Chay Kee-Oh, Nam Koong Kyoung Young, Hwang Shinae, Kim Jong-Keun, Bae Choon Sang
Department of Biochemistry, Chonnam National University Medical School, Gwangju, Korea.
Department of Anatomy, Chonnam National University Medical School, Gwangju, Korea.
Chonnam Med J. 2017 Sep;53(3):196-202. doi: 10.4068/cmj.2017.53.3.196. Epub 2017 Sep 25.
β-Amyloid peptide (Aβ) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aβ-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aβ-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aβ, a fragment of Aβ with an equivalent neurotoxic effect. Aβ induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aβ increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aβ increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aβ. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aβ-induced neuronal death.
β-淀粉样肽（Aβ）是阿尔茨海默病患者脑内老年斑的主要成分，已知是该疾病的主要致病因素。最近的证据表明，小胶质细胞或星形胶质细胞中NADPH氧化酶（NOX）的激活可能是Aβ诱导的活性氧（ROS）的来源。我们研究了神经元NOX在小鼠混合皮质培养物中Aβ诱导的神经元死亡中的作用。在暴露于具有等效神经毒性作用的Aβ片段后24或48小时，通过测量乳酸脱氢酶向培养液中的外流量来评估细胞死亡。Aβ以浓度和时间依赖性方式诱导具有凋亡特征的神经元死亡。神经元死亡不仅被抗凋亡药物如z-VAD-fmk和环己酰亚胺显著减弱，还被抗氧化剂如曲洛克斯、抗坏血酸和表没食子儿茶素没食子酸酯显著减弱。我们还证明，用20μM Aβ处理可增加混合皮质培养物中的荧光信号，但在存在细胞内ROS指示剂2',7'-二氯荧光素二乙酸酯（DCF-DA）的情况下，在纯星形胶质细胞培养物中仅产生微弱信号。DCF-DA荧光的增加不仅被曲洛克斯显著抑制，还被选择性NOX抑制剂如夹竹桃麻素和AEBSF显著抑制。蛋白质印迹分析显示，Aβ增加了细胞中NOX的主要亚基gp91phox的表达。上述抗氧化剂、夹竹桃麻素和AEBSF显著减弱了Aβ诱导的神经元死亡。此外，基于gp91phox特异性siRNA的NOX敲低显著抑制了神经元死亡。这些结果表明，神经元NOX的激活参与了Aβ诱导的神经元死亡。
