CC BY
4DOI 10.24412/cl-37136-2023-1-82-89
PHOTOBIOMODULATION IMPROVES WOUND HEALING THROUGH ACTIVATION OF THE RAS/MAPK AND PI3K/AKT PATHWAY
NICOLETTE HOURELD1
1Laser Research Centre, University of Johannesburg, South Africa nhoureld@uj .ac.za
ABSTRACT
Diabetes mellitus (DM) is a metabolic non-communicable disease (NCD) that has been declared as a global burden, imposing a significant challenge to the health and well-being of individuals, families, and societies. According to the International Diabetes Federation (IDF), the global estimated prevalence of DM in 2021 stood at 537 million cases (adults aged 20-79; 1 in 10 of the world's population in this age group), with the largest increase seen in low- and middle-income countries [1]. The number of people with DM on the African continent in 2021 stood at 24 million (1 in 22 adults) and is thought to increase to 55 million by 2045 (an increase of 134%) [1]. The reality of these figures is probably much higher; over 1 in 2 people (54%) living with DM on the African continent are undiagnosed. South Africa is among the top 5 countries in the African region for age-adjusted prevalence of people with diabetes (20-79 years), with a prevalence of 10.8%, falling in 4th behind the Comoros (11.7%), Zambia (11.9%), and United Republic of Tanzania (12.3%) [1]. The proportion of deaths related to diabetes among people under the age of 60 in South Africa in 2021 stood between 9-12% [1]. DM is one of the fastest growing global health emergencies of the 21st century [2].
People living with DM are at risk of developing several life-threatening complications, leading to an increased need for medical care. Patients with DM repeatedly suffer from non-healing, chronic and frequently debilitating lower limb ulcers, which often necessitate amputation. Diabetic foot ulcers (DFUs) have a negative impact on patient quality of life and are a major source of preventable morbidity. The lifetime risk of developing a DFU stands at 19-34%, with recurrence rates as high as 65% at 3-5 years, a lifetime lower-extremity amputation incidence of 20%, and 5-year mortality of 50-70% [3]. Diabetic foot and lower limb complications, which affect 40 to 60 million people with diabetes globally, is the leading cause for non-traumatic lower limb amputations. It has been estimated that, globally, a lower limb (or part thereof), is lost to amputation every 30 seconds as a consequence of DM [2]. Several underlying pathologies contribute to the impaired wound healing seen in diabetes. These include, but not limited to, increased oxidative stress, inflammation and infection, and decreased immunity and angiogenesis. There is also decreased fibroblast migration and proliferation, often due to disturbances in essential growth factors and signal transduction pathways involved in the wound healing process. DFUs pose a major physical, social, and economic burden on patients and the public health sector and has caused an increase in the demand for effective and safe treatment modalities.
Current treatments for DFUs have not resulted in consistently lower amputation rates, and treatments are challenging, lengthy, costly, and associated with failure to heal and relapse and there is a demand for efficient wound healing interventions [3]. In recent years great emphasis has been directed at using photobiomodulation (PBM) to stimulate and accentuate cellular processes to contribute to more efficient resolution of wound healing, including DFUs. PBM, previously referred to as low-level laser therapy (LLLT), utilises non-thermal light at specific wavelengths (typically between 600 and 1,200 nm) to induce cellular photochemical and physiological changes and provide therapeutic benefits. PBM has been shown to stimulate cellular migration and proliferation, reduce inflammatory markers and increase the synthesis of various growth factors in vitro, and speed up the healing of wounds in vivo, including diabetic ulcers. Although PBM has been used with great success and no reported side-effects, and despite a significant focus on the photochemical mechanisms underlying PBM, its complex functions are yet to be fully elucidated [4].
PBM has been shown to have several positive effects on hyperglycaemic cells [5-8], stimulating cellular migration and proliferation [8], decreasing oxidative stress and inflammatory markers [7,9], and speeding up the
healing of DFUs [10,11]. Two recent reviews have highlighted the effects of PBM on signalling pathways [12,13]. Jere and colleagues [14] demonstrated that PBM at a wavelength of 660 nm and a fluence of 5 J/cm2 was able to stimulate the release of epidermal growth factor (EGF) and activate the Janus kinase/Signal transducer and activators of transcription (JAK/STAT) signalling pathway. The same authors also showed the up-regulation of genes involved in this pathway [15]. Due to the multitude of transduction signals involved in the process of wound healing, more pathways may be activated in response to PBM.
This study aimed to investigate the effect of PBM at 660 nm on cellular migration, proliferation, and survival through activation of the PI3K/AKT and Ras/MAPK signalling pathway in a diabetic wounded fibroblast cell model. To achieve this, human skin fibroblast cells (WS1) were modeled into a wounded (W) and diabetic wounded (DW) in vitro cell model. A wound was simulated via the central scratch assay (Fig. 1), and a diabetic cell model was created by continuously growing the cells for several passages in high glucose media (22.6 mM glucose) [5-8,16,17]. For experiments (n=3), 6 X 105 cells were seeded into 3.4 cm diameter culture plates and incubated for 24 h. Thirty minutes prior to PBM, a 'wound' was created, and cells received PBM at a wavelength of 660 nm with a fluence of 5 J/cm2 (power output density 11 mW/cm2; energy 47.7 J; irradiation time 454 s). Unirradiated cells served as controls (0 J/cm2). Cellular migration rate, proliferation, and survival (viability) was determined 24 and 48 h post-PBM. Proteins and receptors involved in the PI3K/AKT (PI3K, AKT1, mTORl, and GSK3P) and Ras/MAPK (bFGFR,Ras, MEK1/2, MAPK) signalling pathway were evaluated, as were the growth factors vascular epithelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as activators of the pathways respectively.
