Desenvolvimento e caracterização de um modelo de senescência induzida por D-galactose e avaliação dos efeitos do alfa-terpineol em embriões e larvas de zebrafish (Danio rerio)

Abstract

Cellular senescence, a fundamental mechanism of aging, is implicated in several cardiovascular diseases. The zebrafish model (Danio rerio) is a well-established gerontological model; however, D-galactose (D-gal)–induced aging has not yet been tested in zebrafish larvae. The aim of this study was to select, through in silico screening, compounds present in five fruits from Northeastern Brazil with the highest activity in enzymatic pathways involved in aging- associated cardiovascular diseases, as well as to develop in vivo a D-galactose–induced senescence model and to evaluate the effects of α-terpineol(α-T) on senescence, oxidative stress, and heart rate in zebrafish embryos and larvae. Sixty-seven compounds from typical fruits of Northeastern Brazil were investigated in silico for activity in pathways associated with aging-related cardiovascular disease (NRF2, iNOS, NOX, and COX2). Model performance, proComb and Pact docking activity, and molecular docking and molecular dynamics were evaluated. In studies with D. rerio embryos (n = 100), acute toxicity of α-terpineol (OECD 236), mortality rate, and D-gal–induced morphological alterations were assessed. Subsequently, embryos (n = 60) were treated for six days to evaluate morphological changes induced by D- gal (36.03 mg/mL) and the effects of α-terpineol at concentrations of 0.00046 mg/mL and 0.0046 mg/mL.A senescence model was then developed by exposing zebrafish embryos and larvae to D-gal for 72 hours, divided into the following groups: Control (E3 medium); D- galactose (30 mg/mL); D-gal + low-dose α-terpineol (0.000047 mg/mL); D-gal + high-dose α- terpineol (0.000094 mg/mL); and D-gal + resveratrol (RSV, 0.01 mg/mL). Senescence (n = 15), oxidative stress assessed by catalase activity (n = 50), survival and morphological alterations (n = 100), and heart rate (n = 50) were evaluated in the larvae. Student’s t-test and one-way ANOVA followed by Bonferroni post hoc tests were used for statistical analysis. In silico results revealed four robust enzymatic models, with 48 active compounds targeting two or more enzymes; among these, 16 showed activity ≥ 0.6 for NRF2 and ≥ 0.5 for at least one additional enzyme. α-Terpineol was selected for in vivo assays due to its superior molecular dynamics performance, indicating greater stability. The LC₅₀ values for α-terpineol and D-gal were above 0.1 mg/mL and 45.03 mg/mL, respectively, and the LOAEL for α-terpineol was 0.0062 mg/mL. Initial tests with D-gal + α-terpineol (0.0046 and 0.00046 mg/mL) did not reduce the percentage of morphological alterations compared with the E3 group (p < 0.05), but attenuated the severity of the alterations.The developed D-gal–induced senescence model in zebrafish embryos and larvae resulted in a high survival rate (95%), increased β-galactosidase activity in the D-gal group, and reduced activity in the D-gal + low-dose α-terpineol group (p < 0.05). Catalase activity increased and heart rate decreased in the D-gal–treated groups (p < 0.05) compared with the control; however, α-terpineol treatment was not effective in attenuating these effects. In conclusion, novel compounds exhibited activity in aging-associated cardiovascular pathways, particularly those related to NRF2, with α-terpineol standing out. The D-gal–induced senescence model proved to be functional, as demonstrated by increased β-galactosidase levels, elevated catalase activity, and altered heart rate, and low-dose α-terpineol was able to attenuate senescence. Therefore, the use of the developed model is suggested for future studies, along with further molecular and functional approaches to deepen mechanistic understanding of α- terpineol’s effects.