BACKGROUND Hypoxic-ischemic encephalopathy (HIE) has a high morbidity rate and involves severe neurologic deficits, including cerebral palsy. Therapeutic hypothermia (TH) has been shown to decrease the mortality rate and provide neuroprotection in infants with HIE. However, death and disability rates in HIE infants treated with TH remain high. Although the cellular mechanism of the neuroprotective effect of TH remains unclear, astrocytic erythropoietin (EPO) is known to be a key mediator of neuroprotection under hypoxic conditions. In the present study, we investigated the hypothermia effect on EPO expression in astrocytes and determined whether hypothermia attenuates neuronal damage via EPO signaling. METHODS Astrocytes derived from rat cerebral cortex were cultured under oxygen/glucose deprivation (OGD). The expression of EPO and hypoxia-inducible factor (HIF), a transcription factor of EPO, was assessed. After OGD, astrocytes were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions, and then EPO and HIF expression was assessed. After OGD, rat cortical neurons were cultured in astrocyte-conditioned medium (ACM) derived from the hypothermic group, and neuronal apoptosis was evaluated. RESULTS OGD induced EPO mRNA and protein expression, although at lower levels than hypoxia alone. HIF-1α and HIF-2α protein expression increased under hypoxia alone and OGD, although OGD increased HIF-2α protein expression less than hypoxia alone. EPO gene and protein expression after OGD was significantly higher under hypothermia. Moreover, expression of HIF-1α and HIF-2α protein was enhanced under hypothermia. In the presence of ACM derived from hypothermic astrocytes following OGD, the number of cleaved caspase 3 and TdT-mediated dUTP nick-end labeling-positive apoptotic neurons was lower than in the presence of ACM from normothermic astrocytes following OGD. Blockade of EPO signaling using anti-EPO neutralization antibody attenuated the anti-apoptotic effect of ACM derived from hypothermic astrocytes following OGD. CONCLUSIONS Hypothermia after OGD stabilized HIF-EPO signaling in astrocytes, and upregulated EPO expression could suppress neuronal apoptosis. Investigating the neuroprotective effect of EPO from astrocytes under hypothermic conditions may contribute to the development of novel neuroprotection-based therapies for HIE.

中文翻译：

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在低温条件下，延长的星形胶质细胞来源的促红细胞生成素表达会减弱神经元的损伤。

背景技术缺氧缺血性脑病（HIE）具有高发病率并涉及严重的神经功能缺损，包括脑瘫。治疗性低温（TH）已显示可降低HIE婴儿的死亡率并提供神经保护作用。但是，接受TH治疗的HIE婴儿的死亡率和残疾率仍然很高。尽管尚不清楚TH的神经保护作用的细胞机制，但已知星形胶质细胞促红细胞生成素（EPO）是低氧条件下神经保护的关键介质。在本研究中，我们调查了低温对星形胶质细胞中EPO表达的影响，并确定低温是否通过EPO信号传导减弱神经元损伤。方法大鼠脑皮层星形胶质细胞在缺氧/缺糖（OGD）下培养。评估了EPO和EPO的转录因子低氧诱导因子（HIF）的表达。OGD后，在常温（37°C）或低温（33.5°C）条件下培养星形胶质细胞，然后评估EPO和HIF表达。OGD后，大鼠皮层神经元在来自低温组的星形胶质细胞条件培养基（ACM）中培养，并评估神经元凋亡。结果OGD诱导EPO mRNA和蛋白表达，尽管其水平低于单独的缺氧水平。HIF-1α和HIF-2α蛋白表达在单独缺氧和OGD下增加，尽管OGD增加HIF-2α蛋白表达比单独缺氧少。在低温下，OGD后EPO基因和蛋白质表达显着升高。此外，在低温下HIF-1α和HIF-2α蛋白的表达增强。在OGD后源自低温星形胶质细胞的ACM的存在下，裂解的半胱天冬酶3和TdT介导的dUTP缺口末端标记阳性的凋亡神经元的数量要比OGD后从常温星形胶质细胞存在的ACM的数量少。OGD后，使用抗EPO中和抗体阻断EPO信号传导会减弱源自低温星形胶质细胞的ACM的抗凋亡作用。结论OGD可使星形胶质细胞中的HIF-EPO信号稳定并引起EPO表达上调，从而降低体温。研究在低温条件下来自星形胶质细胞的EPO的神经保护作用可能有助于开发基于HIE的新型基于神经保护的疗法。OGD后常温星形胶质细胞裂解的半胱天冬酶3和TdT介导的dUTP缺口末端标记阳性的凋亡神经元的数目低于存在ACM的正常星形胶质细胞。OGD后，使用抗EPO中和抗体阻断EPO信号传导会减弱源自低温星形胶质细胞的ACM的抗凋亡作用。结论OGD引起的低温使星形胶质细胞中的HIF-EPO信号稳定，而EPO表达上调可抑制神经元凋亡。研究在低温条件下来自星形胶质细胞的EPO的神经保护作用可能有助于开发基于HIE的新型基于神经保护的疗法。OGD后常温星形胶质细胞裂解的半胱天冬酶3和TdT介导的dUTP缺口末端标记阳性的凋亡神经元的数目低于存在ACM的正常星形胶质细胞。OGD后，使用抗EPO中和抗体阻断EPO信号传导会减弱源自低温星形胶质细胞的ACM的抗凋亡作用。结论OGD引起的低温使星形胶质细胞中的HIF-EPO信号稳定，而EPO表达上调可抑制神经元凋亡。研究在低温条件下来自星形胶质细胞的EPO的神经保护作用可能有助于开发基于HIE的新型基于神经保护的疗法。OGD后，使用抗EPO中和抗体阻断EPO信号传导会减弱源自低温星形胶质细胞的ACM的抗凋亡作用。结论OGD引起的低温使星形胶质细胞中的HIF-EPO信号稳定，而EPO表达上调可抑制神经元凋亡。研究在低温条件下来自星形胶质细胞的EPO的神经保护作用可能有助于开发基于HIE的新型基于神经保护的疗法。OGD后，使用抗EPO中和抗体阻断EPO信号传导会减弱源自低温星形胶质细胞的ACM的抗凋亡作用。结论OGD引起的低温使星形胶质细胞中的HIF-EPO信号稳定，而EPO表达上调可抑制神经元凋亡。研究在低温条件下来自星形胶质细胞的EPO的神经保护作用可能有助于开发基于HIE的新型基于神经保护的疗法。