Search

Resumo em Galego disponível nas páginas 35 a 42 Neurological diseases are currently one of the main medical and socioeconomic problems, due to the high morbidity and mortality rate affecting millions of people worldwide. Epilepsy is a chronic neurological disorder, characterized by excessive and abnormal neural discharges into the brain, leading to spontaneous and recurrent crisis episodes. Parkinson's disease (PD) is considered a neurodegenerative, chronic, and progressive disease of the central nervous system (CNS) caused by a marked and gradual decrease in dopamine (DA) production in the nigroestric pathway. It was recently discovered that epileptic seizures are related to cognitive decline and that epilepsy and some neurodegenerative diseases, such as PD, have intrinsically related aetiologies. Both share similar pathological mechanisms, such as neurotransmitter dysregulation, excitotoxicity, mitochondrial dysfunction, increased pro-inflammatory cytokines and pro-apoptotic proteins, among others. Due to the complex etiology of these diseases, today research is being carried out on the development of new drugs that are capable of acting on multiple therapeutic targets. Gamma-decanolactone (GD) is a monoterpenic compound that has been shown in previous studies to have neuroprotective activity in vivo and in vitro. The present doctoral thesis aims to study the neuropharmacological aspects and the mechanism of action of GD in experimental models of epilepsy and PD, to contribute to a greater understanding of GD and its possible clinical application. In this thesis, in silico, in vitro, in vivo and ex vivo studies have been carried out. In silico studies include physicochemical properties, the ability to cross the blood-brain barrier (BBB), and the ability to bind to MAO isoforms. Male albino mice "Mus Musculus" CF-1 or Charles River "CD1-Swiss" with 2-3 months of age and weighing 25 ± 5 grams were used to carry out the in vivo experiments. To evaluate its anticonvulsant properties, the possible action of GD (300 mg/kg) in the purinergic and GABAergic systems was studied, using the models of acute seizure induced by aminophylline (AMINOPH) and that of subchronic seizure (Kindling) induced by pentylenetetrazole (PTZ). To make this research possible, we used two adenosine antagonists, DPCPX (A1 receptor antagonist) and ZM241385 (A2A receptor antagonist) and a GABAergic antagonist, bicuculin (GABAA receptor antagonist). The expression of proteins of the GABAA α1 receptor and the adenosine A1 receptor was evaluated by means of the Western blotting technique in hippocampal samples of mice subjected to the Kindling model. In addition, studies of binding to adenosine A1, A2A A2B and A3 receptors were also performed. To evaluate its possible antiparkinsonian properties, the effect of GD in vitro to inhibit isoforms A and B of the human monoamine oxidase enzyme (hMAO) and its cytotoxicity in the human hepatoma cell line (HepG2) was studied. These in vitro studies were completed with in vivo tests, where motor coordination (Rota-rod test), modification of body temperature and locomotor activity were evaluated in an Open Field Test (OFT). These in vivo tests were performed in reserpinized (1.5 mg/kg, 18:00 before) or non-reserpinized mice, to evaluate the antiparkinsonian effect of GD (300 mg/kg), alone or in combination with levodopa plus benserazide (LD+BZ, 100:25 mg/kg). In the in silico studies, the results show that GD has adequate physicochemical properties and the ability to cross BBB. The results obtained in the evaluation of the anticonvulsant activity show that GD protected the mice against acute seizures induced by AMINOPH and against subchronic seizures induced by PTZ. The adenosine A2A receptor antagonist (ZM241385) and the GABAA receptor antagonist (bicuculin) did not modify the behavior of GD. Administration of the adenosine A1 receptor antagonist (DPCPX) reversed the protective effect of GD in the two models studied. Furthermore, GD promoted an increase in the expression of the α1 subunit of the GABAA receptor in the hippocampus of mice subjected to the Kindling model. GD shows practically no affinity for adenosine receptors. In the results of the evaluation of GD as antiparkinsonian, it was observed that GD preferentially inhibited MAO-B in a partially reversible way, with