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Influence of photobiomodulation with blue light on the metabolism, proliferation and gene expression of human keratinocytes

Becker, Anja

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The skin, which is the largest organ of the human body, serves as a protective barrier between the internal milieu and the environment. It functions as the body’s first line of defense against infection and regulates its temperature and fluid balance. Keratinocytes are present in all the layers of the epidermis, the outermost layer of the skin, and are essentially connected to the pathophysiology of skin diseases such as psoriasis and atopic dermatitis, and play a crucial role in skin wound healing. Keratinocytes are the first cells to be in contact when exposed with external stimuli and are consequently more amenable to non-invasive treatments such as PBM using blue light. The anti-microbial, anti-inflammatory and anti-proliferative effects of blue light are already used for different medical treatments like psoriasis, neonatal jaundice and back pain. However, little is known about the mechanisms transducing the light induced signals from target molecules over downstream processes and/or gene expression to the biological effects and therefore the aim of this project was to examine the photobiomodulary effect of blue light on the immortalized human keratinocyte cell line HaCaT in detail.

Photobiomodulation using blue light irradiation induces a biphasic dose response curve of metabolism in HaCaT cells with an increase in metabolism and proliferation for low doses and a decrease in metabolism and proliferation for higher doses in vitro. For further tests, 7.5min (10.35J/cm²) respectively 30min (41.4J/cm²) were chosen for subsequent experiments to test the blue light effect after different harvesting times in the proliferative phase respectively the anti-proliferative phase of PBM.

Gene expression evaluation of HaCaT cells after 30min (41.4J/cm²) of blue light irradiation revealed an upregulation of “AHR battery genes” leading to production of phase I and phase II enzymes of xenobiotic metabolism. One important action of this downstream process is to provide a delicate hormesis between promoting and preventing ROM-mediated oxidative stress, which is in agreement with our ROS measurements. H2O2 concentrations are increased 30min after blue light irradiation; however, already 1h after irradiation H2O2 is metabolized by the cells leading to an even lower ROS concentration. Furthermore, steroid hormone biosynthesis is activated as a downstream process of “AHR battery gene” expression already 1h after irradiation triggering anti-inflammatory responses. Additionally, inflammation is also decreased due to oxidative stress inhibited NF-κB signaling pathway and interaction with JunB. DNA replication pathway is downregulated resulting in a decrease in cell proliferation due to primary production of ROS, AHR-induced downregulation of CDKN1B and prolongation of S-phase. However, ROS concentrations are not reaching a damaging level as cell survival pathways are enhanced by crosstalk of AHR-ligand complex with EGFR. Moreover, reduction of TNF-signaling pathway and downregulation of TRADD gene expression, which are relevant for apoptotic signaling, are consistent with FACS analysis as 24h after blue light irradiation cells are not showing any sign of apoptosis. Finally, it can be concluded that gene expression after 30min (41.4J/cm²) of blue light irradiation shows a time course after blue light irradiation, with early response genes and pathways leading to the identification of AHR as a possible target for PBM with blue light via photo-oxidation of tryptophan resulting, when using this described dose, in a cell protective effect with decreased proliferation, production of steroid hormones and prevention of inflammatory responses. Moreover, the anti- proliferative effect can be prolonged by consecutive irradiations each 24h.

Photobiomodulation with 7.5min (10.35J/cm²) blue light induced a proliferation increase in HaCaT cells until at least up to 24h after irradiation, which was documented in gene expression analysis with upregulation of DNA replication pathway and genes connected to cell cycle. H2O2 concentrations were increased 30min after blue light irradiation to an even higher level than after a 30min (41.4J/cm2) blue light irradiation; however, already 1h after irradiation H2O2 was metabolized by the cells. The hypothesis was set that even though H2O2 concentrations were higher after a 7.5min (10.35J/cm²) blue light irradiation compared to 30min (41.4J/cm2) the actual oxidative stress was lower. This was explained with the triphasic ROS production-curve induced by PBM described by Huang et al. 2011 and could be linked to gene expression analysis results, where for example oxidative stress dependent Nrf2 transcribed genes were not deregulated. It was not only shown that ROS production was not damaging the cells but even that cell survival pathways were enhanced by crosstalk of EGFR with the AHR-ligand complex. Furthermore, apoptotic signaling was downregulated as TRADD gene expression and TNF-signaling pathway were reduced. Comparable with 30min (41.4J/cm²) blue light irradiation, gene expression analysis revealed an upregulation of “AHR battery genes” after 7.5min (10.35J/cm²) blue light leading to production of phase I and phase II enzymes of xenobiotic metabolism and steroid hormone biosynthesis as a downstream process of “AHR battery gene” expression. However, deregulation of genes and pathways occurred to a smaller extent. Finally, it can be concluded that PBM with blue light, when using 7.5min (10.35J/cm²), activates AHR and results in a cell protective effect with increased proliferation, production of steroid hormones and induction of cell survival pathways. Furthermore, it is suggested not to use consecutive irradiations each 24h if a proliferative effect is desired.

Item Type: Dissertation
Supervisor: Gretz, Prof. Dr. med. Norbert
Date of thesis defense: 6 November 2017
Date Deposited: 14 Nov 2017 07:42
Date: 2017
Faculties / Institutes: Medizinische Fakultät Mannheim > Zentrum für Medizinische Forschung
Subjects: 570 Life sciences
600 Technology (Applied sciences)
610 Medical sciences Medicine
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