| Objective: Study the role of NE in cultured astrocytes at the state of ischemia/reperfusion and the protective effect of Sivelestat Sodium. Investigate whether NE aggravates I/R injury through impaired brain blood barrier and become a new target to cure cerebrovascular diseases.To confirm whether Sivelestat Sodium will be a new neuroprotective additive to treat ischemic cerebrovascular diseases.Methods: Pure culture the rat cerebral cortical astrocytes extracted from 2 days newborn Wistar rats and randomly divided into seven 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). Sivelestat sodium group are divided into three subgroups by different drug concentrations: low, middle and high concentrations. Establish the model of cell ischemia /reperfusion. Observe the cells'morphological changes by inverted microscope,scanning and transmission electron microscope.The viability of cells in hypotonic medium was measured by MTT colorimetric method and LDH assay. The AQP4 protein exlression of cells at the state of ischemia-reperfusion were studied by immunocytochemistry and imaging analysis. Using corning transwell cell culture plate, microvascular endothelial cells and astrocytes were cocultured in a"contact through feet"model, established a vitro model of the blood-brain barrier,a vitro model of oxygen glucose deprivation/reoxygenation (OGD/R) and a model of polymorphonuclear leukocyte injury. Use the same grouping method as before.Quantitatively assay the NE by ELISA, the permeability of Horseradish peroxidase (HRP) through the blood-brain barrier, observe the structural changes by scanning and transmission electron microscopy.Results:(1) The characterization of astrocytes was confirmed based on the morphology and positive by the detection of GFAP antigen. The estab lished in vitro model of BBB has basic characteristics of BBB in vivo;(2) Oxygen glucose deprivation for 4 hours and reoxygenation for 24 hours are the best points of ischemia/reperfusion model in vitro;(3) After ischemia/reperfusion injury, NE secretory volume is increasing, BBBM+PMN groups has significant differences compared with I/R+PMN groups(p<0.05), and I/R+PMN groups has significant differences compared with I/R+PMN+Siv groups(p<0.05); (4) MTT,LDH experiment: I/R groups,I/R+PMN groups have significant differences compared with NC groups(p<0.05),at the same time, I/R groups,have significant differences compared with I/R+PMN groups(p<0.05); I/R+PMN+SivL groups , I/R+PMN+SivM groups ,I/R+PMN+SivH groups and I/R+PMN+EDA groups have significant differences compared with I/R+PMN groups(p<0.05), other aspect, the I/R+PMN+SivL groups,I/R+PMN+SivM groups,I/R+PMN+SivH groups have significant differences compared with each other(p<0.05), and I/R+PMN+SivH groups have no differences compared with I/R+PMN+EDA groups;(5) AQP4 expression, the blood-brain barrier permeability testing, scanning and transmission electron microscopy detected indicates in the ischemia/reperfusion injury that AQP4 protein expression, the blood-brain barrier permeability to macromolecules increse.I/R groups,I/R+PMN groups have statistically significant difference between these groups (p <0.05); I/R+PMN+Siv groups were reduced. I/R+PMN groups,I/R+PMN+SIR groups also have statistically significant difference (p <0.05),Conclusions: In cerebral ischemia/reperfusion injuring, NE injury brain microvascular endothelial cells and astrocytes, causing damage to the integrity of the blood-brain barrier (BBB), exacerbate the injury of brain after the ischemia/reperfusion injuried. NE will become the mew therapy strategy to cure ischemic cerebrovascular disease. Sivelestat Sodium will become a new type of neuroprotective agents to treat cerebrovascular disease.
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