Research On Mechanisms Of The Activation Of Autophagy Mediated By HIF-1? In Hippocampal Neurons Under Intermittent Hypoxia Condition

Objective: OSAHS can cause or aggravate cognitive dysfunction, and its mechanism is related to intermittent hypoxia induced apoptosis of hippocampal neurons. We found that autophagy participated in and promoted the apoptosis of hippocampal neurons induced by intermittent hypoxia. However, the mechanism of intermittent hypoxia induced autophagy activation is not clear. Hypoxia-inducible factor 1 alpha is an important player in activation of autophagy induced by hypoxia or ischemia reperfusion. In addition, the BNIP3, as one of HIF-1 alpha downstream target protein, is closely related to Beclin1 activation. Therefore, we speculate that HIF-1 alpha may mediate the activation of autophagy induced by intermittent hypoxia in hippocampal neural injury. We utilized of many kinds of molecular biology techniques to explore the mechanism of HIF-1 alpha medited the activation of autophagy induced by intermittent hypoxia in hippocampal neurons injury. To further clarify the role and mechanism of autophagy activation in IH induced hippocampal neuronal injury.Methods:(1)The primary hippocampus neuronal cultures were prepared and maintained by using neonatal SD neonatal rats.(2) We stablish an intermittent hypoxia model in vitro level. The hippocampus neurons 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).(3)Pharmacological intervention models were pretreated with YC-1 for 2 hours before IH exposure. In addition, we also established the normal control group, IH group, YC-1 administered alone groups.(4)Immunofluorescence and Western blot were used to examine the protein levels of microtubule-associated protein light chain 3-II(LC3-II), HIF-1?, cleaved caspase-3 and HIF-1?-BNIP3-Beclin1 related proteins in all the experiment groups. TUNEL was applied to assay hippocampal neuronal apoptosis rate.Results:(1)Light microscopy showed that the protrusions can be woven into a network on the seventh day.(2) The effects of intermittent hypoxia on autophagy-related protein and HIF-1? protein expression in primary hippocampus neuronal. The detection by Western blotting and immunofluorescence showed that IH exposure significantly upregulated the protein levels of LC3-II and HIF-1?. Moreover, with the extension of IH duration, the proteins expression were gradually increasing, especially in intermittent hypoxia 12 h group(P<0.05).(3)The effect of YC-1 intervention on autophagy-related protein expression in hippocampal neurons: the detection by Western blotting and immunofluorescence showed that the expression of LC3-II in IH group was significantly higher than normal control group(P<0.05), while the expression in IH+YC-1 intervention group was decreased significantly compared with IH group( P<0.05).(4) The effect of YC-1 intervention on apoptosis-related protein expression in hippocampal neurons: the detection by Western blotting showed that the expression of cleaved-caspase-3 in IH group was significantly higher than normal control group(P<0.05), while the expression in IH+YC-1 intervention group was decreased significantly compared with IH group(P<0.05), which reflected the same results in the detection of apoptotic rate by TUNEL assay(P<0.05).(5) The effect of YC-1 intervention on HIF-1?-BNIP3-Beclin1 signalling pathway proteins expression in hippocampal neurons: the detection by Western blotting showed that the expression of HIF-1?, BNIP3 and Beclin1 in IH group was significantly higher than normal control group(P<0.05), while the expression in IH+YC-1 intervention group was decreased significantly compared with IH group(P<0.05).Conclusion: The HIF-1? mediates activation of autophagy induced by IH. YC-1 intervention can reduce the IH-induced hippocampal neuronal apoptosis and autophagy. YC-1 intervention can reduce the IH-induced hippocampal neuronal HIF-1 alpha-BNIP3-Beclin1 signaling pathway. In summary, the process of expression of autophagy mediated by HIF-1 alpha-BNIP3-Beclin1 signaling pathway may aggravate hippocampal neuronal apoptosis induced by IH.