The Role And Mechanism Of Phosphodiesterase-4 Regulated SIRT1/Akt Pathway In Early Brain Injury After Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage(SAH)is considered to be a serious cerebrovascular disease with high rates of mortality and disability.Intense studies have indicated that early brain injury(EBI)is the primary cause of poor outcome after SAH.The exact mechanisms of EBI remain controversial.Among the multiple mechanisms involved,apoptosis has been identified to play an important role in the pathological process of EBI.Growing bodies of studies have demonstrated that suppressing neuronal apoptosis could attenuate EBI and improve the neurological function after SAH.Thus,more efforts are needed in order to explore the mechanisms of apoptosis and develop novel therapeutic strategies against neuronal apoptosis to improve the outcome of patients with SAH.Phosphodiesterase-4(PDE4)is a high-affinity enzyme that specifically hydrolyzes cAMP,which typically acts as the second messenger and mediates a number of cellular process.Previous studies indicated that PDE4 inhibitor rolipram exerts neuroprotective role in a range of central nervous system insults including spinal cord injury,ischemic stroke,and Alzheimer's disease.In spite of great number of studies have been carried out to define the mechanisms of PDE4 mediated brain injury for years,the underlying mechanisms are not fully understood.Recent study indicated that PDE4 regulates the expression and function of Sirtuinl(SIRT1).SIRT1 is a member of(NAD+)-dependent protein deacetylases.A growing body of evidence indicates that SIRT1 modulates a variety of cellular functions via deacetylating its target proteins,including p53,Fox03,IRS-2,and nuclear factor kappaB(NF-?B).Recent studies revealed that SIRT1-overexpression could increase the activity of Akt.The activation of SIRT1/Akt signaling exerts neuroprotective effects against ischemic stroke via enhancing angiogenesis and neurogenesis.inhibition and the activation of SIRTl/Akt pathway in the pathophysiological process after SAH remains unclearly.The present study,therefore,was aimed to investigate the role of PDE4 inhibition through rolipram against SAH and explore the potential role of SIRT1/Akt pathway in rolipram mediated neuroprotection.MethodsPart 11.We performed endovascular perforation techniquethe to establish the SAH model.All experimental Sprague Dawley rats were divided into sham group,SAH 3hour group,SAH 6hour group,SAH 12hour group,SAH 24hour group,and SAH 72hour group in random.Then we evaluated the brain edema and neurological deficit scores.The expression of PDE4,SIRT1.p-Akt and Akt were evaluated by western blot analysis.Double Immunohistochemistry staining were used to localize the expression of PDE4.Part 21.At 2 hour after SAH onset,we treated experimental Sprague Dawley rats with intraperitoneal injection of vehicle or PDE4 inhibitor rolipram.Experimental Sprague Dawley rats were divided into sham group,SAH+vehicle group,SAH+ rolipram group in random.2.At 24 hours after SAH,we measured neurologic scores and SAH grades in each group.Double immunohistochemistry staining of terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)and NeuN were also evaluated in every group at 24 hours after SAH.The Western blot analysis was used to meature the expression of PDE4,SIRT1,p-Akt,Akt,Bcl-2,Bax and Caspase-3.In all,PDE4 inhibitor rolipram performed the neuroprotective effect.Part 31.Adult male Sprague Dawley rats were administrated with vehicle,PDE4 via I.P or SIRT1 inhibitor sirtinol,Akt inhibitor MK2206 via I.C.V.The rats were divided into SAH+vehicle group,SAH+ rolipram group,SAH+ rolipram+ sirtinol group and SAH+ rolipram + MK2206 group in random.2.SAH grade,neurologic score,and brain water content were measured at 24 hours after SAH in all groups.Double immunohistochemistry staining of terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)and NeuN were also performed in each group at 24 hours after SAH.Western blot analysis was performed to meature the expression of SIRT1?p-Akt?Akt Bcl-2,Bax and Caspase-3.These experiments were set to explore the mechanism of PDE4 inhibitor rolipram against brain injury after subarachnoid hemorrhage.ResultsPart 11.Western blot analysis showed that the expression of PDE4 and p-Akt was significantly degraded after SAH,and lowest at 24 hours after SAH.However the the expression of SIRT1 was significantly elevated three hours after SAH,and lowest at 24 hours after SAH..Double immunostaining of PDE4 with NeuN suggested that PDE4 was significantly expressed in neurons.In addition,PDE4 was slightly expressed astrocyte and microglia.Part 21.Post-SAH treatment of PDE4 inhibitor rolipram significantly reduced the expression of PDE4,ameliorated the neurological deficits after SAH.2.Administering rolipram significantly elevate the expression of SIRT1 compared to the SAH+vehicle group.The ratio of Bcl-2/Bax was significantly reduced,and the expression level of cleaved caspase-3 was significantly increased in the SAH+vehicle group.The administration of rolipram significantly reversed these processes.TUNEL staining localized with the neuronal marker NeuN.The total number of TUNEL and NeuN double-stained cells significantly increased at 24 hours after SAH.Treatment of rolipram significantly reduced the number of TUNEL-positive neurons.The inhibition of PDE4 by rolipram significantly promoted cell survival by increasing the Bcl-2/Bax ratio and reducing cleaved caspase-3 expression.Part 31.Precondition of SIRT1 inhibitor sirtinol aggravated brain edema and the neurological deficits after PDE4 inhibitor rolipram treatment in SAH rats.The increased expression of Bcl-2 and decreased expression of Bax,Cleaved Caspase-3 by rolipram treatment was significantly reversed after sirtinol administration.;Meanwhile,the expression of p-Akt was decreased.2.Post-SAH treatment of Akt inhibitor MK2206 aggravated brain edema and the neurological deficits after PDE4 inhibitor rolipram treatment in SAH rats.The increased expression of Bcl-2 and decreased expression of Bax,Cleaved Caspase-3 by rolipram treatment was significantly reversed after MK2206 administration;Meanwhile,there was no-significant in the expression of SIRT1Conclusion1.PDE4 participated in early brain injury after SAH,and was widely distributed in neurons,slightly in astrocytes and microglias.Moreover,the expression of SIRT1/Akt pathway related protein significantly changed.These results indicated tha PDE4 and SIRT1/Akt pathway may play a key role in SAH induced early brain injury.2.Treatment of PDE4 inhibitor rolipram can significantly increase the expression of SIRT1 and p-Akt,inhibited neuronal apoptosis,improved neurological dysfunction,reduced cerebral edema in early brain injury after SAH.3.Inhibition of PDE4 may have the potential to reduce early brain injury after SAH via SIRT1/Akt related anti-apoptosis pathways.