Ergotioneína como alternativa terapêutica: efeitos antioxidantes e/ou moduladores em células eritroleucêmicas K562
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Data
2021-04-07
Autores
Bernardo, Victoria Simões
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Universidade Estadual Paulista (Unesp)
Resumo
Os eritrócitos são células que apresentam um ambiente interno extremamente pró-oxidante, propenso a constante formação de espécies reativas de oxigênio (ERO) devido a seu papel fisiológico como transportador de oxigênio. Além desse ambiente interno oxidante, fontes extracelulares também contribuem para o estresse oxidativo constante dessas células devido à alta permeabilidade de ERO apresentada pela membrana eritrocitária. Em contrapartida, essas células possuem um sistema de regulação redox altamente eficiente e estritamente comprometido em manter a integridade e funcionalidade celular após a completa maturação, devido a capacidade metabólica limitada apresentada por essas células (ausência de núcleo e organelas). A ergotioneína (ERT) é um antioxidante natural que vem ganhando destaque na comunidade científica como uma potencial alternativa terapêutica no tratamento de diferentes doenças hematológicas humanas com quadros clínicos caracterizados por estresse oxidativo e inflamação crônicos. Isto, pois seu uso apresenta uma série de vantagens, dentre as quais podemos citar a i) existência de transportador altamente específico para sua captação, em diversos tipos celulares, inclusive em células eritróides; ii) alta biodisponibilidade, resultante da alta retenção e baixa excreção; iii) sua capacidade de detoxificar uma série de compostos oxidantes; e iv) não apresentar toxicidade ou efeitos indesejado associados à sua administração, mesmo em altas doses. Neste contexto, o presente estudo objetivou investigar os possíveis efeitos antioxidantes e/ou moduladores da ERT sobre os mecanismos celulares de adaptação redox indispensáveis para homeostase em células eritróides submetidas ao estresse oxidativo, baseados nas vias de sinalização em resposta ao estresse oxidativo, PI3K/AKT/FOXO3 e Keap1/Nrf2/ARE; utilizando células eritroleucêmica K562 sob indução de diferenciação eritróide como modelo experimental. Para tanto, foram avaliados a viabilidade celular e os níveis de transcritos dos fatores de transcrição e seus moduladores supramencionados, bem como de importantes antioxidantes envolvidos na adaptação redox. Assim, o estudo contou com três amostras pseudoreplicadas acompanhadas durante cinco dias de diferenciação celular, com avaliações em três períodos do processo de diferenciação: antes do início (D0), no início de diferenciação (D2) e máximo da diferenciação (D4). Além disso, cada amostra foi dividida nos seguintes grupos: células eritróides sem indução de estresse oxidativo e tratamento antioxidante (Referência); células sob indução de estresse com peróxido de hidrogênio (100 µM H2O2); células tratadas com 1 nM (C1) e com 100 µM (C2) de ERT; por fim, dois conjuntos de células tratados com as mesmas concentrações de ERT associados com a indução do estresse (C1+ H2O2 e C2+ H2O2, respectivamente). Dentre os resultados obtidos, destaca-se o tratamento com a menor concentração de ERT (C1) que se associou com a indução da expressão do FOXO3; já na concentração C2 houve uma redução dos níveis de transcritos no início do processo de diferenciação, se mantendo baixa até o final do experimento. Também foi observada que a menor concentração de ERT testada, na presença do agente estressor, apresentou os maiores níveis de transcritos de Nrf2 (todos os períodos), Keap1 (dia 0) e 14-3-3 (dia 2 e 4), enquanto, no tratamento ERT C2 + H2O2, no dia 2, observou-se um aumento de FOXO3 e MST1 e uma diminuição da 14-3-3 e Nrf2. As análises multivariadas destacaram que a via Keap1/Nrf2/ARE apresentou maior contribuição com o padrão de expressão de PRDX1, SOD1 e CAT, enquanto a via MST1-FOXO3, com PRDX2 e Trx. Conclui-se que ERT apresentou uma ação citoprotetora em células eritróides K562, através da ativação dos fatores de transcrição FOXO3 e Nrf2, sendo que a via de sinalização redox Nrf2-ARE mostrou-se como a principal envolvida na homeostase redox, enquanto a via MST1-FOXO3 parece estar envolvida tanto na homeostase redox quanto na proliferação e diferenciação eritróide. É válido ressaltar que os resultados possibilitaram a proposição e/ou elucidação de um mecanismo de ação das vias estudadas em células eritróides, dependente da concentração de ERT administrada.
