| In China,with aging of population,the incidence of ischemic stroke is high,which is significantly higher than the world average.Especially in Northeast China,the incidence of ischemic cerebrovascular disease is more common due to climate and eating habits and other factors,which not only brings immeasurable harm to the patients,but also result in heavy burdens to the community and the family.Ischemia reperfusion injury is an important path physiological process after ischemic stroke.Recent studies have shown that the occurrence of ischemia reperfusion injury is a complex process,which is related to many factors.In this regard,the study of the mechanism of ischemia reperfusion injury may be the basis for the treatment of ischemic cerebrovascular disease.Mitochondria,as an energy center,are the main sites for cells to produce energy and aerobic respiration,meanwhile,mitochondria are involved in cell differentiation,cell information transmission and apoptosis,and also has the ability to regulate cell growth and cell cycle,it is therefore extremely important to maintain the stability of mitochondria and the function of cells.When ischemic stroke occurs,the mitochondrial homeostasis is destroyed,resulting in the release of cytochrome C,which leads to the occurrence of apoptosis.Omi/HtrA2 is a kind of oligomeric serine proteinase located in the mitochondrial membrane space.Under normal circumstances,it maintains a dynamic balance of the inner mitochondria membrane fluidity.When this balance is broken,apoptosis can be induced by a variety of ways.In this thesis we used Confocal laser scanning microscope,Western bolt and PCR-array and so on to research the mitochondrial injury after focal cerebral ischemia reperfusion.DL-3-n-butylphthalide(NBP)is a kind of drugs developed in China,the main ingredient is racemic-3-butylphthalide.It has been proved by many years of clinical application and experimental research that it can protect nerve cells through various ways with good safety.In this study,we established a rat model of middle cerebral artery ischemia(MCAO)by using the modified classical suture method,a comparative study was made between NBP and Omi/HtrA2 specific inhibitor of UCF101 to observe the expression changes of Omi/HtrA2,X chromosome linked inhibitor of apoptosis protein(XIAP),caspase-3,-9,mitochondrial shaping protein Optic Atrophy 1(OPA1)and Caseinolytic protease(ClpP)in mitochondria;furthermore,genes expressions related to mitochondria and mitochondrial energy metabolism pathway were detected by protein chip technique,so as to investigate the protective effects of NBP on mitochondria,and to provide a theoretical basis for better clinical applications.Part I The Mitochondria injury in focal cerebral ischemia-reperfusion and protective effect of UCF101 on mitochondria and its mechanismObjective:To explore The Mitochondria injury in focal cerebral ischemia-reperfusion and protective effect of UCF101 on mitochondria and its mechanism.Methods:Healthy adult male SD rats were selected and randomly divided into the sham group,MCAO group,and MCAO+UCF-101 group,10 rats in each group.The rat model of MCAO was established by using the modified classical suture method,rats underwent ischemia for 1.5h,and then UCF-101(10umol/kg)and the same amount of normal saline were administrated by intraperitoneal injection 75min after ischemia,respectively.The behavior of rats was evaluated after the rats were fully awake.After reperfusion for 24h,rats were sacrificed and the brain samples were taken.TUNEL staining was used to detect neuronal apoptosis;Omi/HtrA2,caspases-3,caspases-9 and PARL protein changes were determined by laser scanning co focal microscopy;Western blot method was used to detect the expression of Omi/HtrA2,caspases-3,caspases-9,XIAP,PARL,OPA1,CHOP,ClpP.Results Compared with the MCAO group(32.81±3.44),the MCAO+UCF-101 group(18.62±2.28)apoptosis index was significantly reduced p<0.01;The result of Confocal laser scanning microscope show that the expressions of Omi/HtrA2 and PARL MCAO group vs MCAO+UCF-101 group werre inhibited.Western bolt show that the expressions of Omi/HtrA2,caspases-3,caspases-9,XIAP,PARL and