Efeito da inibição da MMP-2 sobre a cardiotoxicidade induzida pela doxorrubicina em ratos
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Data
2020-02-28
Autores
Dantas, Danielle
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Universidade Estadual Paulista (Unesp)
Resumo
Introdução: a cardiotoxicidade é o efeito adverso mais severo provocado pelo quimioterápico doxorrubicina. As metaloproteinases (MMP) são enzimas proteolíticas que degradam a matriz extracelular e sua ativação é mecanismo importante na dilatação cardíaca provocada pela doxorrubicina. Sua atividade é regulada pelos seus inibidores teciduais específicos (TIMP). A doxiciclina é um inibidor não seletivo de MMP e foi capaz de impedir a remodelação ventricular em outros modelos de agressão cardíaca. Diante disso, investigamos a influência da inibição de MMP sobre a cardiotoxicidade induzida pela doxorrubicina em ratos. Material e Métodos: foram utilizados 80 ratos Wistar machos que foram alocados em 4 grupos: controle, doxorrubicina, inibição de MMP e doxorrubicina + inibição de MMP. A inibição das MMP foi realizada por injeção de doxiciclina (5mg/kg, IP) uma vez por semana, por 4 semanas. A doxorrubicina (5mg/kg, IP) foi injetada uma vez por semana, por 4 semanas, sempre 48 horas após a injeção de doxiciclina. Após 4 semanas, foi realizado ecocardiograma, estudo do coração isolado e eutanásia dos animais para coleta do material biológico. Foi realizada análise histológica, avaliação do estresse oxidativo, atividade de MMP-2 e expressão proteica de TIMP-4 e colágeno I por Western blot. Análise estatística: ANOVA de duas vias. Resultados: observamos menor ingestão de ração associada à perda de peso dentre os animais que receberam doxorrubicina. O quimioterápico também foi capaz de induzir alterações morfológicas ao ecocardiograma, com aumento do diâmetro do átrio esquerdo e do diâmetro sistólico do ventrículo esquerdo (VE). Em relação à função diastólica, observamos aumento do tempo de relaxamento isovolumétrico do VE, da onda E e relação da relação E/E’ nos animais tratados com doxorrubicina. Houve prejuízo também da função sistólica nesses animais, com diminuição da fração de encurtamento do VE e da onda S’ ao doppler tissular. A administração do inibidor de MMP associado à doxorrubicina melhorou as variáveis estruturais e a função sistólica ao ecocardiograma. A doxorrubicina causou hipertrofia dos miócitos avaliados pela histologia. Também houve aumento do estresse oxidativo nos animais tratados com o quimioterápico, evidenciado por aumento da concentração miocárdica de malondialdeído, porém a inibição de MMP não influenciou esse resultado. Não houve diferenças no estudo do coração isolado, na atividade de MMP e na expressão de TIMP-4 entre os grupos. No entanto, a administração de doxiciclina diminuiu a expressão de colágeno I tanto nos animais tratados com doxorrubicina quanto nos não tratados. Conclusão: A inibição de MMP atenuou a disfunção sistólica do VE causada pela doxorrubicina, porém não foi efetiva em inibir a ativação de MMP, bem como não melhorou o estresse oxidativo. A melhora da disfunção sistólica dos animais tratados com o inibidor da MMP ocorreu por vias ainda a serem elucidadas.
