| Background and objectiveWith the gradual rejuvenation of cardiovascular diseases,the incidence of cardiovascular diseases increased year by year,and then caused more and more sudden cardiac death.Although the international guidelines for cardiopulmonary resuscitation(CPR)have been upgraded and improved year by year,the mortality rate of patients after cardiopulmonary resuscitation(CPR)is still high,and the success or failure of brain resuscitation has always been the focus and difficulty of clinical attention,the concept of "post-cardiac Arrest Syndrome"(PCAS)is now accepted by the international community of cardiopulmonary resuscitation-related disciplines,it covers a series of exposition of pathophysiology,treatment and prognosis of cardiac arrest patients after restoration of autonomic circulation.The main point is that the recovery is over when the patient regains voluntary circulation after cardiac arrest,but it is also the beginning of another,more complicated stage of recovery.Cardiopulmonary Resuscitation(CPR)can improve the survival rate and neurological prognosis of cardiac arrest(CA)patients by studying the pathophysiological mechanism of CPR and seeking for more effective treatment.After the recovery of the autonomic circulation,there are many factors causing the damage of the nervous system,among which the initial ischemic damage and the reperfusion injury after reperfusion are the main factors.In this process,the body has a strong oxidative stress reaction,neuroendocrine and vasoactive substances have changed dramatically and lead to multiple organ dysfunction.The brain tissue is extremely sensitive to hypoxia and the damage is more prominent,so the brain damage after CA is not only the expression after CPR,it is also one of the main causes of the increase in mortality rate.Most of the survivors were resuscitated into comas or eventually into irreversible chronic disability.For example,most of the patients have some neurological deficits such as language,perception and movement,some of them are even in vegetative state or dead,and the long-term prognosis is not optimistic.Studies have shown that brain injury after CPR is associated with a series of pathophysiological changes following cerebral ischemia-reperfusion.Ischemia and Hypoxia are the initial factors leading to the injury of nerve function after cardiopulmonary resuscitation(CPR),including the release of oxygen free radicals(ROS),calcium overload,excitatory amino acid accumulation and activation of cell death pathway.There is a close relationship between apoptosis and mitochondrial structure and function injury.Mitochondrial membrane permeability transition pore(MPTP)releases Cytochrome C(Cyt C)and Apoptosis-inducing factor,caspase-3(caspase-3)and caspase-9(caspase-9)increase in cytoplasm and induce neuronal apoptosis.Speeds up the nerve cell death,causes the nervous system to have the irreversible damage.The key factors leading to the continuous opening of mitochondrial membrane permeability transition pore are the production of Ros and calcium overload,which shows that the antioxidant mechanism plays a key role in the whole damage process,play an antioxidant role in order to reduce this cascade reaction,reduce neuronal apoptosis.Along with the understanding and research of a series of molecular mechanisms,the researchers also found that the body has formed a complex response system in order to fight against the free radicals and toxic substances produced by these oxidative stress,induce the production of a series of protective proteins to alleviate cell damage.This reflex is regulated by antioxidant response elements(ARE)that encode upstream regulatory regions of Protective Protein DNA.Recent studies have shown that NF-E2-related factors(Nrf2)ARE the most important endogenous anti-oxidative stress pathway,which is regulated by the interaction of NF-E2 and ARE.Nrf2,in combination with the antioxidant reactive element(ARE),activates the expression of a number of downstream molecules,so far,the protective genes encoded by Nrf2/ARE signaling pathway include oxidation protein gene,Phase ? detoxifying enzyme gene,molecular chaperone gene and anti-inflammatory factor gene,the genes that encode the proteins are common antioxidant proteases:Ho-1 Heme oxygenase I;Glutamylcysteine Synthetase;Superoxide dismutase;Reduced Coenzyme ? oxidoreductase,etc..Antioxidant enzymes play an important role in maintaining the balance of oxidation and reduction by catalyzing the conversion of free radicals into non-toxic substances and increasing their water solubility.NRF2 is normally combined with Keapl in the cytoploasm and degraded by ubiquitin.Under oxidative stress,the specific cysteine residues of Keapl are modified,and the existence of cysteine residues is a common characteristic of Nrf2 activating molecules.Nrf2 and Keap1 ARE released into the nucleus,which,when combined with ARE,induces the transcription and expression of protective genes and activates the defense