Sirt1调控自噬/ p53/p21信号通路在蛇葡萄素延缓脑衰老中的机制研究

Reduced autophagy and accelerated apoptosis is critically involved in pathogenesis of the brain ageing, suggesting that the exogenous autophagy activator may have therapeutic value. Ampelopsin, a natural flavonoid compound made from Rattan tea, has been reported to possess anti-inflammatory, an-tioxidative and other activities. However, whether Ampelopsin can change the functional status of autophagy on nerve cells, treat brain ageing related pathology as well as the specific molecular mechanism is unclear. Our previous studies found that ampelopsin can protect PC12 cells from H2O2-induced oxidative stress injury and 6-OHDA-induced neurotoxicity. In addition，ampelopsin can delay brain ageing through activating autophay in D-gal-induced brain ageing model. Based on the above background, this project will further investigate the impact of ampelopsin on SAMP8 mice and D-gal-induce SH-SY5Y cell senescence models as well as the signal transduction and clarify the specific molecular mechanism. The project includes(1) correlation of brain aging and functional status of autophagy（2）the effect of ampelopsin on learning and spatial memory in SAMP8 mice (3) the effect of ampelopsin on autophagy and underlying molecular mechanism（4）using autophagy inhibitor chloroquine or RNA interference means reverse verify the key role of ampelopsin induced autophagy in the treatment of brain aging.

自噬功能低下与细胞凋亡过度参与脑衰老的发生，提示外源性自噬激活剂具有重要治疗价值。黄酮类化合物蛇葡萄素Ampelopsin具有抗炎、抗氧化等生物活性。但其能否通过干预神经细胞的自噬功能状态及相关分子机制治疗脑衰老还未见报道。我们研究发现，Ampelopsin能够抵抗H2O2和6-OHDA诱导的氧化应激和神经毒性；在半乳糖诱导的脑衰老模型中，Ampelopsin能够诱导细胞自噬延缓脑衰老。基于以上背景，本项目将深入分析Ampelopsin对快速老化小鼠SAMP8和半乳糖诱导的神经细胞衰老的影响，阐明具体的分子机制。主要包括（1）脑衰老与自噬功能状态的相关性；（2）Ampelopsin干预后对SAMP8小鼠学习和空间记忆能力的影响；（3）Ampelopsin对细胞自噬的影响及信号通路。（4）运用自噬抑制剂氯喹或者Sirt1RNA干扰手段反向验证Ampelopsin诱导的自噬激活在脑衰老治疗的关

脑衰老老严重危害着老年人健康与生活, 我们团队前期的研究表明双氢杨梅素(AMP/DHM)具有神经细胞保护作用及在氧化应激条件下诱导细胞自噬实现细胞稳态调节性适应与生存的能力，DHM干预有益于脑衰老的预防与治疗,但作用机制还不明确。SAMP8 小鼠在 2月龄后便出现记忆衰退，并随着年龄的增长有所加重，6月龄即进入老年期，表现为空间学习记忆能力下降，是一种比较理想的研究脑衰老和痴呆的模型。本研究基于脑衰老可能由于相对细胞自噬低下与凋亡旺盛的特点, 以快速老化小鼠(SAMP8)为研究对象，DHM干预后对SAMP8小鼠进行行为学测定，海马神经元内线粒体与自噬溶酶体的电镜观察, 自噬、能量代谢、细胞凋亡、衰老相关蛋白的免疫印迹或RNA-Seq分析。研究结果表明：1.SAMP8小鼠脑衰老模型存在细胞自噬低下和细胞凋亡过多的显著特征。2.DHM预处理能减轻AD病理样蛋白( APP、BACE1、Tau蛋白磷酸化)的异常升高。3.DHM预处理能够减少增龄所致的SAMP8小鼠衰老相关蛋白（p53、p21、p16）的表达水平。4.以药源性自噬抑制（氯喹）小鼠为对象进行优化干预，阐明了DHM诱导的自噬激活在脑衰老的防治关键作用。5. 以Sirt1抑制剂Ex527处理SAMP8小鼠，阐明DHM是以上调Sirt1来发挥激活自噬的机制。本研究首次提出了DHM诱导的自噬，下调 AD 样病理蛋白的表达，抑制细胞凋亡，实现对神经元细胞的保护而最终延缓脑衰老的进程。