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Evaluation of the H2S-release and cardioprotective effect exhibited by a natural H2S-donor in H9c2 cells
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As doenças cardiovasculares são a principal causa de morte no mundo, verificando-se um aumento do número de casos ao longo dos últimos 25 anos na Europa. Deste grupo fazem parte a hipertensão, angina de peito e enfarte do miocárdio. Hipertensão é a doença crónica com maior distribuição mundial, afetando cerca de 1,13 biliões de pessoas e a principal causa de morte prematura em todo o mundo. Fatores como o tabagismo, consumo de álcool e colesterol alto continuam a ser os principais fatores de risco para doenças cardiovasculares. No entanto, tem-se verificado um aumento de outros fatores relevantes tais como a prevalência de excesso de peso e diabetes causados predominantemente pelo aumento da esperança média de vida e do sedentarismo das populações. Apesar das estratégias farmacológicas e não farmacológicas existentes atualmente serem numerosas e variadas, combater os sintomas que advêm de doenças cardiovasculares continua a ser um desafio. A descoberta da molécula sulfido de hidrogénio (H2S), um transmissor gasoso endógeno, bem como a sua capacidade de agir como um mediador vasorelaxante e o facto de ser um regulador essencial da homeostase cardiovascular, permitiu que este se tornasse num potencial candidato a um inovador medicamento anti-hipertensivo. As plantas comestíveis da família Brassicaceae, tais como os brócolos e a couve-flor, contêm elevadas quantidades de isotiocianatos (ITC) e são conhecidos pelo seu poder anti-inflamatório natural. De facto, a administração de H2S através de dadores adequados pode ser considerada uma estratégia inovadora e útil no tratamento farmacológico de doenças cardiovasculares. Em vários estudos científicos, diferentes tipos de dadores de H2S exibiram atividade vasorelaxante e capacidade de diminuir a pressão arterial. No entanto, um dador de H2S ideal para aplicação clínica deve obedecer a alguns critérios, conseguindo combinar uma libertação de H2S lenta e duradoura, bem como ser dotado de características farmacológicas indispensáveis, tais como uma boa biodisponibilidade oral, segurança e metabolização em produtos inofensivos para o organismo. Serviu a presente investigação para avaliar, em condições in vitro, o potencial de libertação de H2S de um dador de H2S natural e testar as suas propriedades protetoras em células mioblásticas derivadas do tecido cardíaco de ratos. O primeiro segmento experimental teve como objetivo investigar as propriedades de libertação de H2S de um dador natural de H2S no interior de células H9c2, recorrendo a um corante fluorescente (WSP-1). O dador natural de H2S em estudo foi testado em três concentrações diferentes: 30 µM, 100 µM e 300 µM e a quantidade de H2S produzida por este composto foi posteriormente comparada com a que se origina na presença de apenas veículo e de um dador conhecido de H2S, denominado DADS, numa concentração de 300 µM. Observámos que todas as diferentes concentrações do dador natural de H2S foram capazes de libertar H2S em células H9c2. No entanto, registou-se um aumento significativo de libertação de H2S na presença do dador testado na concentração de 300 µM, quando comparada com as restantes concentrações. Tal indica a existência de uma correlação entre a libertação de H2S em células H9c2 e o aumento de concentração do dador de H2S. De seguida, investigámos o efeito cardioprotetor do dador natural de H2S contra o stress oxidativo induzido por peróxido de hidrogénio, H2O2. Nesta experiência, células H9c2 foram tratadas com três concentrações diferentes de dador de H2S: 0,3 µM, 1 µM e 3 µM. e de seguida foram submetidas a uma lesão causada por H2O2. A viabilidade celular foi medida recorrendo a um corante tetrazólico (WST-1) e registada num espectrofluorímetro. O procedimento foi repetido em células H9c2 unicamente tratadas com veículo. Observámos que as células tratadas com o dador de H2S nas concentrações de 1 µM e 3 µM foram significativamente preservadas quando comparadas com as células tratadas com o veículo. Deste modo, podemos afirmar que o dador natural de H2S testado parece proteger as células H9c2 contra lesões oxidativas, podendo ser um potencial candidato a um novo fármaco no tratamento de doenças cardiovasculares. No entanto, realçamos a necessidade de realizar mais testes e modificações experimentais, de modo a melhor compreender o mecanismo de ação da substância H2S e dos seus dadores naturais pertencentes à família Brassicaceae.
