Department of Anatomy, Dongguk University Graduate School of Medicine, Gyeongju 38066, Republic of Korea.
Department of Biotechnology, Pukyong National University, Namku, Busan 48513, Republic of Korea.
J Ethnopharmacol. 2017 Sep 14;209:100-107. doi: 10.1016/j.jep.2017.07.030. Epub 2017 Jul 19.
Neurologic disorders are frequently characterized by synaptic pathology, including abnormal density and morphology of dendritic spines, synapse loss, and aberrant synaptic signaling and plasticity. Therefore, to promote and/or protect synapses by the use of natural molecules capable of modulating neurodevelopmental events, such as, spinogenesis and synaptic plasticity, could offer a preventive and curative strategy for nervous disorders associated with synaptic pathology. Radix Puerariae, the root of Pueraria monatana var. lobata (Willd.) Sanjappa&Pradeep, is a Chinese ethnomedicine, traditionally used for the treatment of memory-related nervous disorders including Alzheimer's disease. In the previous study, we showed that the ethanolic extracts of Radix Puerariae (RPE) and its prime constituent, puerarin induced neuritogenesis and synapse formation in cultured hippocampal neurons, and thus could improve memory functions.
In the present study, we specifically investigated the abilities of RPE and puerarin to improve memory-related brain disorders through modulating synaptic maturation and functional potentiation.
Rat embryonic (E19) brain neurons were cultured in the absence or presence of RPE or puerarin. At predetermined times, cells were live-stained with DiO or fixed and immunostained to visualize neuronal morphologies, or lysed for protein harvesting. Morphometric analyses of dendritic spines and synaptogenesis were performed using Image J software. Functional pre- and postsynaptic plasticity was measured by FM1-43 staining and whole-cell patch clamping, respectively. RPE or puerarin-mediated changes in actin-related protein 2 were assessed by Western blotting. Neuronal survivals were measured using propidium iodide exclusion assay.
RPE and puerarin both: (1) promoted a significant increase in the numbers, and maturation, of dendritic spines; (2) modulated the formation of glutamatergic synapses; (3) potentiated synaptic transmission by increasing the sizes of reserve vesicle pools at presynaptic terminals; (4) enhanced NMDA receptor-mediated postsynaptic currents, and (5) increased cell viability against naturally occurring cell death. Moreover, upregulation of actin-related protein 2 (ARP2) in RPE and puerarin treated brain neurons suggest that RPE and puerarin induced synaptic plasticity might be associated, at least in part, with ARP2-mediated actin-dependent regulation of spinogenesis.
Our findings indicate that RPE and puerarin might play a substantial role in the morphological and functional maturation of brain neurons and suggest that RPE and puerarin are potentially valuable preventative therapeutics for memory-related nervous disorders.
神经系统疾病常表现为突触病理学，包括树突棘密度和形态异常、突触丢失以及异常的突触信号和可塑性。因此，使用能够调节神经发育事件的天然分子（如 spinogenesis 和突触可塑性）来促进和/或保护突触，可能为与突触病理学相关的神经疾病提供一种预防和治疗策略。葛根，野葛的根，是一种中药，传统上用于治疗与记忆相关的神经系统疾病，包括阿尔茨海默病。在之前的研究中，我们表明葛根的乙醇提取物（RPE）及其主要成分葛根素能够诱导培养的海马神经元的神经突生成和突触形成，从而改善记忆功能。
本研究特别研究了 RPE 和葛根素通过调节突触成熟和功能增强来改善与记忆相关的脑疾病的能力。
在不存在或存在 RPE 或葛根素的情况下，培养大鼠胚胎（E19）脑神经元。在预定的时间，用 DiO 活染或固定并免疫染色以可视化神经元形态，或裂解以收获蛋白质。使用 Image J 软件对树突棘和突触形成进行形态计量分析。通过 FM1-43 染色和全细胞贴片钳分别测量功能前突触和后突触可塑性。通过 Western blot 评估 RPE 或葛根素介导的肌动蛋白相关蛋白 2 的变化。使用碘化丙啶排除测定测量神经元存活率。
RPE 和葛根素均：（1）显著增加树突棘的数量和成熟度；（2）调节谷氨酸能突触的形成；（3）通过增加突触前末梢储备囊泡池的大小来增强突触传递；（4）增强 NMDA 受体介导的后突触电流；（5）增加细胞活力，对抗自然发生的细胞死亡。此外，RPE 和葛根素处理的脑神经元中肌动蛋白相关蛋白 2（ARP2）的上调表明，RPE 和葛根素诱导的突触可塑性可能与 ARP2 介导的肌动蛋白依赖性 spinogenesis 调节至少部分相关。
我们的发现表明，RPE 和葛根素可能在脑神经元的形态和功能成熟中发挥重要作用，并表明 RPE 和葛根素可能是治疗与记忆相关的神经疾病的有价值的预防治疗药物。
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