CC BY
7DOI 10.24412/cl-37136-2023-1-76-81
PHOTOBIOMODULATION AT 660 NM PROMOTES CELL PROLIFERATION THROUGH THE RELEASE OF BASIC FIBROBLAST GROWTH FACTOR AND ACTIVATION OF THE GSK3B PATHWAY IN DIABETIC WOUNDED FIBROBLAST CELLS IN VITRO
SANDY JERE1 AND NICOLETTE HOURELD1
1 Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, South Africa.
sandyj@uj.ac.za
ABSTRACT
Basic fibroblast growth factor (bFGF) is broadly used in the management of cutaneous wounds. Glycogen synthase kinase 3 beta (GSK3P) is a serine/threonine kinase and plays a major role in the control of P-catenin signalling. bFGF is a multipotent growth factor that stimulates cellular signalling for cell growth, proliferation, and migration in various cell types, including fibroblasts. Diabetes mellitus (DM) has been linked to atypical cell signalling processes thereby promoting the alteration of key cellular regulatory factors, membrane receptor proteins, and development of slow to heal wounds. Wound chronicity is common in DM, and is the main cause of non-traumatic limb amputation. Photobiomodulation (PBM) involves exposing wounds to light from lasers or light emitting diodes (LEDs) to induce healing. However, its mechanisms on fibroblast cellular proliferation remain unclear. In this investigation, WS1 skin fibroblast cells were split into diabetic (D) and diabetic wounded (DW) cell models, and were subjected to a continuous wave diode laser at a wavelength of 660 nm, power density of 11 mW/cm2, and fluence of 5 J/cm2. Non-treated cells (0 J/cm2) were used as controls. Following irradiation, cells were incubated for 48 h and evaluated for cell proliferation using the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay, and the release of bFGF, phosphorylation of GSK3P, and P-catenin using the Enzyme-linked immunosorbent assay (ELISA). PBM significantly affected cell proliferation, the release of bFGF and activation of the GSK3p/p-catenin pathway in diabetic wounded fibroblast cells in vitro, suggesting that PBM at 660 nm with 5 J/cm2 may be used to augment diabetic wound healing.
INTRODUCTION
Proliferation involves a series of events that take place within the cell and is regulated by growth factors that bind to their specific cell surface receptors. Cell surface receptors, also known as receptor tyrosine kinases (RTKs), are coupled to intracellular signalling proteins, including transcription factors [1]. The activation of RTKs initiates different signalling pathways that stimulate the expression of proteins to interact with other intracellular factors for cell proliferation. Cells proliferate or remain inactive using signalling pathways that communicate about their environment. Activation of the signalling pathways stimulates progression through the typical cell cycle. Signals initiated by growth factors, deoxyribonucleic acid (DNA) damage, and several other developmental indicators, influence the decision for DNA replication and cell proliferation. Deregulated activation of cell signalling is frequently accompanied with diseases such as diabetes and cancer [2, 3]. Basic fibroblast growth factor (bFGF) signalling advances proliferative, anti-apoptotic, anti-inflammatory, and migration progression in dermal fibroblasts during the wound healing process. In acute wound healing, bFGF and P-catenin interaction advances wound regeneration. The phosphorylation of GSK3P inhibits the degradation of P-catenin, causing cytoplasmic accumulation and nuclear translocation of active P-catenin for gene transcription [4, 5].
Diabetes mellitus (DM) refers to a group of metabolic diseases characterised by elevated blood sugar (hyperglycaemia) due to deficiencies in insulin action, secretion, or both, and over time, may lead to damage of the heart, eyes, blood vessels, kidneys, and nerves [6]. DM has become an increasingly prevalent disease worldwide. According to the International Diabetes Federation (IDF), approximately 540 million adults aged 20-80 (10%) were diabetic in 2021, and this number is expected to rise to approximately 645 million (11%) by 2030, and 785 million (12%) by 2045 [7]. Diminished healing of wounds affects approximately 25% of all
patients with DM, often leading to amputation of a lower limb [8]. Wound chronicity in diabetic patients develops due to several factors induced by high blood glucose (Fig 1). The presence of imbalanced matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) affects the release and function of growth factors and blocks several signalling pathways, leading to poor wound and tissue restoration [9]. Treatment of chronic diabetic wounds with synthetic growth factors have the potential to mediate wound healing at different phases of the healing process, stimulating changes in molecular and cellular reactions including the formation of granulation tissue, angiogenesis, and re-epithelization. However, their bioavailability and action in the wound milieu is diminished due to increased action of MMPs which continuously degrade both growth factors and their cell surface receptors [10].
Figure 1: Factors affecting wound healing in diabetic patients.