Erythrocytes are cells that present an extremely pro-oxidant internal environment, prone to the constant formation of reactive oxygen species (ROS) due to their physiological role as oxygen transporter. Besides this oxidizing internal environment, extracellular sources also contribute to the constant oxidative damage of these cells due to the high permeability of ROS presented by the erythrocyte membrane. Because of the mentioned above, these cells have a highly efficient redox regulation system and are strictly committed to maintaining cell integrity and functionality after complete maturation, due to the limited metabolic capacity presented by these cells (absence of nucleus and organelles). Ergothioneine (ERT) is a natural antioxidant that has been gaining notability in the scientific community as a potencial therapeutic alternative in the treatment of different human hematological diseases with clinical conditions characterized by oxidative stress and chronic inflammation. Due to a series of advantages of its use, as i) existence of a highly specific transporter for its uptake, in several cell types, including erythroid cells; ii) high bioavailability, resulting from high retention and low excretion; iii) ability to detoxify a series of oxidizing compounds; and iv) does not present toxicity or adverse effects associated with its administration, even in high doses. In this context, the present study aimed to investigate the possible antioxidant and/or modulating effects of ERT on the cellular redox adaptation mechanisms indispensable for homeostasis in erythroid cells subjected to oxidative damage, based on the signaling pathways in response to oxidative stress, PI3K/AKT/ FOXO3, and Keap1/Nrf2/ARE; using erythroleukemic K562 cells under the induction of erythroid differentiation as an experimental model. For that, the cell viability, and the levels of transcripts of the transcription factors and their aforementioned modulators were evaluated, as well as of important antioxidants involved in redox adaptation. Thus, the study had three pseudo-replicated samples followed for five days of cell differentiation, with assessments in three periods of the differentiation process: before the start (D0), at the beginning of differentiation (D2), and maximum differentiation (D4). Also, each sample was divided into the following groups: erythroid cells without inducing oxidative damage and antioxidant treatment (Reference); cells under stress induction with hydrogen peroxide (100 µM H2O2); cells treated with 1 nM (C1) and 100 µM (C2) of ERT; finally, two sets of cells treated with the same concentrations of ERT associated with stress induction (C1 + H2O2 and C2 + H2O2, respectively). Among the main results, we can highlight that the treatment with the lowest concentration of ERT (C1) promoted the induction of FOXO3 expression, whereas in the C2 concentration there was a reduction in the levels of transcripts at the beginning of the differentiation process, remaining low until the end of the experiment. It was also observed that the lowest concentration of ERT, tested with the stressor, showed the highest levels of Nrf2 transcripts (every day), Keap1 (day 0), and 14-3-3 (day 2 and 4). Meanwhile, in the ERT C2 + H2O2 treatment, on day 2, was observed an increase in FOXO3 and MST1, concomitantly with a decrease in 14-3-3 and Nrf2. Multivariate analyzes of the degree of association between the transcript levels of the antioxidant enzyme genes, highlights that the Keap1 / Nrf2 / ARE pathway presented a greater contribution or involvement in the production of PRDX1, SOD1 and CAT mRNAs and that the MST1-FOXO3 pathway seems to be more associated with the PRDX2 and Trx transcripts in K562 erythroid cells. In conclusion, ERT presented a cytoprotective action on K562 erythroid cells, through the activation of FOXO3 and Nrf2 transcription factors, with the redox signaling pathway Nrf2-ARE being the main one involved in redox homeostasis, while FOXO3 is involved in both homeostasis redox and erythroid proliferation and differentiation. It is noteworthy that those results made it possible to propose a mechanism of redox adaptation of the pathways studied, in an ERT concentration-dependent manner.