system.Activation of the phosphorylated signal transduction pathway may also stimulate Nrf2 dependent cellular defense,leading to conformational changes,Nrf2 is immune to ubiquitination degradation,and Nrf2 phosphorylates with the involvement of phosphorylated products such as PI3K,with the further study of this pathway in our experimental group,we found that Nrf2 phosphorylation is induced by phosphorylated product(PI3K)during the activation of this pathway,which plays an important role in regulating transcription,the phosphorylated product PI3K is the product of activation of PI3K/AKT pathway.So it is true that phosphorylated PI3K/AKT pathway participates in the activation of NRF2/ARE signaling pathway.It is clear that protein kinase C(PKC)causes the phosphorylation of Ser-40 in Nrf2 domain,which results in the dissociation of Nrf2 and Keap1.Tyrosine kinase resulted in the Nrf2 molecules being phosphorylated by Tyr 568,allowing Nrf2 to move out of the nucleus.So we hypothesized that when oxidative stress occurs in the body,whether we can use drugs that promote phosphorylation of the PI3K/AKT pathway and thus increase the Nrf2/ARE signaling pathway to a greater extent,playing an anti-oxidative stress role,to protect the brain?With such research ideas and purposes,we review a large number of literature found that Sufentanil this drug has a stronger role in promoting the PI3K/AKT pathway phosphorylation.It mainly acts on opioid receptor,lipophilicity is high,compared with fentanyl has more easily through the blood-brain barrier and other advantages.Numerous experimental studies have demonstrated that Sufentanil can activate pi3kakt signaling pathway(Phosphatidylinositol kinase threonine kinase)and inhibit the opening of mitochondrial transmembrane pore.And many experiments have proved that sufentanil plays an important role in myocardial ischemia-reperfusion,reducing myocardial cell apoptosis,and plays a cell protection role,however,its specific role and molecular mechanism in the treatment of cerebral ischemia-reperfusion injury after cardiopulmonary resuscitation(CPR)have not been reported.At present,a few reports have found that remifentanil can reduce the expression of myelin basic protein after ischemia-reperfusion in aged rats,and then reduce the injury of neurons.Therefore,it is inferred that remifentanil has brain protection effect,the exact mechanism is still unclear.We found the intrinsic relationship between Nrf2/ARE pathway and PI3K/AKT pathway,and the phosphorylated product PI3K after activation of PI3K/AKT pathway is involved in the up-regulation of Nrf2/ARE pathway,thus,the expression of antioxidant stress protective proteins such as NQO-1 and SOD was increased,the oxidative stress injury was decreased,the integrity of mitochondrial membrane was preserved,the opening of Mitochondrial permeability membrane transition pore(MPTP)was inhibited,and apoptosis was reduced,to protect the brain.Therefore,sufentanil is presumed to have a brain protective effect.So sufentanil upregulates the nrf2 are signaling pathway by activating the PI3K/AKT pathway,it is possible to improve the pathophysiology and prognosis of the cardiopulmonary resuscitation brain by reducing the production of oxygen free radicals and the pathway of neuronal apoptosis.Objective:1.To establish a reliable and stable rat model of cardiopulmonary resuscitation(CPR)by asphyxia.2.Rats were treated with sufentanil in one group and Pi3k/akt phosphorylation specific inhibitor in the other group after successful resuscitation,the expression of Nrf2/ARE pathway related protein was compared with the content of phosphorylated products in rat hippocampal neurons,proteins expressed through the nrf2 are pathway included the determination of the antioxidant stress product oxidoreductase and Superoxide dismutase;In addition,8-iso-prostaglandin F2(8-iso PGF2)and 8-hydroxy-2'-2'-deoxyguanosine(8-ohdg)were measured to further investigate whether Sufentanil upregulates nrf2 are pathway expression in rat brain after cardiopulmonary resuscitation,then,the protective effect of the brain is exerted and the possible mechanism is discussed.3.To examine the occurrence of neuronal apoptosis and the changes of caspase-3 and caspase-9 in sufentanil-treated rats after cardiopulmonary resuscitation,the levels of Bcl-2 and Bax,the levels of IL-1,IL-6 and TNF-and the scores of mNSS and spatial learning ability were measured,to investigate the effect of sufentanil on neuronal apoptosis after cardiopulmonary resuscitation(CPR)in rats and its possible mechanism.Chapter ?:Establishment of rat model of cardiac arrest by asphyxiaObjective:to establish cardiac arrest(CA)model in rats by asphyxiaMethods:In the Animal Laboratory of the Jilin University,30 healthy adult male SD rats weighing 260-320g were selected.Cage rearing,4.The rats were kept at room temperature of 23±2 C,humidity of 40%-70%,12 h light/dark cycle,and fed freely and drank water during the experiment.All Rats were reared for 