调控线粒体钾通道拮抗淀粉样β肽25-35的毒性效应

doi:10.3969/j.issn.1673-5374.2013.01.007

摘要/Abstract

摘要：

实验采用0-20µM的淀粉样β肽25-35干预24h诱导PC12细胞毒性，发现5-20µM的淀粉样β肽25-35均可明显降低PC12细胞活力，而钾离子通道开放剂二氮嗪能抑制淀粉样β肽25-35诱导的PC12细胞活力下降。同时二氮嗪能保护PC12细胞抵抗淀粉样β肽25-35诱导的线粒体膜电位升高及细胞内活性氧的增加，此效应能被钾通道阻断剂5-羟基葵酸盐所逆转。诱导性一氧化氮合成酶抑制剂Nω-nitro-L-arginine亦能保护PC12细胞抵抗淀粉样β肽25-35诱导的线粒体膜电位升高及细胞内活性氧的增加。但是，过氧化氢酶不能逆转淀粉样β肽25-35诱导的细胞内活性氧的增加。同时淀粉样β肽25-35作用于PC12细胞24h未导致细胞凋亡和坏死，表明淀粉样β肽25-35诱导的线粒体膜电位升高及细胞内活性氧增加早于细胞死亡事件的发生。说明淀粉样β肽25-35的细胞毒性作用与线粒体ATP敏感钾通道及一氧化氮有关，调控线粒体钾通道可在早期拮抗淀粉样β肽25-35诱导的PC12细胞神经毒性。

关键词: 神经再生, 神经退行性疾病, 淀粉样β肽25-35, PC12细胞, 钾离子通道开放剂, 诱导性一氧化氮合成酶抑制剂, 线粒体膜电位, 活性氧, 神经保护, 基金资助文章, 图片文章

Abstract:

In this study, we treated PC12 cells with 0–20 µM amyloid-β peptide (25–35) for 24 hours to induce cytotoxicity, and found that 5–20 µM amyloid-β peptide (25–35) decreased PC12 cell viability, but adenosine triphosphate-sensitive potassium channel activator diazoxide suppressed the decrease in PC12 cell viability induced by amyloid-β peptide (25–35). Diazoxide protected PC12 cells against amyloid-β peptide (25–35)-induced increases in mitochondrial membrane potential and intracellular reactive oxygen species levels. These protective effects were reversed by the selective mitochondrial adenosine triphosphate-sensitive potassium channel blocker 5-hydroxydecanoate. An inducible nitric oxide synthase inhibitor, Nω-nitro-L-arginine, also protected PC12 cells from amyloid-β peptide (25–35)-induced increases in both mitochondrial membrane potential and intracellular reactive oxygen species levels. However, the H₂O₂-degrading enzyme catalase could not reverse the amyloid-β peptide (25–35)-induced increase in intracellular reactive oxygen species. A 24-hour exposure to amyloid-β peptide (25–35) did not result in apoptosis or necrosis, suggesting that the increases in both mitochondrial membrane potential and reactive oxygen species levels preceded cell death. The data suggest that amyloid-β peptide (25–35) cytotoxicity is associated with adenosine triphosphate-sensitive potassium channels and nitric oxide. Regulation of adenosine triphosphate-sensitive potassium channels suppresses PC12 cell cytotoxicity induced by amyloid-β peptide (25–35).

Key words: neural regeneration, neurodegenerative diseases, amyloid-&beta, peptide (25–35), PC12 cell, adenosine triphosphate-sensitive potassium channel, inducible nitric oxide synthase, mitochondrial membrane potential, reactive oxygen species, grant-supported paper, photographs-containing paper, neuroregeneration

. 调控线粒体钾通道拮抗淀粉样β肽25-35的毒性效应[J]. 中国神经再生研究(英文版), 2013, 8(1): 56-63.

Min Kong, Maowen Ba, Hui Liang, Peng Shao, Tianxia Yu, Ying Wang. Regulation of adenosine triphosphate-sensitive potassium channels suppresses the toxic effects of amyloid-beta peptide (25–35)[J]. Neural Regeneration Research, 2013, 8(1): 56-63.

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引言

材料方法

Design

A comparative in vitro cell culture experiment.

Time and setting

The experiments were performed at a laboratory in the Shanghai Jiao Tong University School of Medicine from December 2010 to June 2011.

