ATG12对缺氧缺血性脑病小鼠神经细胞凋亡和自噬的影响

doi:10.3877/cma.j.issn.2095-1221.2019.03.005

目的

探讨自噬相关蛋白12 （ATG12）对缺氧缺血性脑病（HIE）小鼠细胞凋亡和自噬的影响及分子机制。

方法

通过尾静脉注射腺相关病毒构建ATG12低表达小鼠模型，将40只小鼠分为假手术组、HIE模型组、对照病毒模型（NC-HIE）组和ATG低表达病毒模型（ATG12 shRNA-HIE）组，HIE模型组小鼠左侧颈动脉结扎后低氧（8﹪氧气+92﹪氮气）处理2.5?h，假手术组不予结扎和低氧处理。缺氧处理后，荧光定量PCR检测脑组织ATG12 mRNA表达水平。比色法检测各组小鼠大脑神经细胞SOD和MDA水平；通过Tunel法检测各组小鼠大脑神经细胞凋亡水平；通过Western Blot检测各组小鼠大脑神经细胞LC3A/B、ATG12和SQSTM1/?p62蛋白表达水平。采用t检验和单因素方差分析对实验数据进行统计分析。

结果

与假手术组小鼠脑组织ATG12 mRNA水平（1.00±0.14）相比，HIE模型组小鼠脑组织ATG12 mRNA水平（5.23±0.37）显著升高（t?= 33.60，P?< 0.01）；与假手术组小鼠脑组织超氧化物歧化酶（SOD）活性[（103.60±4.84）?U/?mgprot]和丙二醛（MDA）含量[（42.40±3.17）?μmol/?mgprot]比较，HIE模型组小鼠脑组织SOD活性[（62.60±3.44）?U/?mgprot]显著降低，MDA含量[（83.80±4.39）?μmol/?mgprot]显著升高，与NC-HIE组小鼠脑组织SOD活性[（61.20±4.39）?U/mgprot]和MDA含量[（85.20± 2.70）?μmol/?mgprot]比较，ATG12 shRNA-?HIE组小鼠脑组织SOD活性[（93.80± 5.43）?U/?mgprot]显著升高，MDA含量[（49.20±3.49）?μmol/mgprot]显著降低，差异具有统计学意义（F?= 222.7，P?< 0.01；F?=?415.8，P?<?0.01）。Tunel结果显示，与假手术组比较，HIE模型组小鼠脑组织凋亡程度升高，与NC-HIE组比较，ATG12 shRNA-HIE组小鼠脑组织凋亡程度降低。Western Blot结果显示，与假手术组小鼠脑组织ATG12、LC3A/B和SQSTM1/?p62蛋白（0.14±0.03，0.13±0.02，0.53±0.03）比较，HIE模型组小鼠脑组织ATG12和LC3A/?B蛋白表达（0.49±0.04，0.45±0.03）显著升高，SQSTM1/p62蛋白表达（0.24±0.03）显著降低，与NC-?HIE组小鼠脑组织ATG12、LC3A/B和SQSTM1/p62蛋白（0.46±0.03，0.45±0.03，0.25±0.03）比较，ATG12 shRNA-HIE组小鼠脑组织ATG12和LC3A/B蛋白表达（0.13±0.02，0.16±0.03）显著降低，SQSTM1/p62蛋白表达（0.53±0.04）显著升高，差异具有统计学意义（F?= 432.9，P?< 0.01；F?= 437.5，P?< 0.01；F?= 301.9，P?< 0.01）。

结论

降低ATG12表达能够抑制小鼠大脑神经细胞氧化应激，缓解HIE小鼠大脑神经细胞凋亡和自噬水平，抑制脑损伤。

关键词: ATG12, 缺氧缺血脑损伤, 氧化应激, 细胞自噬

Objective

To investigate the effect of autophagy-related protein 12 (ATG12) on apoptosis and autophagy in mice with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism.

Methods

The ATG12 low expression mouse model was constructed by injecting adeno-?associated virus (AAV) into the tail vein. Forty mice were divided into a sham operation group, HIE model group, negative control virus HIE model (NC-HIE) group and ATG low expression virus HIE model (ATG12 shRNA-HIE) group. The mice in the HIE model group were treated with hypoxia (8﹪ oxygen + 92﹪ nitrogen) for 2.5?h after ligation of the left carotid artery. The mice in the sham operation group were not treated with ligation and hypoxia. After hypoxia and hypoxia treatment, the expression level of ATG12 mRNA in the brain tissues was detected by real-time PCR. The levels of SOD and MDA in the brain tissues of each group were detected. The apoptosis level of the brain tissues was detected by Tunel method. The expressions of LC3A/B, ATG12 and SQSTM1/?p62 protein in the brain tissues of each group were detected by Western Blot. Statistical analysis of experimental data was performed by t-test and one-way ANOVA.

