Mitochondrial Mechanisms Of Extracellular Signal Kinase Inhibitor PD98059 In Attenuating Brain Injury After Cardiopulmonary Resuscitation In Rats

Background and objectiveBrain injury after cardiopulmonary resuscitation(CPR)is an important factor for the high mortality and disability rate in cardiac arrest(CA)patients after successful resuscitation.It is actually cerebral ischemia-reperfusion injury.Mitochondria are important targets in cerebral ischemia-reperfusion injury.Extracellular signal-regulated kinase(ERK)is a mitogen-activated protein kinase,which can regulate cell growth,division,proliferation,apoptosis and other physiological processes through phosphorylation.It can be activated in various stress states.Our previous studies have confirmed that ERK was highly activated in rat CA/CPR model and ERK inhibitor PD98059 showed the effects of increasing survival rate,improving neurological function and inhibiting apoptosis in rats.However,the mechanism of these phenomena which is related to the protective effect of PD98059 on the structure and function of brain mitochondria under ischemia-reperfusion injury remains unclear.Based on the previous work,this study aims to establish a CA/CPR rat model,to focuse on the effects of PD98059 on the structure and function of brain mitochondria at different time points after resuscitation and to explore its molecular mechanism of inhibiting brain injury after resuscitation.MethodSD male rats were randomly divided into five groups:sham operation group,CPR group,dimethylsulfoxide(DMSO),PD98059 0.15 mg/kg group,PD980590.3 mg/kg group.In addition to sham-operated group,the other four groups of rats were induced into CA by esophageal electrical stimulation and then CPR.One minute after resuscitation,rats were randomly administered in the same volume via femoral vein by 0.9%saline,DMSO,0.15mg/kg PD98059 or 0.3mg/kg PD98059,respectively.The 24 hours and 48 hours after resuscitation were selected as observation time points to detect related indictors in each group.The experiment was carried out in two stages.Firstly,the effects of PD98059 on survival rate and neurological function of resuscitated rats were observed at macro level.Secondly,we observed the effects of PD98059 on the structure and function of mitochondria in cerebral cortex of resuscitated rats and the related molecular mechanisms.The detection indicators include the following aspects:1.The survival rate of rats in each group was calculated and the neurological deficit score was used to evaluate the neurological impairment of surviving rats in each group.2.Detection of the structure and function of mitochondria in the cerebral cortex of surviving rats in each group.The damages of mitochondria in cerebral cortex were observed under transmission electron microscopy.Adenosine triphosphate(ATP)contents were detected in rat fresh cortical tissue.Mitoehondria of eerebral eortex were extracted by differential eentrifugation and used to determine the activities of mitochondrial respiratory chains ?,?,?,?and mitochondrial succinate dehydrogenase activities,respectively.3.Detection of molecular indicators of mitochondrial-related apoptotic pathways in cerebral cortex of surviving rats at two time points.The rat brain paraffin sections were prepared and apoptosis was detected by TUNEL kit;Mitochondrial permeability transition pore(MPTP)in cerebral cortex was determined by calcein fluorescence detection.The expressions of cytochrome C(CytC),BCL-2,BAX and cleave-caspase 3 were determined by western blotting.4.Detection of molecular indicators of autophagy associated with mitochondria and co-expression of autophagy and apoptosis in cerebral cortex of surviving rats at two time points.Autophagy was observed under transmission electron microscopy.The expression of LC3,Beclin-1 and P62 in cerebral cortex was detected by Western blotting.The expression of LC3 in the cerebral cortex was observed by immunofluorescence method and the co-expression of TUNEL and LC3 was detected by immunofluorescence double staining.5.Detection of mitochondrial dynamic proteins in cerebral cortex of surviving rats at two time points and