联合应用PARP-1与Caspase-3抑制剂对脊髓损伤大鼠神经细胞凋亡的影响

Effect of combined PARP-1 and Caspase-3 inhibitors on neurocyte apoptosis after spinal cord injury in rats

中文关键词:  脊髓损伤  PARP-1抑制剂  Caspase-3抑制剂  凋亡诱导因子  大鼠

英文关键词:Spinal cord injury  PARP-1 inhibitor  Caspase-3 inhibitor  Apoptosis-inducing factor  Rat

中文摘要:

  【摘要】 目的：探讨联合应用多聚腺嘌呤二核苷酸核糖聚合酶-1（PARP-1）抑制剂3-氨基苯甲酰胺（3-aminobenzamide，3-AB）和Caspase-3抑制剂Z-DEVD-FMK对脊髓损伤大鼠神经细胞凋亡的影响。方法：120只成年健康SD大鼠随机分为假手术组（A组）、模型组（B组）、PARP-1抑制剂组（C组）和联合用药组（D组），每组30只。以Allen′s打击法制备大鼠脊髓损伤模型，每组分别于造模后1d、3d、7d取5只大鼠行BBB评分，处死后利用免疫组化方法检测损伤部位脊髓内PARP-1、凋亡诱导因子（AIF）、Caspase-3及Bcl-2的表达；各时间点各组剩余大鼠处死后利用Western blotting检测PARP-1、Caspase-3蛋白表达水平，实时荧光定量PCR检测PARP-1、AIF、Caspase-3及Bcl-2的mRNA水平，采用原位末端标记（TUNEL）法检测神经细胞凋亡情况。结果：造模后1d时B、C、D组大鼠BBB评分均为0分；3d时三组间的评分无统计学差异；7d时D组及C组明显高于B组，且D组最高（P＜0.05）。免疫组化及Western blotting结果显示，脊髓损伤后1~7d，B组脊髓组织中PARP-1、AIF及Caspase-3表达逐渐增强，Bcl-2表达逐渐减弱（P＜0.05）；与B组比较，D组及C组的PARP-1、AIF、Caspase-3表达均显著降低，且D组最低（P＜0.05）；而D组及C组的Bcl-2表达显著高于B组，且D组最高（P＜0.05）。实时荧光定量PCR检测各目的基因表达水平与其蛋白水平一致。TUNEL结果显示，B组脊髓损伤后3d凋亡细胞最多，7d时数量减少，但仍保持在较高水平（P＜0.05）；D组及C组凋亡细胞指数均显著低于B组，且D组最低（P＜0.05）。结论：联合应用PARP-1抑制剂3-AB和Caspase-3抑制剂Z-DEVD-FMK可有效抑制大鼠脊髓损伤后神经细胞凋亡，其机制可能与PARP-1、AIF、Caspase-3的表达抑制及Bcl-2的表达上调有关。

英文摘要:

  【Abstract】 Objectives: To discuss the influence of combined PARP-1 inhibitor 3-aminobenzamide(3-AB) and Caspase-3 inhibitor Z-DEVD-FMK on the spinal neurocyte apoptosis after spinal cord injury in rats. Methods: 120 healthy adult SD rats were randomly divided into sham operation group(group A), model group(group B), PARP-1 inhibitor group(group C) and the combination of 3-AB and Z-DEVD-FMK group(group D) with 30 rats in each group. Then the rats of each group were divided into 1, 3, 7 days. Spinal cord injury models were established by Allen′s method. The BBB locomotor testing score was used to evaluate the rats′ locomotor function of hindlimbs in four groups after operation. The immunostaining was performed to detect the expressions of PARP-1, apoptosis-inducing factor(AIF), Caspase-3 and Bcl-2 in all groups. Western blotting was used to measure the expressions of PARP-1 and Caspase-3 protein after spinal cord injury, and real-time quantitative PCR was further used to detect the expressions of PARP-1, AIF, Caspase-3 and Bcl-2 mRNA in all groups following spinal cord injury. The level of cell apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay. Results: The BBB scores of rats in group D and C were significantly higher than that in group B at 7 days after SCI. Furthermore, the score in group D was higher than that in group C(P＜0.05). Immunohistochemistry results and Western blotting results showed that, from 1d to 7d after SCI, the expressions of PARP-1, AIF and Caspase-3 increased in group B, while the expressions of Bcl-2 gradually weakened(P＜0.05). Compared with group B, the expression of PARP-1, AIF and Caspase-3 in group D and C after SCI decreased significantly, and the expression in group D was lower than that in group C(P＜0.05). While compared with group B, the expression of Bcl-2 in group D and C obviously increased, and the expression in group D was higher than that in group C(P＜0.05). The results of real-time quantitative PCR corroborated the immunohistochemistry and Western blotting detection results. TUNEL result showed that, the apoptotic cells in group B peaked at 3d and decreased at 7d after SCI(P＜0.05). The apoptotic index of group D and C significantly decreased than that of group B, and the rate in group D was lower than that in group C(P＜0.05). Conclusions: Combined PARP-1 inhibitor 3-AB and Caspase-3 inhibitor Z-DEVD-FMK can reduce the apoptosis of cells after SCI, which may be related to the inhibition of the expression of PARP-1, AIF and Caspase-3, and the up-regulation of the expressions of Bcl-2.