Efeito modulatório do barbatimão (Stryphnodendron adstringens (Mart.) Coville) em marcadores citofuncionais de fibroblastos humanos senescentes: estudo in vitro

Abstract

Introduction: the skin, with aging, undergoes a decrease in functions responding more slowly and partially to maintenance and regeneration processes. Because it is a complex phenomenon, many issues are involved in the onset and progression of aging, in which the shortening of telomeres, decrease in the synthesis of sirtuins, uncontrolled levels of reactive oxygen species (ROS) that may lead to oxidative stress, increased apoptosis rates via super regulation of the p53 protein and also to chronic inflammatory conditions. Since human populations are increasing their life expectancy, identifying potential factors that slow down the aging of the skin can be considered a highly relevant aspect. In Brazil there are many plants that have cicatrizant action and that could also have potential action in the modulation of factors associated with aging of the skin. This is the case of barbatimão (Stryphnodendron adstringens (Mart.) Coville), a plant native to the Cerrado and Amazonian Biomes that has its chemical matrix rich in biomolecules, such as tannins. Objectives: To evaluate in vitro the modulatory effect of the aqueous extract of barbatimão on cytofunctional markers of a commercial line of senescent human fibroblasts. Methodology: the human fibroblast lineage (HFF-1) was purchased from the ATCC-EUA via Cell Bank of Rio de Janeiro. In the Laboratory of Biogenomics (UFSM), to obtain a senescent phenotype, the lineage was cultured under controlled conditions (37°C and 5% CO2 saturation) during several passages until it showed a decrease in the proliferation rate in 72h cultures and cyto-morphological modifications of the cell monolayer. A concentration response curve of barbatimão extract (0.49, 0.99, 1.99, 3.92 mg/mL) was initially performed in the senescents fibroblasts, where a potential effect on viability was investigated. From this curve, the concentrations with potential efficacy and safety were chosen for the continuation of the analyzes. The cells cultured again were exposed to these concentrations and after 24 and 72 hours and through spectrophotometric, fluorimetric, and gene modulation tests by qRT-PCR technique, complementary analyzes were conducted in relation to parameters related to cell viability and proliferation, including fibroblast growth factor (FGF) and keratinocytes (KGF), morphology, inflammatory markers such as interleukins (IL-1β, IL-6, IL-10), tumor necrosis factor (TNF-α) and interferon gamma (INF-γ), cell apoptosis, such as caspases (Casp 3 and 8) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Expression of the telomerase, sirtuin and p53 genes was used as a marker of senescence. Results: from the initial curve, concentrations of 0.49 mg/mL and 0.99 mg/mL were chosen to evaluate the effect of barbatimão on the modulation of senescents fibroblasts. Cell proliferation was increased only at the concentration of 0.99 mg/mL. However, the increase in FGF and KGF levels, the reduction of inflammatory cytokines (IL-1β, IL-6, TNF-α, INF-γ), an increase in anti-inflammatory activity (IL-10) and a decrease in Casp 3 and 8 and 8-OHdG were dose-dependent of barbatimão extract. In gene expression, telomerase and sirtuin genes were also super-expressed in a dose-dependent way, whereas p53 was down regulated. Despite the methodological limitations associated with in vitro protocols, the observed results point to the modulatory capacity of barbatimão for the senescence of senescent human dermal fibroblasts (HFF1-old). These results suggest that barbatimão could be used in cosmetic and/or dermatological preparations aimed at slowing down the aging process of the skin.