Caspase抑制剂对体外氧糖剥夺损伤大鼠海马神经元的保护作用

Protective effects of Caspase inhibitor on in vitro hippocampal neurons of rats with oxygen glucose deprivation injury

目的 探讨Caspase抑制剂对体外氧糖剥夺损伤大鼠海马神经元的保护作用及机制. 方法 新生SD乳鼠海马神经元经体外原代培养7d后分为对照组、氧糖剥夺/复氧组、Caspase抑制剂苄氧羰基-缬氨酰-丙氨酰-门冬氨酰氟甲基酮（Z-VAD-FMK）组和二甲基亚砜（DMSO）组,其中氧糖剥夺/复氧组氧糖剥夺6h后复糖复氧,Z-VAD-FMK组在氧糖剥夺复氧过程中加入20 μmol/L Z-VAD-FMK,DMSO组在氧糖剥夺复氧过程中加入1‰药物溶媒DMSO.复氧复糖24 h后光镜及电镜下观察各组海马神经元的形态学变化,MTT比色法测定细胞存活率和培养液中乳酸脱氢酶（LDH）含量,Annexin V-FITC/PI双染后流式细胞仪检测神经元凋亡率,比色法测定神经元Caspase-3活性,Western blotting法分析Caspase-3活性蛋白表达. 结果 氧糖剥夺/复氧组细胞胞体肿胀,部分胞膜破裂,细胞完整性破坏,细胞核水肿,线粒体肿胀,嵴排列混乱;Z-VAD-FMK组细胞损伤较氧糖剥夺/复氧组减轻,大多数神经元形态完整,细胞内颗粒增多,细胞器肿胀减轻,线粒体嵴结构存在,分布较规则.与氧糖剥夺/复氧组比较,Z-VAD-FMK组海马神经元吸光度值（0.204±0.019 vs 0.303±0.018）及细胞存活率（0.481％±0.045％vs 0.704％±0.040％）明显增高,LDH含量[（406.500±21.286） U/L vs（326.000±20.278） U/L]明显降低,细胞凋亡率（49.700％±3.100％vs 29.820％±2.839％）明显降低,反应Caspase-3活性片段的吸光度值明显降低,Caspase-3活性蛋白表达（1.070±0.077 vs 0.785±0.058）明显下降,差异均有统计学意义（P＜0.05）. 结论 Caspase抑制剂可通过抑制Caspase-3蛋白活性,减轻海马神经元的体外氧糖剥夺损伤.

Objective To explore the protective effects of Caspase inhibitor on in vitro hippocampal neurons of rats with oxygen glucose deprivation （OGD） injury.Methods The primary hippocampal neurons from newborn Sprague-Dawley rats were cultured for 7 days,and then,divided into control group,OGD/re-OG treatment group,Caspase inhibitor （Z-VAD-FMK） treatment group and DMSO treatment group; and OGD/re-OG was performed in the later three groups for OGD duration of 6 h.Through the experimental process,20 μmol/L Z-VAD-FMK was given to the Z-VAD-FMK treatment group,while 1‰ DMSO was added into culture medium in the DMSO group.The morphology of the neurons was observed 24 hours after re-OG under light microscopy and electron microscopy.MTT assay was used to determine the rate of survived cells and lactate dehydrogenase （LDH） content in culture medium.The neurons apoptosis rate was measured by using flow cytometry （Annexin V-FITC/PI）.Caspase-3 activity of neurons was analyzed by colorimetry.The protein expression of Caspase-3 wasassessed by Western blotting.Results Cells in the OGD/re-OG treatment group showed swelled body,some having ruptured membrane,cell integrity being damaged,cell nucleus edema,mitochondrial swelling and cristae ordered chaos; cells in the Z-VAD-FMK treatment group showed decreased injury as compared with the OGD/re-OG treatment group,having cell integrity; as compared with the OGD/re-OG treatment group,Z-VAD-FMK treatment group had significantly higher absorbance value （0.204±0.019 vs.0.303±0.018） and cell survivalrate （0.481%±0.045%vs.0.704%±0.040%）,obviously decreased lactate dehydrogenase （LDH） content （[406.500±21.286] U/L vs.[326.000±20.278] U/L）,apoptosis rate （49.700%±3.100% vs.29.820%±2.839%）,absorbance value of the Caspase-3 active area and Caspase-3 protein expression （1.070±0.077 vs.0.785±0.058）,with statistically differences （P〈0.05）.Conclusions Caspase inhibitor may protect rat hippocampal neurons from oxygen glucose deprivation injury by inhibiting Caspase-3 activity.