an IC50 of 55.95 ± 9.06 μM. It is also a safe compound, since only at the highest concentration (100 μM) did the viability of HepG2 cells decrease. Finally, in the in vivo studies, GD partially reversed ataxia in reserpinized mice. Concomitant treatment of GD with LD+BZ showed potentiation of ataxia reversal and also facilitated reversal of hypothermia caused by reserpine for all times measured (p < 0.01 vs. vehicle), except at 24:00 h. In contrast, GD did not reverse hypokinesia in the OFT. Las enfermedades neurológicas son actualmente uno de los principales problemas médicos y socioeconómicos, debido a la alta tasa de morbilidad y mortalidad que afectan a cientos de millones de personas en todo el mundo. La epilepsia es un trastorno neurológico crónico, caracterizado por descargas neuronales excesivas y anormales en el cerebro, que conducen a episodios de crisis epilépticas espontáneas y recurrentes. La enfermedad de Parkinson (EP) es considerada una enfermedad neurodegenerativa, crónica y progresiva del sistema nervioso central (SNC) causada por una disminución marcada y gradual en la producción de dopamina (DA) en la vía nigroestriada. Recientemente se descubrió que las crisis epilépticas están relacionadas con el deterioro cognitivo y que la epilepsia y algunas enfermedades neurodegenerativas, como la EP, tienen etiologías que están intrínsecamente relacionadas. Ambas comparten mecanismos patológicos similares, como la desregulación de neurotransmisores, la excitotoxicidad, la disfunción mitocondrial, el aumento de las citocinas proinflamatorias y de las proteínas pro-apoptóticas, entre otros. Debido a la compleja etiología de estas enfermedades, hoy se investiga en el desarrollo de nuevos fármacos que sean capaces de actuar sobre múltiples dianas terapéuticas. La gamma-decanolactona (GD) es un compuesto monoterpénico que en estudios previos demostró tener actividad neuroprotectora in vivo e in vitro. La presente tesis doctoral tiene como objetivo estudiar los aspectos neurofarmacológicos y el mecanismo de acción de la GD en modelos experimentales de epilepsia y de la EP, para contribuir a un mayor conocimiento de la GD y su posible aplicación en clínica. En esta tesis se han llevado a cabo estudios in silico, in vitro, in vivo y ex vivo. Los estudios in silico incluyen las propiedades fisicoquímicas, la capacidad para atravesar la barrera hematoencefálica (BHE) y la capacidad de fijación a las isoformas de la MAO. Para la realización de los experimentos in vivo se utilizaron ratones albinos machos "Mus Musculus" CF-1 o Charles River "CD1-Swiss" con 2-3 meses de edad y un peso de 25 ± 5 gramos. Para evaluar sus propiedades anticonvulsivas, se estudió la posible actuación de la GD (300 mg/kg) en los sistemas purinérgico y GABAérgico, utilizando los modelos de crisis aguda inducida por la aminofilina (AMINOPH) y el de crisis subcrónicas (Kindling) inducidas por el pentilentetrazol (PTZ). Para hacer posible esta investigación, utilizamos dos antagonistas de adenosina, el DPCPX (antagonista del receptor A1) y el ZM241385 (antagonista del receptor A2A) y un antagonista GABAérgico, la bicuculina (antagonista del receptor GABAA). La expresión de proteínas del receptor GABAA α1 y del receptor de adenosina A1 se evaluó mediante la técnica de Western blotting en muestras de hipocampo de ratones sometidos al modelo de Kindling. Además, también se realizaron estudios de unión a los receptores de adenosina A1, A2A A2B y A3. Para evaluar sus posibles propiedades antiparkinsonianas, se estudió el efecto de la GD in vitro para inhibir las isoformas A y B de la enzima monoamino oxidasa humana (hMAO) y su citotoxicidad en la línea celular de hepatoma humano (HepG2). Estos estudios in vitro se completaron con los ensayos in vivo, donde se evaluó la coordinación motora (prueba de Rota-rod), la modificación de la temperatura corporal y la actividad locomotora en una prueba de campo abierto. Estos ensayos in vivo se realizaron en ratones reserpinizados (1,5 mg/kg, 18:00 h antes) o no reserpinizados, para evaluar el efecto antiparkinsoniano de la GD (300 mg/kg), sola o asociada con levodopa más benserazida (LD+BZ, 100:25 mg/kg). En los estudios in silico los resultados ponen de manifiesto que la GD presenta unas adecuadas propiedades fisicoquímicas, capacidad para cruzar la BHE y para unirse de forma