Data_Sheet_1_Pharmacological Disruption of Phosphorylated Eukaryotic Initiation Factor-2α/Activating Transcription Factor 4/Indian Hedgehog Protects Intervertebral Disc Degeneration via Reducing the Reactive Oxygen Species and Apoptosis of Nucleus Pulposus Cells.docx

Excessive reactive oxygen species (ROS) and apoptosis in nucleus pulposus (NP) cells accelerate the process of intervertebral disc degeneration (IDD). Here, we integrated pathological samples and in vitro and in vivo framework to investigate the impact of phosphorylation of eukaryotic initiation factor-2α (eIF2α)/activating transcription factor 4 (ATF4)/Indian hedgehog (Ihh) signaling in the IDD. From the specimen analysis of the IDD patients, we found phosphorylated eIF2α (p-eIF2α), ATF4 and Ihh protein levels were positively related while the NP tissue went degenerative. In vitro, tumor necrosis factor (TNF)-α caused the NP cell degeneration and induced a cascade of upregulation of p-eIF2α, ATF4, and Ihh. Interestingly, ATF4 could enhance Ihh expression through binding its promoter region, and silencing of ATF4 decreased Ihh and protected the NP cells from degeneration. Moreover, ISRIB inhibited the p-eIF2α, which resulted in a suppression of ATF4/Ihh, and alleviated the TNF-α-induced ROS production and apoptosis of NP cells. On the contrary, further activating p-eIF2α aggravated the NP cell degeneration, with amplification of ATF4/Ihh and a higher level of ROS and apoptosis. Additionally, applying cyclopamine (CPE) to suppress Ihh was efficient to prevent NP cell apoptosis but did not decrease the ROS level. In an instability-induced IDD model in mice, ISRIB suppressed p-eIF2α/ATF4/Ihh and prevented IDD via protecting the anti-oxidative enzymes and decreased the NP cell apoptosis. CPE prevented NP cell apoptosis but did not affect anti-oxidative enzyme expression. Taken together, p-eIF2α/ATF4/Ihh signaling involves the ROS level and apoptosis in NP cells, the pharmacological disruption of which may provide promising methods in preventing IDD.

髓核（nucleus pulposus, NP）细胞内过量的活性氧（reactive oxygen species, ROS）积累与过度凋亡会加速椎间盘退变（intervertebral disc degeneration, IDD）的进程。本研究整合病理样本与体内外实验体系，探究真核翻译起始因子-2α（eukaryotic initiation factor-2α, eIF2α）/激活转录因子4（activating transcription factor 4, ATF4）/印度刺猬因子（Indian hedgehog, Ihh）信号通路的磷酸化修饰在IDD发生发展中的作用。对IDD患者的标本分析显示，随着NP组织退变程度加重，磷酸化eIF2α（p-eIF2α）、ATF4及Ihh的蛋白表达水平呈正相关。体外实验中，肿瘤坏死因子（tumor necrosis factor, TNF）-α可诱导NP细胞退变，并触发p-eIF2α、ATF4及Ihh的表达上调级联反应。值得注意的是，ATF4可通过结合Ihh的启动子区域增强其表达；而敲低ATF4则可下调Ihh的表达，从而保护NP细胞免于退变。此外，ISRIB可抑制p-eIF2α的活化，进而阻断ATF4/Ihh信号轴，同时减轻TNF-α诱导的NP细胞ROS生成与凋亡。反之，进一步激活p-eIF2α则会加重NP细胞退变，伴随ATF4/Ihh信号轴的激活增强，以及更高水平的ROS生成与细胞凋亡。另外，环巴胺（cyclopamine, CPE）可通过抑制Ihh有效阻断NP细胞凋亡，但无法降低ROS水平。在不稳定诱导的小鼠椎间盘退变模型中，ISRIB可抑制p-eIF2α/ATF4/Ihh信号通路，通过保护抗氧化酶系统、减少NP细胞凋亡来延缓IDD进展；而CPE仅能抑制NP细胞凋亡，对抗氧化酶的表达无影响。综上，p-eIF2α/ATF4/Ihh信号通路参与调控NP细胞的ROS水平与凋亡过程，针对该通路的药理学干预有望为IDD的防治提供新的潜在策略。