Li Shi-Ping, Zhu Jiang-Hu, Zhao Feng-Yan, Zheng Zhen, Mu De-Zhi, Qu Yi
Department of Pediatrics, Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second Hospital, Sichuan University, Chengdu 610041, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2017 Aug;19(8):938-944. doi: 10.7499/j.issn.1008-8830.2017.08.017.
To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia.
Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique.
The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05).
Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.
探讨缺氧缺血性脑损伤后新生大鼠神经元自噬基因和昼夜节律基因的表达情况以及缺氧/缺血诱导神经损伤的机制。
将12只Sprague-Dawley（SD）大鼠随机分为缺氧缺血（HI）组和假手术组，每组6只。通过结扎右侧颈总动脉并进行缺氧处理建立缺氧缺血性脑损伤模型。采用蛋白质免疫印迹法检测皮质和海马中昼夜节律蛋白Clock的表达。将大鼠神经元进行体外培养并随机分为氧糖剥夺（OGD）组和对照组。OGD组神经元用不含葡萄糖和血清的DMEM培养基处理以模拟缺血状态，并进行缺氧处理以建立体外缺氧缺血性脑损伤模型。采用蛋白质免疫印迹法检测不同时间点自噬相关蛋白Beclin1、LC3和Clock蛋白的表达。运用小干扰RNA技术抑制神经元中Clock蛋白表达后，检测Beclin1和LC3表达的变化。
体外培养的神经元中自噬相关蛋白Beclin1和LC3Ⅱ的表达呈现节律性波动；OGD处理后，Beclin1和LC3Ⅱ的表达随处理时间逐渐增加，不再具有节律性波动。与假手术组相比，HI组皮质和海马中Clock蛋白的表达显著降低（P<0.05）。OGD处理后，体外培养的神经元中Clock蛋白的表达显著降低（P<0.05）。与阴性对照组相比，Clock基因抑制组Beclin1和LC3Ⅱ的表达显著降低（P<0.05）。
缺氧/缺血诱导自噬相关蛋白Beclin1和LC3的表达节律紊乱，其机制可能与昼夜节律蛋白Clock参与调控Beclin1和LC3的表达有关。