preferente a la isoforma B de la MAO. Los resultados obtenidos en la evaluación de la actividad anticonvulsivante muestran que la GD protegió a los ratones contra las crisis agudas inducidas por AMINOPH y contra las crisis subcrónicas inducidas por PTZ. El antagonista del receptor de adenosina A2A (ZM241385) y el antagonista del receptor del GABAA (bicuculina) no modificaron el comportamiento de la GD. La administración del antagonista del receptor de adenosina A1 (DPCPX) revirtió el efecto protector de la GD en los dos modelos estudiados. Además, la GD promovió un aumento en la expresión de la subunidad α1 del receptor GABAA, en el hipocampo de los ratones sometidos al modelo de Kindling. La GD prácticamente no muestra afinidad por los receptores de adenosina. En los resultados de la evaluación de la GD como antiparkinsoniana se apreció que esta inhibió preferentemente la MAO-B de forma parcialmente reversible, con una CI50 55,95 ± 9,06 μM. También es un compuesto seguro, ya que solamente a la concentración más alta (100 μM) disminuyó la viabilidad de las células HepG2. Finalmente, en los estudios in vivo, la GD revertió parcialmente la ataxia de los ratones reserpinizados. El tratamiento concomitante de la GD con la LD+BZ mostró una potenciación de la reversión de la ataxia y también facilitó la reversión de la hipotermia causada por la reserpina para todos los tiempos medidos (p < 0.01 vs. vehículo), excepto a las 24:00 h. Por el contrario, la GD no revirtió la hipocinesia en el ensayo de campo abierto.

Gama-decanolactona (GD) é um composto monoterpeno com estrutura semelhante às lactonas naturais, que estão presentes em diferentes espécies e são usadas como terapia farmacológica na região amazônica. GD mostrou um efeito anticonvulsivo em ambos os modelos de pentilenotetrazol (PTZ) agudo e crônico e atua como um antagonista de glutamato não-competitivo. Considerando estudos anteriores sobre atividades biológicas de GD, o presente estudo teve como objetivo explorar o seu perfil anticonvulsivante em diferentes modelos de epilepsia em camundongos e sobre parâmetros de estresse oxidativo, neuroinflamação e genotoxicidade em células microgliais N9. A atividade anticonvulsiva de GD (100 e 300 mg/kg) foi investigada em convulsões induzidas por aminofilina, isoniazida, picrotoxina, 4-aminopiridina (4-AP) e pilocarpina em camundongos machos. Os animais receberam uma administração de solução salina (SAL), GD ou diazepam (DZP 2 mg/kg), de controle positivo e após 30 min receberam os seguintes agentes convulsivos: aminofilina, isoniazida, picrotoxina, 4-AP ou pilocarpina. Os parâmetros avaliados durante 1h foram a latência para a primeira convulsão, a porcentagem de convulsões e a taxa de mortalidade. A fim de avaliar a neurotoxicidade de GD 100 e 300 mg/kg, foi realizado o teste de desempenho de rotarod. O tempo para cair do rotarod foi gravado em diferentes momentos após a administração GD. E por último foi avaliado o desempenho de GD 100 e 300 mg/kg no teste de sono induzido por DZP (17 mg/kg). Os resultados demonstraram que GD foi capaz de aumentar a latência para a primeira convulsão induzida pela aminofilina, isoniazida, 4-AP e pilocarpina, mas não para picrotoxina. No teste de rotarod, GD 300 mg/kg reduziu o tempo de latência para cair da barra após 30 min de administração, mas não após 60, 90 ou 120 min. GD 300 mg/kg aumentou a latência do sono induzido pro DZP. Os resultados acima revelaram que GD apresentou um efeito anticonvulsivo nos modelos de epilepsia utilizados neste estudo, principalmente na dose de 300 mg/kg, bem como o tempo de latência para a primeira convulsão, sugerindo que GD pode modular as vias da adenosina e afetar os canais de potássio, direta ou indiretamente. O papel de GD sobre o estresse oxidativo em status epilepticus (SE) foi investigado medindo a produção de espécies reativas de oxigênio (EROS), superóxido dismutase (SOD), catalase (CAT), teor de nitrito, e os danos do DNA induzidos por pilocarpina no córtex cerebral de camundongos. GD foi capaz de aumentar as atividades de SOD e CAT, diminuir a produção de EROS, NO e danos no DNA durante o estabelecimento de SE no córtex cerebral. Estes dados sugerem que as enzimas SOD e CAT são o principal sistema de eliminação de radicais livres, e GD fornece neuroproteção contra o aumento do estresse oxidativo no cérebro. Um