Elucidating the time course of the transcriptomic response to photobiomodulation through gene co-expression analysis
Introduction
Photobiomodulation (PBM) – the irradiation of cells or tissues with low-intensity red to near infrared light (600–1100 nm) in order to modulate biological processes – accelerates tissue repair and wound healing, reduces inflammation and alleviates pain [[1], [2], [3]]. There is now substantial evidence that PBM also has neuroprotective effects, mitigating abnormalities in nervous system structure and/or function in pre-clinical models of Alzheimer's disease, Parkinson's disease, traumatic brain injury, stroke, retinal disease and multiple sclerosis, as reviewed elsewhere [4,5].
Despite its demonstrated efficacy, the mechanisms underpinning the protective effects of PBM in various neurodegenerative and other disease states are yet to be fully elucidated. The biphasic dose-response relationship of PBM [6] is consistent with the phenomenon of hormesis, whereby an intervention that is inhibitory or detrimental at a high dose is also stimulatory or beneficial at a low dose [7]. The observation of hormesis in biological systems generally indicates the activation of an adaptive stress response [8], providing clues to the mechanisms underlying these wide-ranging effects.
Past research suggests PBM conveys its beneficial effects throughout the body via two complementary avenues, which can be categorised as the intracellular (direct) and systemic (indirect) mechanisms of PBM [9]. Most relevant to this article are the direct, intracellular effects, which are associated with a host of molecular changes, some of which persist long after light exposure has ceased [10,11]. These can be further categorised into the ‘primary’ and ‘secondary’ intracellular effects of PBM.
The mitochondria appear key in mediating the ‘primary’ intracellular effects of PBM. A large body of evidence suggests the primary photoacceptor of red to near infrared light is cytochrome C oxidase, a key enzyme in mitochondrial electron transport chain [[12], [13], [14]]. The absorption of photons by cytochrome C oxidase is proposed to cause a redox change in the enzyme, leading to increased mitochondrial membrane potential, increased ATP production, the liberation of nitric oxide and a transient burst of reactive oxygen species (ROS) [10,15].
While these primary intracellular effects of PBM are fairly well established, they alone are unlikely to account the broad and potent cytoprotective effects of PBM. The benefits of PBM have been shown to persist following cessation of treatment, implicating the activation of downstream “secondary” effectors to establish this prolonged effect [15,16]. Molecules such as nitric oxide, ROS and ATP have been implicated in the activation of transcription factors such as cyclic AMP response element binding protein, activator protein−1 and nuclear factor kappa B [10], which in turn modulate expression of a range of genes that feed into a variety of molecular pathways. It is this widespread regulation of the transcriptome, which occurs as a downstream consequence of the primary effects of PBM within the mitochondria, that we propose is the main contributor to the observed protective effects of PBM.
In order to gain a clear and comprehensive view of the molecular mechanisms underlying the secondary intracellular effects of PBM, a holistic approach is required: one that moves beyond investigations of candidate molecules to instead consider entire molecular systems and the timing of their involvement. In this study, we aimed to gain insights into the secondary intracellular effects of PBM by screening the transcriptomic response to a therapeutic dose of PBM over a 24 h time course. To retain important information about the temporal dimension of this response, we developed a co-expression analysis approach to identify coordinated changes in molecular pathways over the 24 h period.
Section snippets
Cell Culture and Treatment
Human neuroblastoma SH-SY5Y cells (ATCC® CRL-2266™), a transformed cell line commonly used to model neuronal-like cells [17], were cultured in Dulbecco's Modified Eagle's Medium containing 10% (v/v) Foetal Bovine Serum and 0.1 μg/mL penicillin/streptomycin. Cells seeded into separate 6-well plates and grown to ~80% confluence were treated with 670 nm PBM, delivered from a WARP75 LED panel (Quantum Devices, Barneveld, WI, USA). The LED panel was positioned 13.6 cm from the base of the plate,
Overview of Microarray Results
To gain insights into the temporal intracellular molecular response to PBM, RNA was isolated from cells at various time points over a 24 h period (15 and 30 min and 1, 2, 4, 8 and 24 h) post-PBM treatment, and the transcriptome comprehensively screened using RNA microarray.
