Mecanismos envolvidos no efeito cicatrizante de feridas cutâneas do lupeol isolado das cascas do caule de Bowdichia virgilioides Kunth. (Fabaceae) em modelos experimentais in vivo e in vitro
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Data
2019-03-01
Autores
Beserra, Fernando Pereira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A dificuldade de cicatrização de feridas é uma das principais complicações decorrentes do Diabetes mellitus (DM), que torna o processo cicatricial mais lento e o tratamento mais dispendioso. O presente estudo teve como objetivo investigar o efeito e os mecanismos do lupeol envolvidos no processo de cicatrização de feridas cutâneas experimentais in vitro e em ratos normoglicêmicos (RN) e hiperglicêmicos (RH). Foi investigado o efeito do lupeol (0,1, 1, 10 e 20 μg/mL) em ensaios in vitro de cicatrização de feridas utilizando queratinócitos e fibroblastos de prepúcio neonatal humano. Para os estudos in vivo, ratos Wistar machos (n=8) foram randomicamente divididos nos seguintes grupos experimentais usando RN: creme base, colagenase 1.2 U/g, creme à base de lupeol 0,1%, 0,2% e 0,4% tratados durante 3, 7 e 14 dias. O estudo realizado em RH, os grupos foram divididos em: creme base, insulina 0.5 U/g e creme à base de lupeol 0.2% (menor dose efetiva em RN) em RH tratados durante 14 dias. A indução experimental de feridas cutâneas foi realizada na região dorsal dos ratos com o auxílio de um punch de 2 cm de diâmetro. Foram investigados parâmetros macroscópicos, histológicos, imunohistoquímicos, imunoenzimáticos e moleculares. Os resultados in vitro mostraram que, baixas concentrações do lupeol foi capaz de estimular a migração e proliferação celular de queratinócitos, efeito contrátil em fibroblastos dérmicos embebidos em um solução gel de colágeno, possivelmente através das vias PI3k/Akt, MAPK/p38, vias sinalizadoras envolvidas na proliferação/migração celular, angiogênese e reparo tecidual. Nossos resultados in vivo mostraram potencial cicatricial do lupeol após 7 e 14 dias de tratamentos em feridas de ratos normoglicêmicos. Lupeol mostrou atividade anti-inflamatória, com significativa redução de TNF-α, IL-6 e NF-κB, e aumento nos níveis de IL-10, atividade proliferativa por estimular a expressão de Ki-67, aumento do processo angiogênico por aumento da expressão de VEGF, estímulo da reepitelização observados pelo aumento de EGF e remodelação da matriz extracelular por estimular a síntese de colágenos. Os resultados em RH, mostraram um aumento na porcentagem de retração da lesão a partir do 11º dia de tratamento após da indução da lesão. As análises histopatológicas revelaram uma diminuição da infiltração de células inflamatórias, aumento da proliferação de fibroblastos, vascularização e deposição de fibras colágenas no tratamento com lupeol. Os resultados do ELISA revelaram níveis de TNF-α e IL-6 reduzidos e níveis de IL-10 sobreregulados, mostrando atividade anti-inflamatória. Os resultados moleculares e imunohistoquímicos confirmaram o potencial anti-inflamatório do lupeol através da redução de NF-κB e do estresse oxidativo por aumentar a expressão de enzimas antioxidantes, como HO-1 e SOD-2, bem como mecanismos de proliferação celular, angiogênese e remodelamente de matriz extracelular estimulados por FGF-2, TGF-β1, HIF-1α e colágeno III. Nossos achados permitem concluir que o lupeol pode servir como uma nova opção terapêutica para o tratamento de feridas cutâneas normais e em condições hiperglicêmicas, regulando os mecanismos envolvidos nas fases inflamatória, proliferativa e de remodelação tecidual.
