| BackgroudStroke is an acute cerebral blood flow circulatory disorder in the central nervous system.It is recognized as the third most common deadly disease in the world,causing a severe psychological and economic burden on patients and greatly affecting the quality of lives for its sufferers.Among circulatory disorders,the ischemic cerebrovascular disease accounts for 60%to 80%of the total number,which is the main type of cerebrovascular disease.Brain damage caused by ischemic cerebral disease(ICVD)includes primary ischemic injury and secondary cerebral ischemia-reperfusion injury(Cerebral Ischemia-Reperfusion Injury,CIRI).During perioperative period,a variety of surgical trauma may lead to CIRI,which may lead to long-term learning,memory and cognitive dysfunction,such as cerebral perfusion insufficiency caused by intraoperative continuous hypotension and extracorporeal circulation,cerebrovascular injury caused by neurosurgery and other conditions.Clinically available in the treatment window Internal measures such as thrombolysis provide timely symptomatic treatment to restore blood supply,but for secondary CIRI,current clinical treatment methods and effects are far from expected.Therefore,the study on the pathogenesis and prevention and treatment of perioperative brain CIRI has become a scientific problem to be solved urgently.CIRI is a pathophysiological process of complex cascades involving multiple mechanisms such as oxidative stress theory,inflammatory injury theory,energy metabolism disorder theory,mitochondrial damage theory,intracellular calcium overload theory,etc.It is a single action,and it can also alternately cycle,generating feedforward,feedback and superposition effects with each other,causing each other to cause and effect,affecting the occurrence,development,and outcome of the disease.The damage of oxidative stress and inflammatory injury to nerve cells during the development of CIRI has become a hot topic in current research.There are enzyme systems that inhibit oxidative stress in normal organisms,such as superoxide dismutase(SOD),glutathione peroxidase,and catalase.Among them,SOD is the main body.Antioxidant enzyme-superoxide radical scavenger,in the process of CIRI development,a large amount of reactive oxygen species(ROS)is produced and released,resulting in lipid peroxidation of unsaturated fatty acids in cell membrane phospholipid molecules.Oxides,after being catalyzed by oxidase,produce toxic products such as malondialdehyde(MDA).MDA changes the structure of cell membrane phospholipids,and the membrane is severely damaged,causing necrosis and apoptosis of nerve cells.Tumor necrosis factor(TNF)is a key cytokine in inflammatory response activity and is considered to be the initiator of the systemic inflammatory response,which can directly lead to vascular endothelial cell dysfunction,increased vascular permeability,and reduced circulatory resistance.It induces the release of interleukin(IL)-1,IL-6,IL-8,and other cytokines and adhesion molecules,which constitutes a cascade effect of the inflammatory injury.The transcription factor NF-?B is the central link of the inflammatory response during cerebral ischemia-reperfusion injury.Many studies have confirmed that NF-?B can interfere with apoptosis by regulating the expression of apoptosis-related genes and improve the survival rate of nerve cells.Autophagy is a stress response of the body under the condition of ischemia and hypoxia.Autophagy is the process by which cellular components are transported by vesicles to lysosomes for degradation.In eukaryotic organisms,autophagy is involved not only in physiological processes such as growth and development,metabolism and immunity,but also in pathological processes such as cerebral ischemia,myocardial ischemia and renal ischemia.Several studies have showed that autophagy not only has a neuroprotective effect,but also participates in the process of nerve cell death.Therefore,focal cerebral ischemia reperfusion can activate autophagy.In different mechanisms of CIRI-induced neuronal damage,the expression of different kinds of gene proteases plays an important role in cell apoptosis or survival outcome:(1)B-cell lymphoma-2,Bcl-2 gene family members Bel-2 and Bax genes are currently well-defined functionally opposite apoptosis-regulating genes;(2)caspase family is another large class of cells Apoptosis regulator,Caspase-3 is the most important apoptosis-executing