基于金钗石斛生物碱调节细胞自噬的抗阿尔茨海默病机制研究

The prevalence of Alzheimer's disease (AD) gets increasingly prominent in elderly people, but there is still lack of effective drugs for the treatment of AD. Recently, some studies suggest that decrease in autophagy results in deposition and accumulation of Aβ, abnormal tau protein, and damaged organelles, which is critical in the pathogenesis of AD. However, whether the drug can ameliorate AD via autophagy remains unclear. Our previous research has demonstrated that dendrobium nobile lindl alkaloids (DNLA) ameliorates AD in APP/PS1 transgenic mice through upregulation of autophagy. The present study aims to investigate the relationship between autophagy and the function of learning and memory, as well as pathological changes such as Aβ deposition and tau phosphorylation in APP/PS1 transgenic AD mice of different ages (month). Also, the various inflammatory pathways including AMPK/Sirt1, NF-κB/cytokines and Nrf2/antioxidase were examined. Utilizing autophagy inducer metformin and autophagy inhibitor hydroxychloroquine as positive and negative control respectively, the effects and the molecular mechanisms of DNLA on autophagy as well as learning and memory function of APP/PS1 transgenic mice were investigated. This project will provide pharmacological basis for application of DNLA into the treatment of AD, and offer new sights for designing autophagy-based AD treatment strategies.

阿尔茨海默病(AD)在老年疾病谱中的地位日益重要，目前尚缺乏有效治疗药物。近年来发现自噬功能减弱导致沉积的Aβ、异常Tau蛋白及受损细胞器不能及时清除是诱发AD的关键病因之一，但是否可通过调节自噬改善AD仍不甚清楚。我们前期发现金钗石斛生物碱可通过促进自噬发挥抗AD的作用，但详细机制不明。本项目拟进一步探讨不同月龄APP/PS1转基因AD小鼠自噬及学习记忆功能与Aβ沉积、Tau蛋白磷酸化等病理改变的相关关系，考察海马及皮质AMPK/Sirt1、NF-κB/相关炎症因子、Nrf2/抗氧化酶等信号通路的变化，以自噬诱导剂二甲双胍和自噬抑制剂羟氯喹为正反对照，从调控自噬的角度探讨金钗石斛生物碱改善APP/PS1转基因AD小鼠学习记忆功能的详细机制，为金钗石斛生物碱用于AD的治疗提供实验依据，并为基于调节自噬的AD治疗新策略打下基础。

阿尔茨海默病(AD)在老年疾病谱中的地位日益重要，目前尚缺乏有效治疗药物。近年来发现自噬功能减弱导致沉积的Aβ及受损细胞器不能及时清除是诱发AD的关键病因之一，但是否可通过调节自噬改善AD仍不甚清楚。本研究组发现金钗石斛总生物碱（DNLA）具有抗AD的作用，但其机制尚未完全阐明。本项目进一步从调控自噬的角度探讨了DNLA的抗AD作用机制。结果发现，DNLA可改善Aβ25-35诱导的AD小鼠学习记忆障碍，减少神经元的凋亡，减轻突触病变及丢失，增加神经元内的尼氏体，增加海马和皮质内BDNF、CNTF和GDNF的表达；DNLA能减轻APP/PS1转基因小鼠学习记忆减退，抑制NF-κB和p38活化和上调PSD-95、SYP表达，可明显减少APP/PS1小鼠海马神经元内突触的丢失及神经元变性，减少海马组织中Aβ的表达，减少海马神经元内的自噬小体，促进甘露糖-6-磷酸受体和Cat D的分离，成熟的Cat D表达增加，增加海马内v-ATPase A1的表达，促进溶酶体酸化。DNLA可降低Aβ25-35诱导的大鼠原代海马神经元轴突变性模型的变性指数，增加细胞活力，提高PSD95和SYN蛋白水平；提高LC3BⅡ/LC3BⅠ比值和Beclin1、ATG5蛋白水平，促进自噬体与溶酶体融合，降低溶酶体腔的pH值激活CTSB和CTSD，促使DQ-RED-BSA和内源性SQSTM1/p62降解。DNLA主要成分——石斛碱能够改善Aβ25-35所致的PC12细胞活力降低，通过激活mTOR/ULK1信号通路诱导PC12细胞自噬流发生。综上所述，表明DNLA可通过诱导自噬保护Aβ25-35诱导的神经细胞损伤、减轻AD小鼠神经元和突触的病变和丢失，从而发挥抗AD作用。本研究系列发现为DNLA用于AD的治疗提供了实验依据，并可为基于调节自噬的AD治疗新策略打下基础，具有潜在的临床应用前景。

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