Materials

Aβ_25-35 and Aβ_35-25 purchased from Sigma (St. Louis, MO, USA) were dissolved in sterile distilled water at 500 μM and incubated at 37°C for 4 days.

Methods

PC12 cell culture and differentiation

Rat PC12 pheochromocytoma cells (Chinese Academy of Sciences, Shanghai, China) were cultured at 37°C in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 5% horse serum (Gibco), 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Sigma), 100 IU/mL penicillin (Sigma), and 100 μg/mL streptomycin (Sigma) in a humidified atmosphere containing 5% CO₂ in air. Neuronal-like differentiation of PC12 cells was induced by 7-day treatment with neural growth factor (100 ng/mL) (Gibco) (Figure 5).

Grouping and intervention

Aggregated Aβ_25-35 (0.1, 1, 5, 10, 20 µM) or Aβ_35-25 (0.1, 1, 5, 10, 20 µM) were applied to the differentiated cells (at a density of 6 × 10⁴/cm²) for 24 hours. Diazoxide (Sigma) was prepared as a stock solution of 50 mg/mL in dimethyl sulfoxide (final concentration 0.08%) and added to the media for a final concentration of 1 mM. Cells were preincubated for 1 hour with 1 mM diazoxide and then co-treated with Aβ_25-35 and diazoxide for 24 hours. The Nω-nitro-L-arginine (Roche, Basel, Switzerland) was dissolved in 0.2 mM PBS (pH 7.4) and added to the media for a final concentration of 500 µM. Cells were co-incubated with Nω-nitro-L-arginine and Aβ_25-35 for 24 hours.

Cellular viability as detected by MTT

The viability of cultured cells exposed to various drugs for 24 hours was evaluated using the MTT colorimetric assay^[30]. A total of 10 μL MTT reagent (Sigma) was added to plated cells for 2 hours until a purple precipitate was visible. A total of 100 μL detergent reagent was added at room temperature in the dark for 2 hours. MTT reduction was quantified at 570 nm using a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The ratio of MTT reduction in samples treated with various drugs to the MTT reduction in control samples was used to determine cellular viability.

Determination of the mitochondrial membrane potential using JC-1 fluorescence

Mitochondrial membrane potential was quantified using the ratiometric probe JC-1^[18]. In apoptotic cells, in which the mitochondrial membrane potential had collapsed, monomeric JC-1 remained in the cytosol and appeared green. Following exposure to 5 μM Aβ_25-35 or Aβ_35-25 in the presence or absence of 1 mM diazoxide or 1 mM diazoxide plus 500 μM 5-hydroxydecanoate (Sigma) or 500 μM Nω-nitro-L- arginine for 24 hours, cells were incubated in culture medium-free serum containing 10 µg/mL JC-1 (solubilized in N, N-dimethylformamide, 1% v/v; Molecular Probes Europe, Leiden, Netherlands) for 30 minutes at 37°C under 5% CO₂, and then washed with 0.1 M PBS twice. Laser confocal microscopy (Leica, Wetzlar, Germany) semi-quantitative analysis of JC-1 fluorescence was used to determine the average intensity of green (527 nm) fluorescent monomers and J-aggregates with orange-red (590 nm) fluorescence. The ratio of fluorescence at 590 nm/fluorescence at 527 nm was used to monitor changes in mitochondrial membrane potential.

Measurement of reactive oxygen species by DCFH-DA fluorescence

The production of reactive oxygen species was estimated by flow cytometry using the oxidation-sensitive fluorescent probe DCFH-DA^[31], a cell-permeable dye that, once inside the cell, is cleaved by intracellular esterase into its non-fluorescent form DCFH. This form, which is no longer membrane permeable, may be further oxidized by H₂O₂ or OH^–to its fluorescent form, DCF. Following exposure to 5 μM Aβ_25-35 or Aβ_35-25 in the presence or absence of 1 mM diazoxide or 1 mM diazoxide plus 500 μM 5-hydroxydecanoate or 500 μM Nω-nitro-L-arginine or 100 mg/mL catalase (Sigma) or 1 µM H₂O₂ (Sigma) for 24 hours, the cultured cells were incubated in serum-free culture medium containing 10 µM DCFH-DA (Molecular Probes Europe) for 30 minutes at 37°C and were then washed with 0.2 M PBS once. Cells were then trypsinized and washed, followed by flow cytometer detection using a FACS Calibur cytometry instrument (BD Biosciences, San Jose, CA, USA). Fluorescence emission from DCF (green) was detected at a wavelength of 527 nm.

