ+NAR) were employed. After 30 days of treatment, the rats were sacrificed and blood and testes were obtained. Blood glucose, HbA1c and serum testosterone were determined. Glutathione content (GSH) and catalase (CAT) activity were quantified in testis homogenates. In testicular sections, the expression of the inducible enzyme nitric oxide synthase (iNOS) was evaluated by immunohistochemistry and cell apoptosis by TUNEL. The results were analyzed by ANOVA/Tukey (p<0.05). The project was approved by the CICUAL from the School of Medicine, UNC (50/17). The STZ rats exhibited increase in the serum glucose and HbA1c values. The different doses of NAR did not affect these variables. Serum testosterone levels were lower in STZ rats. NAR40 and NAR80 treatments partially prevented this decrease (C: 5.05±0.72; STZ:1.27±0.07*; STZ+NAR20:1.55±0.13*; STZ+NAR40:2.82±0.16*#; STZ+NAR80:2.56±0.36*# ng/mL, *p<0.05 vs C, #p<0.05 vs STZ and STZ+NAR20). Therefore, the chosen treatment dose was 40 mg/kg b.w. STZ rats had lower GSH content and higher CAT activity than controls. NAR normalized these parameters. The percentage of Leydig cells with positive iNOS staining was higher in diabetic rats and NAR prevented this increase (C:35.88±7.56; STZ:77.12±6.74*; STZ+NAR:28.69±8.09, *p<0.001 vs C and STZ+NAR). The number of TUNEL (+) cells was significantly increased in the STZ group. NAR treatment blocked this enhancement (C:5.83±1.13; STZ:71.07±28.36*; STZ+NAR:5.42±3.92; *p<0.05 vs C and STZ+NAR). The results indicate that NAR prevents the cell death by apoptosis of the Leydig cells in the experimental DM1 avoiding, at least in part,  the increase in the testicular oxidative and nitrosative stress.El hipogonadismo es una complicación asociada a la Diabetes mellitus tipo 1 (DM1). La hiperglucemia y la falta de insulina desencadenan estrés oxidativo y nitrosativo afectando a las células de Leydig, productoras de testosterona. Nuestro objetivo fue evaluar el efecto del flavonoide naringina (NAR) como protector de las células de Leydig en un modelo experimental de DM1. Se emplearon ratas Wistar machos controles, tratadas con 60 mg de estreptozotocina/kg peso corporal (pc) (STZ) y ratas STZ tratadas con NAR (20, 40 u 80 mg/kg pc, STZ+NAR). A los 30 días de tratamiento, las ratas se sacrificaron y se obtuvo sangre y testículos. Se determinó glucemia, HbA1c y testosterona sérica. En homogeneizados de testículos se cuantificó el glutatión (GSH) y la actividad de catalasa (CAT). En cortes testiculares se evaluó la expresión de la enzima óxido nítrico sintasa inducible (iNOS) por inmunohistoquímica y apoptosis celular por TUNEL. Los resultados se analizaron mediante ANOVA/Tukey (p<0,05). Aprobación de CICUAL (50/17). Las ratas STZ presentaron valores de glucemia y HbA1c por encima de los valores normales. NAR no logró evitar tales efectos en ninguna de las dosis estudiadas. La testosterona sérica disminuyó en las ratas diabéticas. La administración de NAR 40 u 80 mg/kg pc logró prevenir parcialmente esta disminución (C:5,05±0,72; STZ:1,27±0,07*; STZ+NAR20:1,55±0,13*; STZ+NAR40:2,82±0,16*#; STZ+NAR80:2,56±0,36*#, ng/mL, *p<0,05 vs C, #p<0,05 vs STZ y STZ+NAR20). Por lo tanto, la dosis de tratamiento elegida fue la de 40 mg/kg pc. Las ratas STZ presentaron menor contenido de GSH y mayor actividad de CAT que las controles. NAR normalizó dichos parámetros. El porcentaje de células de Leydig con tinción de iNOS positiva fue mayor en las ratas diabéticas y NAR evitó este aumento (C:35,88±7,56; STZ:77,12±6,74*; STZ+NAR:28,69±8,09, *p<0,001 vs C y STZ+NAR). El número de células de Leydig TUNEL positivas aumentó significativamente en el grupo STZ. El tratamiento con NAR impidió este aumento (C:5,83±1,13; STZ:71,07±28,36*; STZ+NAR:5,42±3,92; *p<0,05 vs C y STZ+NAR). Los resultados indican que en la DM1 experimental NAR previene la muerte por apoptosis de las células de Leydig a través de evitar, al menos en parte, el incremento del estrés oxidativo y nitrosativo testicular. &nbsp

Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus x Graomys centralis male hybrids.

Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis maleswere studied.Testes fromG. centralis, G. griseoflavus and hybridswere compared at different ages.Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D28k staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-old hybrid animals, respectively. Pachytene spermatocyte death in adult hybrids occurs via apoptosis, as revealed by high caspase-3 expression. Calbindin was highly expressed in spermatocytes of adult hybrids, in which massive cell death occurs via apoptosis. Calbindin co-localisation with TUNEL or Fas, Bax and cytochrome c was very limited, suggesting an inverse regulation of calbindin and apoptotic markers. Hybrid sterility is due to breakdown of spermatogenesis at the pachytene spermatocyte stage. Both extrinsic and intrinsic pathways are involved in apoptosis of spermatocytes, which are the most sensitive cell type to apoptotic stimuli

Disfunción arterial asociada a la diabetes mellitus tipo 1 experimental, efecto protector de naringina

Diabetes mellitus (DM) is a risk factor for the development of systemic atherosclerosis and vascular calcification (VC). Chronic inflammation and oxidative stress might be the involved mechanisms in the VC. Hyperglycemia produces oxygen- and nitrogen-derived free radicals (ROS) and triggers endothelial cell (EC) dysfunction. Treatment with natural antioxidants is likely to prevent these complications. Aim: To evaluate the effect of Naringin (NAR) as a CV protector in an experimental model of type 1 DM. Wistar rats were divided in controls (C), diabetic (STZ, treated with 60 mg of streptozotocin/kg b.w.) and diabetic treated with NAR (40 mg/kg b.w.). CICUAL:16/08/2018. The animals were sacrificed 30 days post-treatment after blood extraction and the aortas were obtained. Serum alkaline phosphatase (AP) activity and ROS level in red blood cells were determined. In histological sections of aorta, the morphology of the vascular wall was analyzed with H&E. In cultured aortic ECs, the NO• content, an indicator of vascular health, and the expression of the enzyme inducible nitric oxide synthase (iNOS) were quantified by immunohistochemistry. ANOVA and Tukey were used for statistical analysis (p<0.05). AP activity and ROS level increased in STZ rats and returned to control values with NAR (AP: C:198.50±28.78; STZ:515.20±54.23*; NAR:227.80±46.43 IU/L; *p<0.001vs C and NAR. ROS: C:224.66±1.45; STZ:327.33±41.28*; NAR:218.33±17.37 AU; *p<0.05 vs C and NAR). NO• content and iNOS expression in ECs from diabetic rats decreased by 33 and 25%, respectively. NAR only normalized NO• values. Aortas from all groups were exposed to