The Mechanism For HCN2 Channel-mediated Neuroprotection Through The Regulation Of Autophagy Under Oxygen Glucose Deprivation/Reperfusion In HT22

Objective: Autophagy plays an important role in cerebral ischemia.Studies have indicated that HCN channel is aslo closely related to ischemic brain injury.However,during oxygen glucose deprivation / reperfusion,the correlation between HCN channel and autophagy,and the mechanism behind it have not been well understood.Methods: OGD-Rep model was applied to mimic cerebral ischemia reperfusion injury.CCK8 and Flow cytometer test were used to evaluate the cell viability and apoptosis of HT22 and detect the effect of ZD7288 on OGD-Rep injury.Lentiviral transfection was utilized to specifically knockdown HCN2 subunits.Immunofluorescence was to detect the expression of HCN2 in HT22 subjected to lentivirus and monitor changes of autophagy in HT22 suffering from OGD-Rep injury.Western blotting was applied to analyse the expression of autophagy related proteins.Results: OGD(6h)-Rep(12h)resulted in a moderated morphology and cell viability changes with specifically upregulating HCN2 expression in HT22.Immunofluorescence and Western blotting results showed that lentivirus paticularly knockdown HCN2 subunits without influence on HCN1 subunits.In addition,OGD-Rep significantly induced the expression of autophagy related proteins including upregulation of LC3 ?and ATG5 and downregulation of p-m TOR and p62,but HCN channel blocker,ZD7288,reversed the change of autophagy in HT22 induced by OGD-Rep.,sh HCN2 also had the same effect with ZD7288.However,lysosomal inhibitors,CQ,abolished the influence of ZD7288 and sh HCN2 on OGD-Rep induced autophagy.Conclusion: The current findings suggested that the inhibitory effect of HCN2 subunits relieved the OGD-Rep injury by accalerating autophagy-lysosome degradation.