Efeito da ciclosporina a na expressão da sirtuína 1 e no estresse oxidativo/nitrosativo em células mesangiais imortalizadas de camundongos
Data
2024-10-29
Autores
Torres Júnior, José Rodrigues 
Orientadores
Higa, Elisa Mieko Suemitsu
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A ciclosporina A (CsA) é uma droga imunossupressora desempenha um papel crucial no campo do transplante de órgãos, reduzindo as taxas de rejeição e melhorando a sobrevivência dos pacientes submetidos a transplantes de órgãos e enxertos. Apesar de ser um importante agente na imunossupressão em transplantes renais, seu uso está associado a efeitos colaterais, incluindo nefrotoxicidade. A fisiopatologia da lesão renal causada pela CsA ainda não foi totalmente elucidada. Evidências indicam que a CsA induz estresse oxidativo (EO) e nitrosativo (EN), contribuindo para danos estruturais e funcionais nos rins. A sirtuina 1 (SIRT1), uma enzima pertencente à família Sir2 parte do grupo das histonas desacetilases (HDACs), é conhecida como um regulador da morte/sobrevivência celular e pela resposta ao EO. A SIRT1 promove a expressão da superóxido dismutase (SOD) e catalase (CAT) aumentando a resistência ao EO, e a desacetilação promovida pela SIRT também pode estar envolvida na melhora da sobrevida celular ao inibir a apoptose relacionada ao gene Trp53 induzida pela CsA. Objetivo: Estudar o efeito da CsA na expressão da SIRT1 e sua correlação com fatores epigenéticos e o EO/EN, em células mesangiais imortalizadas de camundongos (CMiC). Métodos: As CMiC foram alocadas nos grupos: controle e CsA (20 µg/mL), na presença ou ausência de 25 µM de resveratrol (RSV), um agonista de SIRT1. Avaliamos a viabilidade e proliferação celular, o óxido nítrico (NO) e espécies reativas de oxigênio (ROS). Analisamos as proteínas SIRT1, HDAC e SOD, CAT, nitrotirosina, caspase-3 e calpaína por imunocitoquímica. Os resultados foram descritos como média ± erro padrão, com significância estatística para p < 0,05. Resultados: Nos grupos tratados com CsA houve uma diminuição da viabilidade celular, assim como aumento de ROS, indicando EO. Também evidenciamos a regulação positiva da expressão de SIRT1, HDAC1 e CAT, indicando possível mecanismo compensatório provocado pelo estresse induzido pela CsA, sugerindo que a SIRT1 e HDAC1 podem inibir a apoptose por p53. Os níveis elevados de CAT nos grupos tratados com CsA podem ser indicativos de uma possível ativação desta enzima pela SIRT1. Os grupos tratados com RSV demonstraram aumento da viabilidade celular, porém, observamos redução na expressão SOD1 e elevação dos níveis de nitrotirosina e calpaína, sugerindo possível EN na dose utilizada. Conclusão: A CsA induz o EO em CMICs, diminuindo a viabilidade celular e aumentando a expressão da SIRT1 como mecanismo compensatório.