OPA1,CHOP and ClpP proteins were inhibited in MCAO+UCF-101 group.Part II Protective effect of DL-3-n-butylphthalide on focal cerebral ischemia reperfusion injury in ratsObjective:To explore the protective effect of DL-3-n-butylphthalide(NBP)on focal cerebral ischemia reperfusion injury in ratsMethods:Healthy adult male SD rats were selected and randomly divided into the sham group,model(MCAO)group,low-dose group(20mg/kg),and high-dose group(80mg/kg),7 rats in each group.The rat model of MCAO was established by using the modified classical suture method,rats underwent ischemia for 1.5h,and then NBP(20 mg/kg and 80 mg/kg)and the same amount of normal saline were administrated by intraperitoneal injection 1h after ischemia,respectively.The behavior of rats was evaluated after the rats were fully awake.After reperfusion for 24h,rats were sacrificed and the brain samples were taken,HE staining was then used to observe the neuron damage;TTC staining was used to observe the changes of infarct volume;apoptosis was detected by TUNEL staining;the above experimental operating procedures were completed to evaluate the protective effect of NBP on focal cerebral ischemia reperfusion injury in rats.Results:Results:Compared with the MCAO group(6.27±0.78);no matter which 20mg NBP group(10.14±1.36)or 80mg NBP group(13.28±1.42)had higher scores of behavior and smaller infarct size p<0.01,and 80mg NBP group was better than 20mg NBP group.The BIVP of 20mg NBP group(22.28±2.47)and 80mg NBP group(16.42±2.76)VS MCAO group(28.42±1.91)were decreased p<0.01.The morphology and apoptosis of nerve cells were significantly better than that of the model group.Besides,the effect was found in a dose-dependent manner,which was more obvious in the high-dose group(80mg/kg).PartⅢ Protective effect of NBP on mitochondria and its possible mechanismObjective:To determine the protective effect of NBP on mitochondria and its possible mechanism.Methods:Healthy adult male SD rats were selected and randomly divided into the sham group,model(MCAO)group,low-dose group(20mg/kg),and high-dose group(80mg/kg),7 rats in each group.The rat model of MCAO was established by using the modified classical suture method,rats underwent ischemia for 1.5h,and then NBP(20 mg/kg and 80 mg/kg)and the same amount of normal saline were administrated by intraperitoneal injection lh after ischemia,respectively.After reperfusion for 24h,rats were sacrificed and the brain samples were taken.Omi/HtrA2,caspases-3,caspases-9 and PARL protein expressions were evaluated using laser scanning co focal microscopy and Western blot;effects of NBP on the mRNA levels of genes related to mitochondrial function and energy metabolism was examined by PCR Array.Results:Compared with MCAO group,the expression of Omi/HtrA2,caspases-3,caspases-9,PARL and other proteins were significantly inhibited in NBP group;PCR Array results showed that compared with the MCAO group,NBP group was greatly improved in both mitochondrial function and mitochondrial energy metabolism;The experimental results showed that the effect of 80mg treatment group was better than that of the 20mg treatment group.Results of PCR Array indicated that mitochondrial and energy metabolism of rats in MCAO group decreased significantly compared with the sham group;and the effects of NBP on the mRNA levels of genes related to mitochondrial and energy metabolism was raised。Conclusion:1.The multiple influences to mitochondria after focal cerebral ischemia reperfusion injury and Omi/HtrA2 specific inhibitor of UCF-101 can reduce the apoptosis of neurons through a variety of ways which in the focal cerebral ischemia reperfusion injury in rats.2.Early application of NBP can significantly improve the neurological behavioral score of rats after focal cerebral ischemia reperfusion,reduce infarct volume,inhibit the apoptosis of nerve cells,and reduce cerebral ischemia reperfusion injury.3.NBP can reduce apoptosis by inhibiting the expression of Omi/HtrA2;at the same time,NBP can significantly improve the function and energy metabolism of mitochondria after cerebral ischemia reperfusion injury. |
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