Cysteamine Treatment Of Ischemic Stroke By Inhibiting Oxidative Stress On Endothelial Cells

In most countries,stroke especially ischemic stroke,various kinds of heart diseases and malignant tumors become the main three major cause of death.According to the pathogenesis of stroke divided into cerebral thrombosis,cerebral embolism and hemodynamic mechanisms caused by cerebral infarction.In the clinical,the cerebral thrombosis is the most common pathophysiological process of cerebral infarction.Cerebral thrombosis has close relationship with endothelial cell injury.Normal vessel wall was covered with vascular endothelial cells,normal endothelial cells itself has the function of inhibiting platelet aggregation,inhibit thrombosis.When endothelial cells lesions by oxidative stress,it caused by internal and external stimulation,will lead to abnormal apoptosis of endothelial cells,making the subcutaneous tissue exposed,resulting in platelet aggregation and thrombosis,leading to blood flow interruption supply area,eventually lead to ischemia and anoxic deformation necrosis.The endothelial cells of brain blood vessels was damaged,lead to orgburo brain ischemia,and then produce the signs and symptoms of nerve defect.Therefore,inhibiting apoptosis of endothelial cells caused by oxidative stress become a new way of thinking for the treatment of ischemic stroke.Experiment purpose1.To explore the protective effect of cysteamine on endothelial cells from oxidative stress injury and its mechanism.2.Discussion of cysteamine on ischemic stroke in rats and its molecular mechanism.Research MethodsAccording to the principle of cerebral thrombosis,we simulate the process of endothelial cell injury in vitro.Cultured the human umbilical vein endothelial cells(HUVECs)and divided the cells into five groups as control group,rhodamine B group,rhodamine B and light combination group,cysteamine group,cysteamine protection group.The morphology and density changes were observed the phase contrast microscope.The cell viabilities were detected by trypan blue staining.Cell cycles and apoptosis were measured flow cytometry.Caspase activity was detected by fluorescence quantification.DCFH assay detect intracellular ROS.Use the Western blotting method to detect the related proteins.The model of the cerebral focal ischemia was carried out by photochemical method.The mice were randomly divided into three groups as sham group,vehicle group and cysteamine treated group.To evaluate the neruonogical behavior,the Zea Longa's scoring,foot-fault test and the cylinder test were carried out at 1d,2d and 3d after the operation repectively.To observe the infraction of the brain and determine the volume of the infraction,TTC staining were applied.Brain edema was evaluated by wet-dry method.To illustrate the mechanism of neuroprotective effect,the expression of MPO and AQP4 in the brain were detected by immunofluorescent staining.ResultsUnder phase contrast microscope observation,found that cysteamine protection group compared with photo-oxidative injury group,significant changes in cell morphology,cell death was significantly reduced.Trypan blue showed that,compared to normal cells,photooxidative injury group cell activity decreased,cysteamine can increase cell activity,the differences were statistically significant(P<0.05).Flow cytometry results showed that photo-oxidative injury group was significantly apoptosis,but cysteamine protection group Sub-G1 peak decreased.DCFH-DA staining discovery that in photo-oxidative injury group ROS increased,after protecttion of cysteamine ROS level was lower.Western blotting results showed that cysteamine protection group,DNA injury associated protein ATM,ATR,p53 expression decreased,the mitochondrial apoptosis pathway associated proteinmediated caspase-3,-7,-9 expression decreased,MAPK / AKT signaling pathway related protein p38 / JNK expression decreased,while ERK / AKT expression increased.Cysteamine treated group compared with vehicle group,all the neurological deficits were significantly improved at 1d,2d and 3d,the infarct volumes and the degrees of edema were all decreased,that results were significant differences in statistics.By immunofluorescence staining and qualitatively observation showed that the sham brain tissue seen in a small number of scattered cells AQP4 and MPO expression,while massive AQP4 and MPO expression were observed in vehicle mice,AQP4 and MPO expression was remarkably attenuated in cysteamine-treated mice.ConclusionWe established the stable endothelial cell injury model in vitro by photochemical method.Cysteamine can inhibit photooxidation-induced apoptosis of endothelial cells by inhibiting ROS-mediated DNA damage and regulation of MAPK and AKT signaling pathways.Cysteamine has a protective effect on early cerebral ischemia in mice.Cysteamine can reduce post-ischemic stroke neurological deficit,it can significantly reduce infarct volume and cerebral edema after stroke.Cysteamine is expected to be a high efficiency,low toxicity and mechanism clearly drug,it can be widely used in the treatment and prevention of stroke.