Tsumura Research Laboratories, Kampo Scientific Strategies Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
J Ethnopharmacol. 2013 Aug 26;149(1):360-70. doi: 10.1016/j.jep.2013.06.052. Epub 2013 Jul 6.
Yokukansan, a traditional Japanese (Kampo) medicine, composed of seven medicinal herbs has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Recently, this medicine has been reported to improve the behavioral and psychological symptoms of dementia (BPSD) that often become problematic in patients with Alzheimer's disease (AD).
Amyloid β (Aβ) oligomers, which are extremely toxic to neurons, are involved in neurodegeneration in AD. In animals, yokukansan has been proven to improve memory impairments and BPSD-like behavior in transgenic mice overexpressing amyloid precursor protein and mice intracerebroventricularly injected with Aβ oligomers. These results suggest that yokukansan is potentially able to reduce the neurotoxicity of Aβ oligomers. Therefore, the present study aimed to explore the improving effects brought by yokukansan that consists of seven herbs for Aβ oligomer-induced neurotoxicity in vitro and to identify the candidate herbs in yokukansan's action.
Primary cultured rat cortical neurons were used. Neurotoxicity induced by Aβ oligomers (3µM) and improving effects of yokukansan (300-1000 µg/mL) and its constituent herbs were evaluated in MTT assay, DNA fragmentation analysis, and electron microscopic analysis at 48h after treatment with Aβ oligomers and drugs. Moreover, changes in expression of genes related to endoplasmic reticulum (ER) stress and in caspase-3 activity that is the enzyme closely related to apoptosis were analyzed to investigate the underlying mechanisms.
Yokukansan ameliorated Aβ oligomer-induced neuronal damage in a dose-dependent manner in the MTT assay. This drug also suppressed DNA fragmentation caused by Aβ oligomers. Electron microscopic analysis suggested that yokukansan reduced karyopyknosis and the expansion of rough ER caused by Aβ oligomers. However, neither Aβ oligomers nor yokukansan affected the mRNA expression of any ER stress-related genes, including CHOP and GRP78. On the other hand, yokukansan dose-dependently suppressed Aβ oligomer-induced activation of caspase-3. Among the seven constituents of yokukansan, Glycyrrhiza and Uncaria Hook (60-200 µg/mL) suppressed Aβ oligomer-induced neuronal damage, DNA fragmentation, karyopyknosis, and caspase-3 activation to almost the same extent as yokukansan.
The present results suggest that yokukansan possesses an ameliorative effect against Aβ oligomer-induced neuronal apoptosