Papel regulatório das prostaglandinas produzidas a partir das vias COX-1 e COX-2, sobre a neovascularização durante a regeneração muscular esquelética, induzida pela injeção de veneno de serpente Bothrops asper
Data
2022
Autores
Correia, Melissa Rodrigues 
Orientadores
Moreira, Vanessa
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Um evento essencial no reparo do tecido muscular esquelético após injúria, é o restabelecimento da microcirculação local, importante no suprimento de oxigênio e nutrientes, e remoção de metabólitos e debris celulares. Apesar do conhecimento da regulação dos mediadores pró-inflamatórios sobre o processo de angiogênese, pouco se conhece a influência dos mediadores derivados do ácido araquidônico, como as prostaglandinas (PGs) produzidas pelas vias das ciclooxigenases (COX)-1 e -2. O uso de venenos de serpentes do gênero Bothrops constitui um modelo complexo para estudos de regeneração muscular, pois contêm toxinas que, além de inflamação e miotoxicidade local, comprometem a estrutura de vasos sanguíneos, afetando a qualidade do reparo tecidual. O objetivo do trabalho foi avaliar a influência das prostaglandinas das vias COX-1 e- 2 sobre a neovascularização do tecido muscular esquelético, após injúria induzida pelo veneno de Bothrops asper. Para tanto, grupos distintos de camundongos Swiss machos (CEUA 9047230920) receberam VBa i.m. (2.5 mg/kg/50μL) no músculo gastrocnêmio direito ou solução salina (SS) no músculo contralateral (controle). Após 30 min, 2 e 6 dias foram tratados por via oral com lumiracoxibe (LUM/inibidor seletivo de COX-2/20mg/kg), indometacina (IND/inibidor não seletivo de COXs-/5 mg/kg) ou solução veículo (TW/Tween 1%). Após 24 h, 7 e 21 dias (d), os músculos foram coletados e analisados quanto ao grau de hemorragia (sol. Drabkin), isquemia (laser doppler), produção de fator de crescimento endotelial vascular (VEGF) e de metaloproteinases de matriz (MMPs) (ELISA), bem como expressão proteica de CD31 e COX-2 (western blotting). Na fase degenerativa (24h), os camundongos tratados com VBa/IND e VBa/LUM apresentaram diminuição significativa
(p<0,05) do fluxo sanguíneo (39±4 e 43±2%, respectivamente) quando comparados ao VBa/TW (58±3%). Na fase regenerativa (21d) animais VBa/IND e VBa/LUM apresentaram aumento significativo (p<0,05) no fluxo sanguíneo do membro posterior (116±7 e 110±4%, respectivamente) em comparação aos camundongos VBa/TW (94±6%). Em 24h, camundongos VBa/IND apresentaram teor significativo de hemoglobina tecidual (22±2 mg/g), quando comparados aos animais VBa/TW (17±1 mg/g). No início da regeneração (7d), animais VBa/IND (424±42, 733±70 e 3247±150 ng/mL/mg, respectivamente) e VBa/LUM (427±10, 676±109 e 2919±256 ng/mL/mg, respectivamente) apresentaram diminuição significativa (p<0,05) nas concentrações de MMP-9, -10 e -13 quando comparados aos animais VBa/TW (588±46, 1172±98 e 4596±427 ng/mL/mg, respectivamente). Nesta fase (7d), animais VBa/IND (19±3 pg/mL/mg) apresentaram aumento significativo (p<0,05) na produção de VEGF, comparados ao grupo VBa/TW (12±1 pg/mL/mg). Já em 21d, animais VBa/LUM e VBa/IND apresentaram aumento significativo (p<0,05) nos níveis de VEGF (12±1 e 12±2 pg/mL/mg, respectivamente), em comparação ao grupo VBa/TW (6±0.03 pg/mL/mg). De outra parte, na fase degenerativa (24h) animais VBa/TW apresentaram diminuição significativa
(p<0,05) na expressão de COX-2 e CD31, em comparação ao controle (SS/TW), enquanto os tratados com VBa/LUM e VBa/IND apresentaram expressão de COX-2 e CD31 igual aos respectivos grupos SS/TW. No início da regeneração (7 d) animais tratados com VBa/IND apresentaram diminuição significativa (p<0,05) de COX-2, comparados aos VBa/TW. Por
outro lado, animais tratados com VBa/LUM, apresentaram aumento significativo (p<0,05) da expressão CD31, quando comparados aos respectivos animais VBa/TW. No período mais avançado da regeneração (21d) os animais VBa/LUM e VBa/IND, apresentaram aumento significativo de CD31 (p<0,05), quando comparado aos animais VBa/TW. Os resultados
indicam que as prostaglandinas das vias das ciclooxigenases, especialmente a COX-2, estimulam mecanismos que preservam a lesão vascular causada pelo VBa, e são responsáveis pela regulação dos fatores angiogênicos como MMP-9,-10 e -13 e VEGF durante a regeneração tecidual. Ainda, a inibição da produção destes mediadores no início da regeneração desencadeia mecanismos supra regulatórios que aceleram o processo de angiogênese neste modelo.
An essential event for skeletal muscle regeneration after injury is the microcirculation restoration, important in providing oxygen and nutrients, in addition to removal of metabolites and cellular debris. Despite the knowledge of pro-inflammatory mediator regulation in angiogenesis, the influence of arachidonic acid-derived mediators, as prostaglandins, generated by cyclooxygenase (COX)-1 and -2 pathways, is still poorly investigated. Experimental models using bothropic snake venoms are ideal for broad characterization of muscle regeneration, because they contain toxins that, in addition to inflammation and myotoxicity, compromise the vascular structure, affecting the quality of tissue repair. The aim of this study was to evaluate the influence of prostaglandins from COX-1 and –2 pathways on the neovascularization of skeletal muscle after injury induced by Bothrops asper venom (BaV). Different groups of male Swiss mice (CEUA 9047230920) received BaV i.m. (2.5 mg/kg/50μL) in the right gastrocnemius muscle or saline solution (SS) in the contralateral muscle (control). After 30 min, 2 and 6 days, animals received lumiracoxib (LUM/selective COX-2 inhibitor/20mg/kg), indomethacin (IND/non-selective COXs inhibitor/5 mg/kg) or vehicle solution (TW/Tween 1%). After 24 h, 7 and 21 days (d), muscles were collected and analyzed for hemorrhage assessment (Sol. Drabkin), ischemia (laser doppler), vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) production, as well as protein expression of CD31 and COX-2 (western blotting). In the degenerative phase (24h), mice treated with BaV/IND and BaV/LUM showed a significant decrease (p<0.05) in blood flow (39±4 and 43±2%, respectively) when compared to BaV/TW (58±3%). In the regenerative phase (21d) BaV/IND and BaV/LUM animals showed a significant increase (p<0.05) in hind limb blood flow (116±7 and 110±4%, respectively) compared to BaV/TW mice (94±6%). At 24h, BaV/IND mice had a significant tissue hemoglobin content (22±2 mg/g), when compared to BaV/TW mice (17±1 mg/g). At the beginning of regeneration (7d), animals BaV/IND (424±42, 733±70 and 3247±150 ng/mL/mg, respectively) and BaV/LUM (427±10, 676±109 and 2919±256 ng/mL/mg, respectively) showed a significant decrease (p<0.05) in MMP-9, -10 and -13 concentrations when compared to BaV/TW animals (588±46, 1172±98 and 4596±427 ng/mL/mg, respectively). In this phase (7d), BaV/IND animals (19±3 pg/mL/mg) showed a significant increase (p<0.05) in VEGF production, compared to the BaV/TW group (12±1 pg/mL/mg). At 21d, BaV/LUM and BaV/IND animals showed a significant increase (p<0.05) in VEGF levels (12±1 and 12±2 pg/mL/mg, respectively), compared to the BaV/TW group. (6±0.03 pg/mL/mg). On the other hand, in the degenerative phase (24h) BaV/TW animals showed a significant decrease (p<0.05) in the expression of COX-2 and CD31, compared to the control group (SS/TW), while those treated with BaV/LUM and BaV/IND showed expression of COX-2 and CD31 equal to their respective SS/TW groups. At the beginning of regeneration (7 d) animals treated with BaV/IND showed a significant decrease (p<0.05) of COX-2, compared to BaV/TW. On the other hand, animals treated with BaV/LUM showed a significant increase (p<0.05) in CD31 expression, when compared to their respective BaV/TW group. In the late period of regeneration (21d) the BaV/LUM and BaV/IND animals showed a significant increase in CD31 (p<0.05), when compared to the VBa/TW animals. These results indicate that the prostaglandins of the cyclooxygenase pathways, especially COX-2, stimulate mechanismsthat preserve the vascular injury caused by BaV, and are responsible for the regulation of angiogenic factors such as MMP-9, -10 and -13 and VEGF during tissue regeneration. Furthermore, the inhibition of the production of these mediators at the beginning of regeneration triggers supraregulatory mechanisms that accelerate the process of angiogenesis.
