Objective To investigate the effect of 810 nm low-level laser on neuronal axonal regeneration of mice with spinal cord injury and its related mechanism.Methods In vivo experiment:20 Balb/c mice were randomly divided into the spinal cord injury group(SCI group)and the 810 nm low-level laser irradiation group(low-level laser group)after spinal cord injury according to the random number table method,with each group containing ten mice.A mice SCI model was established through clamp injury and the low-level laser group continuously irradiated the damaged area with weak 810 nm low-level laser with selected parameters(continuous wave with wave length 810 nm,power density 2 mW/cm2,spot are 4.5 cm2,irradiation time 50 minutes,energy 6000J/cm2).Then immunofluorescence staining was used to observe the M1 macrophage marker-inducible nitric oxide synthase(iNOS),the M2 macrophage marker arginase 1(Arg-1)and the universal marker F4/80 of macrophages after 14 days.Furthermore,in the in vitro experiment,standardized low-level laser-macrophage irradiation model was established.Another 20 Balb/c mice were used to obtain primary bone marrow-derived macrophages which were induced into M1 macrophages using lipopolysaccharide(LPS)and interferon-gamma(INF-γ).The M1 macrophages were randomly divided into the M1 macrophage group(M1 group)and the low-level laser therapy group(M1 + low-level laser group)equally according to the random number table method.The M1 group was not treated,and the M1 + low-level laser group was treated with low-level laser of selected parameters.RT-qPCR and ELISA were used to detect the expression of interleukin-1 receptor antagonist(IL-1RA)and interleukin-10(IL-10)in M1 macrophages 24 hours after irradiation.Western blot was used to analyze the expression of iNOS,Arg-1,differentiation antigen cluster 206(CD206),protein kinase B(AKT),phosphorylated protein kinase B(p-AKT),cyclic adenosine response element binding protein(CREB)and phosphorylated cyclic adenosine response element binding protein(p-CREB)in M1 macrophages 48 hours after irradiation.Dorsal root gangtion neurons(DRG)were cultured in two groups of macrophage conditioned medium,and the length of DRG axon growth was measured 48 h later to evaluate the effect of low-level laser on neuronal axon growth.Results In the in vivo experiment,compared with mice with spinal cord injury alone,the fluorescence intensity of F4/80+ iNOS+ in the spinal cord injury area decreased(1.00±0.08vs. 0.06±0.04)(P< 0.05)and the fluorescence intensity of F4/80 + Arg-1 + increased after low-level laser(1.00±0.07vs.2.15±0.12)(P<0.01).In the in vitro experiment,compared with the M1 group,the expression of the M1 macrophage marker iNOS in the M1 + low-level laser