线粒体Omi/HtrA2信号通路在颅脑损伤后神经细胞凋亡中的作用

doi:10.3877/cma.j.issn.1674-6880.2018.02.007

目的

探讨大鼠颅脑损伤后线粒体Omi/HtrA2信号通路在大鼠神经细胞凋亡中的作用。

方法

90只大鼠按随机数字表法分成假手术组、颅脑损伤组和UCF-101干预组，每组30只，然后每组按照12、24、48 h三个时间点分成三个亚组，每个亚组10只。使用大鼠自由落体颅脑损伤动物模型，UCF-101干预组大鼠在模型形成前0.5 h按1.5 μmol/kg的剂量腹腔注射UCF-101，其余两组大鼠腹腔注射1 mL的等渗NaCl溶液。按照12、24、48 h三个时间点每组分批断头处死10只大鼠，分离海马组织。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）测定法检测各组大鼠海马组织神经细胞凋亡情况，Western-blotting检测Omi/HtrA2、X染色体连锁凋亡抑制蛋白（XIAP）、pro-caspase-3、pro-caspase-9和剪切聚腺苷二磷酸-核糖多聚酶（PARP）蛋白表达，四肽荧光底物法检测caspase-3和caspase-9蛋白活性。

结果

三组大鼠海马神经细胞凋亡比例，Omi/HtrA2、pro-caspase-3、pro-caspase-9、剪切PARP和XIAP蛋白表达以及caspase-3和caspase-9蛋白活性比较，差异均有统计学意义（F=217.742、107.139、145.748、133.970、103.029、65.112、142.772、96.187，P均< 0.05）。12、24、48 h时颅脑损伤组大鼠凋亡的海马神经细胞比例，Omi/HtrA2、pro-caspase-3、pro-caspase-9和剪切PARP蛋白表达以及caspase-3和caspase-9蛋白活性均较假手术组显著升高（P均< 0.05）；而UCF-101干预组大鼠凋亡的海马神经细胞比例，Omi/HtrA2、pro-caspase-3、pro-caspase-9和剪切PARP蛋白表达以及caspase-3和caspase-9蛋白活性均较颅脑损伤组显著下降（P均< 0.05）。各时间点颅脑损伤组大鼠海马组织XIAP蛋白表达均较假手术组显著下降（P均< 0.05），而UCF-101干预组大鼠海马组织XIAP蛋白表达均较颅脑损伤组显著升高（P均< 0.05）。

结论

Omi/HtrA2介导的线粒体途径可能参与大鼠颅脑损伤后神经细胞凋亡的发生过程，而UCF-101可有效抑制神经细胞凋亡。

关键词: 大鼠, 颅脑损伤, 神经细胞凋亡, Omi/HtrA2, UCF-101

Objective

To investigate the role of the mitochondrial Omi/HtrA2 signaling pathway in neuronal apoptosis after craniocerebral injury in rats.

Methods

A total of 90 rats were randomly divided into the sham operation group, craniocerebral injury group and UCF-101 intervention group, 30 rats in each group; at 12, 24 and 48 h, each group was further divided into three subgroups at random, 10 rats in each subgroup. The animal model of free-fall brain injury in rats was used. Rats in the UCF-101 intervention group were intraperitoneally injected with UCF-101 at a dose of 1.5 μmol/kg 0.5 h before the formation of the model, and rats in the other two groups were intraperitoneally injected with 1 mL sodium chloride solution. At 12 h, 24 h and 48 h, 10 rats in each group were sacrificed to isolate the hippocampal tissues. Terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL) assay was used to detect neuronal apoptosis in hippocampal tissues of rats in each group, the Western-blotting method was used to detect the expressions of Omi/HtrA2, X-linked inhibitor of apoptosis protein (XIAP), pro-caspase-3, pro-caspase-9 and shearing poly-ADP-ribose polymerase (PARP), and the tetrapeptide fluorescence substrate method was used to detect the activity of caspase-3 and caspase-9.

Results

The ratio of apoptotic hippocampal neurons, the expressions of Omi/HtrA2, pro-caspase-3, pro-caspase-9, shearing PARP and XIAP, and the activity of caspase-3 and caspase-9 were all statistically significantly different in the three groups (F=217.742, 107.139, 145.748, 133.970, 103.029, 65.112, 142.772, 96.187; all P < 0.05). At 12 h, 24 h and 48 h, the ratio of apoptotic hippocampal neurons, the expressions of Omi/HtrA2, pro-caspase-3, pro-caspase-9 and shearing PARP, and the activity of caspase-3 and caspase-9 were significantly higher in the craniocerebral injury group than in the sham operation group (all P < 0.05), while they were significantly lower in the UCF-101 intervention group than in the craniocerebral injury group (all P < 0.05). The expressions of XIAP in hippocampi in the craniocerebral injury group at different time points were significantly lower as compared to the sham operation group (all P < 0.05), while the expressions of XIAP in the UCF-101 intervention group were significantly higher as compared to the craniocerebral injury group (all P < 0.05).

Conclusion

The mitochondrial pathway mediated by Omi/HtrA2 may be involved in the process of neuronal apoptosis after craniocerebral injury in rats, and UCF-101 can effectively inhibit the apoptosis of nerve cells.