protease in the Caspase-activated cascade.Activated Caspase-3 cleaves cytoskeletal proteins,DNA-dependent protein kinases,and other Caspase-associated substrate proteins.Altering the cell structure,eventually leading to the occurrence of apoptosis;(3)autophagy is a way of apoptosis,its activation is mainly related to the activity of the ULK complex,and the activity of the ULK complex is mainly caused by the mammalian rapamycin.Mammalian target of rapamycin(mTOR)regulation;(4)C-Jun N-terminal kinase(JNK)is a member of the mitogen-activated protein kinase superfamily.The JNK signaling pathway can be activated by cytokines,growth factors,stress and other factors,and is involved in various physiological processes such as cell proliferation and differentiation,cell apoptosis,and stress response.In addition,JNK signaling pathway is closely related to the activation of autophagy and plays an important role in the occurrence and development of neurodegenerative diseases and ischemia reperfusion injury.Hypothermia treatment is currently one of the most frequent treatments for critically ill patients in critically ill patients.It is also used in critically ill patients during the perioperative period.While reducing the body's energy consumption,it can protect brain cells and neuronal damage and apoptosis by hypothermia,which may be related to the following mechanisms:(1)increase the expression of Bcl-2 in neurons and decreases the expression of Bax,thus reduce Neuronal apoptosis significantly;(2)decrease caspase-3 expression and apoptotic cells,inhibit the activation of caspase-3 by inflammatory factors(cytochrome C,free radicals,NO),thereby reduce caspase-3 after injury Expression,which plays a protective role on brain nerve cells;(3)promote P-ERK1/2 expression,but only has a small up-regulation effect on JNK expression,thereby increase the survival rate of ischemia-reperfusion neurons;(4)promote P-Akt expression,which inhibits apoptosis.The above different mechanisms are interrelated,mutually influential and beneficial.At present,mild low temperature(33?35 ?)and moderate low temperature(28-32 C)are collectively referred to as sub-low temperature(MH).After years of animal research and clinical application,the hypothermia technology has been gradually matured and widely used in the treatment of cardiac arrest,severe craniocerebral injury and stroke,and has received positive clinical effects.3-n-butylphthalide(NBP),also known as celery A,is the first new drug with independent intellectual property rights in the field of cerebrovascular disease treatment in China.A large number of domestic and foreign studies have shown that NBP can block multiple aspects of ischemic reperfusion injury and has an effective therapeutic effect:(1)It can improve the activity of Na+/K+-ATPase and Ca2+-ATPase in mitochondria and protect mitochondrial function;(2)reduce the release of cytochrome c,weaken the role of apoptosis protease;(3)inhibit calcium overload;(4)reduce the release of excitatory amino acids;(5)reduce the degree of damage of the blood-brain barrier;(6)improve microcirculation and energy metabolism in the ischemic area;(7)prolong the time window of thrombolytic therapy.Dexmedetomidine(Dex),as a new type of highly selective 2 adrenoceptor agonist,has a good sedative effect and is a commonly used sedative in clinical practice.Studies have found that dexmedetomidine can alleviate ischemia reperfusion injury in rats by reducing oxidative stress and reducing the release of inflammatory mediators.However,there is no study that focuses on the mechanism of dexmedetomidine post-conditioning in inflammatory response and autophagy of focal cerebral ischemia reperfusion injury in rats.Another focus of this study is the protective mechanism of Dex post-treatment to alleviate cerebral ischemia reperfusion injury in rats by inhibiting inflammatory response and neuronal autophagy.In conclusion,the study on the protective mechanism of perioperative cerebral ischemia reperfusion injury is divided into two parts:(1)The rat model of cerebral ischemia-reperfusion injury was established to study the application of combination therapy of hypothermia and NBP.By detecting the content of oxidative damage and inflammation-related factors and the expression of inflammation-related proteins,the combination of MH and NBP was applied to rats.