Role Of ERK Pathway Mediated Golgi Stress In Cerebral Ischemia-reperfusion Injury

Background:Cerebral ischemia-reperfusion injury(CIRI),characterized by aggravated nerve damage and dysfunction,is the most common complication occurs in the stroke patients following thrombolytic therapy.Previous studies have demonstrated that oxidative stress plays a key role in the pathophysiology of CIRI through the overproduction of free oxygen radicals,which results in substantial cell apoptosis and death.In the process of oxidative stress,Golgi apparatus(GA)act as a key organelleby participating in signal transduction,homeostasis and cell apoptosis;this effect is named as Golgi Apparatus Stress(GAS).A growing number of evidence has revealed that activation of ERK pathway contributes to the neuronal damage caused by oxidative stress.Furthermore,previous research reported that the ERK mediate phosphorylation of the Golgi reassembly and stacking protein 65(GRASP65),thereby resulting in cisternae disaggregation.However,most previous studies are in vitro cell test,whether the ERK pathway can be activated on GA in CIRI in vivo remains unknown.Objective:This study was aimed to verify whether the ERK pathway can activate the phosphorylation of Golgi body structure protein GRASP65 on GA in CIRI both in vitro and in vivo.In addition,Effect of NBP and its underlying mechanisms were explored.Methods:Cultured mouse cerebral cortical neurons and an oxygen glucose deprivation reperfusion(OGD/R)model were used.Changes of Golgi body morphology were observed with immunofluorescence laser confocal to determine the existence of Golgi stress in CIRI.On this foundation,the experiment was divided into two parts.Part one was in vitro cell test where mouse cerebral cortical were divided into control group,OGD/R group and drug pretreatment group.The latter two groups were divided into subsets according to different observation time at 4h,12h and 24h.DL-3-n-butylphthalide(NBP)was used as pretreatment drug.Immunofluorescence laser confocal was used to observe the expression of GRASP65、pGRASP65 and the morphological changes of Golgi apparatus.Western blot was adopted to measure the expression of ERK,pERK,GRASP65 and pGRASP65.Part two was in vivo animal test where the Pulsinelli’s four-vessel occlusion method was used to establish the mice model of Cerebral ischemia.A total of 100ICR micewere divided into sham-operation group,control group,pre-treated group,treat group and pre-treated plus treat group.The intervention drug was NBP.HE staining was used to observe the morphological changes of neurons in each group on Day 5 after reperfusion.TTC staining was used to estimate the global cerebral ischemia range of mice in each group on Day 5 after reperfusion.Theneurological severity score(NSS)was used to evaluate the neurological damage of mice in each group on Day 5 after reperfusion.Xanthine oxidase method and TBA colorimetric method were used to determine the content of SOD and MDA in brain tissue of mice in each group on Day 5 after reperfusion.Western blot was used to measure the expression of ERK,pERK,GRASP65 and pGRASP65 in each group after reperfusion 5d.Results:In OGD/R model,the morphological changes of Golgi bodies observed with immunofluorescence laser confocal showed that Golgi stress existed in CIRI.In vitro cell test,western blot results showed that expression of ERK and GRASP65 has no significant difference across different groups(P>0.05).Meanwhile,expression of pERK significantly increased(P<0.01)at 12h、24h point in OGD/R group,compared with the control group,indicating that ERK activation occurred in CIRI.NBP intervention weakened the pERK expression at 12h、24h point compared with that in OGD/R group(P<0.01),Showing that NBP intervention successfully suppressed ERK activation in CIRI.Immunofluorescence confocal studies demonstratedin control group that GRASP65 markered Golgi apparatus was dense,whereas pGRASP65 was not significant expressed.In OGD/R group,expression of GRASP65 showed no significant difference with that in control group,while pGRASP65 expression was significantly increased.In OGD/R 4h subgroup,pGRASP65 expression was increased.In OGD/R 12h subgroup,pGRASP65 expression was further increased.In OGD/R 24h subgroup,There was no significantly change of pGRASP65 expression compared with that in 12h subgroup.Meanwhile,the structure of Golgi apparatus was loose and the Golgi apparatus fragmentation was visible in OGD/R group.After pretreated with NBP,pGRASP65 expression was significantly weakened than that in OGD/R group at all time points,suggesting that NBP intervention inhibited GRASP65 phosphorylation.Meanwhile,the structure of Golgi apparatus in NBP group was better than in OGD/R group.It was suggested that inhibition of ERK activation can improve the morphological changes of Golgi apparatus in CIRI.As for vivo animal test,on Day 5 after reperfusion,HE staining showed in sham-operated group,cells were densely arranged and had normal nuclear morphology with uniform stain.whereasin control group,cells were loosely arranged and the neurons were denatured.After NBP intervention,cell morphology of was significantly improved compared with the control group(P<0.05).Pre-treated plus treat group was superior to the pre-treated group and treat group in cell morphology.TTC results showed in sham-operated group that brain tissue was uniformly stained and exhibited no significant infarction.Control group had significantly increased infarct size than sham-operated group and NBP intervened group(P<0.01).Furthermore,Pre-treated plus treat group had significantly lower infarct size than pre-treated group and treat group(P<0.01).Pre-treated group had further lower infarct size compared with treat group(P<0.01).NSS score results demonstrated that pre-treated group(P<0.05)and pre-treated plus treat group(P<0.01)had lower neurological severity scores than control group while treat group showed no significant difference compared with the control group(P>0.05).The results of SOD content in the brain tissue showed that SOD of the control group was significantly lower than that of the sham group(P<0.01).NBP intervention significantly decreased the level of SOD content in pretreat group(P<0.01 and P<0.05).The results also showed that the MDA content of the control group was significantly increased than that of the sham operation group(P<0.01).Meanwhile,pretreatment plus treatment and pretreatment but not treat group(P>0.05)significantly increased(P<0.01 and P<0.05,respectively)the MDA content,compared with the OGD/R group.Expression of ERK and GRASP65 were determined with western blot and has no significant difference among all groups(P>0.05).Meanwhile,expression of pERK significantly increased(P<0.01)in control group compared with sham-operated group group,and expression of pERK significantly increased(P<0.01 and P<0.05)in control group compared with NBPintervention group.The fluctuation trend of pGRASP65 in each group was in accordance with that of pERK.Conclusion:ERK pathway was activated during ischemia-reperfusion and was involved in CIRI,perhaps via phosphorylation of GRASP65.Inhibition of the activation of ERK has protective effects againstCIRI.Golgi apparatus underwent irregular morphological changes and fragmentation in CIRI,which may be associated with ERK mediated GRASP65 phosphorylation.NBP may alleviate by inhibiting ERK mediated phosphorylation of GRASP65.