姜黄素对脊髓损伤大鼠神经保护作用及其机制

Neuroprotective effect of Curcumin on spinal cord injury in rats and its molecular mechanism

目的探讨姜黄素对脊髓损伤大鼠神经保护作用及其机制。方法30只SD大鼠采用随机数字表法分为假手术组、模型组和姜黄素组。假手术组大鼠仅切开椎板,不做其他处理。模型组大鼠采用改良Allen’s捶击法建立脊髓损伤模型,姜黄素组大鼠在建立脊髓损伤模型后给予50 mg/kg的姜黄素。治疗4周后采用Basso Beanie Bresnahan(BBB)量表进行运动功能评分,斜板试验最大角度来评估脊髓功能恢复情况,苏木精-伊红(HE)染色观察脊髓组织病理形态变化,酶联免疫吸附试验(ELISA)法检测损伤段脊髓组织肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)水平,生化酶学检测大鼠脊髓组织谷胱甘肽过氧化物酶(GSH-px)、总超氧化物歧化酶(SOD)和丙二醛(MDA)水平;蛋白质免疫检测脊髓组织磷酸化p65和半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)蛋白表达水平。组间比较采用单因素方差分析,组间两两比较采用LSD法。结果姜黄素组大鼠斜板实验最大角度[(47.23±4.51)°]明显高于模型组大鼠[(31.35±4.56)°],差异有统计学意义(t=7.833,P<0.05)。姜黄素组大鼠BBB评分[(11.48±2.22)分]明显高于模型组大鼠[(5.88±0.86)分],差异有统计学意义(t=7.430,P<0.05)。模型组大鼠脊髓组织结构被损伤,细胞排列凌乱,灰质和白质界限模糊,且伴随大量胶质细胞的增生;姜黄素组大鼠脊髓结构明显好转,脊髓坏死范围明显降低,灰质和白质界限好转,且可见少量神经元。姜黄素组大鼠外周血GSH-px和SOD活性[(97.74±5.54)、(89.42±5.43)U/ml]明显高于模型组大鼠[(73.41±6.79)、(60.69±8.91)U/ml],差异有统计学意义(t=8.733、8.711,P<0.05)。姜黄素组大鼠外周血MDA水平[(5.43±0.49)U/ml]明显高于模型组大鼠[(7.14±0.60)nmol/L],差异有统计学意义(t=6.940,P<0.05)。姜黄素组外周血TNF-α、IL-6和IL-1β水平[(107.70±10.88)、(69.11±5.76)、(122.01±15.99)pg/ml]明显低于模型组大鼠[(169.95±12.32)、(100.08±10.17)、(223.15±12.97)pg/ml],差异有统计学意义(t=11.980、8.378、15.530,P<0.05)。姜黄素组大鼠脊髓组织磷酸化p65和剪接Caspase-3蛋白水平(0.91±0.07、0.66±0.06)明显低于模型组大鼠(1.44±0.15、1.27±0.14),差异有统计学意义(t=10.180、12.580,P<0.05)。结论姜黄素可显著下调脊髓损伤大鼠氧化应激和炎症水平,改善脊髓损伤大鼠的神经功能,主要通过抑制核因子-κB和Caspase-3实现。

Objective To investigate the neuroprotective effect of Curcumin on spinal cord injury in rats and its molecular mechanism.Methods Totally,30 SD rats were randomly divided into Sham surgery group,model group and Curcumin group.In the Sham surgery group,only the vertebral lamina was cut without any other treatment.The spinal cord injury model of rats in the model group was established by the modified Allen’s hammering method,and the rats in the Curcumin group were given 50 mg/kg Curcumin after the establishment of the spinal cord injury model.After 4 weeks of treatment,the motor function score was analyzed by the Basso Beanie Bresnahan(BBB)scale.The recovery of spinal cord function was evaluated by the maximum angle of inclined plate test.The pathological changes of spinal cord tissue were analyzed by hematoxylin and eosin(HE)staining.The tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and IL-1βin the injured spinal cord tissues were analyzed by enzyme-linked immunosorbent assay(ELISA).The levels of glutathione peroxidase(GSH-px),total superoxide dismutase(SOD)and malondialdehyde(MDA)in spinal cord tissues of rats were measured by biochemical enzymology.The expression levels of phosphorylated p65 and cysteinyl aspartate-specific protease-3(Caspase-3)proteins in the spinal cord tissue were detected by Western blotting.Single factor analysis of variance was used for inter-group comparison,and LSD method was used for inter-group pairwise comparison.Results The maximum angle of the inclined plate experiment in the Curcumin group[(47.23±4.51)°]was significantly greater than that in the model group[(31.35±4.56)°,t=7.833,P<0.05].The BBB score of rats in the Curcumin group(11.48±2.22)was significantly higher than that in the model group(5.88±0.86,t=7.430,P<0.05).The spinal cord tissue structure of the model group was damaged,with disordered cell arrangement,blurred boundaries between gray and white matter,and accompanied by proliferation of a large number of glial cells.In the Curcumin group,the structure of spinal cord was significantly improved,the extent of spinal cord necrosis was significantly reduced,the boundaries of gray matter and white matter were improved,and a small number of neurons were visible.The activities of GSH-px and SOD[(97.74±5.54),(89.42±5.43)U/ml]in peripheral blood of rats in the Curcumin group were significantly higher than those in the model group[(73.41±6.79),(60.69±8.91)U/ml,t=8.733,8.711,P<0.05].The level of MDA in peripheral blood of rats in the Curcumin group[(5.43±0.49)U/ml]was significantly higher than that in the model group[(7.14±0.60)nmol/L,t=6.940,P<0.05].The levels of TNF-α,IL-6 and IL-1βin peripheral blood of Curcumin group[(107.70±10.88),(69.11±5.76),(122.01±15.99)pg/ml]were significantly lower than those in the model group[(169.95±12.32),(100.08±10.17),(223.15±12.97)pg/ml,t=11.980,8.378,15.530,P<0.05].The phosphorylated p65 and spliced Caspase-3 protein levels in spinal cord tissue of rats in the Curcumin group(0.91±0.07,0.66±0.06)were significantly lower than those in the model group(1.44±0.15,1.27±0.14,t=10.180,12.580,P<0.05).Conclusion Curcumin can significantly reduce the levels of oxidative stress and inflammation in rats with spinal cord injury,and improve the neurological function of rats with spinal cord injury,mainly by inhibiting nuclear factor-κB and Caspase-3.