The Effects Of Chloroquine Inhibited Autophagy On Hippocampal Neuron(HT22 Cells) Exposed To Hypoxia-reoxygenation Injury

Objective:This study simulated cerebral ischemia-reperfusion injury,replication hippocampal neurons(HT22 cells) hypoxia-reoxygenation model. Observevation of hypoxia-reoxygenation impact on hippocampal neurons autophagy.To investigate the role of autophagy in the injury of hippocampal neurons induced by hypoxic-reoxygenation.Method:1.According to the literatures, hypoxic cell culture was used a closed device. After HT22 cells reached 80% confluency, put the cells into the closed device, and inject into the mixed gas, including95%N2+5%CO2.Experiment is divided into three groups,with the development of the corresponding time normal controls,observation of hypoxia 1h 、 3h and 6h impact on HT22 cells autoghagy and function.Detection indicators includes, Western blot autophagy-related protein Beclin-1, LC3 Ⅱ / Ⅰ change ratio and P62 expression levels, MTT colorimetric detection of cell viability, lactate dehydrogenase(LDH) kit colorimetric assay HT22 cells function. 2.Oxygen recovery after 1hoxygen supply, copy of hypoxia-reoxygenation injury model.Divided into three groups: compared with corresponding time of normal training and lack of oxygen, oxygen observed after 1h, 3h, 6h effects on autophagy HT22 cells. 3. With hypoxia 1h after reoxygenation 1h for hypoxia-reoxygenation model。Observe the effect of autophagy inhibitor chloroquine hippocampal neuronal(HT22 cells) function.To divide the cultured HT22 cells into four groups:(1)control group(2)control +chloroquine group(3)H/R group(4)H/R+ chloroquine group. We detect the function of cell after treating with chloroquine by LDH method and test the autophagy-related protein by western blot in H/R group and H/R+chloroquine group.Result:1.In the HT22 cells hypoxia 1h、3h and 6h,autophagy-related protein Beclin-1 expression increased,the ratio of LC3II/I increased,P62 protein expression were reduced.Cell viability decreased,LDH release were increased;autophagy extent to which the most prominent group of hypoxia 1h. 2.HT22 cells reoxygenation 1h, 3h,6h, and the corresponding time of cultivation and lack of oxygen time compared with normal group autophagy related proteins Beclin 1 increased, the ratio of LC3II/I increased, P62 protein expression were reduced,Reoxygenation 1h autophagy level most prominent. 3.Chloroquine inhibition of HT22 cells by hypoxia-reoxygenation induced autophagy, autophagy-related protein Beclin-1 expression was decreased, the ratio of expression of LC3II/I andP62 protein increased cell damage mitigation.Conclusions:1.Hypoxia induced autophagy and causing the cell injury.2.HT22 cells hypoxia reoxygenation induced autophagy more significant.3.Autophagy inhibitors CQ may be reduce the cell damage in reoxygenation, suggesting that autophagy might have weakened the protection of ischemic-reperfusion effect.