Atividade protetora do carvacrol na senescência endotelial do corpo cavernoso induzida por D-galactose: investigações in vitro e in silico

Abstract

Aging is a natural, complex, and multifactorial phenomenon characterized by the decline of physiological functions at the molecular and cellular levels over time. In the cardiovascular system, this process results in the progressive accumulation of senescent endothelial cells, which exhibit abnormal function and a loss of proliferative capacity, leading to endothelial dysfunction — a key mechanism in the development of various disorders, including erectile dysfunction (ED). The ED is the inability to achieve and/or maintain a penile erection sufficient for satisfactory sexual intercourse. Increasing evidence suggests that oxidative stress is one of the main mechanisms responsible for endothelial senescence. Thus, reducing the levels of reactive oxygen species (ROS) may be an important factor in reversing oxidative damage and restoring endothelial function. Antioxidant compounds, such as carvacrol, limit this damage and therefore offer benefits in preventing endothelial senescence. Therefore, the aim of this study was

to evaluate the senoprotective activity of carvacrol in cavernous endothelial senescence induced by D- galactose, through in vitro and in silico investigations. For this, rat cavernous endothelial cells (RCEC)

were cultured and treated for 48 hours, in the absence and presence of D-galactose, an agent that

induces premature senescence, and carvacrol. Initially, was evaluated cell viability, β-galactosidase- associated senescence (SA-β-galactosidase), and the antioxidant activity of carvacrol. In the D- galactose model, treatment with carvacrol restored cell viability and, under basal conditions, did not

show toxic effects. Regarding SA-β-galactosidase, carvacrol was able to reduce senescence both in the presence and absence of D-galactose. Furthermore, in the senescence model, carvacrol was capable of reducing oxidative stress. Oxidative stress is often accompanied by a lower production of nitric oxide (NO), which may weaken its protective effect. Therefore, we evaluated whether the decrease in oxidative stress caused by carvacrol could be increasing the bioavailability of NO. This analysis demonstrated that carvacrol, in the presence of D-galactose, increases the bioavailability of NO, indicating an improvement in endothelial health. Apotosis was also evaluated, revealing that carvacrol is able to increase cell survival and decrease early and late apoptosis in the presence of D-galactose, suggesting that, by acting as an antioxidant and increasing NO bioavailability, it could be reducing the induced oxidative damage. Senescent cells are generally considered to be permanently arrested in the G0/G1 phase of the cell cycle, therefore, we evaluated the proportion of RCEC in each phase of the cycle. Carvacrol, in the presence of D-galactose, reduced the proportion of these cells in the G0/G1 phase and increased their presence in the S and G2/M phases, probably by acting as an antioxidant, since oxidative damage is one of the main mechanisms responsible for cell cycle arrest in senescent cells. Finally, in order to further understand the mechanisms involved in these effects, key molecular targets of senescence pathways were selected for interaction analysis with carvacrol, through in silico analysis. These studies revealed interactions with all seven evaluated targets, with emphasis on p53 and NADPH oxidase, which presented highly stable interactions, which may be even better than the standard ligand. Thus, the antioxidant activity and reduction of cell cycle arrest in the G0/G1 phase of carvacrol may be related to the negative modulation of NADPH oxidase and p53, respectively.