Department of Neurochemistry, Institute for Nautical Medicine, Nantong University, Nantong 226001, PR China.
Neurochem Int. 2013 Jan;62(2):157-64. doi: 10.1016/j.neuint.2012.11.007. Epub 2012 Nov 28.
Although ischemic preconditioning (IP) can provide powerful protection on brain against ischemic insult, it is rarely used in clinic to prevent the occurrence of ischemic stroke because of safety concerns. It is therefore necessary to seek the safer stimuli to initiate pharmacological preconditioning. Our previous work demonstrated that ginkgolide B (GB) could protect neurons against ischemia-induced apoptosis. Astrocytes are the most numerous cells in mammalian central nervous system and there is a close bi-directional communication between neurons and astrocytes in brain. Besides neurons, whether GB can exert the role of preconditioning on astrocytes through which to further improve neuronal survival under ischemic condition is not yet known. In the present study, primary cultured astrocytes were treated with GB for 24h or short-term ischemia (ischemia for 3h, as ischemic preconditioning/IP), and then cultured back to normoxia and normal medium for 24h to induce the preconditioning response. Astrocyte-conditioned medium (ACM) was then collected and used to incubate the cultured neurons for 24h before neurons were subjected to severe ischemia. Our results demonstrated that not only GB and IP increased astrocytic viability in ischemia, but also the conditioned medium from astrocytes treated with GB or IP increased cell viability and decreased the number of apoptosis of neurons in ischemia. We also found that GB and IP significantly stimulated astrocytes to express and secrete erythropoietin (EPO) into ACM, and the addition of anti-EPO antibody blocked the protective effect of GB or IP-treated astrocytes culture medium on neurons in ischemia. Further study of above protection revealed that ACM from astrocytes treated with GB or IP induced the inactivation of proapoptotic factor Bad by phosphorylation at serine 136 and 112 ((136)p-Bad and (112)p-Bad) in neurons. Together, our results suggest that GB is capable of preconditioning on astrocytes as IP and then protects neurons against ischemia-induced apoptosis, which is mediated by EPO.
虽然缺血预处理 (IP) 可以为大脑提供对抗缺血性损伤的强大保护，但由于安全性问题，它很少用于临床预防缺血性中风的发生。因此，有必要寻找更安全的刺激来启动药物预处理。我们之前的工作表明，银杏内酯 B (GB) 可以保护神经元免受缺血诱导的细胞凋亡。星形胶质细胞是哺乳动物中枢神经系统中数量最多的细胞，神经元和星形胶质细胞之间存在着密切的双向通讯。除了神经元，GB 是否可以通过星形胶质细胞发挥预处理作用，进一步提高神经元在缺血条件下的存活，目前尚不清楚。在本研究中，原代培养的星形胶质细胞用 GB 处理 24 小时或短暂缺血（缺血 3 小时，作为缺血预处理/IP），然后培养回常氧和正常培养基 24 小时，以诱导预处理反应。然后收集星形胶质细胞条件培养基 (ACM)，用其孵育培养的神经元 24 小时，然后使神经元遭受严重缺血。我们的结果表明，不仅 GB 和 IP 增加了缺血时星形胶质细胞的活力，而且用 GB 或 IP 处理的星形胶质细胞的条件培养基也增加了神经元的活力并减少了缺血时神经元的凋亡数。我们还发现，GB 和 IP 显著刺激星形胶质细胞将促红细胞生成素 (EPO) 表达和分泌到 ACM 中，并且添加抗 EPO 抗体阻断了用 GB 或 IP 处理的星形胶质细胞培养基对缺血神经元的保护作用。对上述保护的进一步研究表明，GB 或 IP 处理的星形胶质细胞的 ACM 诱导神经元中促凋亡因子 Bad 的磷酸化失活，Ser136 和 Ser112 位点磷酸化的 Bad（(136)p-Bad 和 (112)p-Bad）。总之，我们的结果表明，GB 能够像 IP 一样对星形胶质细胞进行预处理，然后通过 EPO 保护神经元免受缺血诱导的细胞凋亡。
