目的和意义： 神经系统作为生命的中枢，是人体最为复杂和最为重要的组织器官，随着年龄结构老龄化等多种因素的影响，以脑卒中和老年痴呆症为代表的神经细胞损伤和退行性病变，已经成为了危害社会公共健康的重要因素。新橙皮苷(neohesperidin)是一种在柑橘属植物的果实中含量丰富的黄酮类化合物，体外实验证实新橙皮苷具有显著的抗氧化和神经保护能力，作为药食同源植物为来源的天然产物，新橙皮苷具有极大的研究与开发价值。但是，新橙皮苷具体的神经保护作用，以及分子机制仍不明确。因此，本课题组针对新橙皮苷是否具有抗脑缺血再灌注损伤，以及抗神经元退行性损伤的保护作用开展了研究，并对潜在的分子机制与信号通路进行了深入的探讨。 方法与结果： 在本研究中，我们采用脑中动脉栓塞(MCAO)动物模型来考察新橙皮苷抗脑缺血再灌注损伤的作用，动物脑缺血2h，再灌注24h，手术前72，48，24h，0h腹腔注射新橙皮苷(40㎎／㎏)。实验结果显示新橙皮苷能够明显抑制脑梗死面积和改善神经功能。分子作用机制研究表明，新橙皮苷具有显著的抗氧化应激作用，并抑制脑缺血再灌注损伤引起的凋亡相关通路信号分子Bax上调，以及线粒体凋亡通路中细胞色素C (cytochrome C)释放，并且减弱了半胱天冬酶家族中caspase-9和caspase-3的活化，以及抗凋亡相关蛋白Bcl-2的下调。进一步的信号通路研究显示，新橙皮苷能够激活蛋白激酶B(Akt)／转录因子Nrf2(nuclear factor erythroid-2-related factor 2，Nrf2)这条信号通路，增加抗氧化酶血红素氧合酶1(HO-1)的表达，发挥抗氧化应激作用。因此，本研究证明了作为一种食物来源天然产物，新橙皮苷可能在防治脑卒中的过程中扮演着重要的角色，并具有潜在的开发价值。另一方面，本研究通过采用β淀粉样蛋白肽段25-35(amyloid-beta protein，Aβ〓)损伤大鼠元代海马神经元模型检测了新橙皮苷对退行性病变损伤的保护作用，实验结果首次证明了新橙皮苷能够显著性的逆转Aβ〓引起的元代海马神经元的凋亡。进一步的机制研究显示新橙皮苷主要通过减弱错误折叠蛋白反应(unfolded protein response，UPR)和内质网应激(endoplasmic Reticulum Stress，ERs)，进而缓解Aβ〓的毒性作用。深入的分子机制研究证明，Aβ〓作用会引发原代海马神经元细胞中蛋白质二硫键异构酶(protein disulfide isomerase， PDI)的硝基化水平升高，从而阻断PDI对于UPR和ERs作用的缓解，加剧细胞凋亡的产生，而新橙皮苷正是通过逆转Aβ〓引起的PDI硝基化水平的升高，从而减弱其引发的凋亡作用。 研究结论： 脑卒中和退行性病变，不仅严重危害人类的生命健康，而且两种病理损伤又存在着极大地相关性，流行病学研究证明卒中极大地增加了患者预后痴呆的发病率，这提示了对于缺血性损伤和Aβ〓损伤同时具有保护作用的天然产物新橙皮苷拥有着潜在的开发价值。而本课题的研究结果不仅揭示了新橙皮苷通过调控Akt／Nfr2与内质网应激过程的神经保护作用的分子机制，并且为后续的医学转化奠定了坚实的基础。 关键词：新橙皮苷；神经保护；缺血性损伤；退行性病变；硝基化作用 论文类型：应用基础 *本研究得到陕西省自然基金面上项目：柚皮芸香甙调控Nrf2／ARE通路抗缺血再灌注分子机制研究(编号：2014JM2-8163)资助。

 Purpose and significance: As the vital center of the body, nervous system is the most complex and crucial tissue and organ of the human body. With the influence of various factors such as aging and stressing, neurons damage and degenerative diseases have become the serious factors that harm the public health, such as stroke and Alzheimer's disease. Neohesperidin, a dihydroflavone abundantly detected in citrus flavonoids, has been demonstrated to have ROS-scavenging activity, and showed the neuroprotective effects against H₂O₂-caused and Aβ-induced cytotoxicity. Therefore, it presents an underlying assumption that the promising preventive effect of neohesperidin for cerebral ischaemic injury and Aβ-induced neurotoxicity might improve the pathological state of neurons damage and retrogression. However, the in-depth investigation of the molecular mechanisms of neuroprotective effects of neohesperidin and the molecular mechanisms remain unclear. Therefore, our present studies confirm whether neohesperidin could alleviate cerebral ischemia-reperfusion injury and neuronal degeneration injury, and explore the potential molecular mechanisms and signaling pathways. Content and results: In this study, we used an animal model of middle cerebral artery occlusion (MCAO) to investigate the effect of neohesperidin on cerebral ischemia-reperfusion injury.Animals were treated with cerebral ischemia for 2 h,reperfusion for 24 h, and intraperitoneal injection of neohesperidin (40 ㎎/㎏) at 72, 48, 24 h and 0 h before surgery. The results showed that neohesperidin could significantly inhibit cerebral infarct size and improve neurological function. Molecular mechanism also showed that neohesperidin inhibited the MCAO-induced upregulation of Bax, cytochrome c, and cleaved caspase-9 and -3, as well as the downregulation of Bcl-2. Interestingly, NH treatment upregulated heme oxygenase-1 (HO-1) in a concentration-dependent manner, which was due to the NH-mediated activation of the protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. NH also abolished the MCAO-induced inhibition of the Akt/Nrf2 pathway. Therefore, this study demonstrated that, as a natural source of food, neohesperidin could play an important role in the prevention and treatment of stroke, and had potential development value. On the other hand, in this study, the protective effect of neohesperidin on degenerative lesions was examined by using hippocampal neuron model in rats with Amyloid-beta protein 25-35 (Aβ〓). The experimental results demonstrated for the first time that neohesperidin could significantly reverse the apoptosis of hippocampal neurons induced by Aβ〓. Further mechanistic studies had shown that neohesperidin mainly attenuates the toxic effects of Aβ〓 by attenuating the unfolded protein response (UPR) and endoplasmic reticulum stress (ERs) via enhancing the expression of ER chaperones, GRP78, GRP94 and PDI, inhibiting dissociation of GRP78 from PERK and IREα. In-depth molecular mechanism studies had shown that Aβ〓 could trigger an increase in the level of nitration of protein disulfide isomerase (PDI) in primary hippocampal neurons, thereby blocking the relief of PDI on UPR and ERs and aggravating the generation of apoptosis. Neohesperidine reduces the level of PDI nitration caused by reversing Aβ〓, thereby attenuating its apoptotic effect. Conclusion: Stroke and degenerative disease not only seriously endanger human life and health, but also have a great correlation between the two pathological injuries. Epidemiological studies have shown that stroke greatly increases the incidence of Alzheimer's disease, which suggests, the natural product neohesperidin, has both protective effects on blood damage and Aβ〓 damage. It has potential developmental value. The results of this study not only revealed the molecular mechanism of neohesperidin by regulating the neuroprotective effects of Akt/Nfr2 and endoplasmic reticulum stress, but also laied a solid foundation for subsequent medical transformation. KEY WORDS: Neohesperidin; Neuroprotection; Ischemic injury; Degenerative disease; Nitration TYPE OF DISSERTATION: Application Fundamentals