The Effect Of Neutrophil Elastase On Brain Microvascular Endothelial Cells In Ischemia/Reperfusion Injury

BackgroundIschemic cerebrovascular disease is a kind of common and frequent disease that severely affects people's lives and health. It is necessary to study the pathology and physiology mechanism deeply and search the pointed therapy strategy of ischemic cerebrovascular disease. It is important significance for economy and social.Brain ischemia/reperfusion (I/R) has relationship with many factors, such as free radicals, the excitotoxicity, free calcium over-loading, the dysfunction of mitochondria, inflammation and apoptosis. Recent years, people gradually think highly of the roles of inflammation in brain ischemia/reperfusion injury. It is confirmed that the blood vessel inflammatory reaction by leucocyte (especially polymorphonuclear leukocyte ) and inflammatory cell factors make a important effect in I/R. The effect of polymorphonuclear leukocyte in I/R has two aspect:(1) mechanism blockage (2) releasing mediators of inflammation and proteolytic enzyme(neutrophil elastase) , aggraving the injury of brain tissue. Neutrophil elastase(NE)is a kind of proteolytic enzyme which has intensive hydrolization and the most optimistic substrate is elastin. Previous study shows that neutrophil elastase either participates many inflammatory disease for example chronic bronchitis, bronchiectasis, gential tract infect or makes a important role in the I/R injury of tissue and organic.At the same time, it is has proved that the damage of brain microvascular endothelial cells (BMEC) is the earlier pathological changes and basic remote cause in ischemic cerebrovascular disease. Therefore, we can further confirm that NE participate the ischemia /reperfusion injury,and the NE inhibitor Sivelestat Sodium likely to cerebral ischemia / reperfusion injury protection through the study of BMEC in oxygen glucose deprivation/reoxygenation.Objective(1) To isolate and to observe the morphology of brain microvascular endothelial cells from Wistar rat.(2) To establish a simple, stable and reliable model of oxygen glucose deprivation/reoxygenation (OGD/R) of microvascular endothelial cells in vitro.(3) To investigate whether NE aggtavate I/R injury though impairing brain blood capillary. So NE will become the new pointed therapy strategy in the therapy of ischemic cerebrovascular disease. And the Sivelestat sodium will be a new nerves protective additive in the therapy of cerebrovascular disease.MethodsBMEC of 5～7 days Wistar rats were primary cultured. The BMEC were affirmed by immunocytochemical method, morphologies under light microscope and ultramicrostructure under electron microscope. We observed the growth curve of BMEC by MTT assay. We established a model of oxygen glucose deprivation/reoxygenation (OGD/R) of microvascular endothelial cells in vitro. The experiment has been divided 5 groups: normal control group (NC group); ischemia/reperfusion group (I/R group); ischemia/reperfusion + polymorphonuclear leukocyte group (I/R+PMN group); ischemia/reperfusion + polymorphonuclear leukocyte + Sivelestat sodium group (I/R+PMN+Siv group); polymorphonuclear leukocyte + Edaravone group (I/R+PMN+EDA group). We quantitative assayed the NE by ELASA; estimate cells survival and degree of injury by MTT and LDH; assayed the endothelin-1 secretory volume by radioactive immunoassay (RIA); detected the intercellular adhesion molecule-1 (ICAM-1) by Immuneeocytochemical method ,and assayed the angiogenin receptor-2 mRNA (Tie-2 mRNA) by RT-PCR.Results(1) The characterization of BMEC was confirmed based on the morphology and positive by the detection of factorⅧantigen.(2) Oxygen glucose deprivation for 4 hours and reoxygenation for 24 hours can simulate the ischemia/reperfusion injury of BMEC in vivo.(3)NE,MTT,LDH,ET-1,ICAM-1 and Tie-2experiment show: in the process of oxygen glucose deprivation/reoxygenation ,BMEC have subjected great damagement, I/R groups have significant differences compared with NC groups (p<0.05);polymorphonuclear leukocyte can aggregrate this injury, I/R+PMN groups have significant differences compared with I/R groups (p<0.05); after Sivelestat Sodium treatment, the effect of I/R injury has been decreased, I/R+PMN+Siv groups have significant differences compared with I/R+PMN groups (p<0.05), and NE,MTT,LDH experiment show that the three dose groups there was a significant dose-effect relationship. The protective effect on the BMEC has a dose-dependent manner.Conlcusions(1) We isolated and cultured BMEC in vitro successfully. The methods provided a useful way to study the cerebrovascular disease in vitro.(2) We have established a simple, stable and reliable model of I/R of vascular endothelial cells in vitro successfully.(3) It is confirmed that NE injury brain blood capillary and aggravate I/R brain injured through increasing the expression of ET-1,ICAM-1,Tie-2. So NE will become the new pointed therapy strategy in the therapy of ischemic cerebrovascular disease. And this will lay a foundation for the Sivelestat sodium as a new nerves protective additive in the therapy of cerebrovascular disease.