Photobiomodulation (PBM) is initiated by low-level light from lasers or light emitting diodes (LEDs) or both, and interacts with cellular chromophores to stimulate or inhibit biological activities [9]. The explanation of the effect and mechanism of PBM at a molecular, cellular, and tissue level remains indefinable. It is proposed however, that its impact is affected by the fluence, wavelength, output power, and time. Photoreceptors in the mitochondria, such as cytochrome c oxidase, and other cellular partitions absorb the light, converting it into photochemical energy with a subsequent increase in the production of adenosine triphosphate (ATP), synthesis of proteins, and cell proliferation [11]. PBM has shown to have stimulatory, anaesthetic, and anti-inflammatory effects in tissue and wound regeneration [12]. In addition, PBM is capable of altering the action of MMPs and enhance collagen production in chronic diabetic wounds [13]. Ayuk et al., [14] reported a significant rise in cell proliferation and viability in diabetic wounded cells irradiated at a wavelength of 660 nm with a fluence of 5 J/cm2 in vitro. The aim of this study was to assess the effect of PBM at a wavelength of 660 nm and a fluence of 5 J/cm2 on proliferation, the release of bFGF, and activation of the GSK3p/p-catenin pathway in diabetic wounded fibroblast cells in vitro.
METHODOLOGY
WS1 human skin fibroblast cells (ATCC®, CRL-1502™) were cultivated using standard culture procedures. Two cell models were used in the study, namely diabetic (D) and diabetic wounded (DW). An in vitro diabetic model was achieved by continuously cultivating WS1 cells in supplemented minimum essential medium (MEM) with an additional 17 mM D-glucose, thereby mimicking a hyperglycaemic condition [15]. To perform experiments, cells (6 X 105) were cultivated in 3.4 cm diameter tissue culture plates and incubated at 37°C in 5% CO2 for attachment. After 24 h, a central scratch was performed 30 min pre-irradiation in the wounded cell model (DW), thereby creating a cell free zone bordered by cells on both sides of the "wound" in the confluent monolayer [16]. Cell culture plates, with the lids off, were exposed to laser light from above in the dark. Table 1 shows the laser parameters used. Cells were irradiated at a wavelength of 660 nm and a fluence of
5 J/cm2, after which cells were incubated for 48 h. Cells were analysed for cell proliferation using the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay (Sigma Aldrich, 11465007001), and the release of bFGF into the culture media (Human FGF basic ELISA Kit (FGF2), BIOCOM Africa, Abcam, ab99979), phosphorylation of GSK3P (p-GSK3 beta (Ser9) InstantOne ELISA™ Kit, ThermoFisher Scientific, 85-86172), and P-catenin (Human Beta Catenin ELISA Kit, BIOCOM Africa, Abcam, ab275100) using the enzyme-linked immunosorbent assay (ELISA). Unirradiated cells were used as controls (0 J/cm2). All experiments were repeated three times (n=3), and for ELISA, tests were done in duplicate, the average of which was used. SigmaPlot version 14 (Systat Software, Inc.) was used for statistical analysis. Statistical differences between groups was determined by the Student t test. Analysis of variance (ANOVA) followed by Dunnett's test was used to compare differences between D and DW cell models. Results are shown as standard error of the mean (SEM), and statistical significance is shown in the graphs as *p<0.05, **p<0.01 and ***p<0.001.
Table 1. Laser parameters.
Light source Wavelength (nm) Emission
Power output (mW)
2
Power density (mW/cm )
2
Spot size (cm )
2
Energy density (J/cm ) Irradiation time Energy (J)
Diode laser 660
Continuous wave 101 11
9.1
5
7 min 35 s 46
RESULTS
In this study, PBM at a wavelength of 660 nm with a fluence of 5 J/cm2 significantly reduced cellular proliferation in PBM treated D cells (p<0.01) and significantly increased proliferation in DW cells (p<0.05) when compared to their control cells at 48 h. There was a significantly increased cellular release of bFGF in PBM treated D cells (p<0.05) and DW cells (p<0.01) when compared to their control cells at 48 h (Figure 1). When compared to non-treated D cells using ANOVA, a significant decrease in proliferation was noted in non-treated DW cells (p<0.01), and a significant increase in treated DW cells when compared to treated D cells (p<0.05). There was no difference in the release of bFGF in non-treated and treated DW cells when compared to non-treated and treated D cells, respectively.
Figure 1: Cell proliferation and the release of basic fibroblast growth factor (bFGF) in the media were assessed by the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay and the enzyme-linked
immunosorbent assay (ELISA), respectively at 48 h in non-treated (0 J/cm2) andphotobiomodulation (PBM)
2 2 2 2 2 treated (5 J/cm ) diabetic (D 0 J/cm ; D 5 J/cm ) and diabetic wounded (DW0 J/cm ; DW5 J/cm ) cells.
Significant probability is shown as **P<0.01 and *P<0.05 ±SEM.
