Efeito do ânion superóxido em marcadores de senescência proliferativa e funcional de fibroblastos dérmicos humanos

Abstract

Aging is an irreversible process that is associated with the decline of certain biological functions, including those visible on the skin. Oxidative stress, resulting from the formation of excess reactive oxygen species (ROS), such as the superoxide anion (O2●), can be considered the central mechanism of skin aging. Although the relevance of oxidative metabolism in aging is well established, investigations on the subject do not address the specific role of different ROS in triggering changes related to senescence. In this sense, it is possible that changes in mitochondrial O2● levels induce proliferative and functional changes in dermal fibroblasts, contributing to skin aging. To test this hypothesis, the present study evaluated the in vitro effect of the pharmacological imbalance of superoxide anion, induced by acute exposure to rotenone, on the modulation of markers of replicative and functional senescence of young dermal fibroblasts. For that, cultures of the commercial strain of human fibroblasts (HFF-1) were exposed to rotenone for 72 hours and compared with the control group in two complementary protocols. The first protocol evaluated five different concentrations of rotenone in order to identify the concentration capable of triggering senescence changes in β-galactosidase activity, cell proliferation, apoptosis and superoxide anion markers. In a second moment, the effect of the rotenone concentration chosen in the previous protocol was evaluated in chromosomal alterations by micronucleus test, oxidative stress markers, fibroblast interconnections and intra and extracellular collagen deposition. The impact of rotenone on the expression of genes that participate in collagen metabolism was also investigated here. The results showed that, although rotenone-treated cells did not express increased superoxide levels, there was a decrease in mitochondrial metabolism activity (concentrations from 0.5 to 1.5 µM) and an increase in βgalactosidase activity levels. (concentrations from 0.5 to 2.5 µM), in addition, none of the concentrations showed cytotoxic activity, nor were they able to induce interruption in the rate of cell synthesis. However, rotenone at a concentration of 1 µM induced an increase in the frequency of apoptotic cells. Based on these results, a 1 µM concentration of rotenone was used in complementary tests showing greater DNA damage, increased levels of oxidative markers, decreased cytoplasmic fibroblast connections and extracellular collagen deposition. Additional analysis showed downregulation of the collagen I genes, FGF-2 and FGF-7, and overexpression of the MMP-1 gene. Despite the methodological limitations inherent to the in vitro study, our results suggest that O2● could be an oxidative molecule that triggers replicative and functional prosenescence changes in fibroblasts, contributing to skin aging.