1 week to adapt to the laboratory environment before the experiment.All animal experiments are conducted in accordance with the national guidelines for the rearing and use of laboratory animals.The experimental group was divided into 6 min group(15 rats)and 7 min group(15 rats).The rats were weighed and anesthetized with a mixture of 2-5%isoflurane and oxygen(100%oxygen containing 2-5%isoflurane).Complete anesthesia was followed by fixation and continuous infusion of saline(at a rate of 0.1 ml/h)through an exposed femoral vein in the inguinal region to maintain baseline blood pressure and fluid balance.Continuous body temperature monitoring and maintenance at 37 ?,with heating pad and external heating lamp.The right side of the carotid artery was cannulated and connected to a pressure transducer for blood pressure monitoring,and ECG monitoring was performed routinely.The trachea was separated and intubated after tracheotomy,and the blood pressure and heart rate were stabilized,in Rats,tracheal intubation was performed with end-of-breath clamping,and pure oxygen mechanical ventilation(frequency 80 Times/min,tidal volume 6 ml/kg)was given after CA occurred.At the same time,the external heart was pressed by mechanical press,the pressing frequency was 200 times/min,at the same time,0.02 mg/kg epinephrine was injected through femoral vein and pressed until the recovery of spontaneous circulation(ROSC).The patients who did not resume spontaneous circulation after CPR 10 minutes failed to locate cardiopulmonary resuscitation(CPR).Results:We successfully induced CA in all 30 rats,15 rats in each group,and the time from the beginning of Asphyxia to CA was(324 ± 38)S.During the whole process of Asphyxia,we found that the carotid blood pressure monitoring decreased rapidly to less than 25 MMHG in all rats after 6 minutes of Asphyxia,and the pulsatile wave disappeared,reaching the standard of CA,the incidence of ventricular fibrillation(VF)was 66.7%(10/15 rats),20%(3/15 rats)and 13.3%(2/15 rats)in mechanical isolation,26.6%(4/15 rats)in ventricular fibrillation(VF)and 46.7%(7/15 rats)in asphyxia for 7 minutes,the rate of complete arrest was 26.7%(4/15 rats),and the number of ventricular fibrillation decreased with the prolongation of asphyxia time,and the number of electromechanic separation and cardiac arrest increased gradually(p<0.05).At the same time,the recovery rate of ROSC was higher in the Group of 6 Min of asphyxia than that in the group of 5 min of Asphyxia.When the Asphyxia reached 7 Min,the majority of rats suffered cardiac arrest,and the time needed for CPR was prolonged accordingly.There was significant difference between them(P<0.05).After successful resuscitation,the 6-hour,3-day and 7-day survival rates of rats in asphyxia group were significantly higher than those in Asphyxia Group for 6 minutes(P<0.05).There was significant difference between them(P<0.05).Conclusion:The model of CA could be induced within 6 minutes,which was an ideal time of asphyxia in the model of cardiopulmonary resuscitation.The successful model of CPR WITHIN 5 minutes had a high proportion of ROSC rats,and the survival rate of Rosc rats was 6 hours,the 3-day survival rate and 7-day survival rate were significantly higher than those of CA 7 min rats.The model of asphyxia for 6 minutes was more stable and convenient for experimental study.Chapter ?:Effect of sufentanil on neuronal apoptosis after cardiopulmonary resuscitation in ratsObjective:To investigate whether Sufentanil can inhibit neuronal apoptosis,improve the spatial learning ability and neuroprotection in rats and its possible mechanismMethods:Healthy adult male SD rats,weight 260-320G.All animals are approved by the Jilin University.Laboratory animals are kept in four cages.The rats were kept at room temperature of 23±2 ?,humidity of 40%-70%,12 h light/dark cycle,and fed freely and drank water during the experiment.All Rats were reared for 1 week to adapt to the laboratory environment before the experiment.All animal experiments are conducted in accordance with the national guidelines for the rearing and use of laboratory animals.The rats were randomly divided into four groups:Control Group 1(N=20):Rats received the same operation,tracheal intubation,no suffocation and no cardiopulmonary resuscitation.Group 2(N=30):CA and CPR were performed and 0.5 ml saline/12h(7 days)was given.Group 3:sufentanil Group(N=30):CA and CPR were performed and sufentanil was injected 1.0 g/kg/12h after CA(7 days).Group 4:Sufentanil+PI3KSpecific Inhibitor Group(N=30):CA and CPR were performed,and Sufentanil of 1.0 g/kg/12h weight was injected after Ca,while Wortmannin 15 g/kg/12h was injected intravenously(7 days).The expression of caspase-3,caspase-9,Bcl-2 and Bax protein in hippocampus of rats was detected by Western Blot and Elisa,and the levels of interleukin-1(IL-1),interleukin-6(IL-6)and Tumor necrosis factors factor(TNF-?)in the hippocampus.The improved neurological severity score(mNSS)and spatial working