The Role Of Therapeutic Hypothermia Via CIRP/Trx1 Pathway On Brain Resuscitation In A Rat Model Of Cardiac Arrest

Backgrounds Sudden cardiac arrest(CA)seriously threatens the safety of human life and is one of the most important causes of death.Despite the continuous improvement of CPR technology,after recovery of spontaneous circulation(ROSC),some CA patients eventually die of brain injury,and permanent cognitive dysfunction may occur in long-term surviving patients.Therefore,after successful CPR,cerebral resuscitation becomes a major problem.Therapeutic hypothermia(TH)is one of the important feasible methods of cerebral resuscitation in clinical treatment.The therapeutic hypothermia(32-34 ℃)can effectively improve the neurological prognosis and improve the survival rate of patients after CA,and has been recognized by the International CPR Association.However,it is not easy to apply TH in clinical practice because of some side effects in treatment,so it is urgent to understand the mechanism of therapeutic hypothermic in order to find supplementary methods of hypothermia treatment.Cold-inducible RNA binding protein(CIRP)is a common cold shock protein in complex life.Under stress conditions,such as low temperature and hypoxia,CIRP expression increases.As an RNA chaperone,it participates in the specific transcription of stress response genes,such as thioredoxin(Trx),and plays an important role in oxidative stress,inflammation,and apoptosis.The previous research of our research group found that CA-induced brain injury can significantly up-regulate the expression of CIRP in neurons.After therapeutic hypothermia treatment,the protein expression further increased.By up-regulating CIRP,the mitochondrial-related oxidative stress response was alleviated and reduced the brain injury after CA.Trx,as an important component of the thioredoxin system,can scavenge oxygen free radicals well,reduce oxidative stress,inhibit apoptosis,and has an effect on neuroprotection.It also suggests that the mechanism of hypothermia treatment to reduce oxidative stress may be related to inducing high expression level of Trx1 and CIRP.The purpose of this study is to establish a rat model of cardiac arrest induced by esophageal ventricular fibrillation,compare and analyze the physiological and pathological changes after hypothermia treatment and CRIP silencing,so as to explore the mechanism of therapeutic hypothermia on brain resuscitation in a rat model of cardiac arrest.Part 1 The physiological and pathological changes of rats in a model of cardiopulmonary resuscitation after cardiac arrestObjective: Observing the physiological and pathological changes of rats in a model of cardiopulmonary resuscitation after cardiac arrest,and for further explore the correlation between TH and CIRP/Trx1.Methods: According to the random number table,male SD rats,weighing 280-320 g,were divided into Control group(Control),operation group(Sham),model group(I/R)and hypothermia treatment group(TH).Control group do not do anything.The Sham group only received tracheal intubation and arteriovenous catheterization.I/R group and TH group all induced cardiac arrest by electrical stimulation the transesophageal.CPR was performed after 4 minutes of cardiac arrest.Among them,n=18,rats were used to detect the changes of proteins Trx1 and CRIP after 6,24,and 72 h by western blot;n= 15,rats were used to observe the seven-day survival rate and the pathological changes in hippocampal CA1 area via light microscope.Results: After experiencing cardiac arrest and cardiopulmonary resuscitation,the survival rate of I/R group was 40 %,and the survival rate of TH group was 60 %,the difference was statistically significant.Compared with the Sham group,the normal neurons in the I/R group and TH group decreased,and the apoptosis rate increased;compared with the I/R group,the normal neurons in the TH group increased,and the apoptosis rate decreased,the difference was statistically significant.Compared with the Sham group,the expression of Trx1 and CIRP decreased in I/R group(P <0.05).Compared with I/R group,the expressions of Trx1 and CIRP in the TH group increased(P <0.05).Conclusions: The mechanism of therapeutic hypothermia treatment in cerebral resuscitation may be closely related to the increased expression of CIRP and Trx1.Part 2 The research of CIRP/Trx1 pathway on a model of cardiopulmonary resuscitation after cardiac arrestObjective: Observing the effect of therapeutic hypothermia and suppression of CIRP expression in brain injury resuscitation,and for further to explore the mechanism of CIRP and Trx1 during therapeutic hypothermic after cardiac arrest.Methods: According to random number table,male SD rats,weighing 280-320 g,were divided into control group(Control),AAV negative group(NC),AAV-CIRP silent group(CIRPi),operation group(Sham),model group(I/R),hypothermia treatment group(TH),hypothermia treatment + AAV-CIRP silent group(TH + CIRPi)and hypothermia treatment + AAV negative group(TH + NC).The model of brain injurywas established by cardiopulmonary resuscitation after cardiac arrest induced by transesophageal electrical stimulation for 4 min.Control,NC,and CIRPi groups were directly sampled for two weeks after injection in the brain area to observe the effect of AAV transfection.RT-PCR was used to detect the expression level of CIRP m RNA,and western blot was used to detect the expression of CIRP protein.The five groups of Sham,I/R,TH,TH + CIRPi and TH + NC were used to detect the activity of ROS and the expression of CIRP,Trx1,and downstream protein such as p-ASK1 in 1 day after the recovery of spontaneous circulation;the rate of apoptosis at 3 days was observed by immunofluorescence.Results: Compared with the Sham group,CIRP expression in the I/R group decreased,and p-ASK1 expression increased(P <0.05);compared with the I/R group,TH group expressions of CIRP and Trx1 were significantly up-regulated,and the expression of p-ASK1 was significantly down-regulated(P <0.05);compared with the TH group,TH+CIRPi group the CIRP and Trx1 expression were significantly reduced,and p-ASK1 was significantly increased(P <0.05).Compared with the Sham group,except that there was no significant difference in p-JNK,the expression levels of protein TXNIP,p-p38,MMP9,and HIF-1α increased in I/R group(P <0.05).Compared with I/R group,the expression levels of above five different proteins in the TH group decreased significantly,and the differences were statistically significant.After the injection of AAV in the brain area inhibited the synthesis of CIRP,compared with the TH group,the expression of TXNIP,p-JNK,p-p38,MMP9,and HIF-1α were significantly up-regulated in the TH + CIRPi group(P <0.05).1 day after ROSC,ROS activity was significantly enhanced compared with the Sham group(P <0.05).Compared with I/R group,the ROS activity in the TH group,TH + CIRPi group,and TH + NC group all decreased(P <0.05).Compared with the TH group,the ROS activity in the TH + CIRPi group was significantly enhanced(P <0.05).After ROSC 3 days,compared with Sham group,other groups of apoptosis rate were significantly increased(P <0.05);compared with I/R group,TH group,TH + CIRPi group and TH+ NC group,the apoptosis rate decreased(P <0.05);compared with the TH group,the apoptosis rate in the TH + CIRPi group increased significantly(P <0.05).Conclusions: Therapeutic hypothermia can up-regulate CIRP to reduce oxidative stress and apoptosis of neurons during brain resuscitation after cardiac arrest.The mechanism may be related to the activation of CIRP/Trx1/ASK1/TXNIP pathway.Summary 1.Therapeutic hypothermic improves the survival rate of rats after cardiac arrest by reducing the damage and apoptosis of neurons.It is closely related to the increased expression of CIRP and Trx1 protein.2.Therapeutic hypothermic can effectively reduce ROS accumulation and reduce neurons damage.After CIRP inhibition,the effect of therapeutic hypothermic was suppressed.Therapeutic hypothermic reduces the oxidative stress damage and neurons apoptosis through the activation of the CIRP/Trx1/ASK1/TXNIP pathway.