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Mechanisms involved in the cutaneous wound healing effect of lupeol isolated from the stem bark of Bowdichia virgilioides Kunth. (Fabaceae) in experimental models in vivo and in vitro

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Fernando Pereira Beserra
Total Authors: 1
Document type: Doctoral Thesis
Press: Botucatu. 2019-05-07.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
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Advisor: Cláudia Helena Pellizzon

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. (AU)