Fluorescent Light Energy (FLE) Acts on Mitochondrial Physiology Improving Wound Healing

Abstract

Fluorescent light energy (FLE) has been used to treat various injured tissues in a non-pharmacological and non-thermal fashion. It was applied to stimulate cell proliferation, accelerate healing in chronic and acute wounds, and reduce pain and inflammation. FLE has been shown to reduce pro-inflammatory cytokines while promoting an environment conducive to healing. A possible mechanism of action of FLE is linked to regulation of mitochondrial homeostasis. This work aims to investigate the effect of FLE on mitochondrial homeostasis in an in vitro model of inflammation. Confocal microscopy and gene expression profiling were performed on cultures of inflamed human dermal fibroblasts treated with either direct light from a multi-LED lamp, or FLE from either an amorphous gel or sheet hydrogel matrix. Assessment using confocal microscopy revealed mitochondrial fragmentation in inflamed cells, likely due to exposure to inflammatory cytokines, however, mitochondrial networks were restored to normal 24-h after treatment with FLE. Moreover, gene expression analysis found that treatment with FLE resulted in upregulation of uncoupling protein 1 (UCP1) and carnitine palmitoyltransferase 1B (CPT1B) genes, which encode proteins favoring mitochondrial ATP production through oxidative phosphorylation and lipid β-oxidation, respectively. These observations demonstrate a beneficial effect of FLE on mitochondrial homeostasis in inflamed cells.

Keywords: fluorescence; fluorescent light energy; gene expression; inflammation; mitochondria; mitochondrial dynamics; wound healing.

Figure 1

Quantification of mitochondrial network 30-min post-treatment. After deconvolution, images were 3D reconstructed and the mitochondrial network was evaluated by automated estimation of (a) volume of the entire mitochondrial network per single cell, (b) number of mitochondria per single cell, and (c) volume of single mitochondrion. Data are expressed as mean ± SD. Multi comparison statistical analysis were performed by using one-way analysis of variance (ANOVA). T tests were performed on all pairwise comparisons between group means. * p < 0.05 from Healthy human dermal fibroblasts (HDFs), † < 0.05 from Inflamed human dermal fibroblasts (HDFs), ns = not significant. (d) Representative images. FLE: fluorescent light energy.

Figure 2

Quantification of mitochondrial network 24-h post-treatment. After deconvolution, images were 3D reconstructed and the mitochondrial network was evaluated by automated estimation of (a) volume of the entire mitochondrial network per single cell, (b) number of mitochondria per single cell, and (c) volume of single mitochondrion. Data are expressed as mean ± SD. Multi comparison statistical analysis were performed by using one-way analysis of variance (ANOVA). T test was to perform all pairwise comparisons between group means. * p < 0.05 from Healthy human dermal fibroblasts (HDFs), † < 0.05 from Inflamed human dermal fibroblasts (HDFs), ns = not significant. (d) Representative images. FLE: fluorescent light energy.

Figure 3

Radial graphs depicting relative changes in mitochondrial networks at (a) 30-min and (b) 24-h post-treatment. All parameters are indexed to Healthy-HDFs (green). Inflamed-HDFs are depicted in red, Light-treated HDFs in blue, FLE-Gel in yellow, and FLE-Matrix in orange. At time point 30-min the FLE-Gel (yellow) and FLE-Matrix (Orange) overlap, and at time point 24-h the Healthy (green) and FLE-Gel (yellow) overlap. MV/c = Mitochondria Volume per cell; MN/c = Mitochondria Number per cell; IMV = Individual Mitochondrion Volume.