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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-19kDa-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 0h组)、pMCAO 3h组、9h组和24h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 3h、9h、24h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 3h和9h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 24h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 3h和9h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 9h组比较,pMCAO 24h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对脑缺血损伤的保护作用。
Objective To investigate the effects of Bcl-2 / adenovirus E1B19kD interacting protein 3 (Bcl-2 / adenovirus E1B-19kDa-interacting protein 3) on brain injury at different time points in permanent middle cerebral artery occlusion (PMCAO) , BNIP3) and mitochondrial autophagy-related protein expression, explore the relationship between BNIP3 and the degree of cerebral ischemia. Methods Healthy male Sprague-Dawley rats were randomly divided into sham group (sham group, pMCAO 0h group), pMCAO 3h group, 9h group and 24h group, 12 rats in each group. The volume of cerebral infarction was detected by TTC staining, the morphology of mitochondria was observed by transmission electron microscope, the expression of BNIP3 and related proteins were detected by Western blot. Results (1) The changes of cerebral infarction volume in each group: TTC staining in sham group showed no infarction, the other three groups had cerebral infarction area. The corrected volume of infarction in the 3 h, 9 h, and 24 h groups of pMCAO were significantly (12.12 ± 2.15)%, (37.00 ± 4.24)% and (51.82 ± 4.39)%, respectively, compared with sham group Ischemic time prolonged and corrected cerebral infarction volume increased (between the four groups for any comparison, P <0.01). (2) The morphological changes of mitochondria in brain tissue of rats in each group: intact bilayer mitochondrial membrane structure was observed by transmission electron microscopy in sham group; typical mitochondrial autophagy was observed in pMCAO 3h and 9h groups: mitochondria with bilayer membrane structure Autophagy lysosomes; Mitochondria in pMCAO 24h group were severely damaged, cristae disappeared, the membrane was damaged and mitochondria were swollen. No mitochondrial autophagy was observed. (3) The expressions of BNIP3 and mitochondrial autophagy in each group: Compared with the sham group, the expression of BNIP3 and autophagy-induced Beclin-1 increased and the ratio of LC3-Ⅱ / Ⅰ increased at 3h and 9h Compared with pMCAO 9h group, the expression of P62 and mitochondrial marker HSP60 and TOM20 decreased (all P <0.01) The expression of BNIP3 and Beclin-1 in pMCAO 24h group decreased, the ratio of LC3-Ⅱ / Ⅰ decreased, the expression of P62, TOM20 and HSP60 increased (all P <0.01). Conclusion BNIP3 may play a protective role in cerebral ischemia injury by promoting mitochondrial autophagy in the early stage of cerebral ischemia.