co-expression of P-DRP1 with apoptosis and autophagy,respectively.The expression of mitofusin 2(MFN2)and Dynamin-related protein 1(DRP1)in cerebral cortex was determined by western blotting.The co-expressions of P-DRP1+TUNEL and P-DRP1+LC3 were detected by immunohistochemical double staining.Result1.Survival rate,neurological function score.?Survival rate.Compared with sham-operated group,the survival rates in CPR group decreased significantly at 24h and 48h post-resuscitation(P<0.01,P<0.001).Compared with CPR group,the survival rates in PD98059 groups increased in a dose-dependent manner at 24h and 48h post-resuscitation,(P<0.05,P<0.001).?Neurological function score.Compared with sham-operated group,the neurological function score in CPR group decreased significantly at 24h and 48h post-resuscitation(P<0.01,P<0.001).Compared with CPR group,the neurological function score in PD98059 groups increased in a dose-dependent manner at 24h and 48h post-resuscitation(PD98059 0.3mg/kg:P<0.01,P<0.001).2.Relevant indicators of mitochondrial structure and function.?Mitochondrial injury score.Compared with sham-operated group,the cerebral cortex mitochondrial injury score in CPR group increased 24 h and 48 h post-resuscitation(P<0.01,P<0.001).Compared with CPR group,the cerebral cortex mitochondrial injury score in PD98059 groups decreased in a dose-dependent manner at 24h and 48h post-resuscitation(PD98059 0.3mg/kg:P<0.01,P<0.001).?ATP content.Compared with sham-operated group,ATP content of cerebral cortex decreased significantly at 24h and 48h post-resuscitation in CPR group(P<0.001,P<0.01).Compared with CPR group,ATP content of cerebral cortex increased in a dose-dependent manner at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.001,P<0.05).?Mitochondrial respiratory chain activities.Compared with sham-operated group,the activity of mitochondrial respiratory chain ? in cerebral cortex decreased at 24h post-resuscitation in CPR group(P<0.01).Compared with CPR group,the activity of mitochondrial respiratory chain ? in cerebral cortex increased in a dose-dependent manner at 24h post-resuscitation in PD98059 groups(P<0.01).?Mitochondrial succinate dehydrogenase activity.Compared with sham-operated group,the activity of mitochondrial succinate dehydrogenase in cerebral cortex decreased at 24h post-resuscitation in CPR group(P<0.05).Compared with CPR group,the activity of mitochondrial succinate dehydrogenase in cerebral cortex increased in a dose-dependent manner at 24h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.05).3.Relevant indicators of mitochondrial apoptotic pathway.?Apoptotic index.Compared with sham-operated group,the apoptotic index of cerebral cortex in CPR group increased at 24h and 48h post-resuscitation(P<0.01,P<0.001).Compared with CPR group,the apoptotic index of cerebral cortex in PD98059 groups decreased in a dose-dependent manner at 24h and 48h post-resuscitation(PD98059 0.3mg/kg:P<0.001,P<0.001).?The opening of MPTP.Compared with sham-operated group,the opening of MPTP in cerebral cortex increased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.01).Compared with CPR group,the opening of MPTP in cerebral cortex decreased in a dose-dependent manner at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.05,P<0.05).?CytC expression in cytoplasm.Compared with sham-operated group,CytC in cerebral cortex cytoplasm increased at 24h and 48h post-resuscitation in CPR group(P<0.05,P<0.05).Compared with CPR group,CytC in cerebral cortex cytoplasm decreased in dose-dependent at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.05,P<0.05)·?BCL-2/BAX.Compared with sham-operated group,BCL-2/BAX in cerebral cortex decreased at 24h and 48h post-resuscitation in CPR group(P<0.05,P<0.05).Compared with CPR group,BCL-2/BAX in cerebral cortex increased in a dose-dependent manner at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.01,P<0.05).? Cleave-caspase 3 expression.Compared with sham-operated group,cleave-caspase 3 in cerebral cortex increased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.01).Compared with CPR group,cleave-caspase 3 in cerebral cortex decreased in a dose-dependent manner at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.15mg/kg:48h P<0.05;PD98059 0.3mg/kg:P<0.05,P<0.05).4.Autophagy-related indicators and co-expression of autophagy and apoptosis.? Electron microscopy of autophagy.The electron microscopy showed that autophagy existed in all groups.However,autophagy at all stages can be found and the number of autophages was the most in CPR group.?LC3?