(2)In the present study,a focal cerebral ischemia model was established in SD rats to explore the effect of dexmedetomidine post-conditioning on the spatial learning and memory ability,cerebral infarction area,the apoptosis and pathological changes of nerve cells in the hippocampus CA1 region as well as its possible molecular mechanism involved in JNK signaling pathway.Part One The protective mechanism of MH and NBP on cerebral ischemia-reperfusion injury in ratsObjectiveThis research mainly studied the neuroprotective effect of MH and NBP combined application on cerebral ischemia reperfusion injury model in rats by establishing the model of cerebral ischemia reperfusion injury,applying MH and NBP combined treatment,and detecting the content of oxidative injury and inflammation-related factors as well as the expression of inflammation-related proteins.MethodsSeventy-five SPF healthy male SD rats(220-250g)were provided by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences.Rats were randomly divided into sham operation group,model group,MH group,NBP group,and MH+NBP group,with 15 rats in each group.Rats in the NBP group and MH+NBP group were given NBP(80 mg/kg)saline for 7 days(once a day)before the operation.Sham group,model group,and MH group were given the same amount of normal saline.Seven days after drug treatment,a rat model of cerebral ischemia-reperfusion was established by modified Pusinelli four-vessel occlusion.A 20G silicone tube was inserted into the bilateral nasal cavity to a depth of 20 mm,and 0.9%sodium chloride solution(4-5?)was pumped through the tube for MH treatment.After the hippocampal temperature dropped to(33.0±0.5)?,the bilateral common carotid arteries were ligated with the arterial clamp for 15 min and kept at a low temperature for 1 h before natural rewarming.Eight hours after ischemia-reperfusion,brain tissue was collected in an ice bath.The supernatant was collected,and MDA,SOD,NO and NOS contents were determined using an MDA test kit,a SOD test kit,a NO test kit,and a NOS assay.The levels of inflammatory factors IL-6 and IL-1? in brain tissue were measured using an enzyme-linked immunosorbent assay(ELISA)kit.Brain tissue was fixed in 10%formalin solution,then paraffin embedded and conventionally sectioned.Nissl staining was performed.The slides were sealed and the morphology of brain tissue neurons was observed under an optical microscope.Results1.Changes in the content of oxidative stress factor in brain tissue The kits ware used to detect the corresponding oxidative stress factors.The results showed that MH,NBP and MH+NBP could reduce the content of oxidative stress factors MDA,NO and NOS and increase the content of SOD when cerebral ischemia reperfusion injury,and MH+NBP could inhibit oxidative damage caused by cerebral ischemia reperfusion better than MH and NBP alone.2.Changes in inflammatory cytokines in brain tissue ELISA results showed that the IL-6 and IL-1? levels in MH group,NBP group and MH+NBP group decreased to different degrees compared with the model group,while the IL-6 and IL-1? levels in MH+NBP group were significantly lower than those in the model group.3.Structural changes of neurons in brain tissueThe results of nissl staining showed that the number of neurons in the MH group and the NBP group was significantly increased compared with that in the model group.The cells are arranged in a regular manner,and part of the nissl substance is missing in the MH group and the NBP group.Compared with the model group,the number of neurons in the MH+NBP group was significantly increased,and the number of neurons in the MH+NBP group was increased compared with that in the MH group and the NBP group.The structure of the neurons was complete and rich in nissl substance.4.Activation of autophagy related signal m-TOR protein Western Blot results showed that the expression level of p-mTOR protein was significantly decreased after MH and NBP combined treatment.5.Expression changes of apoptosis-related proteins Bcl-2 and Bax Western Blot results showed that Bcl-2 protein expression was increased and Bax protein expression was significantly decreased in the model group after MH and NBP treatment.After MH and NBP combined treatment,Bcl-2 protein expression level was further increased and Bax protein expression was decreased.6.Activity of inflammation-related signal NF-?B protein Western Blot results showed that,compared with the model group,the level of p-NF-?B protein in the NH and NBP groups was significantly decreased,and