橙皮素纳米晶体可改善早期阿尔茨海默病细胞模型中的线粒体功能

Hesperetin nanocrystals improve mitochondrial function in a cell model of early Alzheimer disease

Keywords：Alzheimer disease, ROS, Amyloid beta, Hesperetin, Mitochondria, Mitochondria dysfunction, Nanoparticles, Peroxidase activity

关键词：阿尔茨海默病、活性氧、β淀粉样蛋白、橙皮素、线粒体、线粒体功能障碍、纳米颗粒、过氧化物酶活性

细胞系：神经母细胞瘤细胞系（转化的神经嵴衍生细胞）
作者：Babylon Lukas, Grewal Rekha, Stahr Pascal-L, Eckert Ralph W, Keck Cornelia M, Eckert Gunter P

出版期刊：《Antioxidants》（2021/6/23）

Abstract：

Mitochondrial dysfunction represents a hallmark of both brain aging and age-related neurodegenerative disorders including Alzheimer disease (AD). AD-related mitochondrial dysfunction is characterized by an impaired electron transport chain (ETC), subsequent decreased adenosine triphoshpate (ATP) levels, and elevated generation of reactive oxygen species (ROS). The bioactive citrus flavanone hesperetin (Hst) is known to modulate inflammatory response, to function as an antioxidant, and to provide neuroprotective properties. The efficacy in improving mitochondrial dysfunction of Hst nanocrystals (HstN) with increased bioavailability has not yet been investigated. Human SH-SY5Y cells harboring neuronal amyloid precursor protein (APP695) acted as a model for the initial phase of AD. MOCK-transfected cells served as controls. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels of mitochondrial respiratory chain complex genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid β-protein (Aβ1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, peroxidase activity, and cytochrome c activity were determined using a fluorescence assay. Compared to pure Hst dissolved in ethanol (HstP), SH-SY5Y-APP695 cells incubated with HstN resulted in significantly reduced mitochondrial dysfunction: ATP levels and respiratory chain complex activity significantly increased. Gene expression levels of RCC I, IV, and V were significantly upregulated. In comparison, the effects of HstN on SY5Y-MOCK control cells were relatively small. Pure Hst dissolved in ethanol (HstP) had almost no effect on both cell lines. Neither HstN nor HstP led to significant changes in Aβ1-40 levels. HstN and HstP were both shown to lower peroxidase activity significantly. Furthermore, HstN significantly reduced cytochrome c activity, whereas HstP had a significant effect on reducing ROS in SH-SY5Y-APP695 cells. Thus, it seems that the mechanisms involved may not be linked to altered Aβ production. Nanoflavonoids such as HstN have the potential to prevent mitochondria against dysfunction. Compared to its pure form, HstN showed a greater effect in combatting mitochondrial dysfunction. Further studies should evaluate whether HstN protects against age-related mitochondrial dysfunction and thus may contribute to late-onset AD. 

文章摘要：

线粒体功能障碍是大脑衰老和与年龄相关的神经退行性疾病（包括阿尔茨海默病 (AD)）的标志。 AD 相关的线粒体功能障碍的特点是电子传递链 (ETC) 受损，随后三磷酸腺苷 (ATP) 水平降低，以及活性氧 (ROS) 生成增加。已知具有生物活性的柑橘黄烷酮橙皮素 (Hst) 可调节炎症反应、作为抗氧化剂发挥作用并提供神经保护特性。尚未研究通过增加生物利用度来改善 Hst 纳米晶体 (HstN) 线粒体功能障碍的功效。含有神经元淀粉样前体蛋白 (APP695) 的人 SH-SY5Y 细胞可作为 AD 初始阶段的模型。 MOCK 转染的细胞用作对照。使用荧光素酶催化的生物发光测定法测定高能代谢物 ATP。使用克拉克电极通过高分辨率呼​​吸测定法评估线粒体呼吸链复合物的活性。使用定量实时聚合酶链反应（qRT-PCR）测定线粒体呼吸链复合物基因的表达水平。使用均相时间分辨荧光 (HTRF) 测量淀粉样蛋白 β-蛋白 (Aβ1-40) 的水平。使用荧光测定法测定 ROS 水平、过氧化物酶活性和细胞色素 c 活性。与溶解在乙醇中的纯 Hst (HstP) 相比，与 HstN 一起孵育的 SH-SY5Y-APP695 细胞导致线粒体功能障碍显着降低：ATP 水平和呼吸链复合物活性显着增加。 RCC I、IV 和 V 的基因表达水平显着上调。相比之下，HstN 对 SY5Y-MOCK 对照细胞的影响相对较小。溶解在乙醇中的纯 Hst (HstP) 对两种细胞系几乎没有影响。 HstN 和 HstP 均未导致 Aβ1-40 水平发生显着变化。 HstN 和 HstP 均显示显着降低过氧化物酶活性。此外，HstN 显着降低细胞色素 c 活性，而 HstP 对降低 SH-SY5Y-APP695 细胞中的 ROS 有显着影响。因此，似乎所涉及的机制可能与改变的 Aβ 产生无关。 HstN 等纳米类黄酮具有防止线粒体功能障碍的潜力。与其纯形式相比，HstN 在对抗线粒体功能障碍方面表现出更大的作用。进一步的研究应该评估 HstN 是否可以防止与年龄相关的线粒体功能障碍，从而可能导致迟发性 AD。

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