Abstract
Diabetes mellitus (DM) is reported globally as one of the main contributors to mortality. There were approximately 537 million adults living with diabetes between the ages of 20 and 79 years in 2021, with an estimated rise to 643 million by 2030 and 783 million by 2045 (International Diabetes Federation, IDF, 2021). DM is a chronic disease characterised by hyperglycaemia that occurs as a result of impaired insulin utilisation, as well the body's inability to atone with increased insulin production. One of the complications associated with DM is non-healing chronic diabetic foot ulcers (DFUs). Research into wound healing processes has attracted considerable attention in the healthcare sector, especially with factors causing delays or preventing the healing of wounds (Ranjbar & Takhtfooladi, 2016), and significant focus has been directed on diabetes and infections (Boulton et al., 2020). The treatment of chronic diabetic wounds can become costly and may require patients to be absent from work with possible loss of employment as a result of decreased work productivity. Severe cases of chronic diabetic wounds may necessitate amputation, leading to disabilities.
Wound healing is a complex process involving various cell types and their products that interact with the extracellular matrix (ECM) (de Oliveira Gonzalez et al., 2016; Sorg et al., 2017), and is influenced by several proteins and glycoproteins. Acute wound healing has four phases: haemostasis, inflammation, proliferation, and remodelling. Diabetic wounds are chronic due to prolonged inflammatory, proliferative, or remodelling phases, resulting in tissue fibrosis and non-healing ulcers (Houreld, 2015; Spampinato et al., 2020). These wounds are trapped in a persistent inflammatory state, which is characterised by the accumulation of pro-inflammatory macrophages, cytokines, and proteases, and this is why they do not often heal (Salazar et al., 2016). Treatments that can reduce this hyper-inflammatory state aids in their healing and progression in the stages of wound healing. The inflammatory phase in diabetic wound healing is associated with elevated levels of pro-inflammatory cytokines (i.e., Cox-2, TNF-α and IL-6).
Cyclooxygenase-2 (Cox-2) is involved in inflammation, tumour growth, and renal injuries. In diabetes, Cox-2 up-regulates prostanoids (Feng et al., 2017), affecting vasomotor function and apoptotic cell death (Redondo et al., 2011). Interleukin-6 (IL-
v
6) is associated with type 2 diabetes and impaired wound healing. Diabetes is strongly associated with elevated levels of tumour necrosis factor alpha (TNF-α) and insulin resistance. Further research is needed to understand the role of these pro-inflammatory cytokines in diabetes. Diabetes increases the production of advanced glycation end products (AGEs), which can cause inflammation, apoptosis, and oxidative stress (Byun et al., 2017; Rhee & Kim, 2018). These AGEs interact with receptors and contribute to diabetic vascular complications. Hyperglycaemic conditions increase AGE levels, affecting wound healing and triggering inflammatory responses. Elevated AGE levels contribute to impaired wound healing.
The accumulation of AGEs affects a variety of signalling pathways, including activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and increasing reactive oxygen species (ROS), which in turn alter cellular functions.
DM is a global health challenge causing chronic ulcers and wounds in diabetic patients. Current treatments are ineffective, with only a 50% healing rate (Frykberg and Banks, 2015). Research is ongoing to find optimised technologies to increase cell viability and promote wound healing (Sorg et al., 2017). Photobiomodulation (PBM) is one such technology. PBM is a non-invase, athermal modality which utilizes low-powerd light from lasers or light emitting diodes (LEDs) to modulate cellular function and stimulate tissue regeneration and wound helaing. PBM has shown benefits, but its effectiveness has not been optimised. A variety of wavelengths can be selected based on the intended results, with wavelengths within the visible red to near-infrared (NIR) spectrum proving to be most effective in promoting wound healing. Further investigations are needed to improve wound healing processes. The current study sought to investigate the potential impact of PBM at 830 nm with 5 J/cm2 on receptor for AGEs (AGER) and, consequently, the levels of pro-inflammatory mediators (Cox-2, IL-6 and TNF-α), apoptosis, and the levels and nuclear translocation of NF-κB, as well as the effects of PBM on the expression of genes involved in the human inflammatory cytokines and receptors pathway. A wavelength of 830 nm and a fluence of 5 J/cm2 was selected based on previous studies which showed stimulation of WS1 cells (Houreld & Abrahamse, 2008).
