School of Medical Sciences, Faculty of Medicine and Health, International Medical University, Kuala Lumpur 57000, Malaysia.
Department of Human Biology, Faculty of Medicine and Health, International Medical University, Kuala Lumpur 57000, Malaysia.
Mol Med Rep. 2014 Mar;9(3):947-54. doi: 10.3892/mmr.2013.1878. Epub 2013 Dec 24.
Neurodegenerative diseases remain a global issue which affects the ageing population. Efforts towards determining their aetiologies to understand their pathogenic mechanisms are underway in order to identify a pathway through which therapeutic measures can be applied. One such pathogenic mechanism, oxidative stress (OS), is widely considered to be involved in neurodegenerative disease. Antioxidants, most notably flavonoids, have promising potential for therapeutic use as shown in in vitro and in vivo studies. In view of the importance of flavonoids for combating OS, this study investigated the neuroprotective effects of orientin, which has been reported to be capable of crossing the blood‑brain barrier. The maximum non‑toxic dose (MNTD) of orientin against SH‑SY5Y neuroblastoma cells was determined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assay. The effects of the MNTD and the half MNTD (½MNTD) of orientin on cell cycle progression and intracellular reactive oxygen species (ROS) levels, as well as the activity of caspases 3/7, 8 and 9 after exposure to 150 µM of hydrogen peroxide (H2O2) were also determined using flow cytometry, a 2',7'‑dichlorodihydrofluorescein‑diacetate (DCFH‑DA) assay and caspase assay kits, respectively. The results revealed that orientin at ≤20 µM was not cytotoxic to SH‑SY5Y cells. After treatment with orientin at the MNTD, the percentage of apoptotic cells was significantly reduced compared with that in cells treated with 150 µM H2O2 alone. The results also showed that, although orientin at the MNTD and ½MNTD did not reduce intracellular ROS levels, it significantly inhibited the activity of caspases 3/7. Caspase 9 was significantly inactivated with orientin at the MNTD. Findings from this study suggest that the neuroprotection conferred by orientin was the result of the intracellular mediation of caspase activity.
神经退行性疾病仍然是一个全球性问题，影响着老年人口。为了了解其发病机制，人们正在努力确定其病因，以确定可以应用治疗措施的途径。氧化应激（OS）是一种发病机制，被广泛认为与神经退行性疾病有关。抗氧化剂，尤其是类黄酮，在体外和体内研究中显示出有希望的治疗应用潜力。鉴于类黄酮在对抗 OS 方面的重要性，本研究调查了Orientin 的神经保护作用，已有报道称 Orientin 能够穿过血脑屏障。使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐(MTT)测定法确定 Orientin 对 SH-SY5Y 神经母细胞瘤细胞的最大无毒剂量(MNTD)。还使用流式细胞术、2',7'-二氯二氢荧光素-二乙酸酯(DCFH-DA)测定法和 caspase 测定试剂盒分别测定 MNTD 和 Orientin 的半 MNTD(½MNTD)对细胞周期进程和细胞内活性氧物种(ROS)水平的影响，以及在暴露于 150µM 过氧化氢(H2O2)后 caspase 3/7、8 和 9 的活性。结果表明，Orientin 的浓度≤20µM 对 SH-SY5Y 细胞没有细胞毒性。在用 MNTD 处理 Orientin 后，与单独用 150µM H2O2 处理的细胞相比，凋亡细胞的百分比显著降低。结果还表明，尽管 MNTD 和½MNTD 的 Orientin 没有降低细胞内 ROS 水平，但它显著抑制了 caspase 3/7 的活性。MNTD 的 Orientin 使 caspase 9 显著失活。本研究的结果表明，Orientin 赋予的神经保护作用是细胞内 caspase 活性介导的结果。
