缺氧缺血海马组织中自噬相关蛋白Beclin-1和LC3的表达变化以及雷帕霉素对其表达的影响

Changes in expression of autophagy-related proteins, Beclin-1 and LC3, and effects of rapamycin on their expression in hypoxic-ischemic hippocampus

摘要:

目的 观察缺氧缺血性脑损伤(HIBD)新生大鼠在不同时间点海马组织中自噬相关蛋白Beclin-1 及LC3 的表达变化,并探讨雷帕霉素对上述蛋白表达的影响。方法 将生后7 d 的108 只Sprague-Dawley 大鼠随机分为假手术组、HIBD 组和雷帕霉素组,每组36 只。采用改良Rice 法建立HIBD 模型;假手术组分离左侧颈总动脉但不予结扎,不行低氧处理;雷帕霉素组在模型制作前1 h 予腹腔注射雷帕霉素(0.5 mg/kg)。各组大鼠分别于模型制作结束后0、6、12、24、48、72 h 时间点麻醉后断头取脑,采用Western blot 法检测大鼠海马组织Beclin-1 及LC3 蛋白表达的变化。结果 HIBD 组Beclin-1 蛋白及雷帕霉素组Beclin-1、LC3- Ⅱ蛋白表达水平均从0 h 起开始升高,24 h 时达到峰值后回落;HIBD 组LC3- Ⅱ蛋白表达水平从0 h 起开始升高,12 h时达到峰值后回落。与假手术组相比,HIBD 组及雷帕霉素组Beclin-1、LC3- Ⅱ蛋白表达水平在各时间点均显著升高(P<0.05);与HIBD 组相比,除12 h 时LC3- Ⅱ蛋白水平外,雷帕霉素组各时间点Beclin-1、LC3- Ⅱ蛋白表达水平均明显升高(P<0.05)。结论 海马组织细胞经缺氧缺血后出现自噬激活,而雷帕霉素能增强自噬这一表达过程。

Abstract:

Objective To observe changes in the expression of autophagy-related proteins, Beclin-1 and LC3, in the hippocampal tissue of neonatal rats with hypoxic-ischemic brain damage (HIBD) at different time points, and to investigate the effect of rapamycin (Ra) on the expression of the above two proteins. Methods A total of 108 7-dayold Sprague-Dawley rats were randomly divided into sham, HIBD, and Ra groups (n=36 each). The HIBD model was established using the modified Rice method. For sham rats, only the left common carotid artery was separated without ligation or hypoxic treatment. For Ra-treated rats, 0.5 mg/kg Ra was administered by an intraperitoneal injection 1 hour before model establishment. The rats were anesthetized and sacrificed to collect brain tissues at 0, 6, 12, 24, 48, and 72 hours after model establishment. Changes in the expression of Beclin-1 and LC3 proteins in rat hippocampus were examined by Western blot. Results The expression level of Beclin-1 in HIBD rats began to increase at 0 hour, peaked at 24 hours, and then declined thereafter, similar as those of Beclin-1 and LC3-II in Ra-treated rats. The expression level of LC3-II in HIBD rats began to increase at 0 hour, peaked at 12 hours, and then declined thereafter. At all time points, both Beclin-1 and LC3-II expression levels were significantly higher in HIBD and Ra-treated rats than in sham rats (P<0.05); except LC3-II at 12 hours, Beclin-1 and LC3-II expression levels were significantly higher in Ra-treated rats than in HIBD rats (P<0.05). Conclusions Hypoxia-ischemia activates autophagy in rat hippocampal cells, while Ra enhances the expression process of autophagy.