The Role Of EPAC-mediated Apoptosis In The Secondary Brain Injury Induced By Intracerebral Hemorrhage And Its Potential Mechanism

Part Ⅰ:The protein level of EPAC1 and EPAC2 in brain following ICH in ratsObjectiveTo investigate the protein level of EPAC1 and EPAC2 in brain following ICH in rats and to study the relationships with secondary brain injury(SBI).Methods1.Experimental animal groups:54 health male Sprague-dawley(SD)rats were assigned randomly into 9 groups of 6 rats each,a normal group,a sham group and 7 ICH groups arranged by time:3,6,12,24,48,72 and 7 days after ICH.2.Establishment of ICH model in vivo:a rat ICH model was induced by injection of autologous blood into the caudate nucleus of the brain.All the rats in the experiment were put to death at the indicated time point after ICH and cerebral tissue samples around the hematoma were taken for analysis.3.Establishment and grouping of ICH model in vitro:ICH model in vitro is set up by adding OxyHb into primary rat cortical neurons.Neurons were divided into 6 groups:a normal group and five OxyHb groups arranged by time:3,6,12,24 and 48hours.4.Detection methods:The protein levels of EPAC1 and EPAC2 in brain tissue in each group were detected by western blot method.The protein levels of EPAC1 and EPAC2 in neurocyte.The protein levels of phosphorylated EPAC1 were analysed by using immunoprecipitation reaction.Results1.In vivo experimental results:The Western-blot analysis showed that the protein levels of EPAC2 in brain tissue were higher than in the sham group at 3 hours after ICH and peaked at 6 hour.Unlike the elevations of EPAC2 afte ICH,the total protein levels of EPAC1 in brain tissue have no change.But the protein levels of phosphorylated EPAC1 decreased after ICH.The double immunofluorescence staining demonstrated that both EPAC1 and EPAC2 were expressed in neurons and the expression trend was similar to that of Western-blot analysis.2.In vitro experimental results:The Western-blot analysis showed that the protein levels of EPAC2 in primary rat cortical neurons were higher than in the normal group at 3 hours after ICH and peaked at 6 hour.The total protein levels of EPAC1 in primary rat cortical neurons have no change.But the protein levels of phosphorylated EPAC1 decreased after ICH.Conclusions1.EPAC1 and EPAC2 may play a role in the pathological process of SBI after ICH in rats.2.Six hours after ICH may be the time point for further exploring its possible mechanism.Part Ⅱ:Effects of EPAC2 on SBI after ICH in rats and its possible mechanismsObjectiveTo investigate the effect of EPAC2 on secondary brain injury after ICH in rats and its possible mechanism.Methods1.Experimental animal groups:108 health male SD rats were assigned randomly into 6 groups,including a sham group,ICH group,ICH+Vehicle,ICH+ESI-05(2 mg/kg),ICH+ESI-05(4mg/kg)and ICH+ESI-05(8mg/kg).2.Establishment of ICH model in vivo:a rat ICH model was induced by injection of autologous blood into the caudate nucleus of the brain.3.ESI-05 delivery method:In the ICH+ESI-05 low concentration group,rats were injected intraperitoneally at a dose of 2 mg/kg 1 hour before ICH modeling.In the ICH+ESI-05 medium concentration group,rats were injected intraperitoneally at a dose of 4 mg/kg 1 hour before ICH modeling.In the ICH+ESI-05 hige concentration group,rats were injected intraperitoneally at a dose of 4 mg/kg 1 hour before ICH modeling.4.Establishment and grouping of ICH model in vitro:ICH model in vitro is set up by adding OxyHb into primary rat cortical neurons.Neurons were divided into 6 groups,including a normal group,OxyHb group,OxyHb+rehicle group,OxyHb+ESI-05(2.5μM)group,OxyHb+ESI-05(5μM)group and OxyHb+ESI-05(10μM)group.5.Intervention in vitro model:In OxyHb group:Cells were collected after 6 hours of treatment with OxyHb in neurons.In OxyHb+Vehicle group neurons were pretreatmented with Vehicle for 1 hour,then treated by OxyHb for 6 hours.In OxyHb+ESI-05 low concentration group,neurons were pretreatmented with ESI-05(2.5μM)for 1 hour,then treated by OxyHb for 6 hours.In OxyHb+ESI-05 medium concentration group,neurons were pretreatmented with ESI-05(5μM)for 1 hour,then treated by OxyHb for 6 hours.In OxyHb+ESI-05 high concentration group,neurons were pretreatmented with ESI-05(10μM)for 1 hour,then treated by OxyHb for 6 hours.6.Dectection methods and indicators in vivo:In each group,12 experimental animals were assessed for neurological function 6 hours after establishment of ICH model,and 6 animals were killed 6 hours after establishment of ICH model to obtain samples of brain tissue around hematoma.RAPGEF4 ELISA kit was used to detect EPAC2 protease activity.Western-blot was used to detect the changes of BIM,P38 and Caspase 3 protein levels in brain tissue.TUNEL staining was used to detect apoptotic neurons in brain tissue.FLUORO-JADE B staining was used to detect cerebral cortical cell necrosis.7.Dectection methods and indicators in vitro:RAPGEF4 ELISA kit was used to detect EPAC2 protease activity.Western-blot was used to detect the