Effect of photobiomodulation at 830 nm on gene expression correlated with JAK/STAT signalling in wounded and diabetic wounded fibroblasts in vitro

Treatment of chronic diabetic wounds is an ongoing socio‐economic challenge. Dysregulated signalling pathways characterise cells from chronic diabetic wounds. Photobiomodulation (PBM) stimulates healing by eliciting photochemical effects that affect gene regulation. JAK/STAT signalling is a primary signal transduction pathway involved in wound healing. This in vitro study aimed to determine if PBM at 830 nm and a fluence of 5 J/cm2 regulates genes related to JAK/STAT signalling in wounded and diabetic wounded fibroblast cells. A continuous wave diode laser (12.53 mW/cm2) was used to irradiate cells. Forty‐eight hours post‐PBM, RT‐qPCR was used to analyse 84 genes related to JAK/STAT signalling. Five genes were upregulated and four downregulated in wounded cell models, while six genes were downregulated in diabetic wounded models. The results show drastic gene expression differences between wounded and diabetic wounded cell models in response to PBM using 830 nm.


| INTRODUCTION
Globally, diabetes mellitus (DM) remains one of the most common chronic pathologies.In Africa, DM poses a dire challenge in the economic, medical, social, and financial systems due to its unresponsiveness to orthodox therapeutic procedures, necessitating meticulous intervention.In 2018, South Africa spent approximately ZAR 21.5 billion on diagnosed and undiagnosed DM patients, and this figure is expected to rise to ZAR 35 billion by 2030 [1].
Frequently, DM results in the development of non-healing chronic wounds and diabetic foot ulcers (DFUs).If not cautiously treated, these defects may result in chronic pain and irreversible paralysis, limb amputation, impaired quality of life and lowered standard of living [2].
Photobiomodulation (PBM) involves the application of focused light from lasers and/or light-emitting diodes (LEDs) in the red (600-760 nm) or near-infrared (NIR; 780-1000 nm) light spectrum to revitalise injured tissue, attenuate inflammation, and alleviate pain [3].PBM elicits photochemical and photophysical effects by stimulating cytochrome c oxidase (COX), a terminal enzyme in the mitochondrial respiratory chain, resulting in the extrication of crucial inflammatory mediators and the release of growth factors [4].Light absorption at a non-thermal irradiance by mitochondrial COX results in electronic excitation and, subsequently, the acceleration of electron transfer reactions [5].Furthermore, PBMinduced dissociation of nitric oxide (NO) from COX results in elevated oxygen reduction, mitochondrial membrane potential [6], and adenosine triphosphate (ATP) synthesis, and maintenance of reactive oxygen species (ROS) homeostasis [7].Additional proposed cellular physiologies associated with PBM include the regulation of calcium (Ca 2+ ), potassium (K + ), and sodium (Na + ) ion transportation.PBM-influenced regulation of Ca 2+ , cyclic adenosine monophosphate (cAMP), NO, and ROS activates numerous intracellular signalling pathways [6] correlated with cell proliferation, differentiation, survival, enzyme activation, protein synthesis, and antiinflammatory signalling.
Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling is the principal intracellular signal transduction pathway implicated in a plethora of biological processes, including cell differentiation, proliferation, survival, migration, inflammation, and apoptosis [8].The JAK/STAT pathway is one of the signalling pathways that transmit signals from the extracellular milieu (i.e., cell membrane receptors) to the nucleus, and is activated in response to tissue injury.Post-tissue injury, various ligands such as cytokines, hormones, and growth factors bind to their respective cellular receptor subunits, inducing receptor conformational changes into homodimers (e.g., growth hormone), heterodimers, or heteromultimers (e.g., interleukins; ILs) [8].According to Murray [9], 38 protein ligands and 36 receptor subunit complexes have been depicted.Postligand-mediated receptor multimerisation and intracellular domain receptor transphosphorylation there is recruitment and activation of JAK.The JAK family comprises four members: JAK1, JAK2, JAK3 and nonreceptor tyrosine protein-kinase (TYK2).
Activated JAK recruits and phosphorylates dormant cytoplasmic STAT proteins.Mammalian STAT members include STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6.Post-phosphorylation, STATs dimerise in an antiparallel conformation via extended interfaces with the conserved SH2 domain.Dimerised STATs are translocated to the nucleus in an importin α-5 dependent manner through the Ran nuclear import pathway to bind specific DNA regulatory sequences, thus regulating the transcription of target genes [10,11].Activation of the JAK/STAT signalling pathway is normalised by numerous negative regulators, including the classical negative feedback loop mechanism through suppressor of cytokine signalling (SOCS) proteins and protein inhibitors against STATs (PIAS) [12,13].Disturbances in the stringent regulation of the JAK/STAT signalling pathway can lead to a vast array of pathological disorders, including cancer and the development of chronic wounds [12].
Profiling gene expression for cell signalling pathways using pathway-specific polymerase chain reaction (PCR) arrays gives an insight into the activation of such pathways in the wound-healing process.To date, no studies have produced a JAK/STAT pathway gene signature in diabetic wounded fibroblast cells following irradiation at a NIR wavelength.Regulation of gene expression for a particular cell signalling pathway underlies changes in cell activities, including cell growth and survival [14].
The present study focuses on the regulation of genes related to JAK/STAT signalling in wounded and diabetic wounded fibroblast cells following PBM at a wavelength of 830 nm and a fluence of 5 J/cm 2 .

