| Background:Stroke is a cerebrovascular disease with high morbidity and mortality.Most stroke patients are caused by blockage of blood vessels in the brain leading to interruption of cerebral blood flow,which is also called ischemic stroke.Ischemic stroke accounts for 87%of strokes.High cost of medical treatment and poor prognosis lead to the heavy burden of families and society.After ischemia,endothelial cells were damaged by the ischemic stimulation,followed by the damage of the integrity of endothelial cell barrier,and also the increase in permeability of endothelial cells.All these result in the entry of blood-derived toxic substances into the brain parenchyma,resulting in secondary neuronal damage.Besides,endothelial cells also participate in the formation of the anticoagulation barrier between the blood and brain parenchyma.However,in the pathological state of ischemic stroke,the balance between endothelial cells anticoagulation and hemostasis was disturbed,which would promote the formation of thrombus and then aggravate the ischemic injury of endothelial cells.Salidroside has the effect of promoting blood circulation and has a significant therapeutic effect on ischemic stroke.Therefore,to explore the protective effect of salidroside on ischemic stroke endothelial cells and its mechanism of action will help clarify the mechanism of salidroside in the treatment of ischemic stroke partly.Objective:To observe the therapeutic effect of salidroside on ischemic stroke and its content in brain tissue and plasma at the overall animal level;combine both vitro and vivo experiments to observe the effect of ischemia on endothelial cell barrier function,and to explore the effect and mechanism of salidroside on endothelial cell barrier function.Methods:The first part:SD rats were randomly ranged into the following 5 groups,sham,model,Sal high-(10 mg·kg-1),middle-(5 mg·kg-1),and low-dose(2.5 mg·kg-1)group.The rat model of cerebral ischemia-reperfusion was established by suture method;the infraction area,the neurobehavioral evaluation,and cerebral water content were used as indexs for evaluating the efficacy of salidroside in the treatment of ischemic stroke.The second part:SD rats were randomly ranged into the following 3 groups,15 min,40min,and 120 min group.The rat model of cerebral ischemia-reperfusion was established by suture method,and all rats were received caudal vein injection with Sal(15 mg·kg-1).LC-MS was used to detect the content of salidroside in the brain tissue and plasma of model rats at different time points.Brain/blood was used to evaluate distribution characteristics of Sal.The third part:SD rats were randomly ranged into the following 4 groups,sham,model,Sal high-(10 mg·kg-1),and low-dose(2.5 mg·kg-1)group.The rat model of cerebral ischemia-reperfusion was established by suture method.First,the pathological morphology was observed by HE staining,and integrity of endothelial cell barrier was observed by Evans blue leakage,and transmission electron microscope was used to observe the ultrastructure of vascular endothelial cells.NO content,coagulation four values and TF,vWF content were used to observe the improvement effect of salidroside on endothelial cell anticoagulant function;WB and IF were used to observe the effect of salidroside on endothelial cell-cell junctions(ZO-1,Occludin,and claudin-1).The fourth part:A culture system of human cerebral microvascular endothelial cell line was established,and the cell viability was observed by CCK8 method,and so do the effect of salidroside on the cell viability of normal cultured endothelial cells;the endothelial cell injury model induced by OGD was established based on the previous experimental basis and references,and the integrity and permeability of the barrier were observed by endothelial resistance and FITC-dextran leakage;the content of NO in the cell supernatant was detected,and the therapeutic effect of salidroside on OGD-induced endothelial injury was observed;WB and IF were used to observe the effect of salidroside on the expression of ZO-1,Occludin,and claudin-1,which helps to explore the mechanism of salidroside’s protective effect on endothelial cells.Results:The first part:Compared with the sham group,the rats in the model group had obvious infarction and neurological deficits,the brain water content of rats in the model group was significantly increased after ischemia-reperfusion;compared with the model group,the neurobehavioral scores of the rats in the high-and low-dose groups of salidroside were reduced,and the cerebral infarction area of the rats in the high-,medium-and low-dose groups of salidroside was also significantly reduced;the intervention of different doses of salidroside could alleviate the brain edema after ischemia.The second part:15 min after administration,the content of salidroside in rat plasma,the left and the right brain tissue were 7.84 μg mL-1,0.04 μg·mL-1,and 0.02 μg·mL-1.At 120 min,the content of salidroside in plasma and the left and the right brain tissue were 0.380μg·mL-1,0.007 and 0.005 μg·mL-1.There was no significant difference in the content of salidroside in the left and right brain tissues at each time point.The brain/blood of the rats at each time were lower than 0.1.The third part:HE staining,Evans blue leakage,and cortical tissue transmission electron microscopy experiments showed that after cerebral ischemia-reperfusion in the model group,and neuronal necrosis also occurs in cortical tissue,the endothelial cell structure was destroyed,the tight junctions between endothelial cells were disintegrated,thus induced obvious Evans Blue leaks;compared with the model group,the damage of cortical neurons in salidroside dose groups were reduced,the exudation of Evans blue was reduced,and the physiological structure of endothelial cells was relatively complete.In the model group,the serum NO level was significantly decreased,and the plasma expression levels of TF,vWF,and FIB were also significantly increased.TT,PT were significantly shorter than that of the sham group.Compared with the model group,the content of serum NO in the low-dose and high-dose groups of salidroside increased,the levels of TF,FIB,and vWF decreased,and the TT and PT were also prolonged,WB and IF results showed that compared with the sham group;the expression level of MMP-9 in model group was also significantly higher,the expression level of ZO-1,Occludin,and claudin-1 was significantly reduced.Compared with the model group,both high-and low-dose of salidroside can increase the expression of ZO1,Occludin,and claudin-1 to a certain extent;both the high-and low-dose of salidroside interventions significantly inhibited the increase in the expression of MMP-9 after cerebral ischemia-reperfusion.The fourth part:After OGD treatment,the viability of HCMEC cells was reduced,the release of NO was also significantly reduced,the transmembrane resistance of HCMEC cells was significantly reduced,and the leakage of FITC-dextran was increased.After the intervention of salidroside,the viability of HCMEC cells after OGD was significantly improved,and the release of NO from HCMEC cells was also significantly increased.In addition,salidroside can also reverse the increase in HCMEC cell permeability caused by different OGD treatment times.WB and IF results showed that compared with the control group,the expression of ZO-1,Occludin,and claudin-1 in HCMEC cells after OGD was significantly decreased,and the expression of MMP-9 was significantly increased.50 μM salidroside intervention can inhibit the increase of MMP-9 expression and increase the expression of ZO-1,Occludin,and claudin-1.Conclusion:In conclusion,salidroside can inhibit the disintegration of tight junction by increasing the release of no from endothelial cells and inhibiting the expression of MMP-9 after ischemia,improve the damage of endothelial cell barrier function caused by ischemia,protect endothelial cell barrier and play a brain protective role. |
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