ENOPH1在急性缺血性脑卒中血脑屏障内皮细胞凋亡中的功能及机制研究
发布时间：2018-10-24 07:19
【摘要】：烯醇化酶磷酸酶1(Enolase-phosphatase 1 ENOPH1)是甲硫氨酸补救途径的重要蛋白分子,具有磷酸酶功能,属于非典型烯醇化酶活性的双功能酶,在调节氧化应激反应中发挥着重要作用。本次研究探究的是ENOPH1在急性缺血性脑卒中血脑屏障损伤中扮演的角色。前期研究发现:局灶性脑缺血可诱导大鼠脑微血管组织中ENOPH1的mRNA和蛋白表达水平增高。本研究通过乳酸脱氢酶释放法及TUNEL法检测到3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴化物有所增加,提示缺血缺氧条件(oxygen-glucose deprivation,OGD)下,脑微血管内皮细胞(bEND3 cells)内ENOPH1的mRNA和蛋白表达水平上调,同时伴随着细胞凋亡增加。通过质粒转染法敲低ENOPH1和过表达ENOPH1蛋白水平可分别减少和增加OGD诱导下内皮细胞凋亡,甚至,大幅度扩大或减少氧化应激反应内凋亡相关蛋白(如caspase-3,PARP,Bcl-2 and Bax)和内质网应激蛋白(Ire-1,Calnexin,GRP78 and PERK)的表达水平。同样发现OGD条件下还可诱导ENOPH1下游蛋白分子乙酸二羟丙烯醛加双氧酶(acireductone dioxygenase 1,ADI1)表达水平上调,并且OGD可促进ENOPH1与ADI1之间的相互作用。但有趣的是,敲低ENOPH1并未参与OGD诱导下ADI1蛋白表达水平上调,而只是调节ADI1从细胞核向细胞质转运的过程。最后还发现敲低ENOPH1可显著降低OGD诱导的内皮细胞通透性增加。由此,本研究得出的结论是:ENOPH1的激活通过促进氧化应激反应,增加促凋亡相关蛋白的表达,从而加重OGD诱导下内皮细胞凋亡和血脑屏障损伤。因此,ENOPH1是治疗急性缺血性脑卒中新的靶点。
[Abstract]:Enolase phosphatase 1 (Enolase-phosphatase 1 ENOPH1) is an important protein molecule of methionine remediation pathway. It has phosphatase function and belongs to a bifunctional enzyme with atypical enolase activity, which plays an important role in the regulation of oxidative stress reaction. This study explores the role of ENOPH1 in blood-brain barrier damage in acute ischemic stroke. Previous studies have shown that focal cerebral ischemia can induce increased expression of ENOPH1 mRNA and protein in rat brain microvascular tissue. In this study, lactate dehydrogenase release assay and TUNEL assay were used to detect the increase of 3- (4-dimethylthiazole-2) -2-diphenyl tetrazolium bromide, which suggested that the expression of mRNA and protein in cerebral microvascular endothelial cells (bEND3 cells) was up-regulated under ischemia and hypoxia (oxygen-glucose deprivation,OGD) conditions. At the same time, apoptosis was increased. Knockout and overexpression of ENOPH1 protein by plasmid transfection can decrease and increase the apoptosis of endothelial cells induced by OGD respectively. The expression of apoptosis-related proteins (such as caspase-3,PARP,Bcl-2 and Bax) and endoplasmic reticulum stress protein (Ire-1,Calnexin,GRP78 and PERK) in oxidative stress was significantly increased or decreased. It was also found that the expression of dihydroxyacrolein acetate (acireductone dioxygenase _ 1), a protein molecule in the downstream of ENOPH1, could be up-regulated by OGD, and that OGD could promote the interaction between ENOPH1 and ADI1. Interestingly, knockout ENOPH1 was not involved in the up-regulation of ADI1 protein expression induced by OGD, but only in the process of ADI1 transport from nucleus to cytoplasm. It was also found that knockout of ENOPH1 could significantly reduce the increase of endothelial cell permeability induced by OGD. It is concluded that the activation of ENOPH1 increases the expression of pro-apoptosis-related protein by promoting oxidative stress response, thereby exacerbating endothelial cell apoptosis and blood-brain barrier injury induced by OGD. Therefore, ENOPH1 is a new target for the treatment of acute ischemic stroke.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
本文编号：2290703
[Abstract]:Enolase phosphatase 1 (Enolase-phosphatase 1 ENOPH1) is an important protein molecule of methionine remediation pathway. It has phosphatase function and belongs to a bifunctional enzyme with atypical enolase activity, which plays an important role in the regulation of oxidative stress reaction. This study explores the role of ENOPH1 in blood-brain barrier damage in acute ischemic stroke. Previous studies have shown that focal cerebral ischemia can induce increased expression of ENOPH1 mRNA and protein in rat brain microvascular tissue. In this study, lactate dehydrogenase release assay and TUNEL assay were used to detect the increase of 3- (4-dimethylthiazole-2) -2-diphenyl tetrazolium bromide, which suggested that the expression of mRNA and protein in cerebral microvascular endothelial cells (bEND3 cells) was up-regulated under ischemia and hypoxia (oxygen-glucose deprivation,OGD) conditions. At the same time, apoptosis was increased. Knockout and overexpression of ENOPH1 protein by plasmid transfection can decrease and increase the apoptosis of endothelial cells induced by OGD respectively. The expression of apoptosis-related proteins (such as caspase-3,PARP,Bcl-2 and Bax) and endoplasmic reticulum stress protein (Ire-1,Calnexin,GRP78 and PERK) in oxidative stress was significantly increased or decreased. It was also found that the expression of dihydroxyacrolein acetate (acireductone dioxygenase _ 1), a protein molecule in the downstream of ENOPH1, could be up-regulated by OGD, and that OGD could promote the interaction between ENOPH1 and ADI1. Interestingly, knockout ENOPH1 was not involved in the up-regulation of ADI1 protein expression induced by OGD, but only in the process of ADI1 transport from nucleus to cytoplasm. It was also found that knockout of ENOPH1 could significantly reduce the increase of endothelial cell permeability induced by OGD. It is concluded that the activation of ENOPH1 increases the expression of pro-apoptosis-related protein by promoting oxidative stress response, thereby exacerbating endothelial cell apoptosis and blood-brain barrier injury induced by OGD. Therefore, ENOPH1 is a new target for the treatment of acute ischemic stroke.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
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