| Human fibroblast cell culture
Ethical clearance was granted by the University of Johannesburg, Faculty of Health Sciences, Research Ethics Committee (REC-1021-2021).Commercially purchased human fibroblast cells (BJ-5ta, ATCC ® CRL-4001™) were acquired from the American Type Culture Collection (ATCC) and were cultured according to conventional cell culture methods.Cells were cultured in Minimum Essential Medium (MEM) (Sigma-Aldrich, M7278) containing 10% foetal bovine serum (FBS; Sigma-Aldrich, F9665), 1% penicillin-streptomycin (Sigma-Aldrich, P42942), 1% amphotericin B (Sigma-Aldrich, A2942), 0.1 mM nonessential amino acids (NEAA; Sigma-Aldrich, 11 140-035), 1 mM sodium pyruvate (Sigma-Aldrich, S8636), and 2 mM L-glutamine (Sigma-Aldrich, D6429), and incubated at 37 C (5% CO 2 ; 85% humidity).Two cell models namely wounded (W) and diabetic wounded (DW) were designed.To achieve the diabetic wounded models, human fibroblast cells were continuously grown in media with a basal glucose concentration of 5.6 mM.An additional 17 mM D-glucose was added to generate a hyperglycaemic environment at a total glucose concentration of 22.6 mM [15].For experiments, 1 Â 10 6 cells were seeded into 3.4 cm diameter cell culture plates (BD Biosciences, The Scientific Group, BD 353001).Wounded cell models were formed using a disposable sterile 1 mL pipette (BD Biosciences, The Scientific Group, BD 357522) tip 30 min prior to irradiation to allow cells to settle.A wound was simulated by creating a unidirectional central scratch on a confluent cell monolayer, creating the "wounded" cell models [15,16].The central scratch technique is based on the fact that after generating a "cell-free zone" down the middle of the cell monolayer, cells at the wound edges move toward the simulated gap to close/cover it.

| Laser irradiation
A continuous wave diode laser (Fremont, California, RGBlase, TE-CIRL-70G-830 SMA) with a wavelength of 830 nm was used.PBM parameters are summarised in Table 1.PBM was conducted in a darkened room subjected to ambient environments to curb light interference.Light was delivered from above the cell culture plates containing 1 mL of freshly complete growth media, with the culture dish lids off.Cell culture plates were then incubated for 48 h post-PBM, after which they were analysed for real-time reverse transcriptase polymerase chain reaction (RT-PCR).Non-irradiated cell (0 J/cm 2 ) models were used as controls.

| RNA isolation
Forty-eight hours post-PBM, cells were dissociated using TrypLE™ Express (ThermoFisher Scientific, Gibco ® Invitrogen™, 12563-029) and collected as a pellet (not more than 1 Â 10 7 ) by centrifuging for 5 min at 825 Â g.The pellet was rinsed twice with phosphate buffer saline (PBS; Sigma-Aldrich, P4417-50TAB) to ensure the complete removal of the cell culture medium.Biological samples were lysed in 350 μL of a highly denaturing guanidine-thiocyanate-containing buffer (RLT lysis buffer), which immediately inactivates RNases to guarantee the purification of intact RNA.The RNeasy mini QIAcube kit (The Scientific Group, Qiagen, 74104) isolated total RNA in the QIAcube ® according to manufacturer details.Total RNA was homogenised in the QIAcube ® by a QIAshredder homogeniser (The Scientific Group, Qiagen, 79656).