Key words: Rat, Craniocerebral injury, Neuronal apoptosis, Omi/HtraA2, UCF-101

表1 UCF-101对三组大鼠海马神经细胞凋亡比例的影响（%，±s）

组别	例数	12 h	24 h	48 h
假手术组	30	10.1 ± 0.6^a	9.7 ± 0.6^a	10.5 ± 0.8^a
颅脑损伤组	30	32.2 ± 1.5	50.0 ± 2.0	45.4 ± 2.5
UCF-101干预组	30	25.7 ± 1.1^a	39.0 ± 1.4^a	33.5 ± 1.5^a

表1 UCF-101对三组大鼠海马神经细胞凋亡比例的影响（%，±s）

组别	例数	12 h	24 h	48 h
假手术组	30	10.1 ± 0.6^a	9.7 ± 0.6^a	10.5 ± 0.8^a
颅脑损伤组	30	32.2 ± 1.5	50.0 ± 2.0	45.4 ± 2.5
UCF-101干预组	30	25.7 ± 1.1^a	39.0 ± 1.4^a	33.5 ± 1.5^a

表2 UCF-101对三组大鼠海马组织Omi/HtrA2、pro-caspase-3、pro-caspase-9、剪切PARP和XIAP蛋白相对表达值的影响（±s）

组别	例数	Omi/HtrA2			pro-caspase-3			pro-caspase-9
组别	例数	12 h	24 h	48 h	12 h	24 h	48 h	12 h	24 h	48 h
假手术组	30	100.0 ± 1.6^a	101.1 ± 1.5^a	101.5 ± 1.8^a	100 ± 6^a	100 ± 5^a	109 ± 5^a	100 ± 5^a	105 ± 4^a	107 ± 5^a
颅脑损伤组	30	165.6 ± 8.0	218.8 ± 8.8	204.0 ± 8.1	177 ± 4	239 ± 10	203 ± 8	182 ± 10	267 ± 13	204 ± 9
UCF-101干预组	30	129.9 ± 4.7^a	170.3 ± 6.8^a	151.7 ± 8.4^a	157 ± 5^a	193 ± 6^a	175 ± 6^a	155 ± 6^a	209 ± 9^a	176 ± 10^a
组别	例数	剪切PARP			XIAP
组别	例数	12 h	24 h	48 h	12 h	24 h	48 h
假手术组	30	100 ± 7^a	101 ± 6^a	100 ± 7^a	100 ± 4^a	101 ± 4^a	102 ± 3^a
颅脑损伤组	30	278 ± 23	401 ± 32	354 ± 38	65 ± 5	43 ± 7	58 ± 5
UCF-101干预组	30	163 ± 7^a	218 ± 12^a	176 ± 12^a	74 ± 3^a	58 ± 6^a	70 ± 4^a

表2 UCF-101对三组大鼠海马组织Omi/HtrA2、pro-caspase-3、pro-caspase-9、剪切PARP和XIAP蛋白相对表达值的影响（±s）

组别	例数	Omi/HtrA2			pro-caspase-3			pro-caspase-9
组别	例数	12 h	24 h	48 h	12 h	24 h	48 h	12 h	24 h	48 h
假手术组	30	100.0 ± 1.6^a	101.1 ± 1.5^a	101.5 ± 1.8^a	100 ± 6^a	100 ± 5^a	109 ± 5^a	100 ± 5^a	105 ± 4^a	107 ± 5^a
颅脑损伤组	30	165.6 ± 8.0	218.8 ± 8.8	204.0 ± 8.1	177 ± 4	239 ± 10	203 ± 8	182 ± 10	267 ± 13	204 ± 9
UCF-101干预组	30	129.9 ± 4.7^a	170.3 ± 6.8^a	151.7 ± 8.4^a	157 ± 5^a	193 ± 6^a	175 ± 6^a	155 ± 6^a	209 ± 9^a	176 ± 10^a
组别	例数	剪切PARP			XIAP
组别	例数	12 h	24 h	48 h	12 h	24 h	48 h
假手术组	30	100 ± 7^a	101 ± 6^a	100 ± 7^a	100 ± 4^a	101 ± 4^a	102 ± 3^a
颅脑损伤组	30	278 ± 23	401 ± 32	354 ± 38	65 ± 5	43 ± 7	58 ± 5
UCF-101干预组	30	163 ± 7^a	218 ± 12^a	176 ± 12^a	74 ± 3^a	58 ± 6^a	70 ± 4^a

表3 UCF-101对大鼠海马组织caspase-3和caspase-9蛋白活性相对值的影响（±s）

组别	例数	caspase-3			caspase-9
组别	例数	12 h	24 h	48 h	12 h	24 h	48 h
假手术组	30	100 ± 6^a	101 ± 4^a	108 ± 5^a	100 ± 8^a	97 ± 7^a	99 ± 8^a
颅脑损伤组	30	179 ± 8	311 ± 19	215 ± 12	309 ± 26	594 ± 48	446 ± 48
UCF-101干预组	30	149 ± 4^a	228 ± 20^a	173 ± 6^a	210 ± 20^a	403 ± 33^a	289 ± 341^a

表3 UCF-101对大鼠海马组织caspase-3和caspase-9蛋白活性相对值的影响（±s）

组别	例数	caspase-3			caspase-9
组别	例数	12 h	24 h	48 h	12 h	24 h	48 h
假手术组	30	100 ± 6^a	101 ± 4^a	108 ± 5^a	100 ± 8^a	97 ± 7^a	99 ± 8^a
颅脑损伤组	30	179 ± 8	311 ± 19	215 ± 12	309 ± 26	594 ± 48	446 ± 48
UCF-101干预组	30	149 ± 4^a	228 ± 20^a	173 ± 6^a	210 ± 20^a	403 ± 33^a	289 ± 341^a

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Role of the mitochondrial Omi/HtrA2 signaling pathway in neuronal apoptosis after craniocerebral injury

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Role of the mitochondrial Omi/HtrA2 signaling pathway in neuronal apoptosis after craniocerebral injury