Erythrocytes are cells that present an extremely pro-oxidant internal environment, prone to the constant formation of reactive oxygen species (ROS) due to their physiological role as oxygen transporter. Besides this oxidizing internal environment, extracellular sources also contribute to the constant oxidative damage of these cells due to the high permeability of ROS presented by the erythrocyte membrane. Because of the mentioned above, these cells have a highly efficient redox regulation system and are strictly committed to maintaining cell integrity and functionality after complete maturation, due to the limited metabolic capacity presented by these cells (absence of nucleus and organelles). Ergothioneine (ERT) is a natural antioxidant that has been gaining notability in the scientific community as a potencial therapeutic alternative in the treatment of different human hematological diseases with clinical conditions characterized by oxidative stress and chronic inflammation. Due to a series of advantages of its use, as i) existence of a highly specific transporter for its uptake, in several cell types, including erythroid cells; ii) high bioavailability, resulting from high retention and low excretion; iii) ability to detoxify a series of oxidizing compounds; and iv) does not present toxicity or adverse effects associated with its administration, even in high doses. In this context, the present study aimed to investigate the possible antioxidant and/or modulating effects of ERT on the cellular redox adaptation mechanisms indispensable for homeostasis in erythroid cells subjected to oxidative damage, based on the signaling pathways in response to oxidative stress, PI3K/AKT/ FOXO3, and Keap1/Nrf2/ARE; using erythroleukemic K562 cells under the induction of erythroid differentiation as an experimental model. For that, the cell viability, and the levels of transcripts of the transcription factors and their aforementioned modulators were evaluated, as well as of important antioxidants involved in redox adaptation. Thus, the study had three pseudo-replicated samples followed for five days of cell differentiation, with assessments in three periods of the differentiation process: before the start (D0), at the beginning of differentiation (D2), and maximum differentiation (D4). Also, each sample was divided into the following groups: erythroid cells without inducing oxidative damage and antioxidant treatment (Reference); cells under stress induction with hydrogen peroxide (100 µM H2O2); cells treated with 1 nM (C1) and 100 µM (C2) of ERT; finally, two sets of cells treated with the same concentrations of ERT associated with stress induction (C1 + H2O2 and C2 + H2O2, respectively). Among the main results, we can highlight that the treatment with the lowest concentration of ERT (C1) promoted the induction of FOXO3 expression, whereas in the C2 concentration there was a reduction in the levels of transcripts at the beginning of the differentiation process, remaining low until the end of the experiment. It was also observed that the lowest concentration of ERT, tested with the stressor, showed the highest levels of Nrf2 transcripts (every day), Keap1 (day 0), and 14-3-3 (day 2 and 4). Meanwhile, in the ERT C2 + H2O2 treatment, on day 2, was observed an increase in FOXO3 and MST1, concomitantly with a decrease in 14-3-3 and Nrf2. Multivariate analyzes of the degree of association between the transcript levels of the antioxidant enzyme genes, highlights that the Keap1 / Nrf2 / ARE pathway presented a greater contribution or involvement in the production of PRDX1, SOD1 and CAT mRNAs and that the MST1-FOXO3 pathway seems to be more associated with the PRDX2 and Trx transcripts in K562 erythroid cells. In conclusion, ERT presented a cytoprotective action on K562 erythroid cells, through the activation of FOXO3 and Nrf2 transcription factors, with the redox signaling pathway Nrf2-ARE being the main one involved in redox homeostasis, while FOXO3 is involved in both homeostasis redox and erythroid proliferation and differentiation. It is noteworthy that those results made it possible to propose a mechanism of redox adaptation of the pathways studied, in an ERT concentration-dependent manner.
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Genética humana e molecular, Antioxidantes, Metabolismo regulação, Human and molecular genetics, Antioxidants, Regulation metabolism