| RNA quantification
The Qubit™ RNA BR test kit (ThermoFisher Scientific, Invitrogen™, Q10210) was used to determine RNA concentration with the Qubit™ fluorometer according to manufacturer details.

| RNA purity
RNA purity was determined by the ratio between the absorbance values at 260 and 280 nm (A260/A280) on a UV/Vis spectrophotometer.RNA samples were diluted in 10 mM Tris-Cl (pH of 7.5).Pure RNA exhibits an A260/ A280 ratio between 1.9 and 2.1.

| Reverse transcription (cDNA synthesis)
Reverse transcription (RT) was used to synthesise cDNA using the RT 2 First Strand Kit (The Scientific Group, Qiagen, 330404) in accordance with the manufacturer's instructions.The yielded cDNA samples were stored at À20 C.

| RT 2 profiler™ PCR arrays
The RT 2 Profiler™ PCR Array (The Scientific Group, Qiagen, PAHS-039YD) profiled 84 genes linked to JAKand STAT-mediated signalling as shown in Table S1, using SYBR ® green, ROX™ qPCR mastermix-based detection (The Scientific Group, Qiagen, 330 523) performed on the AriaMx Real-time PCR system (Agilent Technologies), according to manufacturer details.RT-PCR thermal cycling was performed with the following settings: 95 C for 10 min, 40 cycles of 95 C for 15 s (denaturation), and 60 C for 60 s (annealing).As indicators of non-specific amplification and gDNA contamination, positive controls, no template control (NTC), and no reverse transcriptase (NRT) were used.The positive PCR control (PPC, 3 wells) consists of a pre-dispensed artificial DNA sequence and the assay that detects it.This control efficiency of the reverse-transcription reaction is also included in the array.These controls allow for intra-plate consistency.

| Data analysis
Experiments were conducted on two models (W and DW) and were repeated three times (n = 3).To calculate the fold change and p-values, the results were analysed online using the Qiagen GeneGlobe Analysis Software.The GeneGlobe Analysis Software calculated fold change/regulation using the delta delta CT method.Results were normalised using an average of the reference genes (HKG; ACTB, B2M, GAPDH, HPRT1 and RPLP0), followed by delta-delta CT calculations (delta CT (Test Group)-delta CT (Control Group)).Fold Change was then calculated using 2^(delta-delta CT) formula.
Results were reported as significant when p ≤ 0.05.

| RESULTS
Variations in the gene expression profile for JAK-and STAT-mediated signalling were determined in irradiated (5 J/cm 2 ) wounded and diabetic wounded cell models (Figure 1) relative to the respective non-irradiated (0 J/cm 2 ) controls at 48 h post-PBM.A fold change of >1.5 indicated biological upregulation, whereas a fold change of <0.67 indicated biological downregulation.PBM at 830 nm affected nine genes in wounded cell models and six in diabetic wounded cell models.Five genes were upregulated in irradiated wounded cell models, including CEBPB, FCGR1A, GATA3, JUN, and SOCS3, while four genes, including CSF1R, ISG15, MYC, and SMAD2 were downregulated.Irradiated diabetic wounded cell models exhibited a significant downregulation of six genes, including EGFR, F2, IL2RG, MPL, MYC, and OAS1, with no genes being upregulated.

