Jia Dong-Pei, Wang Song, Zhang Bao-Chao, Fang Fang
Department of Neurological Rehabilitation, Nanyang City Center Hospital, Nanyang, Henan 473003, P.R. China.
Exp Ther Med. 2015 Aug;10(2):804-808. doi: 10.3892/etm.2015.2531. Epub 2015 May 29.
In previous years, increasing evidence has indicated that paraptosis and mitochondrial-mediated apoptosis may be associated with Alzheimer's disease (AD). However, the association between paraptosis and mitochondrial-mediated apoptosis, and the pathological processes underlying AD, remain elusive. In the present study, the β-amyloid precursor protein gene, and the gene mutations PS1M146L and L286V, were transfected to an SH-SY5Y cell line to establish an AD cell model. Subsequently, an MTT assay was used to examine the cell viability of the AD cell model, while a TUNEL assay was employed to observe the number of positively stained apoptotic cells. Cytoplasmic vacuolization was examined using light microscopy and images were photographed. Furthermore, western blot analysis was utilized to detect the expression of golden biomarkers of the mitochondrial pathway, including Bcl-2 and Bax. The paraptosis inhibitor, cycloheximide, was selected to treat the AD model cells in order to observe the association between paraptosis and mitochondrial-mediated apoptosis. The results indicated that the decrease in the cell viability of the AD cells was initiated at 24 h, as compared with the normal cells (P<0.05). TUNEL-positive stained cells were observed at 48 h, which was later compared with the cell death initiation. In addition, examination of cytoplasmic vacuolization using microscopy indicated that there were a small number of paraptosis cells present at 24 h. The expression levels of Bcl-2 was significantly decreased, while Bax was significantly increased at 48 h. Furthermore, cycloheximide treatment was demonstrated to significantly increase Bcl-2 expression, while decreasing Bax expression (P>0.05). In conclusion, the occurrence of paraptosis was demonstrated in the early pathological stages of AD, which may subsequently damage the mitochondria and trigger mitochondrial pathway-mediated apoptosis. Thus, paraptosis may trigger programmed cell death directly, or indirectly through the regulation of Bcl-2 and Bax protein expression.
在过去几年中,越来越多的证据表明,副凋亡和线粒体介导的凋亡可能与阿尔茨海默病(AD)有关。然而,副凋亡与线粒体介导的凋亡之间的关联以及AD潜在的病理过程仍不清楚。在本研究中,将β-淀粉样前体蛋白基因以及基因突变PS1M146L和L286V转染至SH-SY5Y细胞系以建立AD细胞模型。随后,采用MTT法检测AD细胞模型的细胞活力,同时采用TUNEL法观察凋亡阳性染色细胞的数量。利用光学显微镜检查细胞质空泡化情况并拍照。此外,采用蛋白质印迹分析检测线粒体途径的关键生物标志物(包括Bcl-2和Bax)的表达。选择副凋亡抑制剂环己酰亚胺处理AD模型细胞,以观察副凋亡与线粒体介导的凋亡之间的关联。结果表明,与正常细胞相比,AD细胞的细胞活力在24小时开始下降(P<0.05)。在48小时观察到TUNEL阳性染色细胞,随后将其与细胞死亡起始时间进行比较。此外,通过显微镜检查细胞质空泡化表明,在24小时存在少量副凋亡细胞。Bcl-2的表达水平在48小时显著降低,而Bax在48小时显著升高。此外,环己酰亚胺处理显示可显著增加Bcl-2表达,同时降低Bax表达(P>0.05)。总之,在AD的早期病理阶段证实了副凋亡的发生,这可能随后损害线粒体并触发线粒体途径介导的凋亡。因此,副凋亡可能直接或通过调节Bcl-2和Bax蛋白表达间接触发程序性细胞死亡。