the level of p-NF-?B protein in the NH+NBP group was further decreased.Conclusions1.MH and NBP combination treatment can better inhibit oxidative damage and inflammatory response caused by cerebral ischemia-reperfusion than MH and NBP alone.2.MH and NBP combination treatment can reduce the damage of rat brain cells caused by cerebral ischemia-reperfusion,which is significantly better than MH and NBP alone.3.NH and NBP combination treatment can play a neuroprotective effect on rats with cerebral ischemia-reperfusion injury by inhibiting the phosphorylation of mTOR and NF-?B protein,promoting the expression of Bcl-2 and inhibiting the expression of Bax.Part Two Protective mechanism of dexmedetomidine post-conditioning on cerebral ischemia reperfusion injury in rats ObjectiveIn the present study,a focal cerebral ischemia model was established in SD rats to explore the effect of dexmedetomidine post-conditioning on the spatial learning and memory ability,cerebral infarction area,the apoptosis and pathological changes of nerve cells in the hippocampus CA1 region as well as its possible molecular mechanism involved in JNK signaling pathway to provide a theoretical basis for the postoperative treatment of cerebral ischemia reperfusion injury.Methods180 healthy adult rats were randomly divided into 6 groups(30 rats per treatment group)for various treatments.There were sham-operated(Sham)group,ischemia reperfusion injury(I/R)group,dexmedetomidine post-conditioning(Dex)group,JNK inhibitor(SP600125)group,dexmedetomidine post-conditioning+JNK activator(Dex+Anisomycin)group and positive drug nimodipine control(Nim)group.The model of middle cerebral artery occlusion(MCAO)in rats was established by suture-occluded method.24h after cerebral ischemia reperfusion,the Morris water maze test was conducted to evaluate the spatial learning ability and memory ability of rats.Brain tissues of 6 rats in each group were sectioned and TTC staining was used to detect ischemic brain area.Brain tissues of 6 rats in each group were soaked in 4%paraformaldehyde for fixation and sectioned for TUNEL staining to detect the number of apoptotic cells in brain tissues.Pathological changes in CA1 brain region were observed by HE staining.TNF-?,IL-6 and IL-1? levels were measured by ELISA kit in serum of rats in 6 groups.Brain tissues of 6 rats in each group were immersed in 2.5%glutaraldehyde and fixed.The structural changes of neurons in hippocampal CA1 area were observed by transmission electron microscopy.RNA was extracted from the brain tissues of 6 rats in each group,and the expression of autophagy related genes was detected by qRT-PCR.Total proteins of brain tissues of 6 rats in each group were extracted and autophagy-related proteins and JNK phosphorylation levels were detected by western blot.Results1.Behavioral competence test of ratsMorris water maze experiment was used to evaluate the spatial learning and memory ability of rats.Compared to the Sham group,the escape latency(EL)were markedly prolonged,while the times across platform and the times stayed in the quadrant of the original platform were decreased in other groups.Compared to the I/R group,the EL were markedly shorter,while the times across platform and the during platfonn quadrant were significantly increased in Dex group,SP600125 group,Dex+Anisomycin group and Nim group.Compared to the Dex group,the EL were markedly prolonged,while the times across platform and the during platform quadrant were significantly decreased in Dex+Anisomycin group.2.Determination of the infarct area of brain tissueThe infarct area of brain tissue was determined by TTC staining.White infarcts of different sizes were observed in I/R group,Dex group,SP600125 group,Dex+Anisomycin group and Nim group.The infarct area of brain tissue in rats was expressed as the percentage of infarcted area to non-infarcted area.The infarct areas of brain tissue in the Dex group,SP600125 group,Dex+Anisomycin group and Nim group were significantly decreased compared with the I/R group.Compared to the Dex group,the white infarct increased in the Dex+Anisomycin group.3.Apoptosis of neurons in hippocampal CA1 regionTUNEL assay was used to detect the positive apoptosis of neurons in hippocampal CA1 region of rat brain.The number of positive apoptosis of neurons were significantly increased in other groups compared with the Sham group.Compared to the I/R group,the number of positive apoptosis of