/LC3I.Comparing with sham-operatedgroup,LC3?/LC3I of cerebral cortex increased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.05).Compared with CPR group,LC3II/LC3I of cerebral cortex decreased in dose-dependent at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.05,P<0.05).?Beclin-1 expression.Compared with sham-operated group,Beclin-1 of cerebral cortex increased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.01).Compared with CPR group,Beclin-1 of cerebral cortex decreased in a dose-dependent manner at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.05,P<0.05).?P62 expression.Compared with sham-operated group,P62 of cerebral cortex decreased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.001).Compared with CPR group,P62 of cerebral cortex increased in a dose-dependent manner at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mg/kg:P<0.001,P<0.001).?Immunofluorescence double staining of TUNEL and LC3.It showed that there was co-expression of TUNEL positive cells and LC3 in cerebral cortex.Compared with sham-operated group,the co-expression of TUNEL positive cells and LC3 increased at 24h and 48h post-resuscitation in CPR group(P<0.001,P<0.001),while the co-expression of TUNEL positive cells and LC3 decreased at 24h and 48h post-resuscitation in PD98059 groups(PD98059 0.3mgkg:P<0.001,P<0.001).5.Expression of mitochondrial dynamic proteins MFN2 and P-DRP1 and co-expression of P-DRP1+TUNEL and P-DRP1+LC3.?MFN2 expression.Compared with sham-operated group,MFN2 in cerebral cortex decreased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.05).Compared with CPR group,MFN2 in cerebral cortex increased at 24h and 48h post-resuscitation in a dose-dependent manner in PD98059 groups,(PD98059 0.3mg/kg:P<0.01,P<0.05).?Compared with sham-operated group,P-DRP1 of cerebral cortex increased at 24h and 48h post-resuscitation in CPR group(P<0.01,P<0.01).Compared with CPR group,P-DRP1 of cerebral cortex decreased at 24h and 48h post-resuscitation in a dose-dependent manner in PD98059 groups(PD98059 0.3mg/kg:P<0.01,p<0.05).?Immunohistochemical double staining of P-DRP1 and TUNEL.It showed that there was co-expression of TUNEL positive cells and P-DRP1 in cerebral cortex.Compared with sham-operated group,the co-expression of TUNEL positive cells and P-DRP1 increased at 24h and 48h post-resuscitation in CPR group.Compared with CPR group,the co-expression of TUNEL positive cells and P-DRP1 decreased at 24h and 48h post-resuscitation in PD98059 groups·?Immunohistochemical double staining of P-DRP1 and LC3.It indicated that there was co-localization expression of P-DRP1 and LC3 in cerebral cortex.Compared with sham-operated group,the co-localization expression of P-DRP1 and LC3 increased at 24h and 48h post-resuscitation in CPR group.Compared with CPR group,the co-localization expression of P-DRP1 and LC3 decreased at 24h and 48h post-resuscitation in PD98059 groups.Conclusion:ERK inhibitor PD98059 can inhibit mitochondrial-mediated apoptosis and over-activation of mitochondrial-related autophagy by reducing the opening of MPTP pore,reducing the release of CytC and regulating the expression of mitochondrial dynamic protein,thus maintaining mitochondria function and structural integrity in rat cerebral cortex after CA/CPR and creating a new way to improve the survival rate and reduce neurological damage after CPR.This study confirmed that ERX pathway participated in the pathophysiological process of cerebral ischemia-reperfusion injury after CA/CPR at cellular and molecular levels.Inhibition of ERK pathway could improve the brain injury after CA/CPR,which provides a new idea for the treatment of brain injury after CA/CPR.It may be a new target for the treatment of brain injury after CA/CPR.