Song Ying, Li Meng, Li Ji-cheng, Wei Er-qing
Department of Cellular Biology, School of Medicine, Zhejiang University, Hangzhou 310031, China.
J Zhejiang Univ Sci B. 2006 Sep;7(9):749-56. doi: 10.1631/jzus.2006.B0749.
Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD (oxygen-glucose deprivation)-reperfusion.
Viability of PC12 cells which were injured at different time of OGD injury, was quantified by measuring MTT (2-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide) staining. In addition, PC12 cells' viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by (OGD). Furthermore, apoptotic population of PC12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/PI staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot.
(1) The viability of PC12 cells decreased with time (1 - 12 h) after OGD. We regarded the model of OGD 2 h, then replacing DMEM (Dulbecco's Modified Eagle's Medium) for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC12 cells were in the state of apoptosis after OGD-reperfusion. (2) The viability of PC12 cells preincubated with edaravone at high concentrations (1, 0.1, 0.01 micromol/L) increased significantly with edaravone protecting PC12 cells from apoptosis after OGD-reperfusion injury. (3) Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion.
Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.
依达拉奉已被证实可有效预防缺血性损伤。在本研究中，我们通过观察依达拉奉对细胞抗凋亡、Bcl-2/Bax蛋白表达调控以及氧糖剥夺（OGD）-再灌注后线粒体损伤恢复的影响，来研究其保护作用。
通过MTT（2-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）染色，对在不同OGD损伤时间点受损的PC12细胞活力进行定量分析。此外，PC12细胞在不同浓度依达拉奉中预孵育30分钟后再进行OGD处理，之后对其活力也进行定量分析。此外，通过流式细胞仪分析、电子显微镜和Hoechst/PI染色，确定经依达拉奉预孵育或未经预孵育的OGD-再灌注后PC12细胞的凋亡群体。最后，通过蛋白质免疫印迹法检测Bcl-2/Bax蛋白表达的变化。
（1）OGD后PC12细胞活力随时间（1 - 12小时）下降。在本研究中，我们将OGD 2小时后用DMEM（杜氏改良伊格尔培养基）替换并再培养24小时的模型视为OGD-再灌注。此外，OGD-再灌注后大多数PC12细胞处于凋亡状态。（2）高浓度（1、0.1、0.01微摩尔/升）依达拉奉预孵育的PC12细胞活力显著增加，依达拉奉可保护PC12细胞免受OGD-再灌注损伤后的凋亡。（3）此外，依达拉奉减轻了OGD-再灌注对线粒体的损伤，并调节了OGD-再灌注后Bcl-2/Bax蛋白的失衡表达。
依达拉奉对缺血性或其他脑损伤的神经保护作用可能部分是通过恢复线粒体损伤来抑制Bcl-2/Bax凋亡途径介导的。
