[The 810 nm low-level laser inhibits the polarization of M1 bone marrow-derived macrophages to promote neuronal axon growth of dorsal root ganglion]

Abstract

Objective To investigate the effect of low-level laser on the polarization and secretory phenotype of primary cultured M1 bone marrow-derived macrophages (BMDMs) in neuronal axons of dorsal root ganglion (DRG). Methods BMDMs were isolated and cultured, and lipopolysaccharide (LPS) combined with IFN-γ were used to induce M1 phenotype polarization of BMDMs, and then F4/80 and CD16/32 expression was detected by flow cytometry. The mature M1 type BMDMs were randomly divided into low-level laser group and control group. The laser exposure group was subjected to the laser treatments of 0.4J, 4J and 10J, and no laser was used in the control group. After 24 hours of laser exposure, the mRNA level of inducible nitric oxide synthase (iNOS) of M1 type BMDMs was detected by reverse transcription PCR, and the protein level of iNOS was detected by Western blot analysis. The levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the supernatant of cultured cells were tested by ELISA. DRG neurons were cultured with the supernatant fluid of M1 type BMDMs, and immunofluorescence cytochemistry was employed to detect neuronal nuclei (NeuN) and β-tubulin III expression of DRG neurons for determining the influence on the growth of DRG neuronal axons. Results Compared with the control group, the mRNA level of iNOS in M1 type BMDMs dramatically increased after 24 hours of low-level laser exposure. Among the 3 groups with different energy levels, the decrease of iNOS mRNA level was the most obvious in the group with 4J laser exposure. The protein levels of iNOS in the groups with 0.4J- and 4J- laser exposure were reduced more significantly than that in the control group, and the down-regulation was more prominent in the group with 4J laser exposure than that with 0.4J laser exposure. In addition, the secretion of TNF-α from M1 type BMDMs was reduced more significantly in the groups of 4J- and 10J- laser exposure than that in the control group. With regard to IL-1β, its secretion was inhibited in all the laser exposure groups compared with the control group, and the suppression was more prominent in the groups of 0.4J- and 4J-laser exposure than that in the group of 10J-laser exposure. Furthermore, 4J-laser exposure significantly potentiated the secretion of BDNF and NGF in M1 type BMDMs compared with the control group. Moreover, co-culture with the supernatants from 4J- and 10J-laser exposure groups could significantly promote the growth of axons of DRG neurons. Conclusion Low-level laser exposure can inhibit the polarization of M1 type BMDM and the secretion of pro-inflammatory factor including TNF-α and IL-1β. Besides, low-level laser exposure could contribute to the secretion of neurotrophic factors including BDNF and NGF, and promote the growth of DRG axon, and this effect is dose-dependent.