调更汤介导海马神经元细胞Nrf2/JNK/Aβ氧化应激网络改善认知功能的机理研究

Our previous studies have shown that Tiaogeng Decoction could improve mild cognitive impairment (MCI) including bad memory and protect central nervous system in women during peri-menopausal period. In this study, 8-month-old SAMP8 mice will be employed to simulate hippocampal mild cognitive impairment model, PC12 cells intervented by Aβ1-40 will be used to simulate oxidative stress hippocampal neurons, 17β-estradiol was used as positive control drug. In vivo experiment, the ability of learning and memory of model mice will be detected by Morris water maze experiment, space exploration experiment, positioning and navigation experiment. Morphology of hippocampal neurons and Organelles will observed by Transmission Electron Microscope. Oxidative stress, mitochondrial damage, Aβ deposition in hippocampus will also be monitored. The gene and protein expression of Nrf2, JNK, and APP, which are key targets of Nrf2 / JNK / Aβ oxidative stress network in hippocampus, will be studied. In vitro experiment, study on the regulation of oxidative stress, mitochondrial damage and apoptosis in PC12 cells by Tiaogeng Decoction and its main compounds Icariin and Paeoniflorin will be researched. High-throughput Sequencing of Transcriptome will be employed to check the reactive sites of Nrf2 / JNK / Aβ oxidative stress network. JNK agonist anisomycin and Nrf2 inhibitors ML385 will be used to validate the sites action. The purpose of the study is to illustrate the mechanism of Traditional Chinese Medicine which inhibits oxidative damage and apoptosis of hippocampal neurons via regulating Nrf2 / JNK / Aβ oxidative stress network, and plays a neuroprotective role in the treatment of MCI. The pharmacological mechanisms of the study will provide a scientific basis for the guidance of clinical treatment and have an important academic value.

前期研究显示调更汤有改善女性围绝经期“健忘”等轻度认知功能损害（MCI）和中枢神经保护作用。项目以8月龄SAMP8小鼠模拟海马型MCI，Aβ1-40干预PC12神经细胞株模拟氧化应激海马神经元细胞，17β雌二醇为阳性对照药。体内实验观察调更汤改善模型小鼠学习记忆能力、海马神经元细胞形态学、氧化应激、线粒体损伤、Aβ沉积作用和干预海马Nrf2/JNK/Aβ氧化应激网络关键靶点；体外实验研究调更汤及淫羊藿苷、芍药苷改善PC12细胞氧化应激、线粒体功能、细胞凋亡，转录组高通量测序明确Nrf2/JNK/Aβ氧化应激网络的作用位点并以JNK激动剂Anisomycin、Nrf2抑制剂ML385验证。项目旨在阐释中医药介导Nrf2/JNK/Aβ氧化应激网络抑制海马神经元细胞氧化损伤、细胞凋亡和Aβ沉积及保护中枢神经的作用机理，为中医药治疗MCI和预防AD药理机制提供科学依据，对指导临床有重要的学术价值。

前期研究显示调更汤有改善女性绝经相关“健忘”等轻度认知功能损害(MCI)作用。项目以APP/PS1小鼠模拟MCI状态，H2O2干预PC12细胞模拟海马神经元氧化应激，17β雌二醇为阳性对照药，进一步探讨调更汤及其成分芍药苷、淫羊藿苷的海马神经保护机制。.体内实验首先观察调更汤对APP/PS1小鼠学习记忆能力、海马神经元细胞形态学、氧化应激、线粒体损伤、Aβ沉积、Nrf2、JNK、HO-1蛋白表达的影响，结果表明调更汤可能介导 JNK/Nrf2/HO-1信号通路减轻APP/PS1小鼠海马神经损伤，改善认知功能，减轻氧化应激状态，抑制Aβ沉积及JNK表达水平，促进Nrf2、HO-1表达水平；其次去势建立低雌激素环境下认知功能障碍小鼠模型，通过透射电镜观察小鼠海马CA1区细胞器形态，免疫荧光和硫磺素染色检测两组小鼠海马区Aβ沉积水平，ELISA检测血清雌激素水平和氧化应激指标，western blot测定雌激素受体α、β水平，结果表明低雌激素环境会导致Aβ沉积增加，海马组织雌激素受体α、β水平降低。可见，认知功能障碍可能与低雌激素环境下Aβ异常沉积有关。.体外实验首先验证低中高三种浓度的调更汤通过JNK/Nrf2/HO-1信号通路提高H2O2损伤的PC12细胞活力，降低细胞毒性和凋亡率，抑制氧化应激反应，增加线粒体膜电位水平，发挥神经保护作用；同时使用H2O2损伤的PC12细胞模型，验证最佳剂量调更汤及其成分芍药苷、淫羊藿苷预处理后PC12细胞的损伤情况，结果表明调更汤及其成分芍药苷、淫羊藿苷处理后PC12细胞损伤降低，减轻细胞氧化应激状态，改善PC12细胞Aβ沉积，降低细胞凋亡，同时上调Nrf2、HO-1、Bcl-2的表达，下调JNK、Bax表达，使用JNK激动剂（Anisomycin）后，JNK表达被激动，同时Nrf2、HO-1表达受抑制，表明调更汤、PF、ICA可以抗氧化应激、抗细胞凋亡。因此，调更汤及其成分芍药苷、淫羊藿苷可能通过JNK/Nrf2/HO-1信号通路发挥神经保护作用。.综上所述，调更汤可能通过介导JNK/Nrf2/HO-1氧化应激网络抑制海马神经元细胞氧化损伤、细胞凋亡和Aβ沉积发挥中枢神经细胞保护作用，为调更汤治疗MCI和预防AD药理机制提供科学依据，对指导临床有重要的学术价值。