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Effect Of THBS1 On Cerebral Vascular Endothelial Function Injury In Cerebral Ischemia Reperfusion Mice By Regulating ENOS

Posted on:2024-07-23Degree:MasterType:Thesis
Country:ChinaCandidate:F ZhengFull Text:PDF
GTID:2544307082964109Subject:Physiology
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Background:Stroke is the leading cause of death and acquired disability in adults,of which ischemic stroke poses a significant threat to human health;However,effective treatments are currently limited.Known cardiovascular and cerebrovascular risk factors promote endothelial dysfunction in the brain,leading to impaired vasodilation and increased inflammatory responses,as well as oxidative stress and increased vascular hyperplasia,which culminate in thromboinflammatory responses,a potential cause of ischemic stroke.These events are further exacerbated when blood returns to the brain after a period of ischemia,a phenomenon known as ischemia-reperfusion injury.Vascular endothelium is a key target for ischemia-reperfusion(I/R)injury severity,no matter what tissue or organ is involved.Endothelial cells can catalyze L-arginine synthesis of Nitric oxide(NO)by Endothelial nitric oxide synthases(eNOS).Nitric oxide(NO)/c GMP signaling pathway is a central regulator of angiogenesis and tissue perfusion during ischemia.THBS1 signaling pathway effectively inhibits NO biosynthesis and vascular cell signaling through its receptor CD47.However,the mechanism of THBS1 induced cerebral vascular endothelial dysfunction in ischemia-reperfusion injury has not been fully elucidated.Purpose:To investigate the role and mechanism of THBS1 in cerebral endothelial function injury of cerebral ischemia reperfusion mice at animal level.At the cellular level,the effects of oxygen glucose deprivation and reoxygenated glucose on THBS1 protein expression in BMECs and the regulatory role of THBS1 on eNOS were verified.It provides a new molecular mechanism for the prevention and treatment of cerebral vascular injury caused by cerebral ischemia reperfusion.Method:1.Experimental mice were randomly divided into model group and sham operation group,and the middle artery of mice was blocked by external cervical access method.After 1.5 h of cerebral ischemia and 24 h of reperfusion,the brain was taken for TTC staining and the volume of cerebral infarction was calculated.2.The effects of THBS1 protein and cerebral ischemia-reperfusion injury on cerebral basilar artery diastolic function in rats were detected by angiotonometry.3.Primary cultured rat brain microvascular endothelial cells were identified by immunofluorescence.4.Westen blot was used to detect the effects of oxygen and sugar deprivation and reoxygenation on THBS1 and eNOS protein expression in cerebral microvascular endothelial cells.5.Calcium ion imaging was used to detect the effects of oxygen and sugar deprivation and reoxygenation on calcium inflow in cerebral microvascular endothelial cells.6.The effects of glucose deprivation and reoxygenation on the release of NO in cerebral microvascular endothelial cells were detected by NO fluorescent probe.Results:1.The expression of THBS1 in plasma of stroke patients was significantly increased.2.Compared with mice in WT(Wild type,WT)ischemia group,the cerebral infarction volume of THBS1-/-mice after ischemia was significantly reduced.3.After ischemia-reperfusion,THBS1 expression increased in the basal artery endothelium of WT mice.4.Compared with the control group,THBS1 incubation group significantly weakened endothelium dependent relaxation of basilar artery,but had no significant effect on endothelium independent relaxation.5.Compared with WT mice,THBS1-/-basilar artery endothelium dependent relaxation was significantly enhanced;Both WT and THBS1-/-mice showed significantly lower post I/R endothelium dependent diastole compared with their respective controls.6.Compared with the I/R model of WT mice,THBS1-/-mice showed significantly enhanced endothelium dependent diastole of basilar artery after I/R.7.Sequencing results showed that the expression of eNOS(also known as NOS3)increased significantly after THBS1 knockout,which was verified by immunoblotting.8.In vitro model of ischemia-reperfusion,compared with glucose deprivation,reoxygenated glucose had a greater effect on THBS1 expression in BMECs,and THBS1 expression increased in a time-dependent manner within a certain period of time.9.Compared with WT mice,NO released by BMECs after THBS1 knockout was reduced;Compared with the control group,NO was released by BMECs after OGD/G in WT mice,while NO was released in THBS1-/-mice.10.THBS1 can reduce BMECs SOCE amplitude in WT mice,that is,decrease calcium influx.The expression of Orai1 and STIM1 proteins,the main components of SOCE,increased after THBS1 knockout.Conclusion:In this study,it was found that the expression of THBS1 on the basilar artery endothelium increased after cerebral ischemia reperfusion in WT mice,and the endothelium dependent relaxation of basilar artery was significantly weakened,and exogenous THBS1 could reduce the endothelium dependent relaxation of basilar artery.At the same time,we used THBS1-/-mice to test the correlation between THBS1 and impaired cerebral vascular function after ischemia reperfusion.Then we used RNA-Sequences technology to process the primary cultured BMECs and sequence them to screen out the genes related to vascular endothelial dysfunction(such as eNOS),and verify the influence of THBS1 changes on them.In vitro experiments,we demonstrated the effects of oxygen glucose deprivation and reoxygenated glucose on THBS1 expression and NO release in BMECs.
Keywords/Search Tags:THBS1, eNOS, Cerebralischemiar eperfusion, Endothelium
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