Department of Emergency Medicine, Xi'an No. 3 Hospital, Xi'an, Shaanxi, China.
Eur Rev Med Pharmacol Sci. 2020 Feb;24(3):1524-1536. doi: 10.26355/eurrev_202002_20211.
To investigate the roles and underlying mechanisms of melatonin in oxygen-glucose deprivation/reoxygenation (OGD/R)-insulted SH SY5Y cells.
SH SY5Y cells were cultured for OGD/R stimulation. Cell viability and cytotoxicity were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, lactate dehydrogenase (LDH), and Hoechst 33258/propidium iodide (PI) staining assays. The mRNA levels of high mobility group box-1 (HMGB1), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) were analyzed by quantitative Real Time-PCR assays. Nitric oxide (NO) production was assessed by Griess reagent. Reactive oxygen species (ROS) production was detected by fluorescent probe. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were examined by commercial kits. Cell apoptosis was analyzed by flow cytometry and caspase-3 activity. The protein levels were detected by Western blot.
Melatonin enhanced the viability and reduced the death and LDH release of OGD/R exposed SH SY5Y cells. Melatonin repressed the HMGB1, TNF-α, and iNOS mRNA expression, NO production, and nuclear factor κB (NF-κB) activation in OGD/R challenged SH SY5Y cells. Melatonin reduced the ROS, MDA, 4-HNE, and 8-OHdG contents but further enhanced the levels of the nuclear factor E2-related factor-2 (Nrf2) and heme oxygenase (HO-1). Melatonin-increased viability and melatonin-decreased LDH release were also mediated by the blockage of NF-κB or reversed by Nrf2 or HO-1 knockdown. Melatonin exerted antiapoptotic effect on OGD/R treated SH SY5Y cells partly by activating Akt signaling. OGD/R challenged SH SY5Y cell autophagy was also repressed by melatonin, as evidenced by the decreased levels of LC-II and beclin-1 and the increased phosphorylation of mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP-1).
Melatonin protected SH SY5Y cells from OGD/R induced oxidative stress, inflammation, apoptosis, and autophagy by blocking NF-κB signaling and activating Nrf2/HO-1, Akt, and mTOR/p70S6K/4E-BP-1 pathways, thereby indicating that melatonin is a potential and novel therapeutic drug for ischemic stroke.
探讨褪黑素在氧葡萄糖剥夺/复氧（OGD/R）损伤的 SH SY5Y 细胞中的作用及其机制。
培养 SH SY5Y 细胞进行 OGD/R 刺激。通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐（MTT）、乳酸脱氢酶（LDH）和 Hoechst 33258/碘化丙啶（PI）染色检测细胞活力和细胞毒性。通过实时定量 PCR 检测高迁移率族蛋白 B1（HMGB1）、肿瘤坏死因子 α（TNF-α）和诱导型一氧化氮合酶（iNOS）的 mRNA 水平。通过格里希试剂评估一氧化氮（NO）的产生。通过荧光探针检测活性氧（ROS）的产生。通过商业试剂盒检测丙二醛（MDA）、4-羟基壬烯醛（4-HNE）和 8-羟基-2'-脱氧鸟苷（8-OHdG）。通过流式细胞术和 caspase-3 活性分析细胞凋亡。通过 Western blot 检测蛋白水平。
褪黑素增强了 OGD/R 暴露的 SH SY5Y 细胞的活力，并减少了细胞死亡和 LDH 的释放。褪黑素抑制了 OGD/R 刺激的 SH SY5Y 细胞中 HMGB1、TNF-α 和 iNOS 的 mRNA 表达、NO 的产生和核因子 κB（NF-κB）的激活。褪黑素降低了 ROS、MDA、4-HNE 和 8-OHdG 的含量，但进一步增加了核因子 E2 相关因子 2（Nrf2）和血红素加氧酶（HO-1）的水平。褪黑素增加的活力和降低的 LDH 释放也通过 NF-κB 的阻断或通过 Nrf2 或 HO-1 的敲低来介导。褪黑素对 OGD/R 处理的 SH SY5Y 细胞发挥抗凋亡作用，部分是通过激活 Akt 信号通路。褪黑素还抑制了 OGD/R 刺激的 SH SY5Y 细胞的自噬，这表现在 LC-II 和 beclin-1 的水平降低，以及哺乳动物雷帕霉素靶蛋白（mTOR）、p70 核糖体蛋白 S6 激酶（p70S6K）和真核起始因子 4E 结合蛋白 1（4E-BP-1）的磷酸化水平增加。
褪黑素通过阻断 NF-κB 信号通路和激活 Nrf2/HO-1、Akt 和 mTOR/p70S6K/4E-BP-1 通路，保护 SH SY5Y 细胞免受 OGD/R 诱导的氧化应激、炎症、凋亡和自噬，从而表明褪黑素是缺血性中风的一种潜在的新型治疗药物。
