硫化氢通过特异性调节去乙酰化酶6(HDAC6)改善老年性血管新生障碍的作用及其机制

Protein acetylation extensively regulates fundamental biological process, such as cardiovascular homeostasis and tumorigenesis. Mechanism of protein acetylation and its abnormal changes during diseases has become one of the basic questions in medical research. However, at present, researches are mainly focused on the structure and function of the downstream targets. How physiological and pathological factors (the upstream signal) regulate acetylation is still poorly understood. We previously reported the pro-angiogenic effect of hydrogen sulfide, and its direct effect towards the structure and function of receptor tyrosine kinase. Our preliminary data shows that HDAC6 protein expression and activity is regulated by H2S, and HDAC6 mediates the effect of H2S on endothelial cell migration. Our data also indicates that H2S can alleviate endothelial cell senescence. The hypothesis is by specifically regulating deacetylase HDAC6, H2S promotes endothelial cell migration and angiogenesis, thus ameliorate impaired senile angiogenesis. By using pro-angiogenic effect of H2S as outcome indicator, and the methods such as molecular biology, biochemistry, confocal time-lapse imaging, in vitro and in vivo angiogenesis models, and clinical sample test, we will intensively study the mechanism of HDAC6 regulation by H2S. Our findings will give rise to a new idea of acetylation regulation, and provide a new method to improve wound healing and blood supply in chronic ischemia among aging people.

蛋白质乙酰化修饰对基本生命现象进行广泛调节，如心血管稳态和肿瘤发生。探索乙酰化修饰机制及其在疾病中的异常表现，已成为医学研究的基本问题之一。但目前该领域的研究主要在于乙酰化修饰的靶分子结构与功能，对于如何调节乙酰化的生理病理机制（上游信号）知之甚少。我们前期报道了硫化氢的促血管新生作用，并可直接调节受体酪氨酸激酶的构象和功能。预实验显示去乙酰化酶6(HDAC6)的表达和活性受硫化氢调节，并介导其促内皮细胞迁移效应。还观察到硫化氢可减轻内皮细胞衰老。由此提出假设：硫化氢通过特异性调节HDAC6，促进内皮细胞迁移和血管新生，从而改善老年性血管新生障碍。以硫化氢促血管新生作用为观察指标，综合运用分子生物学、生物化学、动物模型和临床样本检测等手段，对硫化氢调控HDAC6继而影响蛋白质乙酰化修饰的作用机理进行深入研究，提出一种乙酰化调控的新思路，为改善老年伤口愈合和慢性缺血时血运重塑提供新方法。

前期研究显示，气体信号分子硫化氢（H2S）有促血管新生的作用，并可调节去乙酰化酶6(HDAC6)的表达和活性。本项目主要研究内皮细胞老化过程中HDAC6表达以及活性的改变，探索HDAC6的下游靶分子和上游调控机制，并在老年小鼠伤口愈合模型以及老年小鼠下肢缺血模型中观察HDAC6对老年血管新生的可能调控作用。我们的研究显示，内、外源性H2S均有一定的抑制内皮细胞衰老的作用，外源性补充H2S可促进老年内皮细胞体外血管新生能力的回升，且内源性H2S是外源性H2S起效所必须的，HDAC6介导了H2S促内皮细胞迁移的作用。FAK和paxillin是内皮细胞中HDAC6的下游靶点，细胞外基质组成和整合素的表达也受到HDAC6的调控。内源性H2S和缺氧条件均可促进内皮细胞中HDAC6蛋白的表达。外源性H2S能显著提高HDAC6基因启动子活性，延缓HDAC6 mRNA降解速率，提高HDAC6重组蛋白的活性，表现为对全长乙酰化α微管蛋白的去乙酰化作用增强。自然衰老小鼠血浆H2S含量减少，伤口愈合能力降低，外源性H2S可促进老年小鼠伤口愈合。内源性H2S和HDAC6均是小鼠伤口正常愈合所必须的。老年皮肤HDAC6表达降低可能与其伤口愈合速度减慢有关。外源性H2S可促进下肢缺血后的血流恢复。内源性HDAC6是外源性H2S促缺血后血运重建所需要的。老年糖尿病患者血浆H2S水平明显下降，真皮组织结构紊乱，炎症细胞浸润。内源性H2S生成酶在皮肤组织中有表达，以CSE的表达丰度为高。本研究的科学意义在于：阐明了HDAC6是一个潜在的调控老年血管新生障碍的新靶点。开发新型的内源性H2S生成调节剂或缓释剂，改善HDAC6的表达和功能，将有助于老年群体的伤口愈合和缺血组织血供恢复。