[Experimental study on the effects of 810-nm diode low-level laser on oxidative stress and wound healing]

Objective: To investigate the mechanism by which an 810-nm diode low-level laser (LLL) promotes angiogenesis through activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (NRF2-HO-1) signaling pathway, and to provide a theoretical basis for the repair of acute soft-tissue injury. Methods: 1. In vitro experiments, oxidative stress injury in human umbilical vein endothelial cells (HUVECs)was induced by treatment with 100 μmol/L H₂O₂ for 12 h. The cells were divided into a control group, an H₂O₂ group, and low-level laser intervention groups. The intervention groups received 810-nm diode laser irradiation at energy densities of 2 J/cm² (E1 group), 4 J/cm² (E2 group), and 8 J/cm² (E3 group). Cell proliferation was assessed using the CCK-8 assay. Cell migration was evaluated using wound-healing and Transwell assays. Tube formation on Matrigel was performed to assess in vitro capillary-like structure formation. Intracellular reactive oxygen species (ROS) levels were measured using the 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, and mitochondrial membrane potential was detected using the JC-1 probe. Immunofluorescence staining was performed to detect NRF2 nuclear translocation and HO-1 expression. 2. In vivo experiments, SPF male Sprague-Dawley rats (200-250 g) were used to establish a full-thickness skin wound model. The rats were randomly divided into a control group and a LLL therapy (LLLT) group. The LLLT group received 810-nm laser irradiation at an energy density of 40 J/cm² on the wound area, whereas the control group received no laser treatment. Wound area and healing rate were measured using ImageJ software. Histological changes were observed by HE and Masson staining, and angiogenesis was evaluated by CD31 immunofluorescence staining. Results: Compared with the control group, H₂O₂ treatment significantly decreased the proliferative activity of HUVECs, increased intracellular ROS levels, and reduced mitochondrial membrane potential. After LLL irradiation, cell proliferation increased in all treatment groups compared with the H₂O₂ group. In particular, the E2 group (4 J/cm²) showed optical density values of 1.24±0.11, 1.43±0.06, and 1.83±0.14 at 24, 48, and 72 h, respectively, which were higher than those in the H₂O₂ group (P<0.05). The wound-healing assay showed significant differences in migration rates among groups (P<0.001), with the E2 group showing a migration rate of (%56.07±5.61%), compared with (24.83%±4.31%) in the H₂O₂ group. Transwell assay results also showed significant differences in the number of migrated cells among groups (P<0.001); the E2 group had 74.62±5.98 migrated cells, which was higher than the 20.21±6.55 cells observed in the H₂O₂ group. Tube formation analysis showed significant differences in the number of tubes among groups (P<0.05), with the E2 group exhibiting 43.95±3.47 tubes compared with 26.74±4.65 in the H₂O₂ group. ROS levels decreased and mitochondrial membrane potential increased after LLL treatment. Immunofluorescence analysis showed that the NRF2 nuclear/cytoplasmic fluorescence intensity ratio increased to 2.07±0.46 in the LLL group, compared with 1.39±0.26 in the H₂O₂ group (P<0.05), accompanied by increased HO-1 expression. In the animal experiment, wound healing rates in the LLL group at days 3, 7, and 14 were (37.98±1.14)%, (54.15±6.39)%, and (90.25±2.25)%, respectively, which were higher than those in the control group [(23.16±2.86)%, (34.95±0.39)%, and (77.22±6.01)%] (P<0.05). Histological observation showed increased collagen deposition and more capillary-like structures in the LLL group. Conclusions: 810 nm low-level laser may attenuate H₂O₂-induced oxidative stress damage in HUVECs by activating the NRF2-HO-1 signaling pathway, thereby improving cell proliferation, migration, and in vitro tube formation capability, as well as promoting acute wound healing in rats.