黄芪对脊髓损伤小鼠的神经修复作用

Neuroprotective effects of astragalus on spinal cord injury in mice

目的观察黄芪对脊髓损伤(SCI)小鼠的神经修复作用。方法采用自由落体击打法制作SCI小鼠模型,随机将术后小鼠分为对照组和治疗组,对照组不予治疗,术后腹腔注射适量生理盐水,黄芪治疗组术后腹腔注射黄芪注射液,小鼠术后3 d麻醉处死,用干湿称重法测定出脊髓含水量,二氢乙锭(HEt)染色测定脊髓活性氧(ROS)的积累,原位缺口末端标记法(TUNEL)试剂盒检测损伤脊髓附近区域的凋亡细胞数量,酶联免疫吸附试验(ELISA)试剂盒检测炎性因子肿瘤坏死因子(TNF)和白细胞介素(IL)的表达量,术后7、14、21、28 d分别进行神经功能缺损评分评价损伤小鼠的运动功能,糖水偏嗜实验检测小鼠的抑郁状况,斜面实验评价小鼠的后肢力量,免疫荧光染色检测成熟神经元标志物神经元核性蛋白(NeuN)和脊髓白质标志物髓鞘碱性蛋白(MBP)的表达量。结果(1)黄芪治疗组脊髓含水量(67.67±3.34)和对照组(83.33±1.91)比较明显降低,差异有统计学意义(t=4.078,P<0.01)。(2)对照组(2 051.67±38.05)比较黄芪治疗组ROS荧光强度(1 520.00±71.88)明显降低,差异有统计学意义(t=6.537,P<0.01)。(3)黄芪治疗组细胞凋亡数量[(67.67±3.43)个]和对照组[(79.34±2.53)个]比较数量显著减少,差异有统计学意义(t=2.740,P<0.05)。(4)黄芪治疗组抑炎因子IL-10表达量(1 084.33±59.97) pg/ml明显高于对照组(747.67±34.65) pg/ml,而治疗组促炎因子TNF-α的表达量(834.50±29.71) ng/ml明显低于对照组(1 033.00±46.86) ng/ml,差异有统计学意义(t=3.58,P<0.01)。(5)脊髓损伤第7、14、21、28天,BMS评分的结果表明,相对于对照组[(1.13±2.50)、(1.75±2.38)、(2.13±2.80)、(3.00±2.63)分],黄芪治疗组评分[(2.50±1.38)、(3.00±1.25)、(3.75±1.63)、(4.50±1.50)分]升高。(6)损伤第21天,斜面放置实验结果显示治疗组斜面度数[(54.75±8.34)°]高于对照组[(41.50±13.25)°]。(7)脊髓损伤第21天,糖水偏嗜实验表明黄芪治疗组糖水偏嗜度[(64.67±1.43)%]和对照组[(50.17±2.24)%]比较,差异有统计学意义(t=5.45,P<0.01)。(8)比较对照组[(31.00±1.95)个],治疗组[(51.17±2.40)个]的NeuN阳性细胞数量明显增多,黄芪治疗组[(43.33±1.91)%]的脊髓白质面积明显多于对照组[(29.00±2.21)%],差异有统计学意义(t=4.910,P<0.01)。结论黄芪对脊髓损伤小鼠存在神经保护作用。

Objective To observe the neurorestorative effect of astragalus on mice with spinal cord injury. Methods SCI mouse model was established by modified Allen’s striking method. The postoperative mice were randomly divided into control group and treatment group. The control group was not treated. The rats were intraperitoneally injected with appropriate amount of normal saline. The jaundice treatment group was intraperitoneally injected with jaundice. The injection was anesthetized three days after the operation. The water content of the spinal cord was determined by dry and wet weighing method. The accumulation of reactive oxygen species (ROS) in the spinal cord was determined by hematoxylin and eosin (HE) staining. The TdT-mediated dUTP nick end labeling (TUNEL) kit was used to detect the area near the injured spinal cord. The number of apoptotic cells, enzyme-linked immunosorbent assay (ELISA) kit was used to detect the expression levels of inflammatory factors tumor necrosis factor and interleukin. Neurological deficit scores were evaluated on injured mice at 7, 14, 21, and 28 days after surgery. The motor function, the sucrose preference test to detect the depression of the mice, the hind limb strength of the mouse was evaluated by the bevel experiment, and the expression of the mature neuron marker neuronal nuclear protein (NeuN) and the white matter marker myelin basic protein (MBP) was detected by immunofluorescence staining. Results (1) The water content of the spinal cord in the Huangqi treatment group (67.67±3.34) was significantly lower than that in the control group (83.33±1.91), and the difference was statistically significant (t=4.078, P<0.01).(2) Compared with the control group (2 051.67±38.05), the ROS fluorescence intensity (1 520.00±71.88) in the Huangqi treatment group was significantly lower, and the difference was statistically significant (t=6.537, P<0.01).(3) The number of apoptosis in the Astragalus treatment group [(67.67±3.43) cells] was significantly lower than that in the control group [(79.34±2.53) cells], which was statistically significant (t=2.74, P<0.05).(4) The expression of anti-inflammatory factor IL-10 (1 084.33±59.97) pg/ml in the treatment group of Astragalus membranaceus was significantly higher than that in the control group (747.67±34.65) pg/ml, while the expression of tumor necrosis factor-α(TNF-α) in the treatment group was significantly lower (834.50±29.71) ng/ml. The control group (1 033.00±46.86) ng/ml, the difference was statistically significant (t=3.58, P<0.01).(5) On days 7, 14, 21, and 28 of spinal cord injury, the results of BMS scores showed that compared with the control group [(1.13±2.50),(1.75±2.38),(2.13±2.80),(3.00±2.63)] The scores of the jaundice treatment group [(2.50±1.38),(3.00±1.25),(3.75±1.63),(4.50±1.50)] were elevated.(6) On the 21st day of injury, the experimental results of the inclined surface showed that the degree of slope of the treatment group (54.75±8.34)°was higher than that of the control group (41.50±13.25)°.(7) On the 21st day of spinal cord injury, the sucrose preference experiment showed that the glucone preference (64.67±1.43)% of the jaundice treatment group was statistically significant compared with the control group (50.17±2.24)%(t=5.45, P<0.01).(8) Compared with the control group [(31.00±1.95) cells], the number of NeuN positive cells in the treatment group [(51.17±2.40) cells] increased significantly, and the area of white matter in the jaundice treatment group [(43.33±1.91)%] was significantly higher than that in the control group [(29.00±2.21)%]. The difference was statistically significant (t=4.91, P<0.01). Conclusion The neuroprotective effect of astragalus on mice with spinal cord injury.