The Role Of PERK Pathway In The Secondary Brain Injury Induced By Intracerebral Hemorrhage And Potential Mechanism

Part ?: The expression of p-eIF2? /ATF4 /XBP-1s and ATF6 in brain following ICH in ratsObjective To investigate the expression of p-e IF2 ? /ATF4 /XBP-1s and ATF6 in brain following ICH in rats and to study the relationships with secondary brain injury(SBI).Methods 1.Animal groups: 48 health male Sprague-dawley(SD)rats were assigned randomly into 8 groups of 6 rats each,a sham group and 7 ICH groups arranged by time: 4,8,12,16,24,48 and 72 hours after ICH.2.Animal model making: a rat ICH model was induced by injection of 100 ?L fresh arterial,nonheparinized blood into the brain over 5 min.All the rats in the experiment were killed at the indicated time point after ICH and cerebral tissue samples were taken for analysis.3.In vitro experiment: Primary rat cortical neurons were obtained from 17-day-old SD rat embryos.After a week of incubation,neurons were divided into 8 groups: a control group and 7 Oxy Hb groups arranged by time: 4,8,12,16,24,48 and 72 hours after Oxy Hb treated.To mimic ICH,in Oxy Hb group,neurons were treated with Oxy Hb(10 ?M).4.Detection methods and indicators: The brain cortex of six rats in each group was extracted and used for double immunofluorescence analysisand partial brain samples of the other six were frozen in liquid nitrogen for Western blot analysis.By western blot method to evaluate the expression of p-eIF2? /ATF4 /XBP-1s and ATF6 in brain tissue following ICH in rats at different time points.By immunofluorescence method to evaluate the expression of p-eIF2? and ATF4 in brain tissue following ICH in rats at different time points.Results 1.The western blot analysis showed that levels of p-eIF2? and ATF4 expression in neurons were higher than in the sham group at 4 hours after ICH and peaked at 48 hour,and then decreased.However,at 72 hours after ICH,the protein levels of ATF4 were still visibly higher than that in sham group.The levels of XBP-1s expression in neurons were higher than in the sham group at 4 hours after ICH and peaked at 16 hour,and then decreased.The levels of ATF6 expression in neurons were higher than in the sham group at 12 hours after ICH and peaked at 48 hour,and then decreased.However,at 72 hours after ICH,the protein levels of ATF6 were still visibly higher than that in sham group.2.In vitro,the level of p-eIF2? and ATF-4 were increased significantly from 4 hours after neurons were treated with Oxy Hb,reached the peaks at 48 hours,and remarkable increased of the protein levels of p-eIF2? and ATF-4 in 48 hours group compared with 24 hours group and 72 hours group.3.The double immunofluorescence staining results demonstrated that the expression trend was similar to that of Western blot analysis.Conclusions Endoplasmic reticulum stress is involved in SBI after ICH,and the PERK pathway,IRE1 pathway,and ATF6 pathway of UPR play a role,and the PERK pathway may play a major role.After the ICH,the PERK pathway of neurons in the brain tissue surrounding the SBI hematoma was activated.In an in vitro neuronal ICH model,the PERK pathway may play an important role in post-ICH SBI.Part ?: Experimental study of the effect of intervention in the regulation of PERK pathway on SBI after ICHObjective To observe the effect of intervention in the regulation of PERK pathway on SBI after ICH in rats.Methods 1.Animal groups: 36 health male Sprague-dawley(SD)rats were assigned randomly into 6 groups : sham group,ICH group,ICH + vehicle(GSK2606414)group,ICH + GSK2606414 group,ICH+ vehicle(salubrinal)group,ICH+ salubrinal group,each group with 6 rats.2.Animal model making: ICH model was produced by injecting fresh arterial,nonheparinized blood into the brain.3.Drug Administration :First,GSK2606414 was dissolved in dimethylsulphoxide(DMSO)to 90 ?g/?l and then diluted the store solution to 90 ?g/5 ?l by sterile saline,which was injected intracerebroventricularly(90 ?g/5 ?l).Salubrinal was dissolved in DMSO to 96 ?g/?l and injected intraperitoneally(1mg/kg body weight).The PERK inhibitor GSK2606414 was injected intracerebroventricularly at 1 h after ICH and the eIF2? dephosphorylation inhibitor salubrinal,as a agonist of PERK,was injected intraperitoneally at 30 min before ICH,respectively.4.Detection methods and indicators: At 48 hours after