procalcifying medium for 7 days, then decalcified and the released calcium was quantified spectrophotometrically. Calcium content was higher in STZ rats and NAR normalized the values (C:8.7±0.5; STZ:15.1±0.8*; NAR:10.8±0.7 mg Ca2+/dL; *p<0.01 vs C and NAR). NAR partially prevented the decrease in aortas muscle fiber width in STZ group. These results demonstrate that NAR improves vascular health, attenuates CV and restores NO• levels in a diabetes mellitus experimental model.La Diabetes mellitus (DM) es un factor de riesgo para el desarrollo de ateroesclerosis sistémica y calcificación vascular (CV). La inflamación crónica y el estrés oxidativo pueden ser los mecanismos implicados en la CV. La hiperglucemia produce radicales libres derivados del oxígeno (ROS) y del nitrógeno y desencadena disfunción de células endoteliales (CE). Es probable que el tratamiento con antioxidantes naturales evite estas complicaciones. Objetivo: evaluar el efecto de Naringina (NAR) como protector de las CV en un modelo experimental de DM tipo 1.  Ratas Wistar se dividieron en controles (C), diabéticas (STZ, tratadas con 60 mg de estreptozotocina/kg de pc) y diabéticas tratadas con NAR (40 mg/kg pc), (CICUAL:16/08/2018). Los animales se sacrificaron 30 días post tratamiento previa extracción sanguínea y se obtuvieron las aortas. Se determinó actividad de fosfatasa alcalina sérica (FA) y nivel de ROS en glóbulos rojos. En secciones histológicas de aorta se analizó la morfología de la pared vascular con H/E. En cultivo de CE de aortas se cuantificó el contenido de NO•, indicador de salud vascular, y la expresión de la enzima óxido nítrico sintasa inducible (iNOS) por inmunohistoquímica. Se empleó ANOVA y Tukey para análisis estadístico, considerando significativo p<0,05. La actividad de FA y el nivel de ROS aumentaron en ratas STZ y retornaron a valores controles con NAR (FA: C:198,50±28,78; STZ:515,20±54,23*; NAR:227,80±46,43, UI/L; *p<0,001vs C y NAR. ROS: C:224,66±1,45; STZ:327,33±41,28*; NAR:218,33±17,37 UA; *p<0,05 vs C y NAR). El contenido de NO• y la expresión de iNOS en CE de ratas diabéticas disminuyeron un 33 y 25%, respectivamente. NAR sólo normalizó los valores de NO•. Las aortas de todos los grupos se expusieron a un medio procalcificante durante 7 días, luego se descalcificaron y el calcio liberado se cuantificó por espectrofotometría. El contenido de calcio fue mayor en ratas STZ y NAR normalizó los valores (C:8,7±0,5; STZ:15,1±0,8*; NAR:10,8±0,7 mg Ca+2/dL; *p<0,01 vs C y NAR). NAR evitó parcialmente la disminución del ancho de las fibras musculares de aortas del grupo STZ. Estos resultados demuestran que NAR mejora la salud vascular, atenúa la CV y restaura los niveles de NO• en un modelo experimental de diabetes

Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus x Graomys centralis male hybrids.

Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis maleswere studied.Testes fromG. centralis, G. griseoflavus and hybridswere compared at different ages.Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D28k staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-old hybrid animals, respectively. Pachytene spermatocyte death in adult hybrids occurs via apoptosis, as revealed by high caspase-3 expression. Calbindin was highly expressed in spermatocytes of adult hybrids, in which massive cell death occurs via apoptosis. Calbindin co-localisation with TUNEL or Fas, Bax and cytochrome c was very limited, suggesting an inverse regulation of calbindin and apoptotic markers. Hybrid sterility is due to breakdown of spermatogenesis at the pachytene spermatocyte stage. Both extrinsic and intrinsic pathways are involved in apoptosis of spermatocytes, which are the most sensitive cell type to apoptotic stimuli

Quercetina atenúa el estrés oxidativo testicular asociado al Síndrome Metabólico

The Metabolic Syndrome (MS) generates oxidative stress (OS) altering the male fertility. Quercetin (Qc) supplementation could improve this condition due to its antioxidant properties. Objective: to clarify the mechanisms by which MS affects testicular tissue and to determine if these alterations are reversed by treatment with Qc. Four groups of male Wistar rats were used: Control (C, n: 5), C + Qc (n: 4, 50mg / kg / day, orally, 15 days), MS (n: 4, 10% fructose in water, 45 days) and MS + Qc (n: 4, SM 30 days plus 15 days with Qc treatment). The study time was 45 days. Body weight and abdominal circumference were measured and serum glucose, triglycerides, cholesterol, HDL-cholesterol and testosterone were measured. In epididymis samples, vitality, motility and number of spermatozoa were determined. Glutathione (GSH) and catalase activity (CAT) were quantified in the testis homogenates. The histology of the testes was analyzed with hematoxylin / PAS. ANOVA and Bonferroni were performed (significant differences at p <0.05). MS was confirmed by body weight gain, a decrease in waist circumference, blood glucose (mg/dL) (221.6±13.3 vs 143.0±13.0) and triglycerides (392.7±85.7 vs 86.9±21.9) and a decrease in HDL-cholesterol (25.0±1.1 vs 34.8±0.6). Qc normalized the biochemical parameters. Serum testosterone was similar in all groups. MS produced GSH depletion (nmol/mg prot.) (31.7±1.4 vs 45.1±0.5) and increased CAT activity (IU/mg prot.) (16.1±0.6 vs 9.8±1.6). Qc partially restored the tripeptide levels and normalized the enzyme activity. The area of ​​the testicular sections and the thickness of the cell layer in the seminiferous tubules were lower in the MS group. At the end of the study, all groups presented complete spermatogenesis and the same concentration and motility of spermatozoa. The results suggest that MS produces OS and alters testicular tissue. Although complete spermatogenesis occurs during the development of MS, OS could be the mechanism by which fertility would be affected at longer times. Qc normalizes the testicular redox status and improves the general conditions of MS, which could prevent the development of male subfertility.El Síndrome Metabólico (SM) genera estrés oxidativo (EO) generalizado y, a nivel testicular, afecta la fertilidad masculina. La suplementación con Quercetina (Qc), podría mejorar esta condición. Objetivo: esclarecer los mecanismos por los cuales el SM afecta al tejido testicular y determinar si estas alteraciones son revertidas por el tratamiento con Qc. Ratas Wistar machos se dividieron en grupo control (C, n:5), C+Qc (n:4, 50mg/kg/día, vía oral, 15 días), SM (n:4, 10% de fructosa en el agua, 45 días) y SM+Qc (n:4, SM 30 días más 15 días con tratamiento de Qc). El tiempo del estudio fue de 45 días. Se midió peso corporal y circunferencia abdominal y en suero se determinó, glucosa, triglicéridos, colesterol, HDL-colesterol y testosterona. En muestras de epidídimo se determinó vitalidad, motilidad y número de espermatozoides. En testículo se cuantificó glutatión (GSH) y actividad de catalasa (CAT). La histología de los testículos se analizó con hematoxilina/PAS. Se realizó ANOVA y Bonferroni (diferencias significativas, p<0,05). El SM se confirmó por la ganancia de peso corporal (g) (127,0±6,8 vs 38,3±5,4;), perímetro de cintura (cm) (20,7±0,2 vs 16,4±0,2), glucemia (mg/dL) (221,6±13,3 vs 143,0±13,0), trigliceridemia (392,7±85,7 vs 86,9±21,9) y disminución de HDL-c (25,0±1,1 vs 34,8±0,6). Qc normalizó los parámetros bioquímicos. La testosterona sérica fue similar en todos los grupos. El SM produjo depleción de GSH (nmol/mg prot.) (31,7±1,4 vs 45,1±0,5) y aumento de CAT (UI/mg prot.) (16,1±0,6 vs 9,8±1,6) testicular. Qc restauró parcialmente los niveles del tripéptido y normalizó la actividad enzimática. El área de las secciones testiculares y el espesor de la capa celular en los túbulos seminíferos fueron menor en el grupo SM. En el tiempo de estudio, todos los grupos presentaron una espermatogénesis completa, igual concentración y motilidad de espermatozoides. Los resultados sugieren que el SM produce EO y altera el tejido testicular. A pesar que en el tiempo de desarrollo del SM se produce espermatogénesis completa, el EO podría ser el mecanismo por el cual a tiempos más prolongados se afectaría la fertilidad. Qc normaliza el estado redox testicular y mejora las condiciones generales del SM, lo que podría evitar el desarrollo de la subfertilidad masculina. &nbsp