An essential event for skeletal muscle regeneration after injury is the microcirculation restoration, important in providing oxygen and nutrients, in addition to removal of metabolites and cellular debris. Despite the knowledge of pro-inflammatory mediator regulation in angiogenesis, the influence of arachidonic acid-derived mediators, as prostaglandins, generated by cyclooxygenase (COX)-1 and -2 pathways, is still poorly investigated. Experimental models using bothropic snake venoms are ideal for broad characterization of muscle regeneration, because they contain toxins that, in addition to inflammation and myotoxicity, compromise the vascular structure, affecting the quality of tissue repair. The aim of this study was to evaluate the influence of prostaglandins from COX-1 and –2 pathways on the neovascularization of skeletal muscle after injury induced by Bothrops asper venom (BaV). Different groups of male Swiss mice (CEUA 9047230920) received BaV i.m. (2.5 mg/kg/50μL) in the right gastrocnemius muscle or saline solution (SS) in the contralateral muscle (control). After 30 min, 2 and 6 days, animals received lumiracoxib (LUM/selective COX-2 inhibitor/20mg/kg), indomethacin (IND/non-selective COXs inhibitor/5 mg/kg) or vehicle solution (TW/Tween 1%). After 24 h, 7 and 21 days (d), muscles were collected and analyzed for hemorrhage assessment (Sol. Drabkin), ischemia (laser doppler), vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) production, as well as protein expression of CD31 and COX-2 (western blotting). In the degenerative phase (24h), mice treated with BaV/IND and BaV/LUM showed a significant decrease (p<0.05) in blood flow (39±4 and 43±2%, respectively) when compared to BaV/TW (58±3%). In the regenerative phase (21d) BaV/IND and BaV/LUM animals showed a significant increase (p<0.05) in hind limb blood flow (116±7 and 110±4%, respectively) compared to BaV/TW mice (94±6%). At 24h, BaV/IND mice had a significant tissue hemoglobin content (22±2 mg/g), when compared to BaV/TW mice (17±1 mg/g). At the beginning of regeneration (7d), animals BaV/IND (424±42, 733±70 and 3247±150 ng/mL/mg, respectively) and BaV/LUM (427±10, 676±109 and 2919±256 ng/mL/mg, respectively) showed a significant decrease (p<0.05) in MMP-9, -10 and -13 concentrations when compared to BaV/TW animals (588±46, 1172±98 and 4596±427 ng/mL/mg, respectively). In this phase (7d), BaV/IND animals (19±3 pg/mL/mg) showed a significant increase (p<0.05) in VEGF production, compared to the BaV/TW group (12±1 pg/mL/mg). At 21d, BaV/LUM and BaV/IND animals showed a significant increase (p<0.05) in VEGF levels (12±1 and 12±2 pg/mL/mg, respectively), compared to the BaV/TW group. (6±0.03 pg/mL/mg). On the other hand, in the degenerative phase (24h) BaV/TW animals showed a significant decrease (p<0.05) in the expression of COX-2 and CD31, compared to the control group (SS/TW), while those treated with BaV/LUM and BaV/IND showed expression of COX-2 and CD31 equal to their respective SS/TW groups. At the beginning of regeneration (7 d) animals treated with BaV/IND showed a significant decrease (p<0.05) of COX-2, compared to BaV/TW. On the other hand, animals treated with BaV/LUM showed a significant increase (p<0.05) in CD31 expression, when compared to their respective BaV/TW group. In the late period of regeneration (21d) the BaV/LUM and BaV/IND animals showed a significant increase in CD31 (p<0.05), when compared to the VBa/TW animals. These results indicate that the prostaglandins of the cyclooxygenase pathways, especially COX-2, stimulate mechanismsthat preserve the vascular injury caused by BaV, and are responsible for the regulation of angiogenic factors such as MMP-9, -10 and -13 and VEGF during tissue regeneration. Furthermore, the inhibition of the production of these mediators at the beginning of regeneration triggers supraregulatory mechanisms that accelerate the process of angiogenesis.