ICH,behavioral activity was examined in all groups.The brain cortexes of six rats were extracted for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining,fluoro-jade B staining.5.In vitro experiment: Primary rat cortical neurons were obtained from 17-day-old SD rat embryos.After a week of incubation,neurons were divided into 6 groups: control,Oxy Hb,Oxy Hb + vehicle(GSK2606414),Oxy Hb + GSK2606414,Oxy Hb + vehicle(salubrinal)and Oxy Hb + salubrinal.To mimic ICH,in Oxy Hb group,neurons were treated with Oxy Hb(10 ?M);in Oxy Hb + vehicle(GSK2606414)group,cells were pretreated with DMSO(volume equal to GSK2606414)for 1 h and then exposed to 10 ?M Oxy Hb;in Oxy Hb + GSK2606414 group,cells were pretreated with GSK2606414(1 ?M)for 1 h and then exposed to 10 ?M Oxy Hb;in Oxy Hb+vehicle(salubrinal)group,cells were pretreated with DMSO(volume equal to salubrinal)for 1 h and then exposed to 10 ?M Oxy Hb;in Oxy Hb +salubrinal group,cells were pretreated with salubrinal(50 ?M)for 1 h and then exposed to 10 ?M Oxy Hb in fresh medium.After incubation for 48 h,cells were fixed with 4 % paraformaldehyde.After these treatments,cellular morphology was observed by inverted phase contrast microscope.The TUNEL staining show the apoptosis rate of neurons.Results 1.In vivo,the histological evidence of neuronal death of ICH group,ICH + vehicle(GSK2606414)group or ICH +vehicle(salubrinal)group compared with the sham group were shown that remarkable neuronal apoptosis at 48 h after ICH in brain tissues,no significant differences in histological evidence of neuronal death were seen between the ICH group,ICH + vehicle(GSK2606414)group and ICH+vehicle(salubrinal)group.Accordingly,experimental ICH evoked a significant increase in FJB-positive cells at 48 h after ICH.Rats subjected to ICH + GSK2606414 demonstrated a significant suppress of neuronal death compared with the ICH + vehicle(GSK2606414)group.In addition,experimental ICH + GSK2606414 evoked a significant suppress in FJB-positive cells compared with the ICH + vehicle(GSK2606414)group at 48 h after ICH.2.In vivo,rats subjected to ICH + salubrinal demonstrated a significant increase of neuronal death compared with the ICH + vehicle(salubrinal)group.Accordingly,experimental ICH + salubrinal evoked a significant increase in FJB-positive cells compared with the ICH + vehicle(salubrinal)group at 48 h after ICH.3.In vitro,neurons subjected to Oxy Hb + salubrinal demonstrated a significant increase of neuronal death compared with the Oxy Hb + vehicle(salubrinal)group.4.The neurological behavior scoring showed that GSK2606414 induced a significant suppression in neurological behavior scoring and salubrinal induced a significant aggravation in neurological behavior scoring.Conclusions Inhibition of PERK pathway reduced apoptosis,promoted neuronal survival and induced a significant suppression in neurological behavior scoring both in vivo and in vitro.Activation of PERK pathway promoted apoptosis,inhibited neuronal survival via inhibition of e IF2 ? dephosphorylation and induced a significant aggravation in neurological behavior scoring both in vivo and in vitro.Part ?: The mechanism of PERK pathway involved in action of SBI study after ICHObjective To investigate its possible mechanism of PERK pathway which participated in the SBI after ICHMethods 1.Experimental animal groups: 36 health male Sprague-dawley(SD)rats were assigned randomly into 6 groups : sham group,ICH group,ICH + vehicle(GSK2606414)group,ICH + GSK2606414 group,ICH+ vehicle(salubrinal)group,ICH+ salubrinal group,each group with 6 rats.2.ICH model: Adult male Sprague-Dawley(SD)rats(280-330 g)were anesthetized with intraperitoneal injection of 4% chloral hydrate(0.1 m L/kg body weight).After the rats were completely anesthetized,they were fixed in the stereotactic frame.Depending on the rat's response to the pain,additional chloral hydrate should be injected.The position of basal ganglia was 0.2 mm posterior to bregma,3.5 mm lateral to the midline,and 5.5 mm ventral to the cortical surface.Subsequently,100 ?L of autologous blood was collected from the heart using a 100 ?L microinjector.After the microinjector was in position,100?L of autologous blood was injected over 5 min.Bone wax was used to block the drilling,and medical suture line was used to stitch the scalp.Next,put the mouse back in the cage and gave