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Y.-Y. Huang and others FIGURE 9. Biphasic dose response of NF-κB activation (measured by bioluminescence reporter assay) in mouse embryonic fibroblasts 10 hours after different fluences of 810-nm laser light. CHI is control where all protein synthesis has been inhibited. 4. BIPHASIC LLLT DOSE RESPONSE STUDIES IN OUR LABORATORY 4.1. In vitro activation of NF-κB We developed the hypothesis (Chen et al. 2009a) that NIR light (810- nm laser) would activate the transcription factor NF-κB by generating reactive oxygen species from the mitochondria (see section 2.5). We test- ed this in mouse embryonic fibroblasts that had been genetically engi- neered to synthesize luciferase in response to NF-κB activation (Chen et al. 2009a). We used a wide range (four orders of magnitude) of delivered flu- ences by adjusting the laser power so that the illumination time was kept constant at 5 minutes. As shown in Figure 9 there was a biphasic dose dependent activation of NF-κB as measured by luciferase assay 10 hours after the illumination was completed. There was no significant increase at 0.003 J/cm2 compared to the dark control, a small increase at 0.03 J/cm2,, the maximum activation was observed at 0.3 J/cm2, while at 3 J/cm2 and even more so at 30 J/cm2 there was a decrease in NF-κB activation, but the level was still higher than that found at 0.03 J/cm2. The level of luciferase expression was also measured in the presence of cycloheximide (CHI) as a control. CHI is a protein synthesis inhibitor that removes even the back- ground level of luciferase seen in dark control cells, as well as all the increases seen with the different fluences of 810-nm light. We tested the hypothesis that the activation of NF-κB by LLLT was mediated by generation of ROS because NF-κB is known to be a redox- sensitive transcription factor (Schreck et al. 1992) and moreover ROS have previously been shown to be generated during LLLT (AlexandratouPDF Image | BIPHASIC DOSE RESPONSE IN LOW LEVEL LIGHT THERAPY
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