新生鼠缺氧缺血性脑损伤后自噬水平变化及其与细胞凋亡的关系

Autophagy levels in newborn rats with hypoxic-ischemic brain injury and the relationship with apoptosis

目的研究新生鼠缺氧缺血性脑损伤(HIBD)后自噬水平的变化规律,通过对Caspase-3的检测探讨自噬水平变化对细胞凋亡的影响。方法将实验动物分为假手术组、模型组、3-甲基腺嘌呤组及雷帕霉素组,采用Rice法建立HIBD模型,3-甲基腺嘌呤组及雷帕霉素组于造模后0.5h腹腔注射3-甲基腺嘌呤及雷帕霉素。应用免疫组织荧光化学法检测损伤侧大脑海马及皮层组织自噬相关蛋白Βeclin1的表达,Western-blot法检测Βeclin1表达变化规律,免疫组织化学法检测各组Caspase-3的表达。结果免疫组织荧光化学检测结果显示,缺氧缺血性脑损伤后能激活自噬现象发生。Western-blot检测结果显示,HIBD后2hBeclin1蛋白表达开始增加,24h达到高峰,持续到168h。免疫组织化学检测结果显示,3-甲基腺嘌呤组时间点Caspase-3阳性表达显著升高,雷帕霉素组Caspase-3阳性表达显著下降。结论新生大鼠HIBD模型能激活自噬现象发生,自噬水平增高可抑制细胞凋亡,自噬水平降低可增加细胞凋亡。

Objective To investgate the change of the level of autophagy after hypoxic-ischemic brain demage（HIBD） in neonatal rats,and observe the influence between the changes and apoptosis through Caspase-3 detection. Methods The animals were divided into sham operation group, model group, 3-methyladenine group and rapamycin group.The HIBD model was constructed using the method of Rice. The 3-methyadenine and rapamycin were adminstrated 0.5 h after the surgery by intraperitoneal injection. Immunofluorescence was utilized to measure the autophagy-related protein Beclin 1 in the hippocampus and cortex of the injuried side of the brain. Western-blot assay was used to test the variation of the Beclin 1 level. Immunohistochemical staining was to measure apotosis-related protein Caspase-3. Results The expression of Beclin 1 detected by immunofluorescence staining showed that hypoxic and ischemic conditions could activate the autophagy. The expression of Beclin 1 was significantly increased at 2 h,peaked at 24 h,and still could be found at 168 h.The expression of Caspase-3 was significantly increased in the 3-methyadenine group, while it was decreased in the rapamycin group. Conclusion The autophgy can be activated after hypoxic-ischemic brain demage.Increased autophagy levels may probably inhibit apoptosis, while reduced autophagy levels can increase apoptosis.