| Purpose: Obstructive sleep apnea hypopnea syndrome(OSAHS), a highly prevalent disease, is characterized as an risk factor for the occurrence and the development of cognitive dysfunction in the nervous system. Intermittent hypoxia(IH) is a major pathological component of OSAHS. The repeated hypoxia and reoxygenation play a critical role in the induction of the hippocampal neuronal injury. It is likely that IH-induced hippocampal neuronal injury may be a major mechanism to the cognitive dysfunction. Autophagy is an important pathophysiological mechanism for maintaining intracellular homeostasis. Recent years, increasing evidences indicate that in hypoxia and/or ischemia-reperfusion(I/R) cerebral injury, autophagy activation contributes a critical role in neural cells injury. Nevertheless, the cellular and molecular mechanism of IH-mediated autophagy activation and the role of intracellular increased autophagy in nervous system remain unclear. In our study, we established a primary hippocampal neurons intermittent hypoxia model by simulating OSAHS in the purpose of studying the expression of intermittent hypoxia-induced autophagy in vitro level. Then we established pharmacological intervention models to detect autophagy flux, to further investigate the contribution of autophagy in hippocampal neuronal apoptosis after intermittent hypoxia.Methods: The primary hippocampus neuronal cultures were prepared and maintained by using neonatal SD rats(born in 24 hours). After seventh day, we used immunofluorescence to identify the cultured hippocampus neurons. â‘ We stablish an intermittent hypoxia model in vitro level. The hippocampus neurons, which have been cultured to the seventh day, were further divided into 4 groups with different duration of intermittent hypoxia: 0h, 4h,8h, and 12 h. Cultured hippocampus neuronal were either exposed to IH(Episodic cycles of 1.5% O2, 5% CO2, and balance N2 for 5 min and 21% O2, 5% CO2, balance N2 for 10 min) or to normoxia(21% O2, 5% CO2, and balance N2). Immunofluorescence and Western blot were used to examine the protein levels of microtubule-associated protein light chain 3(LC3), Beclin 1 and cleaved caspase-3 after IH. TUNEL was applied to assay hippocampal neuronal apoptosis rate. â‘¡Pharmacological intervention models were pretreated with rapamycin or chloroquine for 12 hours before IH exposure(12h). In addition, we also established the normal control group, intermittent hypoxia group(12h), rapamycin/chloroquine administered alone groups and EBSS group. Intermittent hypoxia group was exposed to IH for 12 h. Administration alone groups were pretreated with rapamycin or chloroquine for 12 hours. Starvation group that set up as IH positive control group was given starvation treatment with EBSS Sugar-free culture medium. We used Western blot to examine the expression of LC3-II/LC3-I and P62. â‘¢ We Establish the same experimental model and experimental groups with â‘¡. We used Western blot to examine the expression of LC3-II,P62 and cleaved caspase-3, while hippocampal neuronal apoptosis rate was assessed by TUNEL. Furthermore, level of LC3 is detected by Immunofluorescence technology.Results: â‘ The effect of intermittent hypoxia on autophagy-related protein expression in primary hippocampus neuronal. IH exposure significantly upregulated the protein levels of LC3-II and Beclin1. Moreover, with the extension of IH duration, LC3-II and Beclin1 expression in the intermittent hypoxia group were gradually increasing, especially in intermittent hypoxia 12 h group(P<0.5). â‘¡The effect of intermittent hypoxia on autophagy flux in primary hippocampus neuronal. Western blot analysis revealed that levels of LC3-II/LC3-I in intermittent hypoxia group was significantly higher than that in normal control group, while P62 was significantly lower than that in normal control group(**P<0.5). Furthermore, compared with the rapamycin/chloroquine alone group, intermittent hypoxia exposure caused an significant upregulation of LC3-II/LC3-I expression and downregulation of the P62 expression in rapamycin/chloroquine group(**P<0.5). Compared with normal control group, the expression of LC3-II/LC3-I in rapamycin alone group was significantly increased, while there was no significant difference of LC3-II/LC3-I expression between normal control group and DMSO group. Compared with normal control group, the expression of LC3-II/LC3-I in EBSS group was significantly increased, while P62 expression was significantly decreased. There was no significant difference of LC3-II/LC3-I and P62 expression between EBSS group and intermittent hypoxia group.â‘¢The effects of autophagy activation on intermittent hypoxia-induced hippocampal neuronal apoptosis. IH exposure significantly upregulated the expression of apoptosis-positive cells and cleaved caspase-3. Moreover, with the extension of IH duration, the cleaved caspase-3 expression and apoptosis-positive cells were gradually increasing, especially in intermittent hypoxia 12 h group(**P<0.05). Pretreatment with rapamycin significantly upregulated the level of cleaved caspase-3 and apoptosis-positive cells after intermittent hypoxia(**P<0.5). On the contrary, the cleaved caspase-3 expression and apoptosis-positive cells of intermittent hypoxia group were significantly downregulated through pretreatment with chloroquine(***P<0.5). Western blot analysis revealed that levels of cleaved caspase-3 and apoptosis-positive cells in rapamycin alone group were significant higher than those in normal control group(*P<0.5). However, there was no significant difference of cleaved caspase-3 expression between normal control group and DMSO group.Conclusion: 1. Intermittent hypoxia can induce an increased expression of autophagy. 2. Intermittent hypoxia can induce the activation of autophagy. 3. Autophagy activation aggravates intermittent hypoxia-induced hippocampal neurons injury... |
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