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外源性EPO通过激活JAK2-STAT5信号通路保护HT22细胞免受间歇性低氧诱导的损伤

齐珂瑢¹, 陈雪¹, 司建超^1,2, 刘青青¹, 杨胜昌^1,3,*

¹河北中医药大学生理学教研室，石家庄 050091；²广州中医药大学基础医学院，广州 510006；³河北省中医药结合氢医学技术创新中心，石家庄 050091

摘要

本研究旨在探讨外源性促红细胞生成素(erythropoietin, EPO)对间歇性低氧(intermittent hypoxia, IH)诱导的神经元损伤的作用及其机制。小鼠海马神经元HT22细胞暴露于不同时间的IH环境(1% O2 7 min/21% O2 3 min，10 min一次循环)，用CCK-8检测细胞活性，用试剂盒检测细胞培养液上清中EPO含量，用Western blot检测细胞EPO蛋白表达水平，用免疫荧光染色法和Western blot检测EPO受体(EPOR)表达，用试剂盒检测细胞线粒体膜电位和凋亡水平，用DCFH探针检测细胞活性氧(reactive oxygen species, ROS)含量，用Western blot检测JAK2-STAT5通路相关蛋白表达水平。结果显示，IH暴露可显著降低HT22细胞活性；EPO及EPOR蛋白表达水平在IH暴露12 h时显著上调，而在24、48 h显著下调。在IH处理的HT22细胞中，外源性给予EPO可显著提高细胞活性和线粒体膜电位，降低细胞ROS和凋亡水平，上调Nrf-2、血红素氧合酶-1 (heme oxygenase 1, HO-1)蛋白表达水平，降低Cleaved-Caspase-3/Caspase-3、Bax/Bcl-2比值，促进JAK2-STAT5通路相关蛋白磷酸化，而JAK2阻断剂AG490和STAT5阻断剂BD750均可逆转EPO的上述神经元保护作用。以上结果提示，外源性EPO可能通过激活JAK2-STAT5信号通路抑制IH诱导的氧化应激和细胞凋亡，从而发挥神经元保护作用。

关键词： 间歇性低氧; 氧化应激; 细胞凋亡; 促红细胞生成素; JAK2-STAT5

Exogenous EPO protects HT22 cells from intermittent hypoxia-induced injury by activating JAK2-STAT5 signaling pathway

QI Ke-Rong¹, CHEN Xue¹, SI Jian-Chao^1,2, LIU Qing-Qing¹, YANG Sheng-Chang^1,3,*

¹Department of Physiology, Hebei University of Chinese Medicine, Shijiazhuang 050091, China；²Institute of Basic Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China；³Hebei Technology Innovation Center of TCM Combined Hydrogen Medicine, Shijiazhuang 050091, China

Abstract

The aim of this study was to investigate the effects of exogenous erythropoietin (EPO) on intermittent hypoxia (IH)-induced neuronal injury and the underlying mechanism. Mouse hippocampal neuron HT22 cells were exposed to IH for different durations (1% O2 for 7 min/21% O2 for 3 min, one cycle for 10 min). Cell viability was detected by CCK-8. EPO content in the supernatant of cell culture medium was detected by ELISA kit, and the protein expression was detected by Western blot. EPO receptor (EPOR) protein expression was detected by immunofluorescence staining and Western blot. Cellular apoptosis and mitochondrial membrane potential were detected by the corresponding kits. Reactive oxygen species (ROS) level was detected by DCFH probe, and expression levels of JAK2-STAT5 signaling pathway-related proteins were detected by Western blot. The results showed that IH exposure significantly decreased HT22 cell activity. EPO and EPOR protein expressions were significantly up-regulated at 12 h of IH exposure, but down-regulated at 24 and 48 h. In IH-treated HT22 cells, exogenous EPO significantly increased cell activity and mitochondrial membrane potential, decreased ROS levels and cell apoptosis, up-regulated Nrf-2 and heme oxygenase 1 (HO-1) protein expression levels, decreased Cleaved-Caspase-3/Caspase-3 and Bax/Bcl-2 ratios, and promoted the phosphorylation of JAK2-STAT5 pathway-related proteins. Whereas JAK2 and STAT5 blockers both reversed these neuronal protective effects of EPO. These results suggest exogenous EPO inhibits IH-induced oxidative stress and apoptosis by activating the JAK2-STAT5 signaling pathway, thus exerting a neuronal protective effect. 

Key words: intermittent hypoxia; oxidative stress; cell apoptosis; erythropoietin; JAK2-STAT5

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通讯作者：杨胜昌　　E-mail:

引用本文：

齐珂瑢, 陈雪, 司建超, 刘青青, 杨胜昌. 外源性EPO通过激活JAK2-STAT5信号通路保护HT22细胞免受间歇性低氧诱导的损伤[J]. 生理学报 2024; 76 (5): 691-702.

QI Ke-Rong, CHEN Xue, SI Jian-Chao, LIU Qing-Qing, YANG Sheng-Chang. Exogenous EPO protects HT22 cells from intermittent hypoxia-induced injury by activating JAK2-STAT5 signaling pathway. Acta Physiol Sin 2024; 76 (5): 691-702 (in Chinese with English abstract).