Introduction: Cyclosporine A (CsA) is an immunosuppressive drug that plays a crucial role in the field of organ transplantation, reducing rejection rates and improving the survival of patients undergoing organ and graft transplants. Despite being an important agent in immunosuppression for kidney transplants, its use is associated with side effects, including nephrotoxicity. The pathophysiology of CsA-induced kidney injury has not yet been fully elucidated. Evidence indicates that CsA induces oxidative stress (OS) and nitrosative stress (NS), contributing to structural and functional damage to the kidneys. Sirtuin 1 (SIRT1), an enzyme belonging to the Sir2 family and part of the histone deacetylase (HDAC) group, is known as a regulator of cell death/survival and the response to OS. SIRT1 promotes the expression of superoxide dismutase (SOD) and catalase (CAT), increasing resistance to OS, and the deacetylation promoted by SIRT1 may also be involved in improving cell survival by inhibiting CsA-induced apoptosis related to the Trp53 gene. Objective: To study the effect of CsA on SIRT1 expression and its correlation with epigenetic factors and OS/NS in immortalized mesangial cells from mice (IMMCs). Methods: IMMCs were allocated into the following groups: control and CsA (20 µg/mL), in the presence or absence of 25 µM resveratrol (RSV), a SIRT1 agonist. We evaluated cell viability and proliferation, nitric oxide (NO), and reactive oxygen species (ROS). We analyzed the proteins SIRT1, HDAC, SOD, CAT, nitrotyrosine, caspase-3, and calpain by immunocytochemistry. The results were described as mean ± standard error, with statistical significance set at p < 0.05. Results and Discussion: In the groups treated with CsA, there was a decrease in cell viability, as well as an increase in ROS, indicating OS. We also observed the upregulation of SIRT1, HDAC1, and CAT expression, indicating a possible compensatory mechanism induced by CsA-induced stress, suggesting that SIRT1 and HDAC1 may inhibit p53-related apoptosis. The elevated CAT levels in the CsA-treated groups may indicate possible activation of this enzyme by SIRT1. The groups treated with RSV showed increased cell viability; however, we observed a reduction in SOD1 expression and elevated levels of nitrotyrosine and calpain, suggesting possible NS at the dose used. Conclusion: CsA induces OS in IMMCs, reducing cell viability and increasing SIRT1 expression as a compensatory mechanism.
Introduction: Cyclosporine A (CsA) is an immunosuppressive drug that plays a crucial role in the field of organ transplantation, reducing rejection rates and improving the survival of patients undergoing organ and graft transplants. Despite being an important agent in immunosuppression for kidney transplants, its use is associated with side effects, including nephrotoxicity. The pathophysiology of CsA-induced kidney injury has not yet been fully elucidated. Evidence indicates that CsA induces oxidative stress (OS) and nitrosative stress (NS), contributing to structural and functional damage to the kidneys. Sirtuin 1 (SIRT1), an enzyme belonging to the Sir2 family and part of the histone deacetylase (HDAC) group, is known as a regulator of cell death/survival and the response to OS. SIRT1 promotes the expression of superoxide dismutase (SOD) and catalase (CAT), increasing resistance to OS, and the deacetylation promoted by SIRT1 may also be involved in improving cell survival by inhibiting CsA-induced apoptosis related to the Trp53 gene. Objective: To study the effect of CsA on SIRT1 expression and its correlation with epigenetic factors and OS/NS in immortalized mesangial cells from mice (IMMCs). Methods: IMMCs were allocated into the following groups: control and CsA (20 µg/mL), in the presence or absence of 25 µM resveratrol (RSV), a SIRT1 agonist. We evaluated cell viability and proliferation, nitric oxide (NO), and reactive oxygen species (ROS). We analyzed the proteins SIRT1, HDAC, SOD, CAT, nitrotyrosine, caspase-3, and calpain by immunocytochemistry. The results were described as mean ± standard error, with statistical significance set at p < 0.05. Results and Discussion: In the groups treated with CsA, there was a decrease in cell viability, as well as an increase in ROS, indicating OS. We also observed the upregulation of SIRT1, HDAC1, and CAT expression, indicating a possible compensatory mechanism induced by CsA-induced stress, suggesting that SIRT1 and HDAC1 may inhibit p53-related apoptosis. The elevated CAT levels in the CsA-treated groups may indicate possible activation of this enzyme by SIRT1. The groups treated with RSV showed increased cell viability; however, we observed a reduction in SOD1 expression and elevated levels of nitrotyrosine and calpain, suggesting possible NS at the dose used. Conclusion: CsA induces OS in IMMCs, reducing cell viability and increasing SIRT1 expression as a compensatory mechanism.
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Citação
TORRES JÚNIOR, José Rodrigues. Efeito da ciclosporina a na expressão da sirtuína 1 e no estresse oxidativo/nitrosativo em células mesangiais imortalizadas de camundongos. 2024. 77 f. Dissertação (Mestrado em Medicina Translacional) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.