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Figure 1: Micrograph showing the 'wound' (central scratch). Magnification x200 [16].
The distance between the wound margins were measured and used to calculate migration rate using the formula
(T0h - Txh)/T0hx100
where T0h is the initial length connecting the borders edges of the 'wound' at 0 h, and Txh is the succeeding measurement between the edges of the 'wound' at 24 or 48 h respectively. Post-PBM at 660 nm with 5 J/cm2, wounded and diabetic wounded cells exhibited a significant increase in cellular migration rate (Table 1), and diabetic wounded cells showed complete 'wound closure' at 48 h, with no gaps visible in the central scratch.
Table 1: Average rate for cellular motility in wounded and diabetic wounded cells. Significant probability as compared with respective control (0 J/cm2) cells is shown *P < 0.05, **P < 0.01 and ***P < 0.001 [5].
Wounded Diabetic Wounded
0 J/cm2 5 J/cm2 0 J/cm2 5 J/cm2
24h 50.50% 66.60%* 20.60% 79.70%***
48 h 83.10% 99.90%* 62% 100%**
Wounded and diabetic wounded cells exposed to PBM at 660 nm with 5 J/cm2 showed a significant increase in actively proliferating cells (S phase) as determined by 5-bromo-2'-deoxyuridine (BrdU) and 7-aminoactinomycin D (7-AAD) staining as measured by flow cytometry 24 and 48 h post-PBM (Fig. 2).
Figure 2: Changes in percentage of cells in the proliferating (S), resting/preparing for DNA synthesis (G0/G1) and mitotic (G2/M) phases of the cell cycle at 24 h (a) and 48 h (b) following PBM at 660 nm with 5 J/cm2 in wounded (W) and diabetic wounded (DW) cells. Significant probability as compared with respective control (0 J/cm2) cells is shown as *P< 0.05 ** P < 0.01 and *** P < 0.001(SEM) [5].
Cellular viability/survival was determined by the Trypan blue exclusion assay at 24 and 48 h post-PBM. Cell viability significantly increased in both the wounded and diabetic wounded models at 24 h, and in the diabetic wounded model at 48 h (Fig. 3).
Figure 3: Percentage cellular viability at 24 and 48 h post-PBM at 660 nm in wounded (W) and diabetic wounded (DW) cells. Significant probability as compared with respective control (0 J/cm2) cells is shown as *
P< 0.05 and ** P < 0.01 (SEM) [5]. The binding of ligands to their receptor stimulates downstream signalling pathways including the PI3K/AKT/mTOR pathway. Upon receptor binding, phosphorylation of phosphatidylinositol 3-kinase (PI3K)
activates the downstream serine/threonine protein kinase B (PKB; also known as AKT). Downstream targets of AKT includes the mammalian target of rapamycin complex (mTOR), forkhead box O1 (FOXO1), and glycogen synthase kinase-3 beta (GSK3P). GSK3P is active in its unphosphorylated form, thus the phosphorylation of GSK3P by AKT results in its inactivation, necessary for wound healing. mTOR controls the expression of cytokines, including VEGF [18]. Activation of the PI3K/AKT/mTOR pathway control different cellular functions such as cell proliferation, growth, metabolism, and survival [19]. Post-PBM at 660 nm, stimulation of the PI3K/AKT signalling pathway was determined by measuring the phosphorylation (activation) of AKT1, mTORl and PI3K by western blotting (Fig. 4). ELISA was used to measure total GSK3P and phosphorylated GSK3P (Ser9) (Fig. 5) and VEGF (Fig. 6). Wounded models showed a significant increase in PI3K at 24 and 48 h post-PBM, in p-AKT at 48 h, and a significant increase in total GSK3P at 24 h. PI3K and p-AKT was significantly increased in diabetic wounded models at both 24 and 48 h, while p-mTOR was significantly increased at 48 h. Diabetic wounded models showed no change in total GSK3P, but showed a significant increase in p-GSK3p at 24 and 48 h, as well as a significant increase in VEGF at both time points.
Figure 4: Phosphorylated (p-) AKT (a), PI3K (b), and p-mTOR (c) was determined post-PBM at 660 nm in wounded (W) and diabetic wounded (DW) cells at 24 and 48 h. GAPDH was utilised as a loading control. Significant probability as compared with respective control (0 J/cm2) cells is shown as *P < 0.05, and **P <
0.01 (SEM) [16].