The difficulty of wound healing is one of the main complications due to Diabetes mellitus (DM), which makes the healing process slower and the treatment more expensive. The present study aimed to investigate the effect of lupeol and mechanisms involved in the healing process of experimental skin wounds in vitro as well as in normoglycemic (NR) and hyperglycemic rats (HR). The effect of lupeol (0.1, 1, 10 and 20 μg/mL) on in vitro wound healing assays using keratinocytes and human neonatal foreskin fibroblasts was investigated. For in vivo studies, male Wistar rats (n=8) were randomly divided into the following experimental groups using NR: lanette, collagenase 1.2 U/g and lupeol 0.1%, 0.2% or 0.4% treated for 3, 7 and 14 days. In the study performed in HR, the groups were divided into: lanette, insulin 0.5 U/g and lupeol 0.2% cream (lowest effective dose in NR) in HR treated for 14 days. Experimental induction of cutaneous wounds was performed on dorsal region of the rats using a punch 2 cm in diameter. Macroscopic, histological, immunohistochemical, immunoenzymatic and molecular parameters were investigated. The in vitro results showed that low concentrations of lupeol were able to stimulate cell migration and proliferation of keratinocytes, a contractile effect on dermal fibroblasts embedded in a collagen gel solution, possibly through PI3k/Akt, and MAPK/p38 signaling pathways involved in cell proliferation/migration, angiogenesis and tissue repair. Our in vivo results showed wound healing potential of lupeol after 7 and 14 days of treatment in normoglycemic rat wounds. Lupeol showed anti-inflammatory activity, with significant reduction of TNF-α, IL-6 and NF-κB, and increased IL-10 levels, proliferative activity by stimulating Ki-67 expression, increased angiogenic process by increased VEGF expression, stimulus of re-epithelialization observed by the increase of EGF and extracellular matrix remodeling by increase the collagens deposition. The results in HR, showed an increase in the percentage of wound retraction from the 11th day of treatment after the lesion induction. Histopathological analyzes revealed a decrease in inflammatory cells infiltration, increased proliferation of fibroblasts, vascularization and deposition of collagen fibers in lupeol treatment. ELISA results demonstrated reduced TNF-α and IL-6 levels and increased IL-10 levels, suggesting anti-inflammatory activity. Molecular and immunohistochemical results confirmed the anti-inflammatory potential of lupeol by reducing NF-κB and oxidative stress by increasing the expression of antioxidant enzymes, such as HO-1 and SOD-2, as well as mechanisms of cell proliferation, angiogenesis and remodeling of extracellular matrix stimulated by FGF-2, TGF-β1, HIF-1α and collagen III. Our findings allow us to conclude that lupeol may serve as a new therapeutic option for the treatment of normal and hyperglycemic skin wounds, regulating the mechanisms involved in the inflammatory, proliferative and tissue remodeling phases.
The difficulty of wound healing is one of the main complications due to Diabetes mellitus (DM), which makes the healing process slower and the treatment more expensive. The present study aimed to investigate the effect of lupeol and mechanisms involved in the healing process of experimental skin wounds in vitro as well as in normoglycemic (NR) and hyperglycemic rats (HR). The effect of lupeol (0.1, 1, 10 and 20 μg/mL) on in vitro wound healing assays using keratinocytes and human neonatal foreskin fibroblasts was investigated. For in vivo studies, male Wistar rats (n=8) were randomly divided into the following experimental groups using NR: lanette, collagenase 1.2 U/g and lupeol 0.1%, 0.2% or 0.4% treated for 3, 7 and 14 days. In the study performed in HR, the groups were divided into: lanette, insulin 0.5 U/g and lupeol 0.2% cream (lowest effective dose in NR) in HR treated for 14 days. Experimental induction of cutaneous wounds was performed on dorsal region of the rats using a punch 2 cm in diameter. Macroscopic, histological, immunohistochemical, immunoenzymatic and molecular parameters were investigated. The in vitro results showed that low concentrations of lupeol were able to stimulate cell migration and proliferation of keratinocytes, a contractile effect on dermal fibroblasts embedded in a collagen gel solution, possibly through PI3k/Akt, and MAPK/p38 signaling pathways involved in cell proliferation/migration, angiogenesis and tissue repair. Our in vivo results showed wound healing potential of lupeol after 7 and 14 days of treatment in normoglycemic rat wounds. Lupeol showed anti-inflammatory activity, with significant reduction of TNF-α, IL-6 and NF-κB, and increased IL-10 levels, proliferative activity by stimulating Ki-67 expression, increased angiogenic process by increased VEGF expression, stimulus of re-epithelialization observed by the increase of EGF and extracellular matrix remodeling by increase the collagens deposition. The results in HR, showed an increase in the percentage of wound retraction from the 11th day of treatment after the lesion induction. Histopathological analyzes revealed a decrease in inflammatory cells infiltration, increased proliferation of fibroblasts, vascularization and deposition of collagen fibers in lupeol treatment. ELISA results demonstrated reduced TNF-α and IL-6 levels and increased IL-10 levels, suggesting anti-inflammatory activity. Molecular and immunohistochemical results confirmed the anti-inflammatory potential of lupeol by reducing NF-κB and oxidative stress by increasing the expression of antioxidant enzymes, such as HO-1 and SOD-2, as well as mechanisms of cell proliferation, angiogenesis and remodeling of extracellular matrix stimulated by FGF-2, TGF-β1, HIF-1α and collagen III. Our findings allow us to conclude that lupeol may serve as a new therapeutic option for the treatment of normal and hyperglycemic skin wounds, regulating the mechanisms involved in the inflammatory, proliferative and tissue remodeling phases.
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Palavras-chave
lupeol, pele, feridas, cicatrização, hiperglicemia, skin, wounds, wound healing, hyperglycemia