OBJECTIVE In this study, the molecular mechanism by which EPO regulates the angiogenesis after cerebral ischemia through AMPK-KLF2 signaling pathway was investigated. METHODS Sixty healthy, male, C57BL/6 mice were randomly divided into three groups of 20 mice: a sham group, the middle cerebral artery occlusion (MCAO) group, and a MCAO+EPO treatment group. The MCAO model was established using a modified ZeaLonga method. Mice in the EPO treatment group were injected with EPO immediately after reperfusion (5000 IU/kg), and EPO was injected the following day. The number of mouse deaths and neurologic function scores were recorded during the experiment. On day 7 after cerebral ischemia, brain tissue proteins were extracted. The following proteins expressions were detected by western blot assay: EPO, vascular endothelial growth factor (VEGE), vascular endothelial growth factor receptor (KDR), adenosine activated protein kinase (AMPK), and alpha HIF-1α alpha (HIF-1α), KLF2 and nitric oxide synthase (eNOS). RESULTS Compared with the MCAO group, the survival rate of mice in the EPO group was significantly improved and neurological function was significantly improved (P < 0.01). Western blot results showed that the content of EPO in brain tissue in MCAO group significantly increased compared with sham group. The content of EPO in the brain tissue of mice in the MCAO+EPO treatment group was significantly higher than in that of the MCAO group, which indicates that EPO increased the content of EPO in mouse brain tissue. Compared with the sham group, the protein expression of vascular endothelial growth factor (VEGE) and its receptor (KDR) in brain tissue of the MCAO group significantly decreased. However, the protein expression of VEGE and its receptor KDR in brain tissue of rats treated with MCAO+EPO was significantly higher than in that of the MCAO group. Thus, in this study, EPO was associated with vascular endothelial differentiation after cerebral ischemia in mice. The results of AMPK and KLF2 showed that the expression levels of AMPK and KLF2 in brain tissues of MCAO group mice significantly decreased compared with the sham group. However, the expression levels of AMPK and KLF2 in brain tissues of mice treated with MCAO+EPO were significantly higher than those in the MCAO group. Thus, EPO can activate AMPK and upregulate the expression of the transcription factor KLF2. The protein expression of HIF-1α in the brain tissue of mice in the MCAO group significantly increased compared with the sham group. However, the expression of HIF-1α in mice brain tissues in the MCAO+EPO treatment group was significantly lower than in that of the MCAO group, indicating that EPO was involved in regulating HIF-1α expression. The eNOS results showed that, compared with Sham group, the protein expression of eNOS in brain tissue of MCAO group mice significantly decreased. In the MCAO+EPO treatment group, the protein expression of eNOS was significantly higher in the brain tissue of the mice than in that of the MCAO group, indicating that EPO was involved in the synthesis of NO and promoted the angiogenesis. CONCLUSION EPO promotes VEGE and its receptor (KDR) expression and participates in the regulation of HIF-1α and eNOS protein expression through the activation of AMPK-KLF2 signaling pathways to promote new vascular development after cerebral ischemia.

中文翻译：

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EPO的分子机制通过AMPK-KLF2信号通路调节脑缺血后的血管生成。

目的研究EPO通过AMPK-KLF2信号通路调节脑缺血后血管新生的分子机制。方法60只健康的雄性C57BL / 6小鼠随机分为三组，每组20只，分别为假手术组，大脑中动脉闭塞（MCAO）组和MCAO + EPO治疗组。MCAO模型是使用改良的ZeaLonga方法建立的。再灌注后立即向EPO治疗组的小鼠注射EPO（5000 IU / kg），第二天再注射EPO。实验期间记录了小鼠死亡的数量和神经功能评分。脑缺血后第7天，提取脑组织蛋白。通过蛋白质印迹分析检测到以下蛋白表达：EPO，血管内皮生长因子（VEGE），血管内皮生长因子受体（KDR），腺苷活化蛋白激酶（AMPK）和αHIF-1αα（HIF-1α），KLF2和一氧化氮合酶（eNOS）。结果与MCAO组相比，EPO组小鼠的存活率明显提高，神经功能明显改善（P <0.01）。Western blot结果显示，与假手术组相比，MCAO组脑组织中EPO含量明显增加。MCAO + EPO治疗组小鼠脑组织中EPO的含量明显高于MCAO组，说明EPO增加了小鼠脑组织中EPO的含量。与假组相比 MCAO组脑组织中血管内皮生长因子（VEGE）及其受体（KDR）的蛋白表达明显降低。但是，MCAO + EPO处理的大鼠脑组织中VEGE及其受体KDR的蛋白表达明显高于MCAO组。因此，在这项研究中，EPO与小鼠脑缺血后的血管内皮细胞分化有关。AMPK和KLF2的结果表明，与假手术组相比，MCAO组小鼠脑组织中AMPK和KLF2的表达水平明显降低。然而，用MCAO + EPO治疗的小鼠脑组织中AMPK和KLF2的表达水平明显高于MCAO组。因此，EPO可以激活AMPK并上调转录因子KLF2的表达。与假手术组相比，MCAO组小鼠脑组织中HIF-1α的蛋白表达显着增加。然而，MCAO + EPO治疗组小鼠脑组织中HIF-1α的表达明显低于MCAO组，表明EPO参与了HIF-1α表达的调节。eNOS结果表明，与Sham组相比，MCAO组小鼠脑组织中eNOS的蛋白表达明显降低。在MCAO + EPO治疗组中，小鼠脑组织中eNOS的蛋白表达明显高于MCAO组，表明EPO参与了NO的合成并促进了血管生成。