Department of Neurosurgery, Ankara City State Hospital, Ankara, Turkey.
Department of Neurosurgery, Polatlı Duatepe State Hospital, Ankara, Turkey.
Mol Neurobiol. 2022 Jun;59(6):3703-3720. doi: 10.1007/s12035-022-02814-6. Epub 2022 Apr 2.
The hypoxia (HPX) acts the brain injury and apoptosis via the Ca influx-induced excessive mitochondria free reactive oxygen species (mitROS) in neurons. The effective treatment of HPX is not possible yet. In addition to the antiviral and antiparkinsonian actions, amantadine (AMN) has been evaluated as a drug in treatments against brain injury. TRPM2 and TRPV4 channels are activated by mitROS. AMN attenuates NMDA receptor-induced Ca influx, mitROS, inflammation, and apoptosis in the brain. However, the molecular pathways underlying AMN's neuroprotection against HPX remain elusive. We investigated the protective role of AMN via attenuation of TRPM2 and TRPV4 on oxidative neurotoxicity, mitochondrial membrane potential (ΔΨm), inflammation, and apoptosis in neuronal cells (SH-SY5Y). The SH-SY5Y and HEK293 cells were divided into six groups as follows: control, AMN (750 µM for 48 h), HPX (200 µM CoCl for 24 h), HPX + AMN, HPX + TRPM2 blockers (25 µM ACA or 100 µM 2APB for 30 min), and HPX + TRPV4 blocker (ruthenium red (RuR)-1 µM for 30 min). The HPX caused to upregulation of Ca influx with an upregulation of ΔΨm and mitROS. The changes were not observed in the absence of TRPM2 and TRPV4 in the HEK293 cells. When HPX induction, TRPV4 agonist (GSK1016790A) and TRPM2 agonists (ADP-ribose and HO)-induced channel activity were diminished by the incubation of AMN and channel antagonists (RuR, ACA, and 2APB). The changes of mitROS, apoptotic markers (caspase-3 and -9), cell death rate, cell viability, cytokine (IL-1β, IL-6, and TNF-α), ΔΨm, and Zn concentrations were also restored by the incubation of AMN. In conclusion, the treatment of AMN attenuated HPX-mediated mitROS, apoptosis, and TRPM2/TRPV4-mediated overload Ca influx and may provide an avenue for protecting the HPX-mediated neurodegenerative and cerebrovascular diseases associated with the upregulation of mitROS, Ca, and Zn concentration.
缺氧 (HPX) 通过钙流入诱导神经元中过多的线粒体游离活性氧物种 (mitROS) 引起脑损伤和细胞凋亡。目前还没有有效的 HPX 治疗方法。除了抗病毒和抗帕金森作用外，金刚烷胺 (AMN) 已被评估为治疗脑损伤的药物。TRPM2 和 TRPV4 通道被 mitROS 激活。AMN 可减轻 NMDA 受体诱导的 Ca 流入、mitROS、炎症和细胞凋亡。然而，AMN 对 HPX 的神经保护的分子途径仍不清楚。我们通过抑制 TRPM2 和 TRPV4 来研究 AMN 的保护作用，以减轻神经元细胞 (SH-SY5Y) 中的氧化神经毒性、线粒体膜电位 (ΔΨm)、炎症和细胞凋亡。SH-SY5Y 和 HEK293 细胞分为六组：对照组、AMN (750 μM 48 h)、HPX (200 μM CoCl 24 h)、HPX+AMN、HPX+TRPM2 阻断剂 (25 μM ACA 或 100 μM 2APB 30 min) 和 HPX+TRPV4 阻断剂 (钌红 (RuR)-1 μM 30 min)。HPX 导致 Ca 流入增加，ΔΨm 和 mitROS 上调。在 HEK293 细胞中不存在 TRPM2 和 TRPV4 时，未观察到这些变化。当 HPX 诱导时，TRPV4 激动剂 (GSK1016790A) 和 TRPM2 激动剂 (ADP-核糖和 HO) 诱导的通道活性被 AMN 孵育和通道拮抗剂 (RuR、ACA 和 2APB) 减弱。在用 AMN 孵育后，mitROS、凋亡标志物 (caspase-3 和 -9)、细胞死亡率、细胞活力、细胞因子 (IL-1β、IL-6 和 TNF-α)、ΔΨm 和 Zn 浓度的变化也得到恢复。总之，AMN 治疗可减轻 HPX 介导的 mitROS、细胞凋亡以及 TRPM2/TRPV4 介导的超载 Ca 流入，并可能为保护与 mitROS、Ca 和 Zn 浓度上调相关的 HPX 介导的神经退行性和脑血管疾病提供途径。
