原花青素基于Nrf2-iNOS-HDAC2轴调节支气管哮喘对激素敏感性的机制研究

Difficult asthma (including severe asthma) is insensitive to glucocorticoid treatment. The molecular mechanisms that contribute to glucocorticoid resistance are complicated and have not been fully clarified. This glucocorticoid resistance is largely caused by inactivation of histone deacetylase-2 (HDAC2), which is critical for the transrepressive activity of the glucocorticoid receptor that mediates the antiinflammatory effect of corticosteroids. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the cellular antioxidant response by upregulating genes encoding many phase II detoxifying or antioxidant enzymes involved in protecting against oxidative and electrophilic stresses. Under oxidative and nitrative stress, increased inducible nitric oxide synthase (iNOS) and decreased antioxidant enzymes precipitate the nitrosylation modification of HDAC2, which impairs its function and its deacetylase enzymatic activity. Oxidative and nitrative stress is augmented in severe asthma with increased iNOS, and the Nrf2 and HDAC2 functions are impaired in these patients. We hypothesized that Nrf2-iNOS-HDAC2 pathway has a critical role in regulating glucocorticoid sensitivity of asthma. Our previous study suggests that proanthocyanidins attenuates airway inflammation and hyperresponsiveness in a murine model of asthma by downregulating iNOS, and our preliminary experiment shows a powerful capability of proanthocyanidins to restore glucocorticoid sensitivity in a glucocorticoid insensitive asthmatic model. Therefore, we will investigate the mechanisms of Nrf2-iNOS-HDAC2 pathway in glucocorticoid sensitivity in asthma and ask whether proanthocyanidins could reinstate glucocorticoid responsiveness via Nrf2-iNOS-HDAC2 axis in a murine model. This may have implications for devising better therapies for difficult asthma.

难治性哮喘（包括重症哮喘）对激素不敏感，其机制较为复杂，目前尚未完全阐明。激素抵抗的分子机制涉及多个方面，其中HDAC2缺陷是重要原因。Nrf2调控多种II相解毒酶和抗氧化基因的表达，在抗氧化和亲电子应激中有重要作用。氧化/硝化应激条件下，iNOS合成增多，抗氧化酶表达减少，使HDAC2发生亚硝基化，进而使HDAC2的功能和活性降低。重症哮喘患者氧化/硝化应激增加，不仅iNOS高表达，而且Nrf2和HDAC2功能存在缺陷，因此我们猜想Nrf2-iNOS-HDAC2信号通路在哮喘激素抵抗机制中有重要作用。我们以前的研究表明原花青素通过下调iNOS抑制哮喘小鼠气道炎症和气道高反应，预实验也发现原花青素可以恢复激素不敏感哮喘小鼠对激素的敏感性。因此，我们拟通过体内、外实验探讨Nrf2-iNOS-HDAC2轴在哮喘对激素不敏感机制中的地位以及原花青素的作用，这对于难治性哮喘的治疗与控制有重要意义。

难治性哮喘（包括重症哮喘）对激素不敏感，其机制较为复杂，目前尚未完全阐明。激素抵抗的分子机制涉及多个方面，其中HDAC2缺陷是重要原因。Nrf2调控多种II相解毒酶和抗氧化基因的表达，在抗氧化和亲电子应激中有重要作用。氧化/硝化应激条件下，iNOS合成增多，抗氧化酶表达减少，使HDAC2发生亚硝基化，进而使HDAC2的功能和活性降低。重症哮喘患者氧化/硝化应激增加，不仅iNOS高表达，而且Nrf2和HDAC2功能存在缺陷。本研究中，我们成功复制了激素不敏感哮喘小鼠模型，运用实时定量PCR、Western blotting和蛋白免疫沉淀等方法发现激素不敏感哮喘小鼠Nrf2功能低下导致iNOS表达增高和GSH表达降低，iNOS高表达使NO产生增加促使HDAC2亚硝基化，而GSH低表达使亚硝基化的HDAC2去亚硝基化降低，两者使HDAC2活性和功能发生障碍，从而导致激素不敏感；分离出重症哮喘患者肺泡灌洗液巨噬细胞和外周血单个核细胞体外培养，发现重症哮喘患者Nrf2-iNOS-HDAC2轴存在缺陷，佐证了动物实验的结果；原花青素可以通过调节Nrf2-iNOS-HDAC2轴，激活Nrf2使iNOS/NO生成减少和GSH表达升高，进而降低HDAC2亚硝基化，从而恢复哮喘对激素的敏感性。综上，本研究初步阐明了Nrf2-iNOS-HDAC2轴在激素不敏感性哮喘病理机制中的作用，明确了原花青素在哮喘对激素敏感性中的调节作用及其机制，对难治性哮喘治疗与控制有重要意义。