转染ALDH2基因对海马神经元的保护作用及其机制
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摘要
研究背景：4-羟基壬烯醛(4-Hydroxynonenal,4-HNE)是一种重要的过氧化应激标志物,在阿尔茨海默氏病(Alzheimers Disease,AD)等神经退行性疾病中大量沉积。大量研究表明4-HNE的神经毒性作用包括抑制神经突起生长,影响神经微管蛋白功能,破坏神经微管；4-HNE还可改变蛋白质的信号转导机制而介导神经细胞凋亡；4-HNE也可通过损伤线粒体而引起氧化应激反应等。4-HNE在神经退行性疾病的发生发展过程中起重要作用,因此对抗4-HNE的神经毒性作用可能成为治疗神经退行性病变的一个靶点。线粒体乙醛脱氢酶(aldehyde dehydrogenase-2,ALDH2)是一个核编码线粒体酶,定位于线粒体基质。ALDH2在中枢神经系统中广泛表达,然而ALDH2在中枢神经系统中的功能以及中枢神经系统相关疾病中的作用目前仍不明确。须然ALDH2和4-HNE代谢之间的关系尚不清楚,但ALDH2在醛类代谢中起关键作用,由此本实验假设可以通过ALDH2基因转染抑制4-HNE的神经毒性,阻止神经元突起损伤和神经元凋亡而起到治疗神经退行性疾病的作用。目的：在体外培养原代海马神经元模型上探讨转染ALDH2基因对4-HNE诱导神经毒性的改善作用及其机制。方法：构建含有ALDH2 cDNA的pEGFP-N2载体,在细胞水平用电穿孔的方法在原代海马神经元中转染。分别进行免疫印迹法、免疫组织化学和免疫荧光染色法、实时定量逆转录聚合酶链反应法、激酶的活性测定、MTT比色法、免疫沉淀法、Axiovision软件分析法来检测过表达ALDH2对4-HNE诱导神经毒性的改善作用及其机制。结果：(1)在体外培养的原代海马神经元中,ALDH2基因转染有效地提高了ALDH2 mRNA和ALDH2融合蛋白的表达；(2)ALDH2基因转染有效地逆转了4-HNE诱导的神经突起损伤；
(3) ALDH2基因转染可使神经元存活率升高,有效地逆转了4-HNE诱导的神经细胞死亡；(4)ALDH2基因转染通过使casepase-3, Bax活性降低的机制,有效地逆转了4-HNE诱导的神经细胞凋亡；(5) ALDH2基因转染通过抑制线粒体膜电位下降和减少氧自由基水平有效地改善了4-HNE诱导的氧化应激反应的机制实现神经保护作用。结论：ALDH2基因转染不仅抑制4-HNE的神经毒性而且还可减少氧化应激损伤,从而有效逆转神经突起损伤和神经元凋亡。ALDH2可作为对抗4-HNE的神经毒性作用这一个靶点,达到预防和治疗神经退行性疾病的目的。
Background:4-Hydroxynonenal (4-HNE) is an important marker of oxidative stress in Alzheimer's disease (AD) and other neurodegenerative diseases, which can also accumulate in these diseases. Numerous studies show that 4-HNE neurotoxicity includes inhibition of neurite growth, damaging the nervous microtubules and neuronal tubulin; 4-HNE can also act directly on the receptor or the catalytic protein signaling pathway, change the protein signal transduction mechanisms which can lead to neuronal apoptosis; 4-HNE can also cause mitochondrial oxidative stress reactions.4-HNE may play an important role in the development process of neurodegenerative diseases, so treatments against the neurotoxicity of 4-HNE may be a therapeutic target for neurodegenerative diseases. Mitochondrial aldehyde dehydrogenase (aldehyde dehydrogenase-2, ALDH2) is a nuclear encoded mitochondrial enzyme, which is located in the mitochondrial matrix. ALDH2 is widely expressed in the central nervous system, but its functions in the central nervous system and roles in central nervous system-related diseases are still uncertain. We are not sure of the intrinsic link between 4-HNE and ALDH2, however ALDH2 plays a key role in the metabolism of aldehydes, so we assume that overexpression of ALDH2 could inhibit 4-HNE-induced neuronal injury and neuronal apoptosis processes, the purpose of treating neurodegenerative diseases achieved. Objective:we transfect ALDH2 gene in primary hippocampal neurons, and study the neuroprotective effects of ALDH2 and its mechanisms. Methods:we construct pEGFP-N2 vector containing ALDH2 cDNA, we use electroporation method to transfect ALDH2 cDNA into primary hippocampal neurons at the cellular level. We use Western blot, immunohistochemistry and immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, kinase activity assay, MTT assay, immunoprecipitation, and Axiovision software analysis method to detect over-expression of ALDH2 that can block 4HNE and reduce neurotoxicity. Results:(1) in cultured primary hippocampal neurons, ALDH2 gene transfection effectively improved ALDH2 ALDH2 mRNA expression and fusion protein expression; (2) ALDH2 gene transfection effectively reversed the 4-HNE-induced nerve neurite injury; (3) ALDH2 gene transfection can increase neuronal survival, effectively reversed the 4-HNE-induced neuronal cell death; (4) ALDH2 gene transfection can decrease casepase-3 and Bax activity, effectively reversing the 4-HNE-induced neuronal apoptosis; (5) In cultured primary hippocampal neurons, ALDH2 gene transfection effectively reverse the 4-HNE induced oxidative stress by inhibiting mitochondrial membrane potential decrease and reducing neuronal ROS level. Conclusions:ALDH2 gene transfection not only inhibits the neurotoxicity of 4-HNE but also to reduce oxidative stress, thus effectively reversing neurite injury and neuronal apoptosis, so ALDH2 can be used as a potential target for prevention and treatment of neurodegenerative diseases.