The majority of expression changes reflected decreased transcript levels following PBM treatment, relative to untreated baseline controls. With respect to the number of total gene expression changes (nominal p < .05, fold
Discussion
This study utilised an unbiased RNA microarray screening strategy to identify intracellular molecular networks that are modulated in response to a cytoprotective dose of PBM, and the temporal sequence of events over a 24-hour time window. By applying a novel bioinformatics pipeline that coupled clustering with two different enrichment analysis platforms, this analysis has revealed temporal gene expression patterns that provide clues to the various signalling cascades and molecular networks that
Conclusions
The current study reports the first comprehensive analysis of gene expression changes occurring across a time course post-PBM. By coupling hierarchical clustering with extensive bioinformatics analysis, we have revealed that PBM induces subtle gene expression changes across multiple intracellular networks, culminating in the coordination of distinct early and late phase effects on cellular stress response systems. The early activation of transcriptional processes and modulation of various
Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding Sources
This study was funded by the University of Sydney BRIG Scheme.
Acknowledgements
We thank the Molecular Biology Facility of the Bosch Institute (University of Sydney) for access to their facilities and the Hunter Medical Research Institute for access to their microarray scanner.
References (32)
Hormesis defined
Ageing Res. Rev.
(2008)- et al.
Remote photobiomodulation as a neuroprotective intervention – harnessing the indirect effects of PBM
Primary and secondary mechanisms of action of visible to near-IR radiation on cells
J. Photochem. Photobiol. B
(1999)- et al.
Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase
J. Biol. Chem.
(2005) - et al.
Remote tissue conditioning - an emerging approach for inducing body-wide protection against diseases of ageing
Ageing Res. Rev.
(2017) - et al.
Biological effects and medical applications of infrared radiation
J. Photochem. Photobiol. B
(2017) - et al.
Infrared a radiation influences the skin fibroblast transcriptome: mechanisms and consequences
J. Investig. Dermatol.
(2010) - et al.
Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells
Mech. Ageing Dev.
(2012) - et al.
Emerging real-time technologies in molecular medicine and the evolution of integrated ‘pharmacomics’ approaches to personalized medicine and drug discovery
Pharmacol. Ther.
(2012) - et al.
Laser photobiomodulation of wound healing: a review of experimental studies in mouse and rat animal models
Photomed. Laser Surg.
(2010)
Mechanisms and applications of the anti-inflammatory effects of photobiomodulation
AIMS Biophys.
The efficacy of low-power lasers in tissue repair and pain control: a meta-analysis study
Photomed. Laser Surg.
Brain photobiomodulation therapy: a narrative review
Mol. Neurobiol.
Turning on lights to stop neurodegeneration: the potential of near infrared light therapy in Alzheimer’s and Parkinson’s disease
Front. Neurosci.
Biphasic dose response in low level light therapy - an update
Dose Response
Defining hormesis
Hum. Exp. Toxicol.
Cited by (7)
Photobiomodulation Using Light-Emitting Diode (LED) for Treatment of Retinal Diseases
2024, Clinical OphthalmologyNeurodegenerative and Neurodevelopmental Diseases and the Gut-Brain Axis: The Potential of Therapeutic Targeting of the Microbiome
2023, International Journal of Molecular SciencesTherapies for Parkinson’s disease and the gut microbiome: evidence for bidirectional connection
2023, Frontiers in Aging NeuroscienceTherapeutic Potential of Photobiomodulation for Chronic Kidney Disease
2022, International Journal of Molecular SciencesRemote Photobiomodulation Treatment for the Clinical Signs of Parkinson's Disease: A Case Series Conducted during COVID-19
2022, Photobiomodulation, Photomedicine, and Laser SurgeryMicrobiome Changes in Humans with Parkinson’s Disease after Photobiomodulation Therapy: A Retrospective Study
2022, Journal of Personalized Medicine
- 1
These authors contributed equally.