Photobiomodulation Promotes Neuronal Axon Regeneration After Oxidative Stress and Induces a Change in Polarization from M1 to M2 in Macrophages via Stimulation of CCL2 in Neurons: Relevance to Spinal Cord Injury

To study the effect of photobiomodulation (PBM) on axon regeneration and secretion change of dorsal root ganglion (DRG) under oxidative stress after spinal cord injury (SCI), and further explore the effect of changes in DRG secretion caused by PBM on the polarization of macrophages. The PBM-DRG model was constructed to perform PBM on neurons under oxidative stress simulated in vitro. And the irradiation conditions were as follows: wavelength, 810 nm; power density, 2 mW/cm2; irradiation area, 4.5 cm2; and irradiation time, 440 s. Then resulted in an energy of 4 J (2 mW/cm2 × 4.5 cm2 × 440 s). About 100 μM H202 was added to the culture medium to simulate oxidative stress after SCI. An ROS (reactive oxygen species) assay kit was used to measure ROS contend in the DRG. The survival level of the neurons was measured using the CCK-8 method, and the axon regeneration of neurons was observed by using immunofluorescence. The secretion level of CCL2 from DRG was determined by RT-qPCR and ELISA. Further culturing macrophages of DRG-conditioned medium culture, the expression level of iNOS and Arg-1 in macrophages was assessed using Western blot analysis. The expression level of TNF-α and IL-1β was determined by ELISA. After adding the neutralizing antibody of CCL2 to the DRG neuron-conditioned medium following PBM irradiation to culture macrophages to observe the effects on macrophage polarization and secretion. PBM could reduce ROS levels in neurons, increase neuronal survival under oxidative stress, and promote neuronal axon regeneration. In addition, PBM could also promote CCL2 secretion by DRG under oxidative stress. By constructing a DRG supernatant-M1 macrophage adoptive culture model, we found that the supernatant of DRG after PBM intervention could reduce the expression level of iNOS and the secretion of TNF-α and IL-1β in M1 macrophages; at the same time, it could also up-regulate the expression of Arg-1, one of the markers of M2 macrophages. Furthermore, these effects could be prevented by the addition of neutralizing antibodies of CCL2. PBM could promote survival and axonal regeneration of DRG under SCI oxidative stress, increase the secretion level of CCL2 by DRG, and this change can reduce the polarization of macrophages to M1, further indicating that PBM could promote spinal cord injury repair.