enough food and water in the cage.The assessment of SBI occurred 48 hours after the onset of ICH.3.The dosing method: First,GSK2606414 was dissolved in dimethylsulphoxide(DMSO)to 90 ?g/?l and then diluted the store solution to 90 ?g/5 ?l by sterile saline,which was injected intracerebroventricularly(90 ?g/5 ?l).Salubrinal was dissolved in DMSO to 96 ?g/?l and injected intraperitoneally(1mg/kg body weight).The PERK inhibitor GSK2606414 was injected intracerebroventricularly at 1 h after ICH and the eIF2? dephosphorylation inhibitor salubrinal,as a agonist of PERK,was injected intraperitoneally at 30 min before ICH,respectively.4.The detection index: ICH animal model rats were executed after 48 hours.The bottom of the temporal cortex of rats' brain tissue were removed as specimens.Western blot was used to analyze tissue marker protein of endoplasmic reticulum stress (CHOP),p-eIF2?,ATF4 and endoplasmic reticulum stress mediated apoptosis protein(active-caspase-12).5.ICH model in vitro: Primary rat cortical neurons were obtained from 17-day-old SD rat embryos.After a week of incubation,neurons were divided into 6 groups: control,Oxy Hb,Oxy Hb + vehicle(GSK2606414),Oxy Hb + GSK2606414,Oxy Hb + vehicle(salubrinal)and Oxy Hb + salubrinal.To mimic ICH,in Oxy Hb group,neurons were treated with Oxy Hb(10 ?M);in Oxy Hb + vehicle(GSK2606414)group,cells were pretreated with DMSO(volume equal to GSK2606414)for 1 h and then exposed to 10 ?M Oxy Hb;in Oxy Hb + GSK2606414 group,cells were pretreated with GSK2606414(1 ?M)for 1 h and then exposed to 10 ?M Oxy Hb;in Oxy Hb+vehicle(salubrinal)group,cells were pretreated with DMSO(volume equal to salubrinal)for 1 h and then exposed to 10 ?M Oxy Hb;in Oxy Hb +salubrinal group,cells were pretreated with salubrinal(50 ?M)for 1 h and then exposed to 10 ?M Oxy Hb in fresh medium.After incubation for 48 h,cells were fixed with 4 % paraformaldehyde.After these treatments,cellular morphology was observed by inverted phase contrast microscope,and the total protein of the cells was collected and stored at-80 °C for western blot analysis.Results 1.In vivo,the analyses of protein expression in brain tissues from rats subjected to ICH,ICH group,ICH + vehicle(GSK2606414)group or ICH +vehicle(salubrinal)group compared with those from sham group revealed a significant increased the protein levels of p-eIF2?,ATF-4,CHOP,and caspase-12 at 48 h after ICH,no significant differences in the protein levels of p-eIF2?,ATF-4,CHOP,and caspase-12 were seen between the ICH group,ICH + vehicle(GSK2606414)group and ICH+vehicle(salubrinal)group.Our results shown that ICH+ GSK2606414 group compared with those from ICH + vehicle(GSK2606414)group revealed a significant suppressed expression of CHOP,caspase-12,p-eIF2?,ATF-4 at 48 h after ICH.2.In vivo,the analyses of protein expression in brain tissues from rats subjected to ICH,ICH+ salubrinal group compared with those from ICH + vehicle(salubrinal)group revealed a significant increased expression of CHOP and p-eIF2? at 48 h after ICH,and promoted the protein levels of ATF-4 and caspase-12 too.3.In vitro,the analyses of protein expression in OxyHb-treated neurons,OxyHb group,Oxy Hb + vehicle(GSK2606414)group or Oxy Hb +vehicle(salubrinal)group compared with those from control group revealed a significant increased expression of p-eIF2?,ATF-4,CHOP,and caspase-12 at 48 h after Oxy Hb-treated,no significant differences in the protein levels of p-eIF2?,ATF-4,CHOP,and caspase-12 were seen between the Oxy Hb group,Oxy Hb + vehicle(GSK2606414)group and Oxy Hb +vehicle(salubrinal)group.The results demonstrated that Oxy Hb + GSK2606414 group compared with those from Oxy Hb + vehicle(GSK2606414)group revealed a significant suppressed expression of CHOP,caspase-12,p-eIF2?,ATF-4 at 48 h after Oxy Hb-treated.4.In vitro,the analyses of protein expression in neurons subjected to Oxy Hb,Oxy Hb + salubrinal group compared with those from Oxy Hb + vehicle(salubrinal)group revealed a significant increased expression of CHOP,p-eIF2?,ATF-4 at 48 h after Oxy Hb-treated,and promoted the protein levels of caspase-12 too.Conclusion Inhibition of PERK suppressed the protein levels of p-eIF2?,ATF4,CHOP,and caspase-12 both in vivo and in vitro.Inhibition of eIF2? dephosphorylation increased the protein levels of p-eIF2?,ATF4,CHOP,and caspase-12 both in vivo and in vitro.