Abstract
The skin is the largest organ in the human body and is prone to injury (Martin & Nunan, 2015). Wounds usually heal in a timely, well-regulated fashion by following a predictable sequence of events (Tsourdi et al., 2013). Chronic wounds, however, take longer than three months to heal, and display unique characteristics such as disrupted patterns of cytokine expression and a prolonged inflammatory phase which is detrimental (Martin & Nunan, 2015; Wetzler et al., 2000). Interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) are both cytokines which indicate inflammation (Barrientos et al., 2008). Diabetes mellitus (DM) is an endocrine disorder that is reaching epidemic proportion and whose prevalence is increasing worldwide (Cho et al., 2018). In DM, chronic wounds called diabetic foot ulcers (DFUs) are a serious complication which often leads to lower limb amputations (Sohn et al., 2010). The treatment of DFUs is notoriously difficult and new therapies are urgently needed to decrease the burden on national health care systems (Cho et al., 2018). Homeopathy is a complementary health system which is effective in treating a variety of disease conditions (Fisher, 2012). The substances Calendula officinalis (C. officinalis), Hypericum perforatum (H. perforatum) and Echinacea purpurea (E. purpurea) effectively stimulates wound healing in herbal form (Parente et al., 2012; Samadi et al., 2010; Rezaie et al., 2013). These remedies are also indicated in the homeopathic literature for the treatment of wounds, however there are limited studies on their effectiveness (Hering, 1997; Boericke, 1999; Sinha, 1981). Photobiomodulation (PBM) is a therapeutic intervention which involves using lowintensity laser light at specific wavelengths to treat a variety of medical conditions (Andrade et al., 2014). PBM is effective in accelerating wound healing in a variety of experimental models, including DFU in DM (Carvalho et al., 2016; Kaviani et al., 2011). The aim of this study was to determine the effects of a 3 cH homeopathic solution containing C. officinalis, H. perforatum and E. purpurea and PBM, both as single therapies and in combination in an in vitro diabetic wounded cell model. v Commercially available human skin fibroblasts (WS1) were used as an in vitro model in this study. Normal cell models were grown in supplemented culture medium and diabetic cell models were grown in the same with additional glucose to generate a final glucose concentration of 22.6 mMol/L. Cells were sub-cultured at confluency in 3.4 cm diameter culture dishes and subjected to the ‘scratch assay’ (Felice et al., 2015) using a sterile pipette tip to simulate a wound. For the PBM models, cells were irradiated using laser light with a wavelength of 660 nm and a dose of 5 J/cm2 . This dose and wavelength is well established within the research centre, and has been shown to stimulate healing in this in vitro diabetic wounded model (Houreld et al., 2018; Ayuk et al., 2016; Jere et al., 2018). For homeopathically treated models a homeopathic solution containing 3 cH C. officinalis, H. perforatum and E. purpurea prepared in 5% ethanol was added to culture medium and incubated for 48 h. In cells treated with both these therapies, the homeopathic solution was added first and then plates were irradiated. Controls were nonwounded cells, and wounded cells that received neither of the treatments, as well as an alcohol control group where 5% ethanol was added to culture medium. Images were captured using a microscope (Olympus CKX41) coupled to a digital camera at 0 h, 24 h and 48 h after treatment to visualise cellular morphology. For biochemical assays, culture medium was collected 48 h post-treatment and cells were detached from culture plates. A proportion of the culture medium was frozen to perform the enzyme-linked immunosorbent assay (ELISA), and a proportion was used immediately to perform the lactate dehydrogenase (LDH) membrane integrity assay to determine cytotoxicity. Detached cells were used to perform the adenosine triphosphate (ATP) luminescence assay and the Trypan Blue exclusion assay, measures of cellular viability. ELISA was performed using commercially available kits to determine the concentration of proinflammatory cytokines IL-6 and TNF-α in culture medium. Morphological changes revealed that both PBM and the homeopathic solution stimulated wound healing in diabetic wounded cells, and that combining these therapies had an even greater effect. Cellular viability was not affected by either homeopathic solution or PBM but combining them caused increased viability as indicated by ATP results. Trypan Blue results revealed decreased viability, contradicting the ATP results. Cytotoxicity was decreased by the homeopathic solution complex and in the combination therapy group. TNF-α concentration remained unchanged with both PBM and homeopathic solution as vi single treatment interventions but combining them increased TNF-α concentration. PBM decreased IL-6 concentration, indicating an anti-inflammatory effect. These cellular changes indicate that combining PBM and a 3 cH solution containing C. officinalis, H. perforatum and E. purpurea holds potential for accelerating wound healing in the context of DFUs. Further research is necessary to establish effects in more complex models.
M.Tech. (Homoeopathy)