Efeito da fotobiomodulação na proliferação e viabilidade de células endoteliais da veia umbilical humana (HUVEC)

Abstract

The aim of this study was to evaluate, through in vitro experiments, the effect of photobiomodulation using low-level laser irradiation on the proliferation and viability of human endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured and divided into four groups: C - control (without irradiation) and L1, L4 and L7.5 groups - cells irradiated with doses of 1, 4, and 7.5 J/cm², respectively, of diode laser (InGaAlP, wavelength of 660 nm, power of 30 Mw, in a single dose). The metabolic activity of the cells was assessed at the 24, 48 and 72 h after irradiation by the Alamar Blue assay. Cell viability and proliferation were also evaluated at 72 h through the Live/Dead, Annexin V/PI assays, cell cycle phase analysis and Ki67 protein immunostaining. Data from the Alamar Blue assay showed that at 72 h all irradiated groups exhibited significantly greater reduction percentages than the non-irradiated control group (p <0.01), the difference being even more evident in L4 and L7.5 (p <0.001). The analysis of cell viability by Live/Dead demonstrated a large number of viable cells in all groups, which was also observed through Annexin V/PI immunostaining, which showed a high percentage of cell viability in the studied groups (C: 95.2%, L1: 96.1%, L4: 96.1%, L7.5: 96.9%). The evaluation of the phases of the cell cycle showed that the irradiated groups exhibited a gradual increase of the percentage of cells in the proliferative phases of the cycle (S and G2/M), in a dosedependent manner (C: 36.9%, L1: 38.0 %, L4: 38.3%, L7.5: 40.4%). Irradiation promoted a significant increase in Ki67 nuclear protein expression, with a more pronounced result when the G7.5 group was compared with the other groups. Taken together, the results of this study demonstrated that photobiomodulation, especially at a dose of 7.5 J/cm², promoted increased proliferation and viability of HUVEC cells, which may suggest the potential use of the laser in the parameters studied as a stimulus to endothelialization in the vascular tissue engineering techniques.