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Abstract

Phototherapy, basically heliotherapy, has already been used for the last 3,000 years. However, major breakthroughs in modern clinical phototherapy were achieved since the beginning of the 20th century, including for instance the invention of lasers. From then on, the use of light as a therapeutic tool grew constantly, demonstrated by the development of new artificial light sources and an increasing number of published studies on a variety of medical, mostly cutaneous, disorders. But although the last decade has witnessed a rapid expansion of photobiomodulation, its scientific acceptance is still debated: first, due to inconsistent experimental outcomes, caused by highly variable study designs and irradiation parameters, and second, due to a lack of mechanistic insights into light-triggered signaling. The latter especially applies for blue light, which is, in comparison to red and (near-)infrared light, rather used for a limited range of medical cutaneous ailments, primarily necessitating inhibitory or even cytotoxic effects, like fibrotic skin diseases. However, also stimulatory effects of blue light have rarely been reported in wound healing studies. Hence, the biomodulatory potential of different blue light doses on cellular activity was examined using primary normal human dermal fibroblasts, NHDF cells, in particular with respect to metabolic processes, cell proliferation and transcriptome changes.

Photobiomodulation using blue light revealed dose-dependent effects on the metabolism of NHDF cells, with low doses resulting in an increased activity, whereas higher irradiation doses induced a decreased and therefore inhibited cell metabolism. Based on the biphasic response, two irradiation doses, each leading to the maximum effect in the respective phase, were selected: 5.4 J/cm² and 21.6 J/cm². Cell metabolism assays performed at different time points after irradiation with either 5.4 J/cm² or 21.6 J/cm², revealed a fast response following the lower dose, while the higher one led to a delayed onset of metabolic inhibition. Nevertheless, both, stimulation and inhibition of cellular metabolism were long-lasting until 24 hours after light treatment. Subsequent studies with repeated irradiations on consecutive days, imitating a 'chronic' exposure model, showed an enhanced inhibition following multiple treatments using 21.6 J/cm² of blue light, whereas two daily treatments over several days were required for an increased stimulation by 5.4 J/cm² of blue light.

The contrary effects on cell metabolism observed for both irradiation doses, were translated into similar, but less pronounced effects in proliferation, which was studied with various assays testing cell cycle distribution, DNA synthesis and cell counts. Also gene expression profiles revealed contrary trends in DNA replication and cell cycle regulation affecting cyclins, CDKs as well as repair checkpoints. Mitochondrial function was assessed by measuring H₂O₂ concentrations and ΔΨm at different time points following irradiation. Both blue light doses revealed a rapid increase of H₂O₂ levels, accompanied by fast decreases in ΔΨm indicating metabolic stress; however, both effects returned towards control levels within 24 hours. Thus, to some extent, light effects were identified to rely on retrograde mitochondrial signaling altering the expression of several transcription factors like NF-κB or Nrf2 via changes in ΔΨm and the Janus-face mediator ROS. Moreover, PRKAA1 and AKT1 coding for primary effectors of metabolic stress, respectively AMPK and AKT, were found significantly expressed exerting antagonistic regulations of FOXO1 and MTOR, which modulate oxidative stress resistance, cell survival and growth. In addition, genes involved in antioxidant defense mechanisms, inter alia transcribed by Nrf2 and AhR, were found up-regulated, showing a higher activation for 21.6 J/cm². Cytoprotection was further promoted by affecting the interplay between pro- and anti-apoptotic genes inducing stress resistance and even a higher proliferative capacity following 5.4 J/cm² of blue light. Prevention of apoptotic signaling was verified by cell viability studies negating cytotoxicity up to a single light dose of 172.8 J/cm². Though, reduced proliferation rates observed after consecutive irradiations with 21.6 J/cm² each were accompanied by a higher occurrence of apoptotic cells, possibly indicating mitotic catastrophe.

In conclusion, blue light seemed to be pro-oxidant in the short term, but anti-oxidant in the long term likely to induce stress resistance, at least following low and controlled amounts of oxidative stress. Driven by metabolic and redox homeostasis, various downstream processes were activated modifying antioxidant defense, survival and proliferation. Moreover, the results indicated that an optimal choice of irradiation parameters, particularly the dose, is important for the effectiveness of the treatment, since doses lower or higher than the optimum can lead to ineffective or even negative outcomes. Using cycled irradiation, the differential effect of blue light doses on NHDF cells might be exploited for novel concepts in advanced wound care, particularly for chronic wounds showing impaired activity of dividing cells and fibrotic skin diseases.