memory performance(spatial working memory performance)were used to evaluate the neurological deficits in rats.Results:1.After sufentanil treatment,the expression of caspase-3 and caspase-9 decreased,the expression of Bcl-2 increased,the expression of Bax decreased,the ratio of Bax to Bcl-2 decreased,and the corresponding neuron apoptosis index decreased in the hippocampus of rats after cardiopulmonary resuscitation treatment,the levels of IL-1,IL-6 and TNF-were significantly lower than those in CPR group(P<0.05),while the expression of caspase-3 and caspase-9 were increased in PI3K specific inhibitor group,the expression of Bcl-2 was decreased,the expression of Bax was up-regulated,the ratio of Bax/bcl-2 was increased,the corresponding neuron apoptosis index was increased,the contents of IL-1,IL-6 and TNF-were increased,compared with sufentanil group(p>0.05).2.The mNSS scores and spatial learning ability of rats in sufentanil group were significantly lower than those in other groups(P<0.05).There was no significant difference between PI3K specific inhibitor group and CA group(p>0.05)Conclusion:Sufentanil can inhibit the pathological changes of neurons in hippocampal CA1 area,inhibit the apoptosis of neurons in this area,decrease the apoptosis index,up-regulate the expression of Bcl-2 and down-regulate the expression of Bax,and decrease the ratio of Bax/bcl-2,down-regulate the expression of Caspase-3,Caspase-9 in hippocampus,decrease the content of IL-1,IL-6 and TNF-,and play a protective role in cerebral ischemia-reperfusion injury.The possible mechanism of its brain protective effect is related to the activation of PI3K/Akt signaling pathway by sufentanil,which activates AKT phosphorylation and changes the activity of a series of apoptotic proteins downstream,thus exerting the brain protective effect.Chapter ?:Effects of sufentanil on PI3K/AKT and NRF2/ARE signaling pathway after cardiopulmonary resuscitation in ratsObjective:1.To investigate whether sufentanil increased the phosphorylation of PI3K/AKT pathway and upregulated the expression of Nrf2/ARE signaling pathway after cardiopulmonary resuscitation(CPR)in rats.2.The changes of phosphorylation products and the relationship between Nrf2/ARE signaling pathway expression were measured after specific phosphorylation inhibitors were used,furthermore,we hypothesized that if sufentanil inhibited the phosphorylation of PI3K/AKT pathway,the role of Nrf2/ARE pathway would also decrease,furthermore,we hypothesized that sufentanil might be involved in the activation of Nrf2/ARE pathway and the phosphorylation of PI3K/AKT pathway.Methods:healthy adult male SD rats,260-320g body weight.All animals are approved by the Jilin University.Laboratory animals are kept in four cages.The rats were kept at room temperature of 23±2?,humidity of 40%-70%,12 h light/dark cycle,and fed freely and drank water during the experiment.All Rats were reared for 1 week to adapt to the laboratory environment before the experiment.All animal experiments are conducted in accordance with the national guidelines for the rearing and use of laboratory animals.The rats were randomly divided into four groups:Control Group 1(N=12):Rats received the same operation,tracheal intubation,no suffocation and no cardiopulmonary resuscitation.Group 2(N=30):CA and CPR were performed and 0.5 ml saline/12h(7 days)was given.Group 3:sufentanil Group(N=30):CA and CPR were performed,and sufentanil was injected 1.0 g/kg/12h after CA(7 days).Group 4:Sufentanil+PI3K Specific Inhibitor Group(N=30):CA and CPR were performed,and Sufentanil of 1.0 g/kg/12h weight was injected after Ca,while Wortmannin 15 g/kg/12h was injected intravenously(7 days).Results:1.The oxidative stress products(8-iso PGF2 and 8-ohdg)were significantly increased in the CPR group without drug intervention,which indicated that the cerebral ischemia-reperfusion injury was serious,and the corresponding oxidative stress products were decreased in the sufentanil-treated group,the content of P-PI3K,P-AKT,P-Nrf2 increased,and the content of NQO1 and SOD,which had antioxidant protection,also increased significantly,compared with the control group and CPR group(p<0.05)2.The content of oxidative stress products 8-iso PGF2 and 8-ohdg increased again,the content of p-PI3K,P-AKT,P-Nrf2 decreased,and the content of antioxidant protective protein NQO1 and SOD also decreased,compared with sufentanil group,there was significant difference(p<0.05).There was no significant difference between the two groups(p>0.05).Conclusion:SUFENTANIL can promote the phosphorylation of PI3K/AKT pathway,increase the production of P-PI3K and P-AKT phosphorylation,increase the content of P-Nrf2,up-regulate the Nrf2/ARE signaling pathway,and increase the expression of protective protein,and that's what protects the brain.When sufentanil inhibited the phosphorylation of PI3K/AKT pathway,the up-regulation of Nrf2/ARE pathway was also inhibited. |
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