Introduction: Cardiotoxicity is the most severe adverse effect induced by doxorubicin. The matrix metalloproteinases (MMP), which activity is regulated by specific tissue inhibitors (TIMP), are proteolytic enzymes that degrade the extracellular matrix and its activation is an important mechanism in cardiac dilatation observed in cardiotoxicity. The doxycycline is a non-selective inhibitor of MMP and was capable to stop ventricular remodeling in other models of cardiac aggression. Therefore, we investigated the influence of MMP inhibition on induced cardiotoxicity by doxorubicin in rats. Materials and Methods: 80 male Wistar rats were classified into 4 groups: control, doxorubicin, MMP inhibition, and doxorubicin + MMP inhibition. The MMP inhibition was made by the injection of doxycycline (5mg/kg, IP) once in a week, for 4 weeks. The doxorubicin (5mg/kg, IP) was injected once a week, for 4 weeks, always 48 hours after the doxycycline injection. After 4 weeks was performed an echocardiogram, isolated heard study, and the euthanasia of the animals to obtain the biological material. We evaluated histological sections, oxidative stress, MMP-2 activity, and TIMP-4, and collagen I protein expression by Western blot. Statistical analysis: two-way ANOVA. Results: we observed less chow ingestion associated with weight loss among the animals which received doxorubicin. The chemotherapy was also capable to induce morphological alterations in the echocardiogram, increasing the left atrium diameter and left ventricle systolic diameter (LV). Regarding the diastolic function, was observed an increase in isovolumetric LV relaxation time, E wave, and wave E/E’ relationship in animals treated with doxorubicin. These animals also presented worse systolic function, showed by the decrease of the LV shortening fraction and the S’ wave obtained by tissue Doppler. The administration of MMP inhibitor associated with doxorubicin improved structural variables and systolic function in the echocardiogram. Also, the doxorubicin caused hypertrophy of the cardiomyocytes evaluated by histology. There was an increase in oxidative stress in animals treated with doxorubicin, as seen by the increased concentration of MDA. There was no difference between groups in the isolated heart study, the MMP-2 activity, and in the TIMP-4 expression. However, doxycycline decreased the expression of type I collagen in all animals treated with doxycycline. Conclusion: The inhibition of MMP attenuated the LV systolic function caused by doxorubicin; however, inhibition of MMP was not effective to inhibit the MMP-2 activation, as well did not improve the oxidative stress. The systolic dysfunction improvement in animals treated with MMP inhibitor occurred throughs pathways to be elucidated yet.
Introduction: Cardiotoxicity is the most severe adverse effect induced by doxorubicin. The matrix metalloproteinases (MMP), which activity is regulated by specific tissue inhibitors (TIMP), are proteolytic enzymes that degrade the extracellular matrix and its activation is an important mechanism in cardiac dilatation observed in cardiotoxicity. The doxycycline is a non-selective inhibitor of MMP and was capable to stop ventricular remodeling in other models of cardiac aggression. Therefore, we investigated the influence of MMP inhibition on induced cardiotoxicity by doxorubicin in rats. Materials and Methods: 80 male Wistar rats were classified into 4 groups: control, doxorubicin, MMP inhibition, and doxorubicin + MMP inhibition. The MMP inhibition was made by the injection of doxycycline (5mg/kg, IP) once in a week, for 4 weeks. The doxorubicin (5mg/kg, IP) was injected once a week, for 4 weeks, always 48 hours after the doxycycline injection. After 4 weeks was performed an echocardiogram, isolated heard study, and the euthanasia of the animals to obtain the biological material. We evaluated histological sections, oxidative stress, MMP-2 activity, and TIMP-4, and collagen I protein expression by Western blot. Statistical analysis: two-way ANOVA. Results: we observed less chow ingestion associated with weight loss among the animals which received doxorubicin. The chemotherapy was also capable to induce morphological alterations in the echocardiogram, increasing the left atrium diameter and left ventricle systolic diameter (LV). Regarding the diastolic function, was observed an increase in isovolumetric LV relaxation time, E wave, and wave E/E’ relationship in animals treated with doxorubicin. These animals also presented worse systolic function, showed by the decrease of the LV shortening fraction and the S’ wave obtained by tissue Doppler. The administration of MMP inhibitor associated with doxorubicin improved structural variables and systolic function in the echocardiogram. Also, the doxorubicin caused hypertrophy of the cardiomyocytes evaluated by histology. There was an increase in oxidative stress in animals treated with doxorubicin, as seen by the increased concentration of MDA. There was no difference between groups in the isolated heart study, the MMP-2 activity, and in the TIMP-4 expression. However, doxycycline decreased the expression of type I collagen in all animals treated with doxycycline. Conclusion: The inhibition of MMP attenuated the LV systolic function caused by doxorubicin; however, inhibition of MMP was not effective to inhibit the MMP-2 activation, as well did not improve the oxidative stress. The systolic dysfunction improvement in animals treated with MMP inhibitor occurred throughs pathways to be elucidated yet.
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Palavras-chave
Doxorrubicina, Cardiotoxicidade, MMP, TIMP, Estresse oxidativo, Matriz extracelular, Doxorubicin, Cardiotoxicity, Oxidative stress, Extracellular matrix