Irradiation at 830 nm stimulates nitric oxide production and inhibits pro‐inflammatory cytokines in diabetic wounded fibroblast cells

Abstract

Wound healing in diabetic patients remains a chief problem in the clinical setting and there is a strong need for the development of new, safe, reliable therapies. This study aimed to establish the effect of irradiating diabetic wounded fibroblast cells (WS1) in vitro on pro-inflammatory cytokines and the production of nitric oxide (NO). Normal, wounded and diabetic wounded WS1 cells were exposed to an 830 nm laser with 5 J/cm(2) and incubated for a pre-determined amount of time. Changes in cellular viability, proliferation and apoptosis were evaluated by the Trypan blue assay, VisionBlue fluorescence assay and caspase 3/7 activity respectively. Changes in cytokines (interleukin--IL-6, IL-1 beta and tumour necrosis factor-alpha, TNF-alpha) were determined by ELISA. NO was determined spectrophotometrically and reactive oxygen species (ROS) was evaluated by immunofluorescent staining. Diabetic wounded WS1 cells showed no significant change in viability, a significant increase in proliferation at 24 and 48 hours (P<0.001 and P<0.01 respectively) and a decrease in apoptosis 24 hours post-irradiation (P<0.01). TNF-alpha levels were significantly decreased at both 1 and 24 hours (P<0.05), while IL-1 beta was only decreased at 24 hours (P<0.05). There was no significant change in IL-6. There was an increase in ROS and NO (P<0.01) 15 minutes post-irradiation. Results show that irradiation of diabetic wounded fibroblast cells at 830 nm with 5 J/cm(2) has a positive effect on wound healing in vitro. There was a decrease in pro-inflammatory cytokines (IL-1 beta and TNF-alpha) and irradiation stimulated the release of ROS and NO due to what appears to be direct photochemical processes.