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

Abstract

Background: Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear.

Methodology/principal findings: 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 NF-kB was observed at fluences higher than 0.003 J/cm(2) and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration.

Conclusion: We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

Figure 1. Laser activates NF-kB in MEF.

(A) Time course of NF-kB activation measured by luminescence after addition of 0.5 ug/mL of LPS (B) Time course of NF-kB activation after 0.3 J/cm2 810-nm laser irradiation. (C) NF-kB measured 6 hours post irradiation with a wide range of 810-laser fluences and effect of protein synthesis inhibitor cycloheximide. * p<0.05; ** p<0.01 compared to baseline. (D) Immunoblotting for Phospho NFκB and IκB α/β following laser irradiation of MEFs at 0.3 J/cm² over time (upper panel) and at various fluences 0.3, 3 and 30 J/cm² at 15 min (lower panel) was performed. Normalization for protein loading with Actin is shown.

Figure 2. Laser increases ROS in MEF.

(A) MEF were treated with 810 nm laser at varying fluencies 0, 0.3, 3 and 30 J/cm² and ROS generation was measured with CM-H₂DCFDA (green). Mitochondrial localization is demonstrated with MitoTracker Red (red) and overlay (yellow) while cell nuclei were strained with DAPI (blue) (B) Amplex Ultrared assay demonstrates quantitative increase in ROS following laser irradiation at varying fluences. * p<0.05.

Figure 3. Laser increases ATP synthesis in MEF.

(A) ATP increase measured at 5 min post LLLT with wide range laser fluences and effect of mitochondrial inhibitors (sodium azide/deoxyglucose). (B) Time course of ATP increase after 0.3 J/cm2 810 nm laser. * p<0.05; ** p<0.01 compared to baseline.

Figure 4. Mitochondrial inhibitors increase ROS and NF-kB activation in MEF but reduce ATP.

(A) NF-kB activation measured at 6 hours after 2 hour incubation with 100 µM inhibitors, (B) ATP levels measured after 2 hours incubation with 100- µM inhibitor. * p<0.05; ** p<0.01 compared to control. Inhibitors (100 µM) were added for 2 hours and CM-H₂DCFDA was used to measure ROS generation at 30 min (C) control, (D) antimycin A, (E) rotenone, (F) paraquat.

Figure 5. Antioxidants abrogate laser-induced NF-kB activation in MEF but not ATP increase.

(A) NF-kB measured 6 hours post irradiation of cells incubated 2 hours with 1 mM N-acetyl cysteine or 100 µM ascorbic acid. (B) ATP measured after 2-hour incubation with 1 mM N-acetyl cysteine. * p<0.05; ** p<0.01 compared to control.