Effects Of β-Arrestin On Vascular Function And Ischemic Stroke

[Objective]Our project was to apply knockout mice to investigate the role of β-Arrestin playing in vessels and brain. And project was divided into three parts:the first one is genotype identifying of β-Arrestin knockout mice and expression of β-Arrestin; the second one is the effect of β-Arrestin in vascular function; the last one is the effect of β-Arrestin on ischemic stroke after MCAO.[Method]Part Ⅰ:Genotype identifying of β-Arrestin knockout mice and expression of β-Arrestin Mating the β-Arrestin land β-Arrestin2heterozygous breeders, and expanding the posterity.4weeks after born, extracting tail DNA of knockout mice, measuring DNA concentration and run realtime-PCR for β-Arrestin las well as PCR for β-Arrestin2to idenfy the genotype respectively.8-12weeks after born, C57BL6and three genotypes of mice were sacrificed with anesthesia, and then thoracic aorta, heart and brain were extracted to analysis the expression of β-Arrestin1and β-Arrestin2by western blot. Part Ⅱ:Study on effects of β-Arrestin in vascular function;Systolic and diastolic function of thoracic aorta extracted fromp-Arrestinl and β-Arrestin2mice was detected in this study.8-12weeks old, three types of genotype (homozygote, heterozygote and wildtype) knockout mice were killed by dislocated and then thoracic aorta was extracted, vascular adventitia and endothelial were excluded quickly and the aorta were cut into several rings. Bathing in isolated organ perfused system, aorta rings were excited by phenylephrine or angiotensin Ⅱ changes in tension were recorded.Part Ⅲ:Study on effects of β-Arrestin on brain after MCAOThis part investigated the effect of β-Arrestin on infarct area and neurological score after permanent MCAO.8-12weeks old, three types of genotype (homozygote, heterozygote and wildtype) knockout mice were performed by permanent MCAO, after24hours, neurological score and infarct area of brain slices were measured. Changes of β-Arrestin1 and β-Arrestin2expression after permanent MCAO in ischemic penumbra of C57BL6mice were detected by western blot. Primary cultured neurons extracted wildtype and homozygote mice were subjected to oxygen-glucose deprivation; cell number, cell viability and cell injury were detected by different methods.[Result]Part Ⅰ:β-Arrestinl is high expression in thoracic aorta, heart and brain extracted from C57BL6, β-Arrestinl heterozygote and wildtype β-Arrestinl mice, but without homozygote mice. β-Arrestin2expresses in brain of C57BL6, heterozygote and wildtype β-Arrestin2mice, less in thoracic aorta, but without in tissues extracted from homozygote β-Arrestin2mice.Part Ⅱ:1, The optimal resting tension of thoracic aorta ring was1.5g.2, Low reactivity to Angiotensin Ⅱ was detected in aorta rings extracted from β-Arrestin1and β-Arrestin2knockout mice, and had no difference between wildtype mice.3, Tension to phenylephrine of heterozygote mice aorta ring was higher than wildtype ring in P-Arrestinl mice, but without statistical difference; there was no statistical difference between homozygote and wildtype aorta ring tension.Part Ⅲ:1, β-Arrestin1and β-Arrestin2were up-regulated in the ischemic penumbra of C57BL6at24hours after MCAO;2, Heterozygote and wildtype β-Arrestinl mice had the similar infarct area and neurological score, but homozygote mice had bigger infarct area and increased neurological score;3, As to β-Arrestin2, heterozygote and wildtype mice had the similar infarct area and neurological score, but infarct area in homozygote mice was smaller with statistical difference, as well as decreased neurological score without statistical difference;4, knockout β-Arrestinl gained the number of dead neuron, decreased the cell viability and increased the cell injury after OGD performance.[Conclusion]1. β-Arrestin didn’t affect the systolic reactivity induced by phenylephrine.2. Mice thoracic aorta ring had low systolic reactivity to Angiotensin Ⅱ, and had no relation to β-Arrestin. 3. β-Arrestin1had protective effect on ischemic stroke, and was an endogenous protective factor.4. Knockout β-Arrestin2could reduce the infarct area but had no effect on the neurological function.