| DISCUSSION
During acute wound-healing, growth factors, cytokines, and extracellular matrix (ECM) proteins initiate the cells response to injury and play a central role in wound closure.These molecules trigger numerous strategic signalling pathways [17].In DM, the wound healing process is disrupted due to abnormalities in cellular mechanisms [18].In addition to decreasing collagen synthesis, deposition, and wound strength, hyperglycaemia impairs fibroblast cell function.In vitro, diabetic fibroblast cells have shown impairments in cellular processes vital for the wound-healing process, including migration, growth factor production, and their reaction to hypoxia, typically governed by a multifaceted network of pathways for signal transduction for gene transcription [19].During wound healing, the activated JAK/STAT signal transduction pathway controls fibroblast cell proliferation and differentiation and induces anti-apoptotic pathways to prevent cell death [20].Managing DM necessitates complex and ongoing medical care.Different approaches have been used to treat diabetic wounds, and when PBM is appropriately used, it elicits biostimulatory effects that speed up the healing process.PBM is principally a wavelength-dependent procedure involving photon-tissue interfaces [21].
Several studies have described conflicting results in the regulation of genes correlated with wound healing when PBM was applied at a fluence of 5 J/cm 2 [22][23][24][25].Li et al. [26] employed PBM at different wavelengths (ultraviolet radiation at 390 nm, blue light at 415 nm and red light at 660 nm) on human fibroblasts to evaluate cell proliferation and RNA sequence analysis.Their results demonstrated pronounced differences in the expression of matrix metalloproteinases (MMPs) in the blue and red light spectrum.A study by Frozanfar et al. [27] assessed the in vitro effect of PBM in the NIR spectrum (wavelength of 810 nm) on human gingival fibroblasts.Their findings demonstrated a significant upregulation of COL1A1, cell proliferation, and migration.
The present study has demonstrated that PBM at a wavelength of 830 nm and a fluence of 5 J/cm 2 significantly modified genes related to JAK/STAT signalling in wounded and diabetic wounded cells.In the wounded cell model, five of the 84 genes related to the JAK/STAT signalling pathway were significantly upregulated, while four were significantly downregulated.However, irradiated diabetic wounded cell models exhibited six significantly downregulated genes.This is in contrast with a similar study performed at a wavelength of 660 nm (with 5 J/cm 2 ), where 19 genes were regulated in wounded cell models (2 genes were upregulated and 17 were downregulated), and 73 genes were regulated in diabetic wounded cell models (46 genes were upregulated and 27 genes were downregulated (Table S2) [25].It should be noted that although the latter study also made use of human fibroblast cells (ATCC ® WS1), they were not the same fibroblast cell line as utilised in the present study (ATCC ® BJ-5ta).
Post-PBM at 830 nm, no genes involved in JAK activity or the STAT family were upregulated in either of the cell models.One encoding regulator of the JAK/STAT signalling pathway was upregulated in irradiated wounded cell models (SOCS3), while none of the regulators were affected post-PBM in the diabetic wounded model.SOCS are a negative regulator of the JAK/STAT pathway, and they exert an influence on cytokines and growth factors [12].Kershaw et al. [28] elucidated that SOCS3 encodes inhibitors of the catalytic activity of JAK2.SOCS3 binds to JAK kinase and cytokine receptors resulting in the inhibition of STAT3 activation, thereby regulating the dissolution of the JAK/STAT signalling pathway [29,30].In this study, the upregulation of SOCS3 in wounded models correlates with the nonsignificant downregulation in JAK2.
Receptors which bind and activate JAK proteins were downregulated in both wounded and diabetic wounded models.CSF1R was downregulated in the wounded model, while EGFR, IL2RG, and MPL were downregulated in the diabetic wounded model.CSF1R encodes for a tyrosine kinase receptor for colony-stimulating factor 1, and participates in influencing the differentiation, production, and survival of macrophages, and is involved in humoral immune response.Ligands such as epidermal growth factor (EGF) bind to EGFR, resulting in multimerisation which incites the recruitment and transphosphorylation of JAKs.IL2RG encodes a signalling component of several IL receptors, as well as cell differentiation and inflammatory immune responses.MPL encodes CD110, which incites the autophosphorylation of JAKs and STATs, leading to tyrosine dimerisation and regulating the transcription of target genes for cell proliferation.In another study, Jere et al. [25] used a diode laser at 660 nm and a fluence of 5 J/cm 2 on WS1 human fibroblast cells.They observed a significant upregulation in EGFR, IL2RG, and MPL in irradiated diabetic wounded cell models, contradicting the results of the present study.The downregulation of these genes again points to the dissolution of the JAK/STAT signalling pathway in response to PBM at 830 nm.
Genes encoding for transcription factors involved in JAK/STAT signalling were included.Three transcription factors were upregulated (CEBPB, GATA3, and JUN), and one downregulated (SMAD2) in wounded models post-PBM, while none were modified in diabetic wounded models.CEBPB encodes for the protein CCAAT enhancer binding protein beta, a protein with the capacity to regulate immune and inflammatory responses, as well as play a significant role as a STAT-interacting transcription factor.GATA3 encodes a protein which belongs to the GATA family of transcription factors, and has the capacity to regulate mammary stem cell differentiation and T-cell promotion [31].GATA3 is also required for the expression of Myc critical for the metabolism, proliferation, and homeostasis of immune cells, and has also been found to be regulated by Wnt and Notch signals [32].JUN encodes a complex of proteins of the activator protein-1 (AP-1) involved in miscellaneous functions such as regulating gene expression and cell proliferation by stimulating the advancement of the G1 phase of the cell cycle [33].Proteins encoded for by SMAD belong to the SMAD family, and are signal transducers and transcriptional modulators.SMAD2 mediates transforming growth factor-beta (TGF-β) signals and is implicated in various biological activity, including cell proliferation, differentiation, and apoptosis.
Several genes induced by STAT proteins were regulated post-PBM in both wounded and diabetic wounded models.MYC and ISG15 was downregulated, while FCGR1A was upregulated in wounded models.MYC and OAS1 were all downregulated in diabetic wounded models.MYC (downregulated in both models) is a protooncogene and encodes a nuclear phosphoprotein which has been identified in cell cycle advancement and/or maintenance, apoptosis and cellular transformation.ISG15 encodes for a ubiquitin-like protein, and is involved in diverse functions such as microbial infections and cellto-cell signalling.FCGR1A encodes for a high-affinity receptor for immunoglobulin gamma Fc receptor I, and is induced by STAT4.OAS1 encodes members of the 2 0 ,5 0 oligoadenylate family, and are thought to play a role in innate antiviral defence, and has also been implicated in other cellular processes such as cell growth and apoptosis [34].F2, a gene associated with apoptosis, was downregulated in diabetic wounded models.No other genes associated with apoptosis were regulated in wounded cell models.F2 encodes the protein prothrombin (also referred to as coagulation factor II), which plays a vital role in the acute-phase response, cell proliferation, cell cycle, and maintenance.Additionally, activated thrombin is essential in haemostasis and thrombosis by facilitating the conversion of fibrinogen to fibrin during coagulation.
All cells develop strategies that are utilised to adapt to environmental changes through cell signalling and rapid reprogramming of transcriptional alteration mechanisms, and understanding the underlying molecular changes in diabetic cells is central to the development of effective treatment methods for chronic diabetic wounds [35].Zhao et al. [36] notes that 20-25% of genes were regulated by hyperglycemia in cells from diabetic mice.The results from the present study shows that PBM at 830 nm with a fluence of 5 J/cm 2 affects the JAK/STAT signalling pathway in irradiated wounded and diabetic wounded fibroblasts in vitro differently.In another in vitro study conducted by Ayuk et al. [23], different gene profiles were noted between cells grown under normoglycaemic conditions and hyperglycaemic conditions post-PBM at 830 nm with a fluence of 5 J/cm 2 .