Results

Compared with the sham operation group (1.00±0.14) , the ATG12 mRNA level (5.23±0.37) in the brain of the HIE model group was significantly increased (t?= 33.60, P < 0.01) . Compared with the sham operation group[ (103.60±4.84) ?U/mgprot, (42.40±3.17) ?μmol/?mgprot], the SOD activity[ (62.60±3.44) ?U/mgprot] in the brain tissues of the HIE model group was significantly decreased, and the MDA content[ (83.80±4.39) ?μmol/mgprot]was significantly increased. Compared with the NC-HIE group[ (61.20±4.39) ?U/mgprot, (85.20±2.70) ?μmol/mgprot], the SOD activity[ (93.80±5.43) ?U/mgprot] in the brain tissues of the ATG12 shRNA-HIE group was significantly increased, and the MDA content[ (49.20±3.49) ?μmol/mgprot] was significantly decreased, and the differences were statistically significant (F?= 222.7, P?< 0.01; F?= 415.8, P < 0.01) . The results of Tunel showed that compared with the sham operation group, the level of brain tissues apoptosis in the HIE model group was significantly higher. Compared with the NC-HIE group, the brain tissues apoptosis level of the ATG12 shRNA-HIE group was significantly lower. Western Blot results showed that compared with the sham operation group (0.14±0.03, 0.13±0.02, 0.53±0.03) , the expression of ATG12 and LC3A/B protein (0.49±0.04, 0.45±0.03) in the brain of the HIE model group was significantly increased, and the expression of SQSTM1/p62 protein (0.24±0.03) was significantly decreased. Compared with NC-HIE group (0.46±0.03, 0.45±0.03, 0.25±0.03) , the expression of ATG12 and LC3A/B protein (0.13±0.02, 0.16±0.03) in the rat brain tissues was significantly decreased, and the expression of SQSTM1/p62 protein (0.53±0.04) was significantly increased, the difference was statistically significant (F?= 432.9, P?< 0.01; F?= 437.5, P?< 0.01; F?= 301.9, P?< 0.01) .

Conclusion

Inhibition of ATG12 expression can inhibit oxidative stress in mice, alleviate brain tissue apoptosis and autophagy levels in HIE mice, and inhibit brain damage.

Key words: ATG12, Hypoxic-ischemia, Oxidative stress, Autophagy

表1 荧光定量PCR引物序列

分组	Seq（5'→3'）
ATG12	F: TCTTAGACTGCTGTGCCTGC
?	R: AACTTGTTTGACGCCTTCGC
GAPDH	F: AGAAGGCTGGGGCTCATTTG
?	R: AGGGGCCATCCACAGTCTTC

表1 荧光定量PCR引物序列

分组	Seq（5'→3'）
ATG12	F: TCTTAGACTGCTGTGCCTGC
?	R: AACTTGTTTGACGCCTTCGC
GAPDH	F: AGAAGGCTGGGGCTCATTTG
?	R: AGGGGCCATCCACAGTCTTC

表2 QRT-PCR法检测不同组别小鼠脑组织中ATG12 mRNA相对表达量（±s）

分组	动物数（只）	ATG12 mRNA表达（2^-△△t）
假手术组	10	1.00±0.14
HIE组	10	5.23±0.37
t值	?	33.60
P值	?	< 0.01

表2 QRT-PCR法检测不同组别小鼠脑组织中ATG12 mRNA相对表达量（±s）

分组	动物数（只）	ATG12 mRNA表达（2^-△△t）
假手术组	10	1.00±0.14
HIE组	10	5.23±0.37
t值	?	33.60
P值	?	< 0.01

图1 光学显微镜观察检测ATG12在HIE小鼠中高表达（免疫组化染色，×200）

表3 分光光度法检测ATG12对不同处理组小鼠脑组织SOD和MDA含量的影响（±s）

分组	动物数（只）	SOD活力（U/mgprot）	MDA含量（μmol/ mgprot）
假手术组	10	103.60±4.84	42.40±3.17
HIE组	10	62.60±3.44^a	83.80±4.39^a
NC-HIE组	10	61.20±4.39	85.20±2.70
ATG12 shRNA HIE组	10	93.80±5.43^b	49.20±3.49^b
F值	?	222.716	415.836
P值	?	< 0.01	< 0.01

表3 分光光度法检测ATG12对不同处理组小鼠脑组织SOD和MDA含量的影响（±s）

分组	动物数（只）	SOD活力（U/mgprot）	MDA含量（μmol/ mgprot）
假手术组	10	103.60±4.84	42.40±3.17
HIE组	10	62.60±3.44^a	83.80±4.39^a
NC-HIE组	10	61.20±4.39	85.20±2.70
ATG12 shRNA HIE组	10	93.80±5.43^b	49.20±3.49^b
F值	?	222.716	415.836
P值	?	< 0.01	< 0.01

图2 激光共聚焦显微镜下观察ATG12对HIE小鼠脑组织凋亡的影响（Tunel染色，×200）

表4 Western Blot检测不同处理组小鼠脑组织蛋白相对表达量（±s）

分组	动物数（只）	ATG12（/GAPDH）	LC3A/B（/GAPDH）	SQSTM1/P62（/GAPDH）
假手术组	10	0.14±0.03	0.13±0.02	0.53±0.03
HIE组	10	0.49±0.04^a	0.45±0.03^a	0.24±0.03^a
NC-HIE组	10	0.46±0.03	0.45±0.03	0.25±0.03
ATG12 shRNA HIE组	10	0.13±0.02^b	0.16±0.03^b	0.53±0.04^b
F值	?	432.926	437.533	301.918
P值	?	< 0.01	< 0.01	< 0.01

表4 Western Blot检测不同处理组小鼠脑组织蛋白相对表达量（±s）

分组	动物数（只）	ATG12（/GAPDH）	LC3A/B（/GAPDH）	SQSTM1/P62（/GAPDH）
假手术组	10	0.14±0.03	0.13±0.02	0.53±0.03
HIE组	10	0.49±0.04^a	0.45±0.03^a	0.24±0.03^a
NC-HIE组	10	0.46±0.03	0.45±0.03	0.25±0.03
ATG12 shRNA HIE组	10	0.13±0.02^b	0.16±0.03^b	0.53±0.04^b
F值	?	432.926	437.533	301.918
P值	?	< 0.01	< 0.01	< 0.01

图3 ATG12对HIE小鼠脑组织自噬的影响

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Effect of ATG12 gene on apoptosis and autophagy in mice with hypoxic-ischemic encephalopathy

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Effect of ATG12 gene on apoptosis and autophagy in mice with hypoxic-ischemic encephalopathy