modelo clássico para investigar se uma droga atua modulando a neuroinflamação é utilizar a linhagem celular N9 (célula neuronal da microglia murina) ativada com o químico lipopolissacárideo (LPS), que sabidamente, provoca inflamação. Assim, investigou-se o efeito da GD sobre parâmetros inflamatórios e apoptóticos nesse sistema. Avaliou-se a expressão protéica por western blotting das seguintes proteínas: óxido nítrico sintetase induzível (iNOS) e fator de necrose tumoral-alpha (TNF-α), p-38 fosforilada e não fosforilada. Avaliou-se também a atividade da caspase 9-clivada e a formação de EROS por citometria de fluxo bem como o dano ao DNA pelo ensaio cometa alcalino. Nos resultados in vitro, GD atenuou a ativação de células N9 microglias, inibiu a formação de EROS intracelular e a expressão de iNOS e TNF-α induzidas por LPS nas células. Além disso, GD bloqueou a fosforilação da p38, inibiu a caspase-9 clivada e o dano ao DNA. Estes dados indicam que GD tem um potencial terapêutico neuroprotetor e que exerce os seus efeitos através da inibição da inflamação. Gamma-decanolactone (GD) is a monoterpene compound similar to natural lactones in structure, which are present in different species and are used as remedy in the Amazonian region. GD shows an anticonvulsant effect in both the acute and chronic pentileneterazole (PTZ) models and acts as a non-competitive glutamate antagonist. Considering previous studies on biological activities of GD, the present study aimed to explore its anticonvulsant profile in different epilepsy models in mice and on oxidative stress parameters in N9 cell. The anticonvulsant activity of GD (100 and 300 mg/kg) was investigated on seizures induced by aminophylline (AMPH), isoniazid (INH), picrotoxin (PCT), 4-aminopyridine (4-AP) an pilocarpine (PIL) in male mice. The animals received one administration of saline (SAL), GD or the positive control diazepam (DZP 2 mg/kg), and after 30 min they received the following convulsant agents: AMPH, INH, PCT, 4-AP or PIL The parameters evaluated during 1h were the latency to first seizure, the occurrence of seizure and the mortality rate. In order to evaluate the neurotoxicity of GD 100 and 300 mg/kg, the rotarod performance test was performed. The time to fall of the rotarod was recorded at different times after the GD administration. The results demonstrated that GD was able to extend the latency to first seizure induced by AMPH, INH, 4-AP and PIL, but not PCT. In the rotarod test GD 300mg/kg reduced the latency to fall of the bar just at 30 min after the administration, but not at 60, 90 or 120 min. The above results revealed that GD presented an anticonvulsant effect in the epilepsy models used in this study, especially at the dosage of 300mg/kg, as well as the latency to the first seizure, suggesting that the GD could modulate the adenosine and GABA pathways and affect potassium channels directly or indirectly. The role of gamma-decanolactone (GD) on oxidative stress in pilocarpine-induced SE was investigated by measuring ROS production, superoxide dismutase and catalase activities, nitrite content, and DNA damage in the mice cerebral cortex. GD was able to increase the superoxide dismutase (SOD) and catalase (CAT) activities, decreased the ROS, NO production, and DNA damage during the SE establishment in the cerebral cortex. These data suggest that the cortex use SOD and CAT enzymes as the major free-radicals scavenging system, and GD provides neuroprotection against the increase of oxidative stress in the brain. Because previous works have consistently demonstrated the N9 microglial-neuronal system for neuroinflammation investigations, these cells are considered appropriate for this kind of research. To investigate the inhibitory effect of GD on the production of ROS and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) - stimulated N9 murine microglial cells through the p38 MAPK signaling pathway. The results in vitro the GD attenuated the activation of N9 cells and inhibited intracellular ROS and the expression of iNOS and TNF-α induced by LPS in the cells. In addition, GD blocked the phosphorylation of p38 and inhibited cleaved caspase-9 and DNA damage. These data indicate that GD has therapeutic potential for the treatment of neurodegenerative diseases, and that it exerts its effects by inhibiting inflammation.