钙通透TRPV3离子通道在脑缺血性损伤中的作用机制研究

The hypothesis of NMDA receptor (NMDAR)-mediated excitotoxicity is based on the observation of excessive rise in intracellular Ca2+ during ischemia stroke and success of NMDAR antagonists in animal models. However, anti-excitotoxic therapies (ATEs) failed in all clinical trials, indicating that non-NMDA based Ca2+ entry pathways play an important role brain ischemic injury. One well-known effect of ischemic stroke is acute acidosis due to increasing lactic acid concentration. Our proposed study aims to understand the mechanism underlying acidosis-induced brain damage. Our recent studies discovered that TRPV3, a non-selective Ca2+-permeably ion channel broadly expressed in the brain, is highly sensitive to acidosis. This new finding suggests that the channel may be a critical target for ischemic stroke to induce cytotoxicity and brain infarction. Based on the strong evidence for a crucial role TRPV3 plays in ischemia-induced tissue damage, in this proposal we aim to test a novel hypothesis that brain acidosis by lactic acid activates TRPV3 to induce sustained Ca2+ influx, leading to cytotoxicity and ischemic injury. Understanding the role of calcium-permeable TRPV3 in brain ischemia injury may lead to validation of TRPV3 as a target and identification of specific inhibitor for potential therapy.

NMDA受体拮抗剂在临床试验治疗脑卒中疾病的失败说明，非NMDA介导的钙超载是神经缺血性损伤的主要机制。因此，探索脑缺血性损伤钙内流超载的非NMDA来源与机制对发现有效抗脑缺血损伤的靶标和潜在的临床治疗具有重要意义。脑缺血性损伤或缺血性再灌注损伤的重要病理特征表现为脑缺血引起的组织酸化和代谢性酸中毒。我们前期研究发现，细胞内氢离子浓度升高即细胞内酸化可特异性激活增强TRPV3通道的功能导致细胞内钙超载并引起凋亡，提示钙通透TRPV3通道在脑缺血性损伤中扮演着重要的角色。本项目拟对TRPV3通道在脑缺血性损伤中的作用与机制进行深入研究，以期探讨TRPV3在缺血性脑卒中疾病模型中的作用和抑制TRPV3的缺血性保护作用，通过筛特异性TRPV3活性小分子抑制剂，进而探讨靶向TRPV3通道特异性抑制剂在脑缺血性损伤保护与治疗的潜在应用价值。

NMDA（N-甲基-D-天冬氨酸）受体拮抗剂在临床试验治疗脑卒中疾病的失败说明，非NMDA介导的钙超载是神经缺血性损伤的主要机制。因此，探索脑缺血性损伤钙内流超载的非NMDA来源与机制对发现有效抗脑缺血损伤的靶标和潜在的临床治疗具有重要意义。脑缺血性损伤或缺血性再灌注损伤的重要病理特征表现为脑缺血引起的组织酸化和代谢性酸中毒。我们前期研究发现，细胞内氢离子浓度升高即细胞内酸化可特异性激活增强TRPV3通道的功能导致细胞内钙超载并引起凋亡，提示钙通透TRPV3通道在脑缺血性损伤中扮演着重要的角色。之后，运用分子生物学、动物模型和高通量钙荧光筛选等方法发现①非NMDA受体机制参与钙超载兴奋毒性引起的缺血性损伤和延迟的神经细胞凋亡。通过基因沉默手段，证实过度的激活TRPV3介导钙离子内流超载，从而导致神经细胞凋亡；②通过行为学评估和脑组织切片，确认TRPV3基因敲除小鼠对大脑缺血性损伤模型(MCAO)的保护作用；③建立了基于活细胞筛选TRPV3通道抑制剂的高通量钙荧光筛选方法，筛选出特异性抑制TRPV3的天然活性小分子化合物；④评估靶向TRPV3通道的特异性小分子抑制剂在脑缺血损伤模型中的保护作用，为TRPV3抑制剂的潜在应用提供药理学依据。