| 张智昊,王选康,巨承,等.光生物治疗降低神经黏蛋白表达促进小鼠脊髓损伤修复的实验研究.骨科,2023,14(2): 177-186. |
| 光生物治疗降低神经黏蛋白表达促进小鼠脊髓损伤修复的实验研究 |
| An Experimental Study of Photobiological Therapy to Reduce Neurocan Expression to Promote Spinal Cord Injury Repair in Mice |
| 投稿时间：2022-12-28 |
| DOI：DOI：10.3969/j.issn.1674-8573.2023.02.016 |
| 中文关键词: 脊髓损伤 光生物调节 神经黏蛋白 轴突再生 JAK2/STAT3通路 |
| 英文关键词: Spinal cord injury Photobiomodulation therapy Neurocan Axon regeneration JAK2/STAT3 pathway |
| 基金项目:国家自然科学基金（81572151）；陕西省重点研发计划项目（2020ZDLSF02-05、2021ZDLSF02-10） |
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| 中文摘要: |
| 目的 明确光生物调节（photobiomodulation，PBM）对脊髓损伤后小鼠抑制性瘢痕成分神经黏蛋白表达的影响，及其对神经再生和功能修复的改善作用，阐明PBM下调神经黏蛋白表达的机制。方法 利用皮下埋置360°近红外激光照射光纤，定期连接激光发生装置照射脊髓损伤小鼠，建立脊髓损伤激光治疗模型。将C57BL/6小鼠按照不同处理方式分为三组：Sham组（假手术）、SCI组（脊髓损伤）和SCI+PBM组。采用荧光共染的方法观察不同处理组冰冻切片组织中神经黏蛋白的表达和轴突再生的关系，并明确脊髓损伤后星形胶质细胞是表达神经黏蛋白的责任细胞。采用Western blot和免疫荧光染色检测脊髓组织和原代星形胶质细胞中神经黏蛋白的表达水平。采用实时荧光定量聚合酶链式反应（Reverse transcription-polymerase chain reaction，RT-PCR）、免疫荧光和Western blot的方法检测PBM治疗后星形胶质细胞245 KD神经黏蛋白分子和130 KD神经黏蛋白分子的表达水平。利用葫芦素Ⅰ抑制原代星形胶质细胞中的Janus激酶2-信号转导和转录活化因子3（Janus Kinase 2-Signal Transducer and Activator of Transcription 3，JAK2-STAT3）通路，反向验证PBM干预神经黏蛋白表达的通路。采用Basso Mouse Scale（BMS）评分和足印记实验观察研究中小鼠的运动功能恢复情况。结果 皮下埋置360°近红外激光光纤后，经过7天光照射治疗改善了脊髓损伤小鼠运动功能（P＜0.05），足印记实验证实PBM显著提高了脊髓损伤小鼠的步长（P＜0.001）；在损伤旁区的脊髓组织中，神经黏蛋白的表达被上调（P＜0.001），且与星形胶质细胞标志物共标；PBM降低了损伤后7天和14天245 KD神经黏蛋白（P＜0.05，P＜0.001）和130 KD神经黏蛋白（P＜0.01，P＜0.01）的表达，伴随轴突再生的增加。分子细胞学实验表明，PBM干预显著降低了脊髓损伤后神经黏蛋白的表达水平（245 KD：P＜0.001；130 KD：P＜0.001），葫芦素Ⅰ的使用抑制了JAK2和STAT3分子的磷酸化水平（P＜0.01，P＜0.0001），同时显著降低了星形胶质细胞神经黏蛋白的表达（245 KD：P＜0.001；130 KD：P＜0.001）。结论 PBM可能通过调节JAK2/STAT3分子轴抑制组织中星形胶质细胞神经黏蛋白的表达，促进小鼠脊髓损伤后轴突再生和运动功能的恢复。 |
| 英文摘要: |
| Objective To determine the effect of photobiomodulation (PBM) on the improvement of nerve regeneration and functional repair in mice after spinal cord injury (SCI) by regulating the expression of neurocan, an inhibitory scar component, and to clarify its potential mechanism. Methods A 360° near-infrared laser irradiation fiber was implanted subcutaneously and connected to a laser generator with periodical irradiation to establish a SCI model. C57BL/6 mice were divided into three groups according to different treatments: sham-operated control group, SCI group and SCI+PBM group. Fluorescent co-staining was used to observe the relationship between the expression of neurocan and axon regeneration in tissue frozen sections of different treatment groups, and to clarify the expression of neurocan in astrocytes after SCI. Western blotting and immunofluorescent staining were used to detect the expression of neurocan in the spinal cord tissue and primary astrocytes. Real-time quantitative polymerase chain reaction, immunofluorescence and Western blotting were used to detect the expression levels of 245 KD and 130 KD neurocan molecules in astrocytes after PBM treatment. Cucurbitacin I was used to inhibit JAK2-STAT3 pathway in primary astrocytes to verify the potential pathway of PBM intervention on the neurocan expression. The Basso Mouse Scale (BMS) score and footprint test were used to observe the recovery of motor function of the mice in this study. Results PBM treatment of 7 days with subcutaneous implantation of 360° near-infrared laser fiber improved the recovery of motor function in mice with SCI (P＜0.05). Foot print experiment confirmed that PBM treatment significantly increased the step length of the SCI mice (P＜0.001). The expression of neurocan was up-regulated (P＜0.0001) and co-labeled with astrocytes marker in the para-injured spinal cord tissue. PBM treatment decreased the 245 KD neurocan expression (P＜0.05, P＜0.001) and 130 KD neurocan expression (P＜0.01, P＜0.01) at 7th day and 14th day after SCI, accompanied with an increase in axonal regeneration. Molecular and cytology experiments showed that the expression level of neurocan was decreased after PBM irradiation (245 KD, P＜0.001; 130 KD, P＜0.001), cucurbitacin Ⅰ inhibited the phosphorylation level of JAK2 and STAT3 (P＜0.01, P＜0.001) and significantly reduced the expression of neurocan in astrocytes (245 KD, P＜0.001; 130 KD, P＜0.001). Conclusion PBM treatment could inhibit the expression of neurocan in astrocytes by regulating the JAK2/STAT3 molecular axis and promote axon regeneration and motor function recovery after SCI. |
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