丙酮酸程序化预处理：脯氨酸羟化酶的差异性调控机制
任昊，翟效月
（中国医科大学，组织胚胎学教研室）
摘要：目的：开发一种新的丙酮酸预处理模拟低氧方法，保护视网膜免受光损伤。方法：6-8周龄的BALB / c小鼠丙酮酸处理。 Western印迹和实时PCR方法用于蛋白质和mRNA的研究。视网膜形态学染色检查。 TUNEL染色和免费核试验研究细胞凋亡。结果：首先，丙酮酸预处理使视网膜中缺氧诱导因子HIF- 1a高度表达。其次，PHD2水平同时增加。与此相反， HIF -2α和PHD1并不明显受丙酮酸影响。 hemoxygenase -1和促红细胞生成素（ EPO ）这两个下游基因的表达起到了重要的保护作用。我们的数据揭示了PHD2 - HIF -1α的反馈回路，反应了丙酮酸上调HIF -1α的作用机制。丙酮酸预处理能够在光损伤时减少视网膜细胞凋亡。结论：这种新的丙酮酸预处理方法的保护作用主要是由于HIF -1α的稳定（而不是HIF -2α）。这种诱导的差异是由于PHD的两种亚型作为Hif的降解酶对丙酮酸的抑制作用不同而产生的。

关键词：人体组织胚胎学； 缺氧诱导因子； 丙酮酸预处理；
中图分类号：R329.1

A novel programmed application of pyruvate: differential stabilization of HIF-1a and HIF-2a
REN Hao, Xiaoyue Zhai
(china medical university, histology and embryology)
Foundations: Specialized Research Fund for the Doctoral Program of Higher Education (No. 20102104120020) Brief author introduction:associate prof.

Abstract: Purpose. To develop a novel hypoxia preconditioning mimicking approach by pyruvate programming in the retina focusing on the interplay between HIFs and prolyl hydroxylases (PHDs). Methods. 6-8 weeks old BALB/c mice was treated with pyruvate in a programmed regime. Western blotting and real time PCR were applied for the protein and mRNA studies. Retinal morphology was 10 checked by toluidine blue staining. TUNEL staining and free nucleosome assay were used to examine apoptosis. Retinal organic culture was developed to investigate the pyruvate mechanism in vitro. Result. As the predominant isomer in the retina, HIF-1a is highly stabilized by pyruvate both in vivo and vitro. This was followed by a concomitant increase of PHD2 level. In contrast, HIF-2a and PHD1 were not affected by pyruvate in vivo. The down-stream genes of hemoxygenase-1 (HO-1) and 15 erythropoietin (EPO) also mirrored the changes of the HIFs respectively. Our data revealed a PHD2-HIF-1a feedback loop, which was blocked by pyruvate and rendered further HIF-1a accumulation. In vitro, HIF-2a was also able to be stabilized by pyruvate inhibition to PHD1, but only happed after oxygen withdrawal. Pyruvate treatment reduced retinal apoptosis not only before but also after the light insult. Conclusion. This novel pyruvate programming was retinal protective not only with preconditioning but also post-conditioning. This protection was mainly due to HIF-1a stabilization but not HIF-2a. This differentiation was due to the specific abundance of PHD isoforms in the retina and importantly their distinct preference to HIFs as degradating enzymes.
Key words: histology and embryology; HIF; pyruvate pre-conditioning