亚硝酸钠对髓过氧化物酶催化氯化反应的抑制作用及其与老年痴呆症的关系研究

Alzheimer's disease (AD) is the most common type of dementia and has become a severe problem for the elderly. Myeloperoxidase (MPO) and MPO-catalyzed chlorinative reaction have been implicated in AD brain, and play important roles in the onset, progression and pathogenesis of the disease. In our previous study, it was found that nitrite could effectively inhibit peroxidase activity of MPO and attenuate MPO-mediated cellular toxicity. Nitrite, therefore, may serve as a potential antioxidant and offer novel protection against AD. However, the possible critical role of nitrite in AD was unknown. Considering the intimate relationship among MPO, chlorinative reaction and AD, and the fact that nitrite level was decreased in AD brain, we hypothesized that nitrite could significantly influence MPO-induced neuronal cell damage through the intervention in chlorinative reaction. To confirm this hypothesis, the effects of sodium nitrite (NaNO2) on the peroxidase and chlorinating activities of MPO were first studied. Then, the important role of NaNO2 in MPO-mediated chlorinative reaction and cytotoxicity were investigated in neuronal cell models. Furthermore, the effect of NaNO2 on MPO-catalyzed tyrosine chlorination and the subsequent alteration of protein structure and function were clarified in vitro. The implementation of this project will demonstrate the novel mechanism of nitrite in neuronal cell damage and AD onset/progression, and consequently open a new window for the prevention and treatment of AD.

老年痴呆症(AD)是影响老人健康的重大疾病，髓过氧化物酶(MPO)及其催化的氯化反应是引起AD发生发展的重要原因之一。我们前期研究发现亚硝酸盐(NO2-)能有效抑制MPO过氧化物酶活性及其诱导的细胞损伤，因而成为AD发生发展潜在的抗氧化因素，但其生理影响尚不清楚。基于AD患者脑中NO2-水平有所下降的现象，结合MPO、氯化反应与AD的密切关系，我们推测NO2-可通过干预MPO催化的氯化反应来影响神经细胞损伤。为证实这个假说，本项目拟先研究亚硝酸钠(NaNO2)对MPO过氧化物酶活性和氯化活性的影响规律，再在神经细胞上探讨NaNO2对MPO催化氯化反应及其引起的细胞损伤的影响。在此基础上，通过体外化学实验，进一步研究NaNO2对酪氨酸氯化的影响及其与蛋白质结构和功能变化之间的关系。本项目的实施，将有助于阐明NO2-影响神经细胞损伤和AD发生发展的新机制，其结果可能为防治AD提供新思路。

分别在神经细胞和体外生化体系中研究了亚硝酸钠(NaNO2)对髓过氧化物酶(MPO)及其催化产生次氯酸(HOCl)的影响，探讨了NaNO2衍生的酪氨酸硝化修饰对蛋白质结构和功能的影响。在人神经母细胞瘤细胞(SH-SY5Y)中，NaNO2的存在可以削弱MPO/H2O2/Cl- 体系和HOCl介导的神经细胞损伤；MPO/H2O2/NaNO2 体系可使脑中具有重要功能的烯醇酶发生酪氨酸硝化修饰，硝化位点主要在 Tyr12和Tyr191。NaNO2通过干预MPO氯化活性来抑制HOCl的生成，或者通过直接清除HOCl，来抑制MPO和/或HOCl介导的氧化损伤，NO2- 进而在神经退行性疾病中发挥潜在的抗氧化作用。在体外生物化学体系中，NaNO2作为还原剂和抗氧化剂可有效抑制HOCl诱导血红蛋白(Hb)的血红素破坏和游离铁释放。并且，NaNO2/HOCl体系能硝化Hb中α链的Tyr42，进而影响Hb过氧化物酶活性。另一方面，NaNO2在低浓度时能显著加剧Hb-H2O2 体系导致的蛋白质氧化和细胞损伤，而在高浓度时显示抑制效果，在NaNO2干预Hb氧化还原反应的过程中，NO2- 含量减少并被氧化生成 •NO2，进而使蛋白质发生酪氨酸硝化修饰。另外，基于Aβ–heme复合物与老年痴呆症(AD)的密切关系，尽管Tyr10不是Aβ–heme复合物的结合位点，Tyr10的突变会降低Aβ–heme复合物的过氧化物酶活性，并降低Aβ自身聚集。在氧化(H2O2)和硝化应激(H2O2/NaNO2)时，Aβ–heme中的Tyr10会发生氧化和硝化修饰，而NaNO2的存在会抑制Tyr10氧化和heme-蛋白质交联物的形成。通过这些神经细胞和体外生化实验结果，说明NaNO2通过抑制MPO和/或HOCl介导的氧化损伤对神经细胞起保护作用，并且NaNO2对Hb和Aβ–heme复合物结构和功能有重要影响。本项目揭示了NaNO2对MPO催化氯化反应的影响机制，为阐明NO2- 在神经细胞损伤和AD病理过程中潜在的抗氧化作用提供理论依据。