(3) ALDH2基因转染可使神经元存活率升高,有效地逆转了4-HNE诱导的神经细胞死亡；(4)ALDH2基因转染通过使casepase-3, Bax活性降低的机制,有效地逆转了4-HNE诱导的神经细胞凋亡；(5) ALDH2基因转染通过抑制线粒体膜电位下降和减少氧自由基水平有效地改善了4-HNE诱导的氧化应激反应的机制实现神经保护作用。结论：ALDH2基因转染不仅抑制4-HNE的神经毒性而且还可减少氧化应激损伤,从而有效逆转神经突起损伤和神经元凋亡。ALDH2可作为对抗4-HNE的神经毒性作用这一个靶点,达到预防和治疗神经退行性疾病的目的。
Background:4-Hydroxynonenal (4-HNE) is an important marker of oxidative stress in Alzheimer's disease (AD) and other neurodegenerative diseases, which can also accumulate in these diseases. Numerous studies show that 4-HNE neurotoxicity includes inhibition of neurite growth, damaging the nervous microtubules and neuronal tubulin; 4-HNE can also act directly on the receptor or the catalytic protein signaling pathway, change the protein signal transduction mechanisms which can lead to neuronal apoptosis; 4-HNE can also cause mitochondrial oxidative stress reactions.4-HNE may play an important role in the development process of neurodegenerative diseases, so treatments against the neurotoxicity of 4-HNE may be a therapeutic target for neurodegenerative diseases. Mitochondrial aldehyde dehydrogenase (aldehyde dehydrogenase-2, ALDH2) is a nuclear encoded mitochondrial enzyme, which is located in the mitochondrial matrix. ALDH2 is widely expressed in the central nervous system, but its functions in the central nervous system and roles in central nervous system-related diseases are still uncertain. We are not sure of the intrinsic link between 4-HNE and ALDH2, however ALDH2 plays a key role in the metabolism of aldehydes, so we assume that overexpression of ALDH2 could inhibit 4-HNE-induced neuronal injury and neuronal apoptosis processes, the purpose of treating neurodegenerative diseases achieved. Objective:we transfect ALDH2 gene in primary hippocampal neurons, and study the neuroprotective effects of ALDH2 and its mechanisms. Methods:we construct pEGFP-N2 vector containing ALDH2 cDNA, we use electroporation method to transfect ALDH2 cDNA into primary hippocampal neurons at the cellular level. We use Western blot, immunohistochemistry and immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, kinase activity assay, MTT assay, immunoprecipitation, and Axiovision software analysis method to detect over-expression of ALDH2 that can block 4HNE and reduce neurotoxicity. Results:(1) in cultured primary hippocampal neurons, ALDH2 gene transfection effectively improved ALDH2 ALDH2 mRNA expression and fusion protein expression; (2) ALDH2 gene transfection effectively reversed the 4-HNE-induced nerve neurite injury; (3) ALDH2 gene transfection can increase neuronal survival, effectively reversed the 4-HNE-induced neuronal cell death; (4) ALDH2 gene transfection can decrease casepase-3 and Bax activity, effectively reversing the 4-HNE-induced neuronal apoptosis; (5) In cultured primary hippocampal neurons, ALDH2 gene transfection effectively reverse the 4-HNE induced oxidative stress by inhibiting mitochondrial membrane potential decrease and reducing neuronal ROS level. Conclusions:ALDH2 gene transfection not only inhibits the neurotoxicity of 4-HNE but also to reduce oxidative stress, thus effectively reversing neurite injury and neuronal apoptosis, so ALDH2 can be used as a potential target for prevention and treatment of neurodegenerative diseases.
引文
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