Zhou Simin, Zhong Zhifeng, Huang Pei, Xiang Bin, Li Xiaoxu, Dong Huaping, Zhang Gang, Wu Yu, Li Peng
Department of High Altitude Operational Medicine, College of High Altitude Military Medicine, Army Medical University, Chongqing, China.
Key Laboratory of Extreme Environmental Medicine, Ministry of Education of China, Army Medical University, Chongqing, China.
Front Physiol. 2021 Oct 21;12:729925. doi: 10.3389/fphys.2021.729925. eCollection 2021.
Neuron apoptosis, regulated by endoplasmic reticulum (ER) stress in the hippocampus, is an essential factor influencing the cognitive impairment induced by hypobaric hypoxia. Hypoxia mainly changes the activating transcription factor (ATF6) pathway of ER stress. However, the role of ATF6 in neuron survival, apoptosis, and upstream regulation is still controversial. We established a hypobaric hypoxia-induced C57BL/6 murine model and cell lines exposed to 1% hypoxia, including PC12 and HT22. First, we tested the expressions of interleukin 6 (IL-6), IL-1β, and IL-10 in C57BL/6 mice's hippocampus under hypoxia using enzyme-linked immunosorbent assay (ELISA). We determined the signal transducer and activator of transcription 3 (STAT3) phosphorylation at tyrosine (Tyr)705 by western blot and the expression of ATF6, 78-kDa glucose-regulated protein (GRP78), and C/-EBP homologous protein (CHOP) related to ER stress by immunofluorescence (IF), western blot, and qRT-PCR; they were then verified on the cell model. Additionally, IL-6 (40 ng/mL) and STAT3 siRNA were used to treat the PC12 cells for 48 and 4 h to activate or silence STAT3, respectively. Subsequently, the cells of siRNA group were exposed to 1% hypoxia for 48 h. Furthermore, the ATF6 and CHOP expressions were detected with western blot and qRT-PCR. Finally, we examined the binding of STAT3 to the ATF6 promoter by chromatin immunoprecipitation (ChIP)-seq. The results showed that IL-6 increased, IL-10 decreased in the hypoxia group, and IL-1β showed no difference between the hypoxia and the normoxia groups. Neuron apoptosis was significantly elevated by exposure to hypoxia for 48h in PC12 cells. The hypobaric hypoxia-induced ER stress proteins, ATF6, GRP78, and CHOP, and the p-STAT3 (Tyr705) expressions increased both in and . Besides, STAT3 silencing significantly promoted the ATF6 expression and inhibited CHOP, while STAT3 activation downregulated the expression of ATF6 and upregulated CHOP in PC12 cells. The ChIP-seq assay demonstrated that p-STAT3 (Tyr705) protein could bind to the ATF6 promoter region in HT22 cells. Phosphorylation of STAT3 at the Tyr705 site contributes to hypoxia-induced neuron apoptosis by downregulating ATF6, which might explain the inflammatory reaction and apoptosis of the hippocampal neurons induced by ER stress.
内质网（ER）应激调控的神经元凋亡是影响低压缺氧所致认知障碍的关键因素。缺氧主要改变ER应激的激活转录因子（ATF6）通路。然而，ATF6在神经元存活、凋亡及上游调控中的作用仍存在争议。我们建立了低压缺氧诱导的C57BL/6小鼠模型以及暴露于1%低氧环境的细胞系，包括PC12和HT22。首先，我们采用酶联免疫吸附测定（ELISA）检测了缺氧状态下C57BL/6小鼠海马中白细胞介素6（IL-6）、IL-1β和IL-10的表达。通过蛋白质印迹法测定酪氨酸（Tyr）705位点信号转导和转录激活因子3（STAT3）的磷酸化水平，并通过免疫荧光（IF）、蛋白质印迹法和qRT-PCR检测与ER应激相关的ATF6、78-kDa葡萄糖调节蛋白（GRP78）和C/EBP同源蛋白（CHOP）的表达；随后在细胞模型上进行验证。此外，分别用IL-6（40 ng/mL）和STAT3 siRNA处理PC12细胞48小时和4小时，以激活或沉默STAT3。随后，将siRNA组细胞暴露于1%低氧环境48小时。此外，通过蛋白质印迹法和qRT-PCR检测ATF6和CHOP的表达。最后，我们通过染色质免疫沉淀（ChIP）-seq检测STAT3与ATF6启动子的结合情况。结果显示，缺氧组IL-6升高，IL-10降低，IL-1β在缺氧组与常氧组之间无差异。PC12细胞暴露于缺氧环境48小时后，神经元凋亡显著增加。低压缺氧诱导的ER应激蛋白ATF6、GRP78和CHOP以及p-STAT3（Tyr705）的表达在小鼠和细胞中均增加。此外，沉默STAT3可显著促进ATF6表达并抑制CHOP，而激活STAT3则下调PC12细胞中ATF6的表达并上调CHOP。ChIP-seq分析表明，p-STAT3（Tyr705）蛋白可与HT22细胞中的ATF6启动子区域结合。STAT3在Tyr705位点的磷酸化通过下调ATF6促进缺氧诱导的神经元凋亡，这可能解释了ER应激诱导的海马神经元炎症反应和凋亡。
