Laser Biostimulation Induces Wound Healing-Promoter β2-Defensin Expression in Human Keratinocytes via Oxidative Stress

Abstract

The innate immune system is the first line of defense of the body composed of anatomical barriers, such as skin and mucosa, as well as effector cells, antimicrobial peptides, soluble mediators, and cell receptors able to detect and destroy viruses and bacteria and to sense trauma and wounds to initiate repair. The human β-defensins belong to a family of antimicrobial small cationic peptides produced by epithelial cells, and show immunomodulatory and pro-healing activities. Laser biostimulation is a therapy widely used to contrast microbial infection and to accelerate wound healing through biological mechanisms that include the creation of oxidative stress. In this paper, we explored laser biostimulation’s ability to modulate the production of two β-defensins, hBD-1 and hBD-2, in human keratinocytes and whether this modulation was, at least in part, oxidative-stress-dependent. Human spontaneously immortalized keratinocytes (HaCaT) were stimulated using laser irradiation at a 980 nm wavelength, setting the power output to 1 W (649.35 mW/cm2) in the continuous mode. Cells were irradiated for 0 (negative control), 5, 10, 25 and 50 s, corresponding to an energy stimulation of 0, 5, 10, 25 and 50 J. Positive control cells were treated with lipopolysaccharide (LPS, 200 ng/mL). After 6 and 24 h of treatment, the cell conditioned medium was collected and analyzed via ELISA assay for the production of hBD-1 and hBD-2. In another set of experiments, HaCaT were pre-incubated for 45 min with antioxidant drugs—vitamin C (Vit. C, 100 µM), sodium azide (NaN3, 1 mM); ω-nitro-L-arginine methyl ester (L-NAME, 10 mM) and sodium pyruvate (NaPyr, 100 µM)—and then biostimulated for 0 or 50 s. After 6 h, the conditioned medium was collected and used for the ELISA analysis. The hBD-1 and hBD-2 production by HaCaT was significantly increased by single laser biostimulation after 6 h in an energy-dependent fashion compared to basal levels, and both reached production levels induced by LPS. After 24 h, only hBD-2 production induced by laser biostimulation was further increased, while the basal and stimulated hBD-1 levels were comparable. Pre-incubation with antioxidative drugs was able to completely abrogate the laser-induced production of both hBD-1 and hBD-2 after 6 h, with the exception of hBD-1 production in samples stimulated after NaN3 pre-incubation. A single laser biostimulation induced the oxidative-stress-dependent production of both hBD-1 and hBD-2 in human keratinocytes. In particular, the pro-healing hBD-2 level was almost three times higher than the baseline level and lasted for 24 h. These findings increase our knowledge about the positive effects of laser biostimulation on wound healing.