EPO activates PI3K-IKKα-CDK1 signaling pathway to promote the proliferation of Glial Cells under hypoxia environment

Abstract Erythropoietin (EPO), supports the function and survival of neurons through astrocytes and has a protective role in neonatal asphyxia brain injury; yet, its mechanism of action remains unclear. As a neuroprotective factor, EPO is also used in the treatment of various diseases, such as neurodegenerative diseases, Parkinson’s disease, traumatic brain injury, by decreasing inflammatory reaction, resisting apoptosis, and lowering oxidative stress. The aim of this study was to examine the effect and mechanism of EPO on promoting human brain glial cell proliferation under hypoxia in vitro. Under CoC12-induced hypoxia, after adding EPO, high-throughput sequencing was used to screen out meaningful up-regulated and significant differentially expressed genes PI3K, IKKα CDK1 related to proliferation, and make further verification by qPCR and western blotting. Under hypoxia, EPO promoted cell proliferation and the expression of PI3K while this effect was inhibited (along with a decrease of downstream genes IKKα and CDK1 decreased) after adding PI3K inhibitor to cell culture. EPO can promote cell proliferation and CDK1 expression, while after inhibiting CDK1 expression, the promotion of EPO on cell proliferation was eliminated. These data proved that EPO promotes the proliferation of U251 cells by activating the PI3K-IKKα-CDK1 signaling pathway under CoC12-induced hypoxia.

摘要 促红细胞生成素（Erythropoietin, EPO）可通过星形胶质细胞支持神经元功能与存活，并在新生儿窒息性脑损伤中发挥保护作用，但其具体作用机制仍未明确。作为一种神经保护因子，EPO还可通过抑制炎症反应、抵抗细胞凋亡、降低氧化应激，用于治疗多种疾病，包括神经退行性疾病、帕金森病、创伤性脑损伤等。本研究旨在探讨体外缺氧环境下，EPO对人脑胶质细胞增殖的影响及其作用机制。在氯化钴（CoCl₂）诱导的缺氧模型中，加入EPO后，本研究通过高通量测序筛选出与增殖相关的差异表达显著上调基因PI3K、IKKα及CDK1，并采用实时荧光定量PCR（quantitative real-time PCR, qPCR）与蛋白质印迹法（Western Blotting）进行进一步验证。缺氧环境下，EPO可促进细胞增殖及PI3K的表达；而在细胞培养液中加入PI3K抑制剂后，该促进作用被抑制，同时下游基因IKKα与CDK1的表达水平也随之降低。EPO可促进细胞增殖及CDK1的表达；而当抑制CDK1的表达后，EPO对细胞增殖的促进作用便会消失。上述实验数据证实，在氯化钴诱导的缺氧环境下，EPO可通过激活PI3K-IKKα-CDK1信号通路促进U251细胞的增殖。