WNK信号通路对高血压合并缺血性脑卒中脑损伤的作用研究

Ischemic stroke is mainly responsible for non-infectious death and disability in China, leading to heavy social burdens. Hypertension is the first risk factor of ischemic stroke. Recently, it is found that the signaling pathway consisting of the with-no-lysine (K) kinases (WNK), Ste20-related proline alanine-rich kinase (SPAK) and the downstream Na-K-Cl cotransporter (NKCC1) is closely associated with pathogenesis of hypertension. Our recent studies have shown that up-regulation of WNK signaling in spontaneously hypertensive rats may contribute to the worsened ischemic stroke brain injury and that blocking WNK signaling pathway could reduce ischemic brain damage. Therefore, we speculate that WNK signaling pathway may be associated with the pathogenesis of ischemic brain damage caused by ischemic stroke accompanied with hypertension. In this proposal, we plan to use the hypertensive mice and patients with ischemic stroke to confirm three important issues as followings: (1) the expression of WNK-SPAK/OSR1-NKCC1 signaling proteins after hypertension and ischemic stroke, and the correlation of the expression level of these proteins with the degree of brain damage; (2) the protective effect of SPAK inhibitor on brain damage caused by ischemic stroke accompanied with hypertension; (3) the correlation of WNK-SPAK/OSR1-NKCC1 signaling pathway and ischemic brain damage in clinical cases. This study will provide new evidences to explain the pathogenesis of ischemic stroke, and may discover new therapeutic targets for treatment of ischemic stroke especially ischemic stroke accompanied with hypertension.

缺血性脑卒中是我国非传染性致死致残的重要原因，造成沉重的社会负担，高血压是其首要危险因素。已证实，WNK-SPAK/OSR1-NKCC1信号通路与高血压密切相关。课题组前期研究发现，自发性高血压大鼠WNK信号表达水平高于正常鼠，其发生缺血性脑卒中后脑损伤趋于严重；抑制WNK信号通路，可减轻脑损伤程度。基于此，提出研究假设：WNK信号通路可能参与高血压合并缺血性脑卒中后脑损伤的致病过程。本项目拟进行以下研究：(1) 分析WNK-SPAK/OSR1-NKCC1通路在高血压和缺血性脑卒中时的表达及与脑损伤的相关性；(2) 探讨SPAK阻断剂对高血压合并缺血性脑卒中所致脑损伤的保护作用；(3) 在临床病例中验证WNK-SPAK/OSR1-NKCC1通路与高血压合并缺血性脑卒中所致脑损伤的相关性。本项目将为明确缺血性脑卒中的发病机制提供新证据，也将为治疗缺血性脑卒中尤其是高血压合并脑卒中提供新靶点。

缺血性卒中是致死和致残的最常见疾病之一，造成沉重的社会负担。课题组前期研究发现，自发性高血压大鼠WNK-SPAK/OSR1-NKCC1信号通路表达水平高于正常血压大鼠，其卒中后脑损伤更严重。布美他尼 (BMT) 可有效抑制卒中脑组织Na+ -K + -Cl-协同转运蛋白1 (NKCC1)。然而，BMT的缺点是其血脑屏障渗透性较差。本研究采用两种新型NKCC1抑制剂: STS5和STS66，观察其对于减轻缺血性脑损伤的效用，结果发现AgII诱导的自发性高血压小鼠SPAK和NKCC1的磷酸化活性明显增高，对于缺血性卒中，尤其是合并高血压的缺血性卒中，新型的NKCC1抑制剂STS66在减轻脑水肿，减小脑梗死体积，减少神经元死亡，恢复神经功能缺损等方面的疗效优于BMT。我们进一步探索WNK-SPAK/OSR1-NKCC1信号通路的上游调控因子-Cullin3（CUL3）。CUL3在外周免疫细胞中的表达与中风的发生高度相关，在本研究中，我们发现CUL3在脑缺血再灌注（I/R）损伤时明显上调，证实CUL3与WNK3相互作用，敲低CUL3抑制了WNK3的泛素化，并加速了OGD/R刺激神经元中OSR1的磷酸化，敲低CUL3减轻了神经元凋亡，发挥神经保护作用。但研究结果发现WNK3可能不参与或不直接参与CUL3介导的脑I/R损伤的主导机制。这些表明，CUL3介导的脑I/R损伤可能不是通过WNK3信号而是通过其他途径实现的。研究发现敲低CUL3抑制泛素介导的Nrf2降解，并通过增加Nrf2的核易位和HO-1和NQO-1的表达水平来激活Nrf2信号。因此，CUL3可能由于其对Nrf2激活的负调节而加剧大脑I/R损伤。同时，我们探究治疗脑I/R损伤的新靶点：PHLDA1，是免疫调节和细胞凋亡的关键决定因素，但其在I/R损伤中的作用仍有待阐明。在本研究中证明脑I/R时PHLDA1的表达显著增加，敲低PHLDA1可减少脑梗死及脑水肿体积，通过抑制PHLDA1切换小胶质细胞M1/M2极化和抑制NLRP炎症小体激活，能有效减轻脑I/R损伤。研究发现PHLDA1在脑I/R损伤中的作用可能与线粒体功能和内质网应激相关： PHLDA1介导PPARγ核易位减轻线粒体功能障碍和缓解内质网应激，从而减轻脑I/R损伤，为治疗缺血性卒中提供了新的治疗靶点。