Caracterização da regeneração, inflamação e estresse oxidativo no músculo de modelo experimental para distrofia muscular de Duchenne com fenótipo exacerbado pelo exercício
Data
2021-05-12
Autores
Lazzarin, Mariana Cruz [UNIFESP]
Orientadores
De Oliveira, Flavia [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A distrofia muscular de Duchenne é uma doença caracterizada por necrose muscular progressiva. O avanço das intervenções terapêuticas e das terapias genéticas tem trazido esperanças quanto à manutenção da funcionalidade motora mesmo em estágios mais tardios da doença. Entretanto, um grande desafio na prescrição de exercícios na reabilitação física para esses pacientes é a ausência de um perfil abrangente da resposta do músculo distrófico ao exercício. Dessa forma, o objetivo desse trabalho foi compreender, em camundongos mdx com fenótipo exacerbado pelo exercício, parâmetros relacionados à regeneração muscular, à inflamação e ao estresse oxidativo. Materiais e métodos: Camundongos C57BL/10 e C57BL/10-DMDmdx, com seis meses de vida, distribuídos em três grupos (n=10 cada): Controle, Mdx e Mdx+Exercício. O protocolo de corrida em declive foi de sete semanas, com frequência de cinco vezes na semana, a uma velocidade máxima particular para cada animal durante 60 minutos. Após eutanásia, o m. gastrocnêmio foi dissecado e realizou-se as análises: histopatológica; imunoistoquímica para marcadores de regeneração muscular (MyoD e Miogenina), de inflamação (COX-2) e para estresse oxidativo (8-OHdG); expressão de genes (qPCR) de NF-kB e NADP(H) Oxidase 2. Resultados: O grupo Mdx+Exercício apresentou a histopatologia do m. gastrocnêmio alterada pela corrida em declive, com maior incidência de fibrose endomisial e menor porcentagem de área de mionecrose. A análise imunoistoquímica revelou aumento da imunoexpressão de Miogenina no grupo Mdx+Exercício, mesmo não influenciando a expressão de MyoD; menor imunoexpressão do 8-OHdG no grupo Mdx e alterou a região histopatológica com imunoexpressão da COX-2. No grupo Mdx, a COX-2 estava relacionada com áreas de regeneração e infiltrado inflamatório, enquanto que no grupo Mdx+Exercício estava associado a regiões de fibrose muscular. Além disso, o exercício não influenciou a expressão gênica de NF-kB, porém aumentou o RNAm de NAD(P)H Oxidase 2. Conclusão: A exacerbação do fenótipo da DMD no músculo gastrocnêmio de camundongos mdx adultos está associada com o aumento da Miogenina, da COX-2 em regiões de fibrose e do 8-OHdG nuclear, bem como da expressão gênica de NAD(P)H Oxidase 2.
Introduction: Duchenne muscular dystrophy (DMD) is a disease characterized by progressive muscle necrosis. The advance of therapeutic interventions and genetic therapies has brought hopes concerning the maintenance of motor functionality in later stages of DMD. One of the major challenges for prescribing physical rehabilitation exercises for DMD patients is associated with the absence of a thorough knowledge og the dystrophic muscle responsiveness to exercise. This study aimed to understand, in Mdx mice with phenotype exacerbated by exercise, the parameters related to muscle regeneration, inflammation, and oxidative stress. Materials and methods: Six months old C57BL/10 and C57BL/10-DMDmdx male mice, were distributed into three groups (n=10 each): Control, Mdx, and Mdx+Exercise. Animals were trained in a downhill running protocol for seven weeks, five times a week, at maximum speed reached by individual animals with a duration of 60 minutes. The gastrocnemius muscle was dissected and submitted to histopathological, muscle regeneration (MyoD and Myogenin), inflammation (COX-2), and oxidative stress (8-OHdG) immunohistochemistry markers, in addition to gene expression. (qPCR) of NF-kB and NADP(H)Oxidase 2 analysis. Results: In the Mdx+Exercise group, the gastrocnemius muscle showed a higher incidence of endomysial fibrosis and a lower myonecrosis percentage area. The immunohistochemical analysis revealed increased immunoexpression of Myogenin in the Mdx+Exercise group, although the MyoD immunoexpression remained unaltered. In the Mdx group, a decrease in 8-OHdG immunoexpression was noticed additionally to changes in the histopathological region with COX-2 immunoexpression. The COX-2 was related to areas of regeneration process and inflammatory infiltrate in the Mdx group, while associated with areas of muscle fibrosis in the Mdx+Exercise group. In addition, NF-kB gene expression was not influenced by exercise, however, observed a NAD(P)H Oxidase 2 mRNA increased. Conclusion: The exacerbation of the disease phenotype in the gastrocnemius muscle of adult Mdx mice was associated with increased myogenin, COX-2 in fibrosis regions, and nuclear 8-OHdG immunoexpression, as well as NAD(P)H Oxidase 2 gene expression.
Introduction: Duchenne muscular dystrophy (DMD) is a disease characterized by progressive muscle necrosis. The advance of therapeutic interventions and genetic therapies has brought hopes concerning the maintenance of motor functionality in later stages of DMD. One of the major challenges for prescribing physical rehabilitation exercises for DMD patients is associated with the absence of a thorough knowledge og the dystrophic muscle responsiveness to exercise. This study aimed to understand, in Mdx mice with phenotype exacerbated by exercise, the parameters related to muscle regeneration, inflammation, and oxidative stress. Materials and methods: Six months old C57BL/10 and C57BL/10-DMDmdx male mice, were distributed into three groups (n=10 each): Control, Mdx, and Mdx+Exercise. Animals were trained in a downhill running protocol for seven weeks, five times a week, at maximum speed reached by individual animals with a duration of 60 minutes. The gastrocnemius muscle was dissected and submitted to histopathological, muscle regeneration (MyoD and Myogenin), inflammation (COX-2), and oxidative stress (8-OHdG) immunohistochemistry markers, in addition to gene expression. (qPCR) of NF-kB and NADP(H)Oxidase 2 analysis. Results: In the Mdx+Exercise group, the gastrocnemius muscle showed a higher incidence of endomysial fibrosis and a lower myonecrosis percentage area. The immunohistochemical analysis revealed increased immunoexpression of Myogenin in the Mdx+Exercise group, although the MyoD immunoexpression remained unaltered. In the Mdx group, a decrease in 8-OHdG immunoexpression was noticed additionally to changes in the histopathological region with COX-2 immunoexpression. The COX-2 was related to areas of regeneration process and inflammatory infiltrate in the Mdx group, while associated with areas of muscle fibrosis in the Mdx+Exercise group. In addition, NF-kB gene expression was not influenced by exercise, however, observed a NAD(P)H Oxidase 2 mRNA increased. Conclusion: The exacerbation of the disease phenotype in the gastrocnemius muscle of adult Mdx mice was associated with increased myogenin, COX-2 in fibrosis regions, and nuclear 8-OHdG immunoexpression, as well as NAD(P)H Oxidase 2 gene expression.
Descrição
Citação
LAZZARIN, Mariana Cruz. Caracterização da regeneração, inflamação e estresse oxidativo no músculo de modelo experimental para distrofia muscular de Duchenne com fenótipo exacerbado pelo exercício. 2021. 91 f. Tese (Doutorado Interdisciplinar em Ciências da Saúde) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2021.