电针配伍穴位通过HIF-1α/VEGF通路对大鼠脊髓损伤后运动功能的影响

关键词：脊髓损伤  缺氧诱导因子1α  血管内皮生长因子  电针  组合穴位

Keywords:spinal cord injury  hypoxia inducible factor 1α  vascular endothelial growth factor  electroacupuncture  acupoint combination

摘要：

      摘要 目的：观察电针配伍穴位对急性脊髓损伤（SCI）后大鼠运动功能的影响及脊髓神经元缺氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF） 表达变化，探讨电针促进大鼠运动功能恢复作用及其可能机制。 方法：108只雌性SD大鼠，随机分为假手术组36只（Sham组）、模型组36只（SCI组）和电针组36只（SCI+EA组）。以T10为中心，用HI—0400脊髓打击器构建大鼠SCI模型；Sham组只去除椎板，不损伤脊髓。SCI+EA组术后1d开始电针干预，穴位配伍为大椎、至阳、命门、夹脊、足三里、三阴交，每日一次，每次30min，干预28d。于损伤后1d、3d、7d、14d、21d、28d 6个时间点，采用BBB评分评估大鼠后肢运动功能变化；HE染色观察损伤脊髓节段组织结构变化；免疫荧光法、Western Blot法检测各组大鼠脊髓组织中HIF-1α、VEGF蛋白定位及表达变化。 结果：①BBB运动功能评分SCI+EA组14d后明显升高，与SCI组比较有差异（P<0.05）；②HE染色Sham组脊髓结构完整，神经元形态清晰。SCI组3d有打击空洞，出血。7d、14d可见坏死神经元，小胶质细胞增多；21d、28d神经元数量减少。SCI+EA组电针3d后，脊髓打击处可见空洞、出血，7d后变性的神经元细胞结构逐渐清楚，细胞核清晰，核膜完整，坏死细胞计数较SCI组少（P<0.05）；③免疫荧光显示HIF-1α、VEGF定位于运动神经元，损伤后7d荧光强度达到高峰，SCI+EA组荧光强度在各时间点均强于SCI组（P<0.05），Sham组荧光强度低于SCI组与SCI+EA组（P<0.05）；④Western Blot结果显示，损伤后7d SCI+EA组HIF-1α、VEGF蛋白表达明显升高，多于SCI组（P<0.05），14d后各对应时间点SCI+EA组蛋白表达均高于SCI组（P<0.05）。 结论：电针可以促进大鼠脊髓损伤后运动功能的恢复，其机制可能与HIF-1α/VEGF信号通路的激活有关。

Abstract:

      Abstract Objective： To observe the effect of electroacupuncture on the motor function of rats after acute spinal cord injury （SCI） and changes in expression of hypoxia-inducible factor 1α （HIF-1α） and vascular endothelial growth factor （VEGF） in spinal cord neurons as its possible mechanism. Method： One hundred and eight female SD rats were randomly divided into sham-operated group （Sham group, n=36)， model group （SCI group, n=36）， and electroacupuncture group（EA group, n=36）. The rat SCI model was constructed with the HI-0400 spinal cord percussor centered on T10； the Sham group only removed the vertebral plates without damaging the spinal cord. The 28-day electro-acupuncture intervention was assigned on all the acupoints, including Dazhui， Zhiyang， Mingmen， Jiaji， Zusanli， Sanyinjia, 30-minutes per section, once a day, since the second day after operation. The changes of motor function in hind limbs of rats were evaluated by BBB score； the structural changes of the spinal cord were observed by HE staining； the protein localization and expression changes of HIF-1α and VEGF in spinal cord tissues of rats were detected by immunofluorescence and Western Blot at 6 time points: 1d， 3d， 7d， 14d， 21d， 28d after injury. Result： The BBB score of the SCI+EA group showed a significant increase after 14 days compared to the SCI group （P<0.05）；In the Sham group, the structure of the spinal cord was intact，and the neuron morphology was clear upon HE staining. Conversely, the SCI group exhibited cavitation and bleeding for 3 days, with necrotic neurons visible on 7d and 14d， and an increase in microglia numbers. Neurons decreased in number on 21d and 28d. In the SCI+EA group，spinal cord holes and bleeding were observed after 3 days of electroacupuncture; however, after 7 days， the structure of degenerated neurons gradually became clear， with clear nuclei and complete nuclear membrane， and fewer necrotic cells than in the SCI group （P<0.05）. Immunofluorescence analysis showed that HIF-1α and VEGF localized in motor neurons, with peak fluorescence intensity at 7 days after injury. The SCI+EA group had stronger fluorescence intensity than the SCI group at all time points （P<0.05），while the Shame group had lower fluorescence intensity than the SCI group and SCI+EA group （P<0.05）. Western Blot analysis revealed that the protein expression of HIF-1α and VEGF in the SCI+EA group was significantly higher than that in the SCI group at 7 days after injury （P<0.05）， and the protein expression remain higher in the SCI+EA group than in the SCI group at each corresponding time point after 14 days（P<0.05）. Conclusion： Electroacupuncture can improve the recovery of motor function after spinal cord injury in rats，the mechanism may related to the activation of HIF-1α/VEGF signaling pathway.