Low level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts

Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation remain unclear. In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810-nm laser radiation. Significant activation of NFkB was observed for fluences higher than 0.003 J/cm[superscript 2]. NF-kB activation by laser was detectable at 1-hour time point. Moreover, we demonstrated that laser phosphorylated both IKK alpha/beta and NF-kB 15 minutes after irradiation, which implied that laser activates NF-kB via phosphorylation of IKK alpha/beta. Suspecting mitochondria as the source of NF-kB activation signaling pathway, we demonstrated that laser increased both intracellular reactive oxygen species (ROS) by fluorescence microscopy with dichlorodihydrofluorescein and ATP synthesis by luciferase assay. Mitochondrial inhibitors, such as antimycin A, rotenone and paraquat increased ROS and NF-kB activation but had no effect on ATP. The ROS quenchers N-acetyl-L-cysteine and ascorbic acid abrogated laser-induced NF-kB and ROS but not ATP. These results suggested that ROS might play an important role in the signaling pathway of laser induced NF-kB activation. However, the western blot showed that antimycin A, a mitochondrial inhibitor, did not activate NF-kB via serine phosphorylation of IKK alpha/beta as the laser did. On the other hand, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that light also upregulates mitochondrial respiration. ATP upregulation reached a maximum at 0.3 J/cm[superscript 2] or higher. We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive transcription factor NF-kB by generating ROS as signaling molecules.United States National Institutes of Health (R01CA/AI838801 and R01 AI050875