| Ischemic stroke is an acute cerebrovascular disease with high morbidity,mortality,and disability,which seriously endangers human life and health.With the development of medical technology and medical equipment,the treatment of stroke has made a great leap,but the ischemia-reperfusion(I/R)injury following vascular recanalization is inevitable,which seriously restricts the effect of treatment and affects the prognosis of patients.Adiponectin(APN)is an adipose-derived plasma protein.Studies have shown that APN has a good therapeutic and protective effect in the process of myocardial ischemia-reperfusion(I/R)injury,while whether the protective effect of adiponectin exists in the process of cerebral ischemia-reperfusion injury and its related detailed mechanisms still need further study.The aim of this study was to investigate the effects of APN on the brain I/R injury and its possible molecular mechanisms.In this study,the middle cerebral artery occlusion(MCAO)in C57BL/6 mice was used as a stroke model to simulate the pathological process of cerebral ischemia.The mice pretreated exogenous adiponectin were used to explore the protective effects of adiponectin on cerebral ischemia-reperfusion injury and its specific molecular pathways and mechanisms.This study will provide new evidence for the neuroprotective effect of adiponectin in the process of cerebral ischemia-reperfusion injury,and provide a new theoretical basis for the clinical application of adiponectin to intervene the prognosis of patients with ischemic stroke.This study is divided into the following two parts:1.Protective effects of adiponectin on cerebral ischemia-reperfusion injury in mice.2.The role and mechanism of the cAMP/PKA pathway in the neuroprotective effect of adiponectin.Part Ⅰ Protective effects of adiponectin on cerebral ischemia-reperfusion injury in miceMethodsC57BL/6 mice were randomly divided into sham operation group,I/R group,vehicle+I/R group and APN+I/R group.APN was administered three times(0 h,12 h,24 h)before middle cerebral artery occlusion(MCAO).After 2 hours of middle cerebral artery occlusion,the filament was pulled out and cerebral blood flow was reperfused for 24 hours to simulate cerebral ischemia-reperfusion injury.After 24 hours of reperfusion,cerebral infarction volume,neurological deficit score,and brain water content were measured to evaluate the brain protective effect of adiponectin.TUNEL staining was used to detect apoptotic cells in the ischemic penumbra of mice brain.At the same time,the brain tissue of ischemic penumbra was extracted 24 hours after reperfusion,and the expression of active caspase-3,Bax and Bcl-2 in the brain tissue of ischemic penumbra was detected by Western blot to evaluate the effect of adiponectin on apoptosis of ischemic penumbra cells.Results1.Compared with the sham group,cerebral infarction volume,neurological deficit score and brain water content increased after cerebral ischemia-reperfusion injury.Compared with vehicle+I/R group,cerebral infarction volume,neurological deficit score and brain water content in APN + I/R group were decreased.2.Compared with sham group,TUNEL positive cells increased after cerebral ischemia-reperfusion injury.Compared with vehicle+I/R group,TUNEL positive cells in APN+I/R group decreased.Compared with sham group,active caspase-3 and Bax increased and Bcl-2 decreased after cerebral ischemia-reperfusion injury.Active caspase-3 and Bax in APN+I/R group were lower than those in vehicle+I/R group,while Bcl-2 in APN+I/R group was higher than that in vehicle+I/R group.ConclusionsAdiponectin preconditioning can alleviate the injury after cerebral ischemia-reperfusion in mice and effectively inhibit cell apoptosis in ischemic penumbra.Part Ⅱ The role and mechanism of cAMP/PKA pathway in the neuroprotection of adiponectinMethodsC57BL/6 mice were randomly divided into sham operation group,I/R group,vehicle+I/R group and APN+I/R group.The expression of cAMP,PKA,P-CREB and BDNF in ischemic penumbra was detected by Western blot and Immunofluorescence after 24 hours of reperfusion.Then the mice were randomly divided into vehicle+I/R group,APN+I/R group,PKA activator group,PKA inhibitor+APN+I/R group.The expression of cAMP,PKA,P-CREB and BDNF in ischemic penumbra was detected by Western blot after 24 hours of reperfusion again under the action of PKA activator and PKA inhibitor.After 24 hours of reperfusion,cerebral infarction volume,neurological deficit score and brain water content were measured to evaluate the effects of PKA inhibitor on the brain protective effect of adiponectin.Cell apoptosis of brain tissue in ischemic penumbra of mice was detected again by TUNEL staining.At the same time,the brain tissue of ischemic penumbra was extracted 24 hours after reperfusion under the action of PKA activator and PKA inhibitor.Western blot was used to detect the expression of active caspase-3,Bax and Bcl-2 in the brain tissue of ischemic penumbra.From which,the effect of PKA inhibitor on the anti-apoptotic effect of adiponectin in ischemic penumbra was evaluated.Results1.Compared with sham group,the expression of cAMP,PKA,P-CREB/CREB and BDNF in ischemic penumbra decreased after cerebral ischemia-reperfusion injury.Compared with vehicle+I/R group,the expression of cAMP,PKA,P-CREB/CREB and BDNF in APN+I/R group increased.2.Compared with vehicle+I/R group,the expressions of cAMP,PKA,P-CREB/CREB and BDNF in APN+I/R group and PKA activator +I/R group increased.Compared with APN+I/R group,the expression of cAMP,PKA,P-CREB/CREB and BDNF in PKA inhibitor+APN+I/R group decreased.3.Compared with vehicle+I/R group,cerebral infarction volume,neurological deficit score and brain water content in APN+I/R group and PKA activator+I/R group decreased.Compared with APN+I/R group,the cerebral infarction volume,neurological deficit score and brain water content of PKA inhibitor+APN+I/R group increased.4.Compared with vehicle+I/R group,TUNEL positive cells in APN+I/R group and PKA activator+I/R group decreased.Compared with APN+I/R group,TUNEL positive cells in PKA inhibitor+APN+I/R group increased.Active caspase-3 and Bax of APN+I/R group and PKA activator+I/R group decreased,while the Bcl-2 increased,when compared with vehicle+I/R group.Compared with APN+I/R group,the active caspase-3 and Bax of PKA inhibitor+APN+I/R group increased,while the Bcl-2 decreased.ConclusionsThe neuroprotective effect of adiponectin may be mediated by cAMP/PKA-CREB-BDNF pathway. |