changes of BIM,P38 and Caspase 3 protein levels in neurons.flow cytometry was used to detect the apoptosis of neurons.Results1.In vivo,compared with sham group:In ICH group,neurobehavioral damage of rats was aggravated(P<0.01);TUNEL-positive neurons increased significantly(P<0.01);FJB-positive cells increased significantly(P<0.01);BIM,P38,Caspase 3 protein levels in brain tissue increased significantly(P<0.01).2.In vivo,compared with Vehicle group,the neurobehavioral damage of rats in ESI-05 medium concentration group was significantly reduced(P<0.01),the number of TUNEL-positive neurons decreased(P<0.01),the number of FJB-positive neurons decreased(P<0.01),and the levels of BIM,P38 and Caspase 3 protein in brain tissue decreased(P<0.05).3.In vitro,the levels of BIM,P38,Caspase 3 protein and the number of apoptotic cells in the OxyHb groups were significantly higher than those in the normal group(P<0.05).4.In vitro,compared with Vehicle group,the levels of BIM,P3 8,Caspase 3 protein and the number of apoptotic cells decreased in ESI-05 medium concentration group(P<0.05).Conclusions1.In vivo,EPAC2 promotes apoptosis through p38/BIM/caspase-3 pathway and participates in the pathological process of SBI after ICH.2.In vitro,EPAC2 promotes neuronal apoptosis through p38/BIM/caspase-3 pathway.3.ESI-05,an inhibitor of EPAC2,may be helpful in alleviating SBI after ICH.Part Ⅲ:Effects of EPAC1 protein on neurons and its possible mechanism in vitro experiment.ObjectiveTo investigate the effect of EPAC1 protein on neurons and its possible mechanism in ICH model in vitroMethods1·Establishment and grouping of ICH model in vitro:ICH model in vitro is set up by adding OxyHb into primary rat cortical neurons.Neurons were divided into 6 groups,including a normal group,oxyHb group,OxyHb+GFP group,OxyHb+WT group,OxyHb+S108E group and OxyHb+S 108A group.2.Grouping method for detecting phosphorylation level of exogenous EPAC1 protein:Neurons were divided into 4 groups,including GFP group,WT group,OxyHb+GFP group and OxyHb+WT group.In GFP group,neurons were treatmented with pEGFP-N2 overexpression plasmid,then incubated for 48 hours.In WT group,neurons were treatmented with EPAC1(WT)-pEGFP-N2 overexpression plasmid,then incubated for 48 hours.In OxyHb+GFP group,neurons were pretreatmented with pEGFP-N2 overexpression plasmid for 48 hours,then treated with OxyHb for 6 hours.In OxyHb+WT group,neurons were pretreatmented with EPAC1(WT)-pEGFP-N2 overexpression plasmid for 48 hours,then treated with OxyHb for 6 hours.3.Grouping method of intervention experiment:neurons were divided into 6 groups,including nornal group,OxyHb group,OxyHb+GFP group,OxyHb+WT group,OxyHb+S 108E group and OxyHb+S 108A group.4.Dectection methods and indicators:The phosphorylation levels of three exogenous EPAC1(WT)-pEGFP-N2,EPAC1(S108E)-pEGFP-N2and EPAC1(S108A)-pEGFP-N2 proteins were analyzed by immunoprecipitation method.Subcellular localization of GFP fluorescence observed by laser confocal microscopy.Western bolt method was used to detect the changes of BIM,P38 and Caspase 3 protein levels in neurons.Results1.Changes of phosphorylation level of exogenous EPACI protein after treatment with OxyHb in EPAC1(WT)-pEGFP-N2 overexpressed neurons for 6 hours:In neurocyte,the exogenous EPAC1(WT)-pEGFP-N2 protein could be phosphorylated at the serine site.Compared with WT group,the phosphorylated protein level in OxyHb+WT group decreased significantly(P<0.01).2.Comparison of phosphorylation degree of three type of overexpressed neuronal cells:Compared with OxyHb+WT group,the phosphorylation level in OxyHb+S108E group was higher(P<0.01);on the contrary,compared with OxyHb+WT group,the phosphorylation level in OxyHb+S108A group was lower(P<0.01).3.Observation of the distribution of GFP fluorescent protein in neurons by laser confocal microscopy:In WT transfection group,green fluorescence aggregated around the membrane of neurons.In the S108E transfection group,no obvious green fluorescence membrane aggregation effect was observed.Membrane aggregation was the most obvious in the S108A transfection group.4.Comparison of BIM,P38 and Caspase 3 protein levels in three overexpressed neurons:Compared with the normal control group,the levels of BIM,P38 and Caspase 3 protein in OxyHb group increased significantly(P<0.01).Compared with OxyHb+WT group,the levels of BIM,P38,Caspase 3 protein in OxyHb+S108E group decreased significantly(P<0.01);on the contrary,compared with OxyHb+WT group,the levels of BIM,P38 and Caspase 3 protein in OxyHb＋S 108A group increased significantly(P<0.01).Conclusions1.In vitro ICH model,EPAC1 regulates neuronal apoptosis through phosphorylation level changes.The phosphorylation of EPAC1 at the serine site hinders the translocation of EPAC1 protein to cell membrane and plays an anti-apoptotic role.On the contrary,the dephosphorylation of EPAC1 is beneficial to the translocation of EPAC1 protein to cell membrane and promotes apoptosis.2.EPAC1-Serl08 phosphorylation site may be a new target for the treatment of ICH.