多巴胺/D2受体轴在脑出血后血脑屏障损伤中的保护作用及机制研究

Intracerebral hemorrhage (ICH) is a fatal stroke subtype of all strokes. Currently, there is no effective therapy to improve survival rate. The failure of Clinical Trials indicate endogenous mediators regulating BBB integrity should be pay more attention to. Our previous work revealed an up-regulation of dopamine secretion and DRD2 expression in experimental ICH model. However, whether the regional dopamine alterations is relative to neuroprotection in response to hemorrhagic injury remains unknown. In the present study, we sought to determine physiological role of endogenous Dopamine-D2 receptor system in maintaining BBB integrity and the potential therapeutic utility of dopamine agonists in ICH mice models. In order to identify the essential role of endogenous dopamine system for the maintenance of BBB structure and function, we plan to first observe the outcome on BBB permeability using DRD2 knockout mice after ICH insult. Secondly, L-DOPA and two D2 receptor agonist Quinpirole and Ropinirole were administered by daily intraperitoneal injection to investigate the therapeutic effect in experimental ICH model. Also, in vivo siRNAs, the Gβγ subunit inhibitor and ERK1/2 inhibitor will be delivered to study the downstream pathways of DRD2. Post-ICH assessment included neurological function tests, brain edema, Evans blue extravasation, zymography assay, western blot, and immunohistochemistry assay. What’s more, the molecular mechanism will be validated in cell lines in vitro. Our investment will uncover a distinct role of endogenous Dopamine-D2 receptor system in maintaining BBB integrity following ICH and can be an attractive choice to improve the efficacy of therapies for ICH.

脑出血（ICH）是临床最常见的脑血管危急重症之一，目前仍缺乏有效的治疗策略。大量药物临床试验的失败提示应更加重视神经系统“自我修复”的内源性的保护机制。本项目在前期发现ICH后内源性多巴胺和D2受体均显著上调的基础上，并结合L-dopa治疗可以缓解BBB损伤的现象，推测多巴胺/D2受体轴可能具有维持脑血管屏障稳定性的生理作用，其机制可能是调控CRYAB/NF-κB抑制MMP9活化，进而减弱内皮连接蛋白的降解。我们拟首先观察DRD2-/-小鼠在ICH后的BBB病理性通透中的表型，以明确内源性DRD2在维持脑微血管屏障稳定性中的生理意义。然后，探索外源性L-dopa、DRD2不同激动剂对ICH后BBB损伤及神经功能障碍的治疗效应；并结合离体细胞模型、基因沉默、抑制剂干预、免疫共沉淀等技术，阐明DRD2调控MMP-9活性的中间分子通路。本项目为探索内源性保护机制和ICH患者临床救治提供新的靶点。

自发性脑出血（Spontaneous intracerebral hemorrhage，ICH），是临床最常见的脑血管危急重症之一。目前临床上除了运用传统脱水药物外，仍缺乏有效的治疗策略来减低死亡率和改善不良预后。因此，探索ICH后早期脑损伤的干预措施变得尤其重要，成为降低致死致残率和改善预后的关键和临床转化研究的热点。然而，针对ICH的各种神经保护药物在大规模临床试验“Clinical Trial”中的纷纷以失败告终，促使大家愈加重视探索脑出血后神经系统“自我修复”的病理生理过程和内源性的保护机制。本项目以脑出血神经功能救治的新靶点——血脑屏障损伤修复为切入点，探讨多巴胺/D2受体轴在维持血脑屏障完整性中的作用及相应机理。主要研究内容包括：1）ICH后DRD2和MMP-9表达变化规律及细胞定位；2）ICH在体模型中DRD2激动剂对血脑屏障损伤及神经功能的效应；3）探讨DRD2调控MMP-9活性的可能分子机制。本研究发现ICH后内源性多巴胺和受体的表达显著升高。同时，外源性DRD2激动剂能够显著改善ICH后血脑屏障失调并改善神经功能预后，其机制是依赖Gβγ-ERK1／2-CRYAB通路减少内皮细胞间隙蛋白的降解。因此，我们的研究阐明了内源性多巴胺在ICH之后对血脑屏障完整性的保护作用，外源性DRD2激动剂疗法有可能成为改善ICH患者血脑屏障功能的可靠的新选择。