自噬在大鼠蛛网膜下腔出血后早期脑损伤中作用的实验研究

Experimental study on role of autophagy in early brain injury after subarachnoid hemorrhage in rats

目的探讨大鼠蛛网膜下腔出血(SAH)模型中,应用激动剂rapamycin(RAP)增强自噬后观察SAH后早期大鼠神经功能评分、脑水肿和血-脑屏障(BBB)通透性的变化,以及应用抑制剂3-Methyladenine(3-MA)减弱自噬后上述指标的变化。探讨自噬在SAH后早期脑损伤(EBI)中可能的作用。方法雄性SD大鼠60只,随机分为对照组、SAH组、SAH+vehicle组、SAH+RAP组和SAH+3-MA组5组,每组12只,于24 h时间点处死动物后取颞底脑皮层,利用western blot法测定自噬标志物LC3和Beclin-1的表达变化,分别观察各组大鼠神经功能评分的变化,并测定各组大鼠脑水肿和BBB通透性的变化。结果 SAH后脑皮层中LC3和Beclin-1表达较对照组明显增加(均P<0.01),同时伴有明显的脑水肿(P<0.01)及BBB通透性破坏(P<0.01);与SAH+vehicle组相比SAH+RAP组LC3和Beclin-1的表达明显增加(均P<0.05),脑水肿指数下降(P<0.01),BBB通透性好转(P<0.01),同时大鼠的神经功能评分明显好转(P<0.01)。而SAH+3-MA组与SAH+vehicle组相比,LC3和Beclin-1的表达明显下降(均P<0.01),大鼠神经功能评分变差(P<0.05)。结论应用RAP干预自噬后,LC3和Beclin-1的表达明显增高,自噬明显增强,同时大鼠神经功能评分明显好转,脑水肿指数明显下降,BBB通透性明显好转。自噬增强后能减轻SAH后的EBI;3-MA的应用可使EBI加重。

Objective To investigate the changes of neurologic scoring, brain water content and blood-brain barrier （BBB） permeability when use autophagy activator and inhibitor in rats and further study the potential role of autophagy in early brain injury （EBI） after subarachnoid hemorrhage（SAH）. Method 60 health male S-D rats were assigned randomly into control group, SAH group, SAH ＋ vehicle group, SAH ＋ RAP group and SAH ＋ 3-MA group. Western blot was performed to detect the changes of LC3 and beelin-1. At 24h after SAH,we investigate the changes of neurologic scoring, brain water content and BBB permeability in each group. Results Compared with control group, LC3 and Beelin-1 in cerebral cortex were significantly increased （all P 〈 0.01 ） following SAH, at the same time rats had significant brain water content （ P 〈 0.01 ） and BBB permeability dysfunetion（P 〈 0.01 ）;compared with SAH ＋ vehicle group, the level of LC3 and Beelin-1 were markedly up-regulated （all P 〈 0.05 ） , the mean value of brain water content was deereased（P 〈0.01）, BBB permeability was getting better（P 〈0.01）, at the same time rats showed better performanee（P 〈 0.01 ） in SAH ＋ RAP group. But in SAH ＋ 3-MA group LC3 and Beelin-1 expression were significantly decreased （ compared with SAH ＋ vehicle group, all P 〈 0. 01 ） , the clinical behavior was getting worse（ compared with SAH ＋ vehicle group, P 〈 0. 05 ） Conclusions When use RAP intervene autophagy, LC3 and Beclin-1 expression were significantly inceased that prompting autophagy activity was much stronger, at the same time ameliorated the brain edema, BBB permeability and clinical behavior which showed strengthen autophagy can alleviate EBI after SAH. Conversely 3-MA can aggravate EBI.