盐敏性高血压的下丘脑神经元可塑性机制及关键分子研究

The salt sensitivity mechanism is the most important phenotype of hypertension, and neuroendocrine dysfunction is the key characteristics of the pathogenesis of salt-sensitive hypertension. For the discovery of a novel treatment of hypertension, it is important to understand the specific CNS mechanisms of salt-sensitive hypertension. The concentration of intracellular chloride ions in neurons is mainly controlled by Na+-K+-2Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 2 (KCC2). When the chloride homeostasis in neurons is disrupted, GABA can switch from functioning as an inhibitory transmitter to evoking an excitatory response. Our previous study demonstrated that GABAergic inhibition in PVN neurons was diminished after chronic infusion of high Na+ aCSF and NKCC1 was upregulated in the PVN of Dahl hypertensive rats. These observations have led us to hypothesize that the chloride homeostasis of PVN neurons is disrupted and caused by increased NKCC1 activity and (or) decreased KCC2 activity following high salt treatment in salt sensitive subject, which contributes to the diminished GABAergic inhibition and increased hypothalamic neuronal plasticity. The present proposal is designed to support/refute this hypothesis by investigating the following four specific aims: (1) determine the changes and locations of NKCC1 and KCC2 expression in the PVN of salt-sensitive hypertension; (2) determine the role of NKCC1 and KCC2 in the hypothalamic neuronal plasticity and increased excitability of the PVN neurons in salt-sensitive hypertension; (3) determine the effects of long-term viral-mediated down-regulation of NKCC1 in the PVN of salt-sensitive hypertension and (4)determine the mechanism of NKCC1 upregulation in the PVN in salt hypertension. The goal of the proposed studies is to show that NKCC1 in the brain is a key regulator of blood pressure, thus providing a novel target for the prevention and treatment of salt-sensitive hypertension.

盐敏性是高血压的重要表型之一，神经内分泌功能失调是盐敏性高血压的关键病理特点。明确盐敏性高血压的中枢机制对于发现新的高血压疗法至关重要。神经元内Cl-浓度稳态的破坏，可引起GABA效应由抑制向兴奋转化，而Na-K-2Cl共转运体1（NKCC1）和K-Cl共转运体2(KCC2)是该过程的关键分子。前期研究发现高钠脑脊液引起室旁核（PVN）神经元GABA抑制效应显著减弱，以及Dahl盐敏性高血压大鼠出现PVN的NKCC1表达上调。在此基础上提出假说：高盐通过上调PVN内NKCC1和（或）下调KCC2表达，改变神经元Cl-浓度稳态，促进突触可塑性形成，引发交感神经活性和血管升压素分泌增加。本课题拟从NKCC1和KCC2在盐敏性高血压的动态表达变化、细胞亚型分布、突触可塑性变化中的作用、长期调节效应、以及高盐引发表达变化的机制等验证假说。该研究将为盐敏性高血压的形成机制提供新的实验证据和防治靶点。

高血压是由环境因素和遗传因素共同作用所致的一种多基因和多因素的复杂性疾病。盐敏性是高血压的重要表型之一，有其自身特有的发病机制和临床特征，近年来虽然进行了大量的研究工作，但是其发病机制尚不明确。神经内分泌功能失调是盐敏性高血压的关键病理特点，明确中枢机制对于发现新的高血压疗法至关重要。本项目使用分子生物学、电生理和药理学等方法，研究了下丘脑阳离子氯离子共同转运体NKCC1和KCC2在盐敏性高血压的动态表达变化、细胞亚型分布、突触可塑性变化中的作用、长期调节效应、以及其在神经元内表达变化的可能信号转导机制。研究发现侧脑室慢性灌流高钠脑脊液模型和Dahl盐敏感高血压模型均出现PVN神经元的GABAA受体功能下调，以及NKCC1在下丘脑PVN和SON的选择性增加而在OVLT、MnPO、SFO未见显著变化。对PVN内NKCC1阳性神经元的亚型分析发现，NKCC1在PVN的前交感神经元和AVP神经元均有表达分布，而且其在两类神经元的表达均可被高盐应激增加。对Dahl盐敏性高血压大鼠的脑片突触功能分析发现，PVN神经元出现兴奋性突触传递增强和抑制突触传递减弱，以及EGABA的去极化偏移，而NKCC1阻断剂可以改善突触可塑性变化和部分恢复EGABA去极化偏移，表明NKCC1在盐敏性高血压模型的神经元内Cl-浓度的平衡稳态维持和突触可塑性中发挥关键作用。应用NKCC1 shRNA病毒载体下调Dahl盐敏性高血压大鼠PVN内NKCC1表达，发现基因沉默NKCC1后可降低盐敏性高血压模型的外周交感神经活性和靶器官损伤。此外，对NKCC1转运体的上游激酶分析，发现高盐可通过激活SPAK 和 OSR1上调NKCC1表达，调节GABA介导的突触传递活动。该项目对盐敏性高血压的发生机制提供了新的实验证据，也为盐敏性高血压的防治提供了新的思路，和开发基因疗法和新型药物提供了新的靶点。