中文摘要：

目的：观察大鼠永久性大脑中动脉闭塞（permanent middle cerebral artery occlusion，pMCAO）不同时间点脑损伤情况、Bcl-2／腺病毒 E1B19kD 相互作用蛋白3（Bcl-2／adenovirus E1B-19 kDa-interacting protein 3， BNIP3）及线粒体自噬相关蛋白的表达，探讨 BNIP3与脑缺血损伤程度的关系。方法将健康雄性 SD 大鼠随机分为假手术组（sham 组，即 pMCAO 0 h 组）、pMCAO 3 h 组、9 h 组和24 h 组，每组12只。分别采用 TTC 染色检测大鼠脑梗死体积、透射电镜观察线粒体形态变化、Western blot 检测 BNIP3及相关蛋白表达。结果（1）各组大鼠脑梗死体积变化：sham 组 TTC 染色显示无梗死发生，其余3组均存在脑梗死区。pMCAO 3 h、9 h、24 h 组矫正脑梗死体积〔分别为（12．12±2．15）％、（37．00±4．24）％和（51．82±4．39）％〕均明显高于 sham 组（均 P＜0．01），且随缺血时间延长而矫正脑梗死体积增加（四组间两两比较，均 P ＜0．01）。（2）各组大鼠脑组织中线粒体形态变化：sham 组透射电镜可以观察到完整的双层线粒体膜结构；pMCAO 3 h 和9 h 组观察到典型的线粒体自噬现象：具有双层膜结构的线粒体自噬溶酶体；pMCAO 24 h 组线粒体受损严重，嵴消失，膜破损，线粒体肿胀加剧，未观察到线粒体自噬现象。（3）各组 BNIP3和线粒体自噬相关蛋白表达：与 sham 组比较，pMCAO 3 h和9 h 组 BNIP3和自噬诱导分子 Beclin-1表达增加，自噬标记分子 LC3-Ⅱ／Ⅰ比值升高，接头蛋白 P62和线粒体标记分子热休克蛋白60（heat shockprotein-60，HSP60）、线粒体外膜易位酶（translocase of outer mitochondrial membrane 20，TOM20）表达下降（均 P ＜0．01）；与 pMCAO 9 h 组比较，pMCAO 24 h 组 BNIP3和 Beclin-1表达下降，LC3-Ⅱ／Ⅰ比值降低，P62和 TOM20、HSP60表达增加（均 P ＜0．01）。结论 BNIP3很可能在脑缺血早期通过促进线粒体自噬来发挥?

英文摘要：

Objective To investigate the brain injury condition in rat with permanent middle cerebral artery occlusion （pMCAO）at different time points,expression of Bcl-2/adenovirus E1B-19 kDa-interacting protein 3 （BNIP3 ）and mitophagy-related proteins in order to explore the relationship between BNIP3 and cerebral ischemia injury degree. Methods Healthy male SD rats were randomly divided into the sham surgery group （0 h group）,pMCAO 3 h,9 h group and 24 h group,there were 12 rats in each group. The brains were dyed with TTC to measure the infarct percentage at different time points. Then,the mitophagy was observed by transmission electron microscope. The expression of BNIP3 and mitophagy-related proteins in brains were determined quantitatively by western blotting analysis. Results （1）The sham group showed no infarction with TTC staining,but the other three groups had cerebral infarction area. The infarct volume of pMCAO 3 h,9 h and 24 h group [（12.12 ±2.15 ）%,（37.00 ±4.24）% and （51.82 ±4.39 ）%,respectively]was significantly higher than that of the sham group （P 〈 0.01,respectively）,and increased with ischemia time prolonged （multiple comparison,P 〈0.01,respectively）. （2 ）The complete bilayer mitochondrial membrane structure was observed by transmission electron microscope in the sham group;The typical bilayer membrane structure of＆amp;nbsp;mitophagy was observed in the pMCAO 3 h and 9 h groups,and the mitochondrial damage was severe in the pMCAO 24 h group, such as mitochondrial crista disappearing, damaged membrane, mitochondrial swelling.Mitophagy was not observed. （3）The western blot results revealed that the expressions of BNIP3 and Beclin-1 increased,the ratio of LC3-Ⅱ/Ⅰ elevated,P62,TOM20 and HSP60 levels declined within 3-9 h compared with the sham group （P 〈0.01,respectively）.In addition,we also found that the protein levels of BNIP3 and Beclin-1 were down-regulated,the ratio of LC3-Ⅱ/Ⅰ decreased,and the protein levels of P62, TOM20 and HSP60 incre