Research On Mechanisms Of The Changes Of Autophagy In Intermittent Hypoxia-induced Hippocampal Neuronal Injury Mediated By Oxidative Stress

Objective: Obstructive sleep apnea hypopnea syndrome(OSAHS) is the most common sleep and breathing disorders. Neurocognitive dysfunction is one of the common complications of OSAHS, which may be associated with intermittent hypoxia(IH), an important pathophysiological characteristic of OSAHS. IH can lead to hippocampal neuronal damage in varying degrees, such as apoptosis and inflammation. Autophagy, as a widespread degradating/recycling system in eukaryotic cells, has a certain link with ischemia/hypoxia-induced neuronal damage. Our previous studies have demonstrated that autophagy might also be involved in IH-induced hippocampal neuronal injury, but the mechanisms remain to be unclear. Oxidative stress is not only considered to be one of the pathological mechanisms of IH-induced multi-system damage, but also an important mechanism inducing autophagy under cellular stresses. Therefore, we hypothesize that oxidative stress may mediate the changes of autophagy in intermittent hypoxia-induced hippocampal neuronal injury. Thus, this study established IH model in vitro through simulating OSAHS in the purpose of observing the effect on levels of autophagy, ROS and the degree of cell damage by IH and antioxidant intervention, to further investigate mechanisms of changes of autophagy in IH-induced hippocampal neuronal injury mediated by oxidative stress and the role of autophagy and ROS in this process.Methods: The hippocampal tissue of the SD mice born in 24 hours was isolated and inoculated to cell suspension. Morphological changes in hippocampal neurons were observed by an optical microscope on the 1,3,5,7 days of cell cultures. â‘ The petri dishes were randomly divided into four groups: normal control group, the IH 4h group, IH 8h group and IH 12 h group. Each IH group was treated with IH(Circulation patterns: normoxia for 21% O2, 5% CO2 and balanced N2, sustained 10min; hypoxia for 1.5% O2, 5% CO2 and balanced N2, continued 5min), the control group were given continuously normoxia for 21% O2. After treatment of IH, MAP-2 was detected by immunofluorescence to be identified. The expression of autophagy marker protein LC3-II and Beclin1 in each group were detected by Western blotting and immunofluorescence, ROS levels were detected by DCF method. The hippocampal neuronal apoptosis rate was detected through TUNEL method; â‘¡The petri dishes were randomly divided into four groups: normal control group, antioxidant intervention group, IH group, IH group+antioxidant intervention group. LC3-II, ROS, neuronal apoptosis rate and detected respectively through the above detection methods.Results: â‘  Light microscopy detection and identification of hippocampal neurons: Light microscopy showed that branch and bipolar protrusions were appeared on the first day of cell culture. On the third day the protrusions were extended outwardly. To the fifth and seventh day, the protrusions can be woven into a network; The cell bodies and projections of hippocampal neurons showed red fluorescence, indicating MAP-2 expression. â‘¡ The detection of autophagy, ROS expression and apoptosis at different time points in IH: The detection by Western blotting and immunofluorescence showed that the expressions of LC3-II and Beclin1 were gradually increased with the extension of IH time, the expressions in IH 12 h group were significantly higher than the control group(P<0.05). ROS levels were detected by DCF immunofluorescence, the results showed that the expression of ROS in IH 12 h group was remarkably higher than the control group(P<0.05). The detection by TUNEL method showed that hippocampal neuronal apoptotic rate in IH 12 h group was significantly higher than the normal control group(P<0.05). â‘¢ The effect of antioxidant intervention on ROS levels, autophagy and apoptosis 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, while the expression in IH+antioxidant intervention group was decreased significantly compared with IH group(P<0.05). The results of DCF immunofluorescence showed that ROS levels in IH group were also significantly higher than normal control group, while the levels in IH+antioxidant intervention group were also decreased remarkably compared with IH group(P<0.05). The detection of the expression of cleaved-caspase-3 in each group by Western blotting showed that the expression of cleaved-caspase-3 was significantly higher in IH group than normal control group, while the expression was significantly lower in IH+antioxidant intervention group than the IH group, which reflected the same results in the detection of apoptotic rate by TUNEL assay(P<0.05).Conclusion: This study demonstrated that â‘  ROS may involve in the changes of autophagy in hippocampal neurons induced by IH. â‘¡Antioxidant intervention can reduce the IH-induced hippocampal neuronal apoptosis and autophagy. In summary, oxidative stress mediates the changes of autophagy in hippocampal neuronal apoptosis induced by IH, and the process of expression of autophagy mediated by oxidative stress may aggravate hippocampal neuronal apoptosis induced by IH.