group decreased(1.00±0.11 vs.0.08±0.01)(P< 0.01);the M2 macrophage marker Arg-1(1.00±0.14vs.2.44±0.16)(P<0.01),and the expression of CD206(1.00±0.12 vs.1.83±0.05)(P<0.01)increased.In addition,IL-1RA expression was increased in the M1 + low-level laser group compared with the M1 group(RT-qPCR:1.00±0.00vs.2.27±0.22)(P<0.01)(ELISA:1435.58±100.48vs.2006.12±123.91(P<0.05);IL-10 expression was also increased in the M1 +low-level laser group compared with the M1 group(RT-qPCR:1.00±0.00 vs. 3.45±0,56)(P<0.05)(ELISA:137.13±4.20 vs.188.29±8.49)(P< 0,01);compared with the M1 group,the macrophage polarization pathway protein in the M1 + low-level laser group increased,AKT(1.07±0.12vs.1.74±0.04)(P<0.01),p-AKT(1.00±0.12 vs.1.64±0.15)(P<0.05),p-CREB(1.00±0.10vs.2.12±0.18)(P<0.01).Compared with the M1 group,the conditioned medium of the M1 + low-level laser group significantly promoted DRG axon growth(567.66±63.59 vs.1068.95±130.14)(P< 0,05).Conclusions The 810 nm low-level laser irradiation can promote neuronal axon regeneration of mice with spinal cord injury,which may be related to the regulation of macrophage polarization phenotype by low-level laser through AKT/CREB pathway.%目的 探讨810nm弱激光对小鼠脊髓损伤(SCI)后神经元轴突再生的作用及其相关机制.方法 体内实验:选取20只Balb/c小鼠,通过钳夹伤方法,建立标准化小鼠SCI模型,按照随机数字表法分为SCI组及SCI后810nm弱激光照射组(弱激光组),每组10只.弱激光组采用选定参数的弱激光(连续波波长810nm,功率密度2mW/cm2,光斑面积4.5cm2,照射时间50min,获得能量密度6000J/cm2)连续照射损伤区,14d后免疫荧光染色观察SCI组及弱激光组损伤区M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、M2型巨噬细胞标志物精氨酸酶1(Arg-1)及巨噬细胞普遍标志物F4/80的表达情况.体外实验:选取20只Balb/c小鼠,体外获取并培养小鼠原代骨髓源性巨噬细胞,而后使用脂多糖(LPS)和γ干扰素(INF-γ)诱导为M1型巨噬细胞.按照随机数字表法将巨噬细胞培养板分为M1型巨噬细胞组(M1组)和弱激光照射组(M1 +弱激光组),每组细胞数目相同.M1组不做处理,M1 +弱激光组采用体外标准化810 nm弱激光-巨噬细胞照射模型进行照射.照射24h后RT-qPCR和ELISA法检测两组白细胞介素1受体拮抗剂(IL-1RA)、白细胞介素10(IL-10)的表达情况.照射48 h后Western blot分析两组iNOS、Arg-l、分化抗原簇206(CD206)、蛋白激酶B(AKT)、磷酸化蛋白激酶B(P-AKT)、环磷腺苷效应元件结合蛋白(CREB)和磷酸化环磷腺苷效应元件结合蛋白(P-CREB)的表达.照射48h后,分别使用两组巨噬细胞条件培养基培养背根神经节神经元(DRG),培养48h后测量DRG轴突生长的长度.结果 体内实验中,相较于SCI组,弱激光组损伤区F4/80+ iNOS+的荧光强度显著下降(1.00±0.08:0.06±0.04)(P<0.05);F4/80+ Arg-l+的荧光强度显著上升(1.00±0.07:2.15±0.12)(P<0.01).在体外实验中,与M1组相比,M1+弱激光组中M1型巨噬细胞标志物iNOS的表达降低(1.00±0.11 :0.08±0.01)(P<0.01);M2型巨噬细胞标志物Arg-l(1.00±0.14:2.44±0.16)、CD206的表达升高(1.00±0.12:1.83±0.05)(P均<0.01).此外,相较于M1组,M1 +弱激光组的IL-1RA的表达升高[(RT-qPCR:1.00±0:2.27±0.22)(P<0.01)(ELISA:1435.58±100.48 :2006.12±123.91)(P<0.05)];相较于M1组,M1+弱激光组IL-10的表达也出现上升[(RT-qPCR:1.00±0.00:3.45±0.56)(P<0.05)(ELISA:137.13±4.20:188.29±8.49)(P<0.01)];相较于M1组,M1 +弱激光组巨噬细胞极化通路蛋白均出现升高[AKT为1.07±0.12:1.74±0.04,p-AKT为1.00±0.12:1.64±0.15,p-CREB为1.00±0.10:2.12±0.18)(P<0.05或0.01).相较于M1组,M1+弱激光组条件培养基DRG轴突生长更长(567.66±63.59:1068.95±130.14)(P<0.05).结论 小鼠SCI后利用810 nm弱激光照射可促进神经元轴突再生,其机制可能与弱激光通过AKT/CREB通路调控巨噬细胞极化表型有关.
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