PBM at a wavelength of 660 nm with a fluence of 5 J/cm2 significantly increased the inactive form of GSK3P in PBM treated D cells (p<0.05) and DW cells (p<0.001) when compared to their non-treated control cells at 48 h. There was also a significant increase in total P-catenin in PBM treated D cells and DW cells (p<0.05) (Figure 2). When compared to non-treated D cells using ANOVA, a significantly decreased inactivated form of GSK3P was noted in non-treated DW cells (p<0.01), and no significant difference was noted in treated DW cells when compared to treated D cells. There was no difference in total P-catenin in both non-treated and treated DW cells when compared to non-treated and treated D cells, respectively.
Figure 2: Phosphorylated (inactivated) glycogen synthase kinase 3 beta (p-GSK3/3), and beta fl3-) catenin were determined by the enzyme-linked immunosorbent assay (ELISA) 48 h post-photobiomodulation (PBM) in non-treated (0 J/cm2) and PBM treated (5 J/cm2) diabetic (D 0 J/cm2; D 5 J/cm2) and diabetic wounded (DW 0 J/cm2; DW 5 J/cm2) cells. Significant probability is shown as ***P< 0.001 and *P<0.05 ±SEM.
DISCUSSION AND CONCLUSION
The cellular environment dictates whether cells proliferate or remain inactive via different cellular signalling pathways that initiate the cell cycle. The decision to enter the synthesis (S) phase of the cell cycle, in which DNA is replicated, is mostly influenced by growth factors, DNA damage, and developmental signals [17]. During cell proliferation, cells divide and increase in number through a tightly controlled and precise mechanism that occurs in both acute as well as chronic wounds [18]. Fibroblast cells are vital in reinforcing the normal wound healing process, and are implicated in major processes including resolving the fibrin clot, and depositing and constructing new extracellular matrix (ECM) and collagen that support other cells associated with wound healing. Fibroblasts help in contracting the wound and respond to injury by proliferating and migrating to the injured sites [19]. Buranasin et al. [20] mentioned that hyperglycaemia impairs fibroblast cell proliferation and migration due to high glucose-stimulated oxidative stress. Chronic hyperglycaemia causes greater stiffness to the skin, such that it becomes less flexible, making it more prone to injury mainly due to differences in the synthesis of collagen and its degradation [20]. These suggestions give explanation to the delayed wound healing in patients with impaired glucose metabolism and alterations in micro- and macro-vascular circulation, advancing the risk of postponed wound healing [21].
Although PBM has demonstrated its capability to treat chronic wounds, the absence of distinct parameters and a complete understanding of its effect at both a cellular and molecular level hinders its adoption as a
treatment method in the management of chronic wounds. This study assessed the effect of PBM at a wavelength of 660 nm and a fluence of 5 J/cm2 on proliferation, the release of bFGF and the activation of the GSK3P and P-catenin signalling pathway in diabetic wounded fibroblast cells in vitro. In this study, unirradiated diabetic wounded cells exhibited a significant decrease in cell proliferation as compared to unirradiated diabetic cells, indicating that wounding contributes to reduced cell proliferation. This study noted a significant increase in cell proliferation in irradiated diabetic wounded cells compared to their unirradiated controls, indicating the effect of PBM. In addition, irradiated diabetic wounded cells exhibited a significant increase in proliferation compared to irradiated diabetic cells. This shows that diabetic wounded cells respond more favourably to PBM than diabetic cells, possibly due to wounding as more stressed cells respond effectively to PBM [16]. Giannakopoulos et al. [22], demonstrated that PBM at 661 nm activates both cell migration and proliferation in vitro. A comparison study by Zhao et al. [23], suggested that LED-mediated PBM (630 nm and 810 nm) stimulates wound healing through the progression of fibroblast cell proliferation and release of growth factors. In agreement to these findings, this study observed a significant increase in the release of bFGF in irradiated cells as compared to their unirradiated controls, indicating that indeed PBM induces the release of growth factors, a critical aspect in wound healing as growth factors are essential for cell proliferation, migration, and vascularisation. In chronic wounds, growth factor levels are reduced due to both decreased production and extreme protease-mediated degradation [24]. bFGF promotes cell proliferation by phosphorylating and thereby inhibiting the activity of GSK3P [25]. This study observed a significant increase in p-GSK3p (inactive form of the protein which allows activation of downstream proteins), as well as its downstream substrate P-catenin in cells that were irradiated. These findings signify an effectual therapeutic approach and provides an experimental basis to support PBM in the visible red spectrum as a possible option to stimulate fibroblast cell proliferation in chronic diabetic wounds. Nevertheless, further studies including clinical, are needed to expose more about the effect initiated by PBM at 660 nm and a fluence of 5 J/cm2 on cellular phenotypic variations at a molecular level.
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