The Protective Effects Of PDE4 Inhibitors Against Cerebral Ischemia-Induced ER Stress And Ferroptosis In Neurons

Objective:Ischemic stroke remains one of the leading causes of human death and adult disability worldwide.Recombinant tissue plasminogen activator（rtPA）is the only FDA-approved drug for ischemic stroke.However,its narrow therapeutic window and the risk of bleeding greatly limit its clinical application.It is extremely urgent to search targets and drugs for the treatment of ischemic stroke.Phosphodiesterase 4（PDE4）is an enzyme highly expressed in neurons,and is responsible for the hydrolysis of the cAMP.Inhibition of PDE4 alleviates infarction caused by cerebral ischemia.However,the underlining mechanism has not been elucidated.Therefore,this manuscript aims to study the protective effect of PDE4 inhibition against cerebral ischemia-induced neuronal death and its underlying mechanism.Methods:（1）SD rats were subjected to 2 h of middle cerebral artery occlusion（MCAO）,followed by 24 h of reperfusion.The cerebral infarction was detected by TTC staining,the neuronal apoptosis in the ischemic penumbra was detected by FJC staining,and the motor dysfunction of rats was measured by the behavioral study.The oxygen-glucose deprivation/reoxygenation（OGD/R）model was established.Cell viability,intracellular oxidative stress,and neuronal apoptosis were detected by the CCK8 assay,CellROX staining,and Western blot.（2）Western blot and immunofluorescence were applied to investigate the effect of Roflumilast（Roflu）on nuclear translocation of nuclear factor erythroid 2-related factor 2（Nrf-2）and endoplasmic reticulum（ER）stress.HT-22 cells were transfected with GSK3β（S9A）plasmid.The effect of GSK3β overexpression on the protective effect of Roflu was investigated.Western blot and immunofluorescence were used to investigate the ER stress in the ischemic penumbra.（3）BODIPY C11 staining was used to detect the level of intracellular lipid peroxidation.Intracellular Fe²⁺ levels were detected using FerroOrange staining.The effect of Roflu on nuclear translocation of Forkhead box 03 A（FoxO3a）and ferroptosis was detected by Western blot.Glutathione peroxidase 4（GPX4）inhibitor RSL3 was used to block the effect of Roflu in ameliorating MC AO-induced cerebral infarction.Results:（1）PDE4 inhibitor,Roflu and FCPR03,alleviates MCAO-induced cerebral infarction and neuronal apoptosis in the ischemic penumbra.Roflu ameliorates MCAO-induced neurological dysfunction in SD rats.Roflu alleviates OGD/R-induced neuronal apoptosis.Roflu alleviates OGD/R-induced oxidative stress in HT-22 cells.Knockdown of PDE4B alleviates OGD/R-induced neuronal apoptosis,while overexpression of PDE4B promotes OGD/R-induced cell death.（2）Roflu alleviates OGD/R-induced ER stress.Overexpression of GSK3β（S9A）blocked the effects of Roflu in promoting Nrf-2 nuclear translocation,decreasing intracellular oxidative stress,and ameliorating ER stress.Roflu alleviates MCAOinduced ER stress in the ischemic penumbra.（3）Roflu ameliorates OGD/R-induced lipid peroxidation and intracellular Fe²⁺accumulation in HT-22 cells.Roflu reverses the change of ferroptosis related protein caused by OGD/R.GPX4 inhibitor RSL3 blocks the protective effect of Roflu in alleviating MCAO-induced cerebral infarction.Conclusion:Inhibition of PDE4 alleviates OGD/R-induced neuronal death,reduces MCAO-induced cerebral infarction,and improves motor dysfunction in SD rats.Mechanistically,inhibition of PDE4 reduces intracellular oxidative stress by activating the AKT/GSK3β pathway and promoting the activation of Nrf-2,thereby alleviating neuronal ER stress caused by cerebral ischemia.In addition,inhibition of PDE4 induces neuronal ferroptosis caused by cerebral ischemia through inhibiting FoxO3a and activating Nrf-2.