A novel mechanism of neurovascular protection in ischemic tolerance

 描述(申请人提供)：神经血管单位(NVU)是大脑的结构和功能元素，对缺血性中风非常敏感。中风是第三大致死原因和长期致残的首要原因，给社会造成巨大的社会和经济负担。尽管FDA已经批准了用于恢复血液流动的组织纤溶酶原激活剂，但还没有针对中风患者的神经保护药物。近年来，缺血预适应(IPC)被证明能够诱导脑对NVU继发性缺血损伤的缺血耐受；然而，其潜在的保护机制尚不清楚。使用体外和体内的方法，我们已经获得了令人振奋的初步结果，表明IPC通过转录因子Nrf2和产生内源性脂质亲电体来保护缺血损伤，从而产生轻微的氧化应激。抑制Nrf2或用N-乙酰半胱氨酸中和亲电体可消除缺血耐受。亲电体强健地激活Nrf2，诱导2相酶，并保护小鼠脑、培养的大鼠原代皮质神经元、小鼠脑微血管内皮细胞(MBMEC)和星形胶质细胞-内皮细胞共培养的细胞免受缺血损伤。此外，用4-HNE或预适应处理MBMEC细胞可减轻缺血引起的血脑屏障(BBB)损伤。本研究的目的是进一步探讨IPC诱导Nrf2激活和神经血管保护的机制。总的假设是，IPC在大脑中引起轻微的氧化应激，导致亚致死水平的脂类亲电体的产生；这些亲电体随后通过抑制Keap1和GSK3？激活Nrf2途径，从而保护NVU成分。提出了三个具体目标：目的1测试假说，即IPC提供了对局灶性脑缺血的长期神经保护，并且Nrf2和亲电体是持续保护所必需的；Aim 2测试假说，脂类亲电体通过抑制NVU成分细胞中的Keap1和GSK3？激活Nrf2；Aim 3测试假说IPC通过上调CDH5来增强BBB，以及Nrf2作用保护缺血脑中的BBB和NVU。这种对IPC保护机制的彻底研究可能有助于开发未来的治疗方法，促进中风患者的内源性调节机制。

 DESCRIPTION (provided by applicant): Neurovascular units (NVU) are the structural and functional elements of the brain and are exquisitely susceptible to ischemic stroke. Stroke is the third leading cause of death and the leading cause of long-term disability, posing an enormous social and economic burden to society. Although the FDA has approved tissue plasminogen activator for restoring blood flow, no neuroprotective medicine is available for stroke victims. Ischemic preconditioning (IPC) has recently been shown to be able to induce ischemic tolerance of the brain against subsequent ischemic injury to the NVU; however, the underlying protective mechanisms are not clear. Using both in vitro and in vivo methods, we have obtained exciting preliminary results suggesting that IPC protects against ischemic injury via the transcription factor Nrf2 and the generation of endogenous lipid electrophiles that create mild oxidative stress. Suppressing Nrf2 or neutralizing electrophiles with N- acetylcysteine eliminates the ischemic tolerance. Electrophiles robustly activate Nrf2, induce phase 2 enzymes, and protect mouse brains, cultured rat primary cortical neurons, mouse brain microvessel endothelial cells (MBMEC), and astrocyte-endothelial cell co-cultures from ischemic injury. Furthermore, treating MBMEC cells with 4-HNE or preconditioning attenuates blood-brain barrier (BBB) damage induced by ischemia. The purpose of this proposal is to further investigate the mechanisms responsible for Nrf2 activation and neurovascular protection induced by IPC. The overall hypotheses are that IPC causes mild oxidative stress in the brain, leading to the generation of sublethal levels of lipid electrophiles; these electrophiles then activate the Nrf2 pathway by suppressing both Keap1 and GSK3ß, thereby protecting NVU components. Three specific aims are proposed: Aim 1 tests the hypotheses that IPC offers long-term neuroprotection against focal ischemia in mice and that Nrf2 and electrophiles are required for the sustained protection; Aim 2 tests the hypothesis that lipid electrophiles activate Nrf2 by inhibiting Keap1 and GSK3ß in NVU component cells; Aim 3 tests the hypothesis that IPC reinforces BBB via upreglulating CDH5 and that Nrf2 action protects the BBB and NVU in ischemic brain. This thorough investigation of the protective mechanisms of IPC may help develop future therapies that boost endogenous regulatory mechanisms in stroke victims.