Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism
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
Abstract：Objective 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.Methods 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.Results 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).Conclusions 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.
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