Efeito do Stryphnodendron adstringens (Mart.) Coville em marcadores inflamatórios e proliferativos da cicatrização de feridas: um modelo bi-celular in vitro

Abstract

The skin is the largest organ in the human body and acts as a barrier protecting the body from the environment. The rupture of its integrity caused by an injury culminates in the activation of a complex healing process, which seeks the closure of the injured area and involves three overlapping stages over time: inflammatory, proliferative and remodeling. Often, the healing process is not efficient, necessitating the use of drugs or herbal medicines. A medicinal plant that has important healing properties is the Stryphnodendron adstringens (Mart.) Coville, popularly known as barbatimão and native of the Brazilian Cerrado biome. However, it is still unclear how this plant acts on the steps of the healing process. Within this context, the present study aimed to evaluate the in vitro effect of Stryphnodendron adstringens on the modulation of inflammatory and proliferative markers of the wound healing process. For this, an independent bi-cellular experimental model was developed using the commercial cell lines of macrophages (RAW 264.7) and dermal fibroblasts (HFF-1). Cells were maintained in cell culture separately, under sterile conditions at 37°C and 5% CO2. Macrophages were activated with the mitogenic agent phytohemagglutinin (PHA) and, after 24 hours, were treated with different concentrations of the barbatimão bark extract (0.24; 0.49; 0.99; 1.99; 3.98 mg/mL). After 72 hours, cell proliferation was evaluated by the test of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolic bromide (MTT). With the result of this test, the concentrations of 0.49 and 0.99 mg/mL of the barbatimão extract were defined for use in the subsequent analyzes. It was then evaluated, after 72 hours of treatment with both concentrations, the effect of the plant on protein levels (via immunoassay) and levels of gene expression (via Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)) of the proinflammatory cytokines interleukin one beta (IL-1β), interleukin six (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-ү) and the anti-inflammatory cytokine interleukin ten (IL-10) in activated and non-activated macrophages, to investigate the inflammatory phase. An additional protocol, which mimics in vitro healing processes, was also used. For this, cultures of fibroblasts with 80% of monolayer confluence were scratched with the aid of a 200 μL tip to mimic an injury and subsequently these cells were treated with both concentrations of the barbatimão extract. Then, after 24 and 72 hours of treatment, the levels of fibroblast growth factor one (FGF-1) and keratinocyte growth factor (KGF) were analyzed in scratched and integral fibroblasts to evaluate the proliferative state. The results showed anti-inflammatory effect of barbatimão on the activated macrophages, through a decrease of the protein levels and the gene expression levels of the proinflammatory cytokines and increase of the levels of the anti-inflammatory cytokine IL-10. In addition, barbatimão also increased the levels of both growth factors in scratched and integral fibroblasts after 24 and 72 hours of treatment. Despite the methodological limitations related to the in vitro studies, the results indicate that barbatimão has an effective action in the inflammatory modulation of macrophages, as well as it is able to induce the increase of the levels of growth factors related to proliferative events of fibroblasts during the process of wound healing.