Efeitos da fotobiomodulação sobre o estresse oxidativo, viabilidade celular e marcadores inflamatórios em macrófagos j774 expostos ao peróxido de hidrogênio

Abstract

Inflammation and oxidative stress are involved in cellular dysfunction, tissue damage, and the persistence of inflammatory responses. Macrophages participate in innate immunity and modulate cytokine production associated with inflammation and redox balance. Hydrogen peroxide (H₂O₂) is widely used in in vitro models to induce controlled oxidative stress, whereas photobiomodulation (PBM) has been investigated for its effects on mitochondrial activity, cell viability, and inflammatory mediators. This study evaluated the effects of 780-nm PBM on metabolic viability, cell morphology, and TNF-α and IL-6 production in J774A.1 macrophages subjected to H₂O₂-induced oxidative stress. Cells were allocated into seven groups: control, H₂O₂ 50 µM, H₂O₂ 100 µM, H₂O₂ 200 µM, H₂O₂ 50 µM + PBM, H₂O₂ 100 µM + PBM, and H₂O₂ 200 µM + PBM. Oxidative stress was induced by exposure to H₂O₂ for 1 hour. Cells were then washed, centrifuged to obtain a cell pellet, and, in the designated groups, irradiated with a 780-nm infrared laser, 70 mW output power, 1.05 J total energy, 26.25 J/cm² radiant exposure, continuous mode, 15 seconds, one irradiation point, and a single session. Non-irradiated groups underwent the same manipulation with the device turned off. After irradiation or sham manipulation, cells were resuspended in fresh culture medium and incubated for 24 or 48 hours. Cell viability was assessed by MTT assay, morphology by light microscopy, total protein by NanoDrop spectrophotometry, and TNF-α and IL-6 levels by ELISA, with cytokine concentrations normalized to total protein in the supernatant. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test, with p<0.05 considered significant. H₂O₂ reduced metabolic viability and induced progressive morphological alterations, especially at higher concentrations. At 24 hours, PBM significantly increased viability in the 50 µM + PBM and 200 µM + PBM groups compared with their respective non-irradiated groups; at 48 hours, no significant differences were observed between H₂O₂ and H₂O₂ + PBM groups. Total protein did not differ among groups. H₂O₂ increased TNF-α levels, whereas PBM significantly reduced this cytokine compared with the respective non-irradiated groups. For IL-6, no differences were observed at 24 hours; at 48 hours, the H₂O₂ 100 µM and 200 µM groups showed reduced levels compared with the control group, whereas PBM-treated groups maintained higher IL-6 levels than their respective non-irradiated groups. In conclusion, PBM with a 780-nm laser modulated the response of J774A.1 macrophages subjected to H₂O₂-induced oxidative stress, with better qualitative preservation of cell morphology, increased metabolic viability under specific conditions, reduced TNF-α levels, and maintenance of IL-6 levels in selected groups after 48 hours.