| CONCLUSION
The present study investigated the effects of PBM at 830 nm and a fluence of 5 J/cm 2 on gene expression correlated with the JAK/STAT signalling pathway in wounded and diabetic wounded fibroblast cells.The modified expression of several genes involved in JAK/SAT signalling has been published using light in the visible red spectrum (660 nm) [25] but has not been studied using a NIR wavelength.This is the first paper to examine the expression profile of 84 genes related to the JAK/STAT signalling pathway in response to PBM using NIR light.At a wavelength of 660 nm, a significant number of genes were modified in wounded and diabetic wounded models, and there was transcriptional activation of the JAK/STAT signalling pathway under hyperglycaemic wounded conditions [25].This study showed the opposite, with an apparent dissolution of the JAK/STAT signalling pathway at a transcription level in response to PBM at 830 nm.Several signalling pathways are activated during wound healing, and should be investigated using NIR light so as to determine the underlying mechanisms of action at this spectrum.Literature has shown differences on the effects of PBM at different wavelengths, and even on different cell types and models.This study also showed clear differences in gene profiles based on cells grown under normoglycaemic conditions and those grown under hyperglycaemic conditions.
the efficiency of the polymerase chain reaction itself (the threshold cycle should be 20 ± 2 and ensures thresholds between plates are constant).A genomic DNA control (GDC) which detects non-transcribed genomic DNA contamination (threshold cycle value of >35), and a reverse-transcription control (RTC) which determines the F I G U R E 1 Profiling gene expression changes in wounded (W) and diabetic wounded (DW) cell models irradiated at 830 nm and a fluence of 5 J/cm 2 .Total RNA from non-irradiated (0 J/cm 2 ) and irradiated (5 J/cm 2 ) W and DW cell models were typified in triplicate, and the relative expression levels of each gene were characterised for each model.A fold difference of >1.5 indicates biological upregulation, whereas a fold difference of <0.67 indicates downregulation.Statistical significance is presented as *p < 0.05 and **p < 0.01.