RESUMO
SUMMARY: The aim of this study was to determine the relationship of autophagy-enhancing rapamycin (RAPA) and autophagy- inhibitor 3-methyladenine (3-MA) with Nitric oxide synthases (NOS) in Cisplatin (CIS)-induced neurotoxicity in rats. Rats were divided into 4 groups (n=10): Control was applied saline, CIS (a single dose of 8mg/kg intraperitoneal (i.p.) on 7th day of experiment), RAPA+CIS (2 mg/kg/i.p. RAPA per day and 8 mg/kg/i.p. CIS on 7th day), 3-MA+CIS (15 mg/kg/i.p. 3-MA per day and 8 mg/kg/i.p. CIS on 7th day). Rats were sacrificed under anesthesia. Brain tissues were evaluated histopathologically. eNOS, Inos, nNOS and MAP 2 immunostaining were performed to determine the expression levels of these proteins among groups. Superoxide dismutase (SOD), Catalase (CAT), Malondialdehyde (MDA) and Interleukin IL-6 levels in brain tissue and serum nitric oxide (NO) level were measured by ELISA assay. In histopathological evaluation, neurodegeneration was seen in the CIS group. There was an increase in eNOS, iNOS and nNOS immunostaining in CIS group. While MAP2 immunostaining of the CIS group decreased. There was a decrease in SOD and CAT levels of brain tissue in CIS group. However, there was an increase in MDA, IL-6 and NO levels of brain tissue in CIS group. We found that antioxidant capacity increase while, inflammation and nitric oxide levels decreased in the RAPA-treated group. 3-MA does not have a significant effect. We suggest that CIS-induced neurotoxicity is more effective than Rapa 3-MA and may also be linked to NOS enzymes.
RESUMEN: El objetivo de este estudio fue determinar la relación de la rapamicina potenciadora de la autofagia (RAPA) y el inhibidor de la autofagia 3-metiladenina (3-MA) con óxido nítrico sintasas (NOS) en la neurotoxicidad inducida por cisplatino (CIS) en ratas. Las ratas se dividieron en 4 grupos (n = 10): grupo control se aplicó solución salina, CIS (una dosis única de 8 mg / kg intraperitoneal (ip) el día 7 del experimento), RAPA + CIS (2 mg / kg / ipRAPA por día y 8 mg / kg / ip CIS el día 7), 3-MA + CIS (15 mg / kg / ip 3-MA por día y 8 mg / kg / ip CIS el día 7). Las ratas se sacrificaron bajo anestesia y los tejidos cerebrales fueron analizados histopatológicamente. Se realizaron inmunotinciones con eNOS, Inos, nNOS y MAP 2 para determinar los niveles de expre- sión de estas proteínas entre los grupos. Se midieron los niveles de superóxido dismutasa (SOD), catalasa (CAT), malondialdehído (MDA) e interleucina IL-6 en el tejido cerebral y el nivel de óxido nítrico (NO) en suero mediante ensayo ELISA. En la evaluación histopatológica, se observó neurodegeneración en el grupo CIS. Hubo un aumento en la inmunotinción de eNOS, iNOS y nNOS en el grupo CIS. Mientras que la inmunotinción de MAP2 del grupo CIS disminuyó. Hubo una disminución en los niveles de SOD y CAT del tejido cerebral en el grupo CIS, sin embargo, hubo un aumento en los niveles de MDA, IL-6 y NO en el tejido cerebral en el grupo CIS. Observamos que la capacidad antioxidante aumentó, mientras que la inflamación y los niveles de óxido nítrico disminuyeron en el grupo tratado con RAPA. 3-MA no tiene un efecto significativo. Sugerimos que la neurotoxicidad inducida por CIS es más eficaz que Rapa 3-MA y también puede estar relacio- nada con las enzimas NOS.
Assuntos
Animais , Masculino , Ratos , Adenina/análogos & derivados , Cisplatino/toxicidade , Óxido Nítrico Sintase/efeitos dos fármacos , Sirolimo/farmacologia , Síndromes Neurotóxicas , Superóxido Dismutase , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Adenina/farmacologia , Catalase , Interleucina-6 , Ratos Wistar , Malondialdeído , Antineoplásicos/toxicidadeRESUMO
Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.
Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.
Assuntos
Animais , Camundongos , Doença de Parkinson , Rotenona/toxicidade , Adenina/análogos & derivados , Autofagia , Mesencéfalo , Adenina/farmacologia , Células Cultivadas , Morte Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , L-Lactato Desidrogenase/análiseRESUMO
Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-ULK1 and MAPK14/p38 and the sustained inhibition of AKT-MTOR. This produced a transient induction of autophagy, which was followed by senescence. However, at the single cell level, this coordinated transition was not observed, and autophagy and senescence were triggered in a very heterogeneous manner. Indeed, at a population level, autophagy was highly negatively correlated with senescence markers, while in single cells this correlation did not exist. The inhibition of autophagy triggered apoptosis and decreased senescence, while its activation increased temozolomide-induced senescence, showing that DNA damage-induced autophagy acts by suppressing apoptosis.
Assuntos
Autofagia , Senescência Celular , Dano ao DNA , Análise de Célula Única/métodos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Glioma/enzimologia , Glioma/patologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Temozolomida , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to ß-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.