810 nm弱激光抑制M1型骨髓源性巨噬细胞极化促进脊髓背根神经元轴突生长

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

目的研究810 nm弱激光对原代培养M1型骨髓源性巨噬细胞(BMDM)表型极化及分泌对背根神经节(DRG)神经元轴突的作用。方法分离培养BMDM,经脂多糖(LPS)联合γ干扰素(IFN-γ)诱导BMDM向M1表型极化,采用流式细胞术检测细胞F4/80和CD16/32表达。将诱导成熟的M1型BMDM随机分为弱激光照射组与对照组。照射组分别采用波长810 nm,0.4J、 4J和10J的弱激光进行照射;对照组不进行照射。照射后24 h,反转录PCR法检测M1型BMDM诱导型一氧化氮合酶(iNOS)的mRNA水平, Western blot法检测iNOS的蛋白水平, ELISA检测培养细胞上清液肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、脑源性神经营养因子(BDNF)、神经生长因子(NGF)的含量,免疫荧光细胞化学染色检测神经元核蛋白(NeuN)和β-微管蛋白Ⅲ(β-tubulinⅢ)的表达,确定M1型BMDM上清液培养对DRG神经元轴突生长的影响。结果与对照组相比,各能量参数弱激光照射后24h, M1型BMDM iNOS mRNA水平均显著下调, 4J弱激光照射组下调最显著;0.4J及4J弱激光照射组iNOS蛋白水平显著下调, 4J弱激光照射组下调水平较0.4J弱激光照射组更显著。4J和10J弱激光照射组M1型BMDM TNF-α的分泌明显减少,各照射组M1型BMDM IL-1β的分泌明显下调, 0.4J及4J弱激光照射组抑制程度较10J弱激光照射组更为显著。4J弱激光照射组明显促进M1型BMDM的BDNF和NGF分泌。采用照射后的BMDM上清液过继培养DRG 24 h后, 4J及10J弱激光照射组上清液可显著促进DRG神经元轴突的生长。结论弱激光照射可以抑制M1型BMDM表型极化及促炎因子TNF-α、 IL-1β分泌,上调神经营养因子BDNF和NGF的分泌,促进DRG神经元轴突的生长,且呈一定的剂量依赖性。

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.