Roflupram Alleviates Cerebral Ischemic Injury Through Suppressing Neuronal Autophagy

Objective:Stroke is an acute brain injury disease.Currently,tissue-type plasminogen activator(tPA)remains the only drug approved by the US Food and Drug Administration(FDA)for the treatment of the cerebral ischemic injury.Due to the limitations of tPA treatment,it is particularly urgent to identify novel treatment strategies.Studies have shown that inhibition of phosphodiesterase 4(PDE4)exerts a protective effect against cerebral ischemic injury.On the other hand,autophagy is involved in the pathology of cerebral ischemia.However,whether neuronal autophagy is involved in the neuroprotective effects of PDE4 inhibition against cerebral ischemia remains unclear.The present study aimed to investigate the potential mechanism of neuroprotection of the PDE4 inhibitor Roflupram(ROF)against cerebral ischima,with a particular focus on autophgy.Methods:(1)Oxygen glucose deprivation(OGD)model was established by using no-glucose DMEM and a hypoxia chamber.HT-22 neuronal cells or primary cortical neurons pre-treated with ROF(20 μM)were exposed to hypoxic conditions for 6 h.Cells were then allowed to recover for 24 h.The cytotoxicity was determined by the LDH assay and the cell viability was determined by the CCK-8 assay after OGD treatment.The levels of synaptic proteins were detected by the Western blotting.TMRE and MitoSOX staining were used to detect the effects of ROF on the mitochondrial function in HT-22 neuronal cells after OGD treatment.Silencing PDE4B was further used to confirm the protective effects of ROF in HT-22 neuronal cells subjected to OGD treatment.(2)We detected the effect of ROF on autophagy-related proteins,such as microtubule-associated protein 1 light chain 3(LC3),sequestosome 1(p62),and Beclin-1 in HT-22 neuronal cells or primary cortical neurons after OGD treatment by the Western blotting.We detected the effects of ROF on acidic vesicles and autophagic vesicles in HT-22 neuronal cells after OGD treatment by LYT and CYTO staining.We detected the effect of ROF on p-AKT/t-AKT and p-mTOR/t-mTOR in HT-22 neuronal cells after OGD treatment by the Western blotting.Explore the effect of autophagy inhibitor 3-MA in HT-22 neuronal cells after OGD treatment by LDH assay and CYTO staining.Explore the effect of AKT inhibitor MK-2206 and mTOR inhibitor Rapamycin(RAPA)in HT-22 neuronal cells after OGD treatment by LDH assay and Western blotting.(3)The middle cerebral artery(MCAO)was established in SD rats by inserting a silicone-coated nylon suture.After 2 h of occlusion,the suture was withdrawn for reperfusion,and rats were received a single intraperitoneal injection of 2 mg/kg ROF.24 h after reperfusion,neurological deficit scores of rats were measured and the brains were removed for TTC staining.We detected the effects of ROF on autophagy-related proteins and synaptic proteins in SD rats subjected to MCAO by the Western blotting.Results:(1)Pre-treatment with ROF(20 μM)significantly reduced the release of LDH from the cytoplasma to the cultured medium.Simultaneously,ROF increased the viability of HT-22 neuronal cells and primary cortical neurons following treatment with OGD.(2)ROF increased the levels of synaptic proteins GAP-43 and synapsin-1 in HT-22 neuronal cells after OGD treatment.(3)ROF increased the fluorescence intensity of mitochondrial membrane potential and decreased the fluorescence intensity of MitoSOX in HT-22 neuronal cells after OGD treatment.(4)Silencing PDE4B decreased the cytotoxicity in HT-22 neuronal cells after OGD treatment.(5)ROF increased the levels of LC3Ⅱ and Beclin-1,decreased the level of p62.We also found that ROF decreased acidic vesicles and autophagic vesicles in HT-22 neuronal cells after OGD treatment.Mechanistically,ROF increased the phosphorylation of AKT and mTOR in HT-22 neuronal cells after OGD treatment.Rolipram(20 μM),a canonical PDE4 inhibitor,was used as a positive control and produced similar effects.(6)The autophagy inhibitor 3-MA decreased the release of LDH,and decreased autophagic vesicles in HT-22 neuronal cells after OGD treatment.The effect of 3-MA is consistent with that of ROF in HT-22 neuronal cells after OGD treatment.(7)AKT inhibitor MK-2206 and mTOR inhibitor RAPA blocked the effect of ROF in HT-22 neuronal cells after OGD treatment.(8)ROF treatment could decreased neurological deficit scores and reduced cerebral infarct volume in SD rats subjected to MCAO.(9)ROF treatment reduced the levels of LC3II and Beclin-1,and increased the levels of GAP-43,Synapsin-1 and PSD95 in SD rats subjected to MCAO.Conclusion:The PDE4 inhibitor ROF attenuated the neuronal damage caused by the OGD treatment.Activation of AKT/mTOR signaling and the subsequent inhibition of autophagy are probably involved in the protection of ROF in neurons.In rats subjected to MCAO,ROF reduced the infarct volume,improved the neurological functions,and increased the levels of synaptic proteins.Consistently,ROF treatment caused an inhibition of autophagy in vivo as well.Together,Our data suggest that ROF is a promising compound for the treatment of cerebral ischemia.