预先修饰半胱氨酸通过抑制4-HNE-TRPA1敏化减少心肌缺血后损伤的保护机制

Myocardial ischemia and reperfusion induces the abundant generation of 4-hydroxy-2-nonenal (4-HNE), which is the toxic product of lip peroxidation. Recent work suggest that 4-HNE contributes to myocardial injury, though the mechanism has not been elucidated. The transient receptor potential ankyrin-1 (TRPA1) is widely distributed throughout the body in nociceptors as well as cardiomyocytes. We have found that the activation of TRPA1 prior to ischemia reduces myocardial ischemia-reperfusion injury, however, the mechanism is still unknown. Reactive compounds activate TRPA1 channels through the covalent modification of specific cysteine residues (Cys). It was demonstrated that 4-HNE are important mediators of hyperalgesia and cellular damage by causing irreversible modification on cysteines of TRPA1 expressed on nociceptors. Therefore, we speculated that shielding the critical cysteines on TRPA1 which are attacked by 4-HNE could limit TRPA1 sensitization and then limit the cellular damage caused by reperfusion injury. Thus, the study is going to investigate the cardioprotection of natural agonist of TRPA1 and the involved cardioprotective mechanism of critical cysteines pre-modification by inhibiting 4-HNE-TRPA1 sensitization. The model of myocardial ischemia-reperfusion injury will be used in rat and TRPA1 knocked mouse, as well as in the cardiomyocytes. Further, we will take the techniques of site-directed mutagenesis, calcium imaging, patch clamp etc. to elucidate the cardioprotective mechanism of Cys633 and Cys856 pre-modification by inhibiting 4-HNE-TRPA1 sensitization. Our results may shed new light on how to reduce the ischemia-reperfusion injury and indicate a new therapeutic target.

心肌缺血后脂质过氧化产物4-羟基-2-壬烯醛（4-HNE）表达增加可加重心肌损伤，但机制不详。瞬时受体电位锚蛋白通道（TRPA1）在伤害性感受神经元和心肌细胞中共同表达。我们前期研究发现，预先激活TRPA1可促进缺血后心肌存活，但机制尚未阐明。TRPA1分子中半胱氨酸残基（Cys）是调控TRPA1活性的关键位点，已知4-HNE能够不可逆结合伤害性感受神经元中TRPA1分子的Cys诱发痛觉过敏并加重组织损伤。因此，我们推测预先修饰Cys可以通过抑制4-HNE-TRPA1敏化减少心肌缺血后损伤。本课题拟利用成年大鼠与基因敲除小鼠整体和细胞心肌缺血/再灌注模型，验证天然TRPA1受体激动剂的心肌保护作用；并采用定点突变、钙成像及膜片钳等技术，阐释预先修饰Cys633和Cys856通过抑制4-HNE-TRPA1敏化发挥心肌保护作用的新机制。本研究将为筛选出干预心肌缺血后损伤的新靶点提供理论依据。

心肌缺血后脂质过氧化产物4-羟基-2-壬烯醛（4-HNE）表达增加可加重心肌损伤，乙醛脱氢酶2（ALDH2）是降解4-HNE主要蛋白酶，但在心肌缺血后如何调节4-HNE机制未充分阐明。瞬时受体电位锚蛋白通道（TRPA1）在伤害性感受神经元和心肌细胞中共同表达。我们前期研究发现，预先激活TRPA1可促进缺血后心肌存活，但机制尚未明确。本课题在大鼠心肌缺血再灌注模型中研究心肌缺血前于大鼠腹部涂抹TRPA1受体激动剂水杨酸甲酯乳膏模拟伤害性刺激的心肌保护作用，结果发现水杨酸甲酯乳IcyHot能够显著减少心肌缺血后损伤，而TRPA1受体拮抗剂能够抑制水杨酸甲酯乳膏的心肌保护作用。此外，在心肌细胞缺氧复氧模型中给予水杨酸甲酯及TRPA1受体阻断剂，同样发现TRPA1受体介导心肌保护作用。为进一步探讨其相关机制，本研究在大鼠心肌缺血再灌注模型中发现TRPA1进一步介导远端创伤预处理的心肌保护作用，其中与TRPA1提高ALDH2活性，减少4-HNE生成相关。目前，关于TRPA1的研究主要集中在神经源性炎症反应、慢性疼痛治疗方面，TRPA1抑制剂未来有望成为新的临床镇痛药物。本研究结果显示伤害性感受刺激能够对缺血心肌产生保护作用，提示疼痛信号通路与心脏保护之间存在关联。本课题更加完善地阐释TRPA1 生物学效能，为临床安全用药提供综合的参考依据；同时揭示一条全新的信号通路，为筛选出干预心肌缺血后损伤的新靶点提供理论依据。