Binding of FGF to its receptor, FGFR results in the phosphorylation of FGFR and stimulation of downstream signalling pathways, including the Ras/MAPK pathway. Phosphorylation of FGFR activates Ras, which results in the phosphorylation of MEK1/2, that in turn phosphorylates and activates MAPK. MAPK is translocated to the nucleus where it activates genes involved in cellular proliferation, migration, differentiation, and angiogenesis. In this study, bFGF, p-Ras, p-MEK1/2 and p-MAPK was determined in wounded and diabetic wounded cell models by ELISA 24 and 48 h post-PBM at 660 nm. p-FGFR was measured by western blotting.
Figure 5: Total GSK3/3 (a) and phosphorylated (p-) GSK3/3 (b) in wounded (W) and diabetic wounded (DW) cells at 24 and 48 h post-PBM at 660 nm. Significant probability as compared with respective control (0 J/cm2) cells is shown as *P < 0.05, and ***P < 0.001 (SEM) [16].
Figure 6: Vascular endothelial growth factor (VEGF) as determined by ELISA in wounded (W) and diabetic wounded (DW) cells at 24 and 48 h post-PBM at 660 nm. Significant probability as compared with respective control (0 J/cm2) cells is shown as *P < 0.05, and **P < 0.01 (SEM).
Post-PBM at 660 nm, there was a significant increase in bFGF in diabetic wounded models at 24 h (Fig. 7a), with a corresponding increase in p-FGFR (Fig. 7b). However, at 48 h there was a significant decrease (Fig 7a) in bFGF released by the cells into the culture media. This decrease is likely due to the consumption of bFGF by the same cells in a paracrine fashion. At 24 h, there was a corresponding significant increase in p-Ras and p-MEK1/2 in the diabetic wounded models (Fig. 8a and 8b, respectively), while the increase observed in p-MAPK (Fig. 8c) at 48 h was insignificant (P=0.084), possibly due to the large error bar in the control cells. Wounded cells showed no change in bFGF and p-FGFR, a significant increase in p-Ras and p-MEK1/2 at 24 h, and no change in p-MAPK.
Currently, treatments for chronic wounds are limited and associated with repeated failure and relapse, and new therapies are required to treat DFUs. Numerous studies have pointed towards the positive effects of PBM,
however the cellular mechanisms after exposure to PBM are still not well understood and further research on the mechanisms of photon-tissue interaction and the specific parameters that establish the therapeutic results are required [4,20]. The multitude of positive effects seen in response to PBM through various studies appear to be dependent on cell type and PBM parameters used. This study unequivocally shows that PBM using visible red light at 660 nm with 5 J/cm2 has a positive, stimulatory effect on diabetic wounded cells in vitro. A wound was simulated in vitro via the central scratch. Creating a scratch in a 2D cell model has limitations in that it lacks the complexity of the wound bed microenvironment, however the scratch assay is well established and provides a cost-effective assay used for measuring cell migration in vitro and allows for recolonisation and monitoring of the scratched region to quantify cell migration [21,22]. The scratch assay also assists in understanding the mechanisms that influence cellular migration in response to stimulus [23], and in this case PBM. Cellular migration rate was determined in wounded and diabetic wounded cell models in response to PBM at 660 nm. As expected, control diabetic wounded cells showed decreased migration rate, viability and proliferation as compared to the wounded cells grown under normoglycaemic conditions, however when exposed to PBM the migration rate of the diabetic wounded cells increased significantly, with complete 'wound closure' at 48 h. The same effect was evident in cell viability and proliferation post-PBM. It is well known that diabetic wounds exhibit decreased growth factors and have disrupted cell signalling pathways necessary for wound healing, and as expected the diabetic wounded control cells exhibited decreased VEGF and bFGF, as well as signalling proteins involved in both the PI3K/AKT and Ras/MAPK pathways as compared to the wounded control cells. Post-PBM at 660 nm, the levels of the measured growth factors released by diabetic wounded cells significantly increased, and there was activation of the PI3K/AKT/mTOR and Ras/MAPK pathways [16,17]. These results illustrate the effectiveness of PBM at 660 nm in activating cellular pathways in deficient diabetic wounded cells to speed up the healing process and has shown that PBM could be advantageous in the treatment of chronic DFUs. They also provide more insight into the cellular mechanisms involved when utilizing visible red light. There is a definite need to generate new treatment modalities to improve diabetic wound healing, and PBM has an unmistakable role to play.
Figure 7: Basic fibroblast growth factor (bFGF)(a) and phosphorylation (p-) of the receptor, FGFR (b) as determined in wounded (W) and diabetic wounded (DW) cells at 24 and 48 h post-PBMat 660 nm. Significant probability as compared with respective control (0 J/cm2) cells is shown as **P < 0.01, and ***P <
0.001 (SEM) [17].
Figure 8: Phosphorylation (p-)Ras (a) p-MEK1/2 (b) andp-MAPK (c)as determined in wounded (W) and diabetic wounded (DW) cells at 24 and 48 h post-PBM at 660 nm. Significant probability as compared with respective control (0 J/cm2) cells is shown as *P < 0.05, and **P < 0.01 (SEM) [17].
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