A human skin fibroblast cell line (WS1, ATCC®, CRL-1502™) was used in this study where five cell models were used, namely normal (N), wounded (W), diabetic (D),
vi
diabetic wounded (DW), and wounded with D-galactose (WG).
D-galactose (100 mM) was added to normal wounded models to induce the production of AGEs (Genuth et al., 2015). This served as a positive control for diabetic and diabetic wounded cell models. To induce a diabetic in vitro model, cells were grown in 22.6 mM D-glucose. For experiments, cells were seeded at a density of 6 × 105 into 3.4 cm diameter culture dishes. The central scratch assay was used to induce a 'wound' in the wounded models. Experimental models were irradiated at a wavelength of 830 nm with a fluence of 5 J/cm2 (power output: 106 mW). Non-irradiated cells (0 J/cm2) served as controls. Cellular responses were analysed immediately (0 h) or incubated for 24 or 48 h post-PBM.
Cellular morphology and migration rate were determined using inverted light microscopy. Post-PBM, different cell assays were performed. These assays included: cell viability using the Trypan blue exclusion assay and measuring adenosine triphosphate (ATP) luminescence; cell cytotoxicity by measuring lactate dehydrogenase (LDH); levels of inflammatory markers (Cox-2, IL-6 and TNF-α) using the enzyme linked immunosorbent assay (ELISA); measuring AGER using the polymerase chain reaction (PCR); nuclear translocation of (NF-κB) by immunofluorescence and NF-κB levels by ELISA; and cell apoptosis using flow cytometry and Annexin V-FITC/PI staining. The expression of 84 genes related to human inflammatory cytokines and their receptors were determined by a PCR profiler array. The experiments were performed three times (n=3).
Cells displayed a flattened, elongated, and spindle-shaped appearance. Wounded models exhibited alterations in growth direction and cellular projections towards the central scratch at 24 h. Irradiated cells demonstrated a higher level of cell confluency and wound closure. A significant increase in cell viability using the Trypan blue exclusion assay post-PBM was observed in irradiated N, W, D, and DW cell models at 0 h; in N, W, and WG models at 24 h; and in N, D, and DW models at 48 h. Results of the CellTiter-Glo® Luminescent cell viability assay indicated a significant increase in viability (ATP) in irradiated N, D, and DW cells at 0 h; in N, D, DW, and WG cells at 24 h; and in N, D, DW, and WG at 48 h post-PBM. The CytoTox 96® Non-Radioactive Cytotoxicity Assay showed a decrease in cellular damage (LDH) in all irradiated cells at 0, 24 and 48 h post-PBM. There was a significant up-regulation in the expression
vii
of AGER in irradiated DW cells 48 post-PBM. A significant increase was seen in TNF-α levels 24 h post-PBM in N and DW cell models. A significant decrease in IL-6 levels was seen in the irradiated N cell model, while an increase was seen in the irradiated DW cell model at 0 h post-PBM. Irradiated DW cells showed considerable nuclear translocation of NF-κB 24 h post-PBM, which corresponds with the significant increase seen in TNF-α and NF-κB levels (nuclear fraction) 24 h post-PBM. NF-κB levels were increased in irradiated W cells at 0 h, in W, D, and DW cells at 24 h, and decreased in W, D, and DW cells at 48 h. A decrease in late apoptosis was observed in both N and W cell models post-PBM at 0 h. Examination was of 84 genes associated with the signalling pathway of human inflammatory cytokines and receptors (PCR Profiler Array), revealed a significant up-regulation of one gene (CXCL8), and down-regulation of one gene (CXCL12) in W cells.
The results obtained from this study show that the application of PBM at 830 nm, with a fluence of 5 J/cm2, may have a beneficial modulatory impact on the viability of DW fibroblast cells, decrease in cell cytotoxicity and cell migratory effects in wounded models. The findings indicate that PBM at a wavelength of 830 nm and a fluence of 5 J/cm2 have an impact in activation of specific signalling pathways such as the NF-κB particularly in wounded cells cultured in hyperglycaemic conditions.