Detection of apoptosis and necrosis using flow cytometry

After exposure to 5 μM Aβ_25-35 or Aβ_35-25 for 24 hours, free-floating cells pooled with cells detached by mild trypsinization were incubated in 100 mL annexin-V- FLUOS solution (Roche) containing 2 mL Annexin-V reagent (Roche) and 2 mL propidium iodide reagent (Roche) for 10–15 minutes at 20°C and then cooled so that the reaction was terminated. FITC-fluorescence was measured with a FACS Calibur flow cytometer at an excitation wavelength of 488 nm.

Statistical analysis

All data are expressed as mean ± SEM. Statistical analyses between controls and samples treated with various drugs were done by SPSS 10.0 software (SPSS, Chicago, IL, USA) using one-way analysis of variance, followed by two-tailed Student’s t-test or multiple comparison test where appropriate. A value of P < 0.05 was considered statistically significant for all analyses.

讨论

文章快阅

延伸阅读

1 中文内容及构思介绍：
阿尔茨海默病是一种进行性发展的致死性的神经退行性疾病(旧称老年前期痴呆)，脑内(大脑皮质及深部灰质) 老年斑是其特征性病理改变。老年斑的主要成分是淀粉样β肽，目前认为，阿尔茨海默病的发病与β-淀粉样肽在脑实质中沉积密切相关。淀粉样β肽在水解酶作用下可生成长短不同的片段, 其中由25到35位氨基酸残基所组成的多肽片段，是一个富含疏水性氨基酸，体外水溶液中可逐步形成稳定的聚集，呈β片层状二级结构，这种聚集与淀粉样β肽的神经毒性直接相关，可导致突触变性及细胞凋亡，因此常用于作体外神经毒实验的阿尔茨海默病模型。此外，有大量证据表明线粒体功能障碍在AD发病机制中的作用，淀粉样β肽可导致线粒体功能障碍、自由基产生，进而损伤ATP酶活性导致线粒体膜去极化。

二氮嗪，一种线粒体ATP敏感钾通道开放剂，是一种有希望的神经保护剂。它能够激化钾通道，保护神经元抵抗神经毒素，如谷氨酸、淀粉样β肽及过氧化氢的损伤。然而，到目前为止，其确切的神经保护机制尚未明了。

因此，实验用淀粉样β肽25-35处理体外培养的PC12细胞, 首先观察了对线粒体膜电位与活性氧系列的影响，进而利用线粒体ATP敏感钾通道开放剂进行干预，观察能否拮抗淀粉样β肽25-35毒性效应。同时，对与线粒体ATP敏感钾通道密切相关的诱导性一氧化氮合成酶作用进行了探讨。

2 国内外同类研究水平的介绍：
淀粉样β肽与阿尔茨海默病等神经退行性疾病发生相关，能导致神经细胞死亡。淀粉样β肽导致神经细胞死亡的病理环节尚不完全清楚，其中线粒体功能障碍与氧化应激是非常重要的环节。深入阐述线粒体功能障碍与氧化应激发生的机制，并寻找指标靶点，是目前国内外研究的热点问题。

3 与其他同类研究的比较：
文章从与线粒体膜电位相关的ATP敏感钾通道入手，阐述淀粉样β肽致线粒体功能障碍的机制，并对与ATP敏感钾通道密切相关的诱导性一氧化氮合成酶作用进行了研究，对其潜在的治疗价值进行了探讨，有别于其他同类研究。

4 专家意见及答疑：
专家意见：文章阐述一个具体科研目的即可，没有必要罗列两个大方面的内容。如可以单独阐述二氮嗪或者Nω-硝基-L-精氨酸对淀粉样β肽25-35作用下的P12细胞的保护作用.
作者答疑：淀粉样β肽25-35毒性作用与其诱导的线粒体膜电位升高及细胞内活性氧系列的增加有关。通过运用二氮嗪（一种线粒体ATP敏感钾通道开放剂）、Nω-硝基-L-精氨酸（诱导性一氧化氮合成酶抑制剂）进行干预，不仅仅为了说明两种药物的保护作用，而且更加深入的阐述淀粉样β肽25-35诱导线粒体膜电位升高及细胞内活性氧系列增加的机制，即与线粒体ATP敏感钾通道及一氧化氮有关。因此，对二者进行阐述较单独阐述更能揭示深层次的内容。