Cardiovascular diseases are the primary cause of death in the world and these include diseases such as hypertension, myocardial infarction and pulmonary embolism. The leading risk factors for the development of these diseases are aging, smoking, alcohol consumption and also high cholesterol. Hypertension is the most widespread chronic disease and the main cause of life-threatening cardiovascular complications and it is the principal risk factor for premature mortality worldwide. In spite of the wide and heterogeneous choice of pharmacological interventions, a significant quote of hypertensive patients cannot achieve blood pressure control. The discovery of hydrogen sulfide (H2S) as an endogenous gasotransmitter, that acts as a vasorelaxing mediator and that is a pivotal regulator of the cardiovascular homeostasis, is proving to be a potential novel antihypertensive and cytoprotective drug candidate. The edible plants of Brassicaceae family, such as broccoli, cauliflower, contain a high amount of isothiocyanates (ITC), endowed with recognized healthy beneficial effects. In past years, these secondary metabolites were found to be exogenous sources of H2S and some of their beneficial effects were linked to their ability to release H2S in the presence of organic thiols. On these bases, the H2S supplementation by suitable H2S-donors can be considered a useful and innovative strategy for the pharmacological treatment of cardiovascular diseases. Different chemotypes of H2S-donors exhibited vasorelaxing activity and blood pressure lowering effects in several scientific reports. Nevertheless, ideal H2S-donor for clinical applications should combine well-balanced H2S-releasing properties such as a slow and long-lasting release, with other indispensable pharmacological “druggability” features, such as good oral bioavailability, safety, well-known biotransformation, safe metabolites, etc. The aim of this study was to evaluation the H2S-releasing ability of a natural H2S-donor and the H2S-mediated cardioprotective effects in H9c2 cells, which result from a myoblast cell line, derived from embryonic rat heart. In the first experiment, we investigated the H2S-releasing properties of a natural H2S-donor in three different concentrations (30 µM, 100 µM and 300 µM) in H9c2 cells. The procedure was repeated for cells with only the vehicle and the results were compared to the ones obtained with a well-known H2S-donor, DADS. We observed that, in all concentrations, the H2S-donor tested was able to release H2S inside H9c2 cells although, cells treated with the H2S-donor in a concentration of 300 µM showed a significant increase when compared with the others concentrations, indicating a dose-dependent response of H2S release in H9c2 cells. Next, we studied the cardioprotective effect of the natural H2S-donor in three different concentrations (0,3 µM, 1 µM and 3 µM) against oxidative stress induced by hydrogen peroxide, H2O2, in H9c2 cells The procedure was repeated with cells only treated with the vehicle and the cell viability was measured with a tetrazolic dye (WST-1) in a spectrofluorometer. We observed that the cells treated with the natural H2S-donor in concentrations of 1 µM and 3 µM were significantly preserved when compared with cells treated with the vehicle. Taken together, it seems that our natural H2S-donor may be a good candidate in becoming a novel antihypertensive and cytoprotective drug. However, further studies and experiments are necessary in order to elucidate the role of H2S and H2S donors belonging to the Brassicaceae family.
Cardiovascular diseases are the primary cause of death in the world and these include diseases such as hypertension, myocardial infarction and pulmonary embolism. The leading risk factors for the development of these diseases are aging, smoking, alcohol consumption and also high cholesterol. Hypertension is the most widespread chronic disease and the main cause of life-threatening cardiovascular complications and it is the principal risk factor for premature mortality worldwide. In spite of the wide and heterogeneous choice of pharmacological interventions, a significant quote of hypertensive patients cannot achieve blood pressure control. The discovery of hydrogen sulfide (H2S) as an endogenous gasotransmitter, that acts as a vasorelaxing mediator and that is a pivotal regulator of the cardiovascular homeostasis, is proving to be a potential novel antihypertensive and cytoprotective drug candidate. The edible plants of Brassicaceae family, such as broccoli, cauliflower, contain a high amount of isothiocyanates (ITC), endowed with recognized healthy beneficial effects. In past years, these secondary metabolites were found to be exogenous sources of H2S and some of their beneficial effects were linked to their ability to release H2S in the presence of organic thiols. On these bases, the H2S supplementation by suitable H2S-donors can be considered a useful and innovative strategy for the pharmacological treatment of cardiovascular diseases. Different chemotypes of H2S-donors exhibited vasorelaxing activity and blood pressure lowering effects in several scientific reports. Nevertheless, ideal H2S-donor for clinical applications should combine well-balanced H2S-releasing properties such as a slow and long-lasting release, with other indispensable pharmacological “druggability” features, such as good oral bioavailability, safety, well-known biotransformation, safe metabolites, etc. The aim of this study was to evaluation the H2S-releasing ability of a natural H2S-donor and the H2S-mediated cardioprotective effects in H9c2 cells, which result from a myoblast cell line, derived from embryonic rat heart. In the first experiment, we investigated the H2S-releasing properties of a natural H2S-donor in three different concentrations (30 µM, 100 µM and 300 µM) in H9c2 cells. The procedure was repeated for cells with only the vehicle and the results were compared to the ones obtained with a well-known H2S-donor, DADS. We observed that, in all concentrations, the H2S-donor tested was able to release H2S inside H9c2 cells although, cells treated with the H2S-donor in a concentration of 300 µM showed a significant increase when compared with the others concentrations, indicating a dose-dependent response of H2S release in H9c2 cells. Next, we studied the cardioprotective effect of the natural H2S-donor in three different concentrations (0,3 µM, 1 µM and 3 µM) against oxidative stress induced by hydrogen peroxide, H2O2, in H9c2 cells The procedure was repeated with cells only treated with the vehicle and the cell viability was measured with a tetrazolic dye (WST-1) in a spectrofluorometer. We observed that the cells treated with the natural H2S-donor in concentrations of 1 µM and 3 µM were significantly preserved when compared with cells treated with the vehicle. Taken together, it seems that our natural H2S-donor may be a good candidate in becoming a novel antihypertensive and cytoprotective drug. However, further studies and experiments are necessary in order to elucidate the role of H2S and H2S donors belonging to the Brassicaceae family.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019
Palavras-chave
Doenças cardiovasculares Hipertensão H2S, Dador de H2S Células H9c2 Mestrado Integrado - 2019