Department of Otolaryngology, University of Miami, Miami, Florida, USA.
Department of Physiology and Biophysics, University of Miami, Miami, Florida, USA.
Ther Hypothermia Temp Manag. 2021 Jun;11(2):77-87. doi: 10.1089/ther.2019.0034. Epub 2020 Apr 17.
Mild therapeutic hypothermia is protective against several cellular stresses, but the mechanisms underlying this protection are not completely resolved. In the present study, we used an model to investigate whether therapeutic hypothermia at 33°C applied following a peroxide-induced oxidative stress would protect PC12 cells. A 1-hour exposure to tert-butyl peroxide increased cell death measured 24 hours later. This cell death was dose-dependent in the range of 100-1000 μM tert-butyl peroxide with ∼50% cell death observed at 24 hours from 500 μM peroxide exposure. Cell survival/death was measured with an alamarBlue viability assay, and propidium iodide/Hoechst imaging for counts of living and dead cells. Therapeutic hypothermia at 33°C applied for 2 hours postperoxide exposure significantly increased cell survival measured 24 hours postperoxide-induced stress. This protection was present even when delayed hypothermia, 15 minutes after the peroxide washout, was applied. Addition of any of the three FDA-approved antioxidants (Tempol, EUK134, Edaravone at 100 μM) in combination with hypothermia improved cell survival. With the therapeutic hypothermia treatment, a significant downregulation of caspases-3 and -8 and tumor necrosis factor-α was observed at 3 and 24 hours poststress. Consistent with this, a cell-permeable pan-caspase inhibitor Z-VAD-FMK applied in combination with hypothermia significantly increased cell survival. Overall, these results suggest that the antioxidants quenching of reactive oxygen species likely works with hypothermia to reduce mitochondrial damage and/or apoptotic mechanisms. Further studies are required to confirm and extend these results to other cell types, including neuronal cells, and other forms of oxidative stress as well as to optimize the critical parameters of hypothermia treatment such as target temperature and duration.
轻度治疗性低温对多种细胞应激具有保护作用，但这种保护的机制尚未完全阐明。在本研究中，我们使用过氧化物诱导的氧化应激后应用 33°C 的治疗性低温模型来研究其是否会保护 PC12 细胞。1 小时暴露于叔丁基过氧化物会增加 24 小时后测量的细胞死亡。这种细胞死亡在 100-1000 μM 叔丁基过氧化物范围内呈剂量依赖性，在 500 μM 过氧化物暴露 24 小时后观察到约 50%的细胞死亡。用 alamarBlue 活力测定法和碘化丙啶/ Hoechst 成像法测量细胞存活/死亡，以计算活细胞和死细胞的数量。过氧化物暴露后 2 小时应用 33°C 的治疗性低温显著增加了过氧化物诱导应激后 24 小时的细胞存活率。即使在过氧化物洗脱后 15 分钟延迟低温时，也存在这种保护作用。与低温联合使用任何三种已获 FDA 批准的抗氧化剂（Tempol、EUK134、Edaravone 为 100 μM）均可提高细胞存活率。在用治疗性低温处理时，应激后 3 小时和 24 小时观察到 caspase-3 和 -8 以及肿瘤坏死因子-α的显著下调。与此一致，与低温联合使用细胞通透性泛半胱天冬酶抑制剂 Z-VAD-FMK 显著增加了细胞存活率。总的来说，这些结果表明，抗氧化剂淬灭活性氧可能与低温一起减少线粒体损伤和/或凋亡机制。需要进一步的研究来确认并扩展这些结果到其他细胞类型，包括神经元细胞，以及其他形式的氧化应激，并优化低温治疗的关键参数，如目标温度和持续时间。