neurons were significantly decreased to varying degrees,and the staining degree were decreased in Dex group,SP600125 group,Dex+Anisomycin group and Nim group.Compared to the Dex group,the number of positive apoptosis of neurons was increased and the pathological staining was aggravated in the Dex+Anisomycin group.4.Pathological changes of neurons in hippocampal CA1 regionHE staining was used to observe the pathological changes of neurons in the hippocampus CA1 region of rats.The morphological structure of neurons in other groups showed different degrees of abnormality compared with the Sham group.Compared to the I/R group,the degree of the pathological changes of neurons was reduced in the Dex group,SP600125 group,Dex+Anisomycin group and Nim group.There were no significant differences among the Dex group,Nim group and SP600125 group,while the degree of the pathological changes of neurons was slightly heavier in the Dex+Anisomycin group.5.Changes in inflammatory factors in serumThe expression levels of inflammatory factors(TNF-?,IL-6 and IL-1?)in serum of rats were determined by ELISA.The contents of TNF-?,IL-6 and IL-1? in other groups were significantly increased compared to the Sham group.Compared to the I/R group,the contents of TNF-?,IL-6 and IL-1? were significantly decreased in the Dex group,SP600125 group,Dex+Anisomycin group and Nim group.Compared to the Dex group,there was no significant difference in the levels of TNF-?,IL-6 and IL-1? between the Nim group and the SP600125 group,while the contents of TNF-?,IL-6 and IL-1? in the Dex+Anisomycin group were increased.6.Autophagy in hippocampal CA1 regionThe autophagy in hippocampal CA1 region was observed by transmission electron microscopy.The auto-phagosomes or auto-phagocytes with bilayer or monolayer membrane in cells under electron microscopy is the morphological feature of autophagy.Compared to the I/R group,the autophagy was reduced to varying degrees,with the degree of edema and cavitation reduced in the Dex group,the SP600125 group,the Dex+Anisomycin group and the Nim group.There were no significant differences among the Dex group,Nim group and SP600125 group,while the autophagy was slightly heavier in the Dex+Anisomycin group.7.Expressions of Beclin-1,Caspase-3,LC3 mRNA and protein of brain tissues in ratsThe expression of autophagy-related mRNA in rat brain tissues was detected by qRT-PCR.Compared to the Sham group,the mRNA expressions of Beclin-1,Caspase-3,LC3 were significantly increased in other groups.The mRNA expressions of Beclin-1,Caspase-3,LC3 in the Dex group,SP600125 group,Dex+Anisomycin group and Nim group were significantly decreased compared with the I/R group.There were no significant differences among the Dex group,Nim group and SP600125 group,while the mRNA expressions of Beclin-1,Caspase-3,LC3 were slightly higher in the Dex+Anisomycin group.Western blot results were shown that compared to the Sham group,the protein expressions of Beclin-1,Caspase-3,and LC3II/I were significantly up-regulated in other groups,and the overall change trend was consistent with the qRT-PCR experiment.8.Detection of the activity of JNK signaling pathwayWestern blot results were shown that the protein expressions of p-JNK1 in other groups were significantly up-regulated compared to the Sham group,and there were no significant changes in the total JNK1 content.Compared to the I/R group,the protein expressions of p-JNK1 were significantly decreased in the Dex group,SP600125 group,Dex+Anisomycin group and Nim group.Furthermore,compared to the Dex group,the protein expression of p-JNK1 was slightly up-regulated in the Dex+Anisomycin group.Conclusions1.Both dexmedetomidine post-conditioning and the JNK pathway inhibitor treatment could improve the learning and memory dysfunction reduce the infarct area caused by focal cerebral ischemia reperfusion injury in rats.2.Both the dexmedetomidine post-conditioning and the JNK pathway inhibitor treatment could reduce the positive apoptosis and pathological changes of neurons in hippocampal CA1 region caused by focal cerebral ischemia reperfusion injury in rats.3.Dexmedetomidine post-conditioning could reduce the inflammatory response and autophagy effect.The mechanism of the effect might be related to the inhibition of JNK pathway activation,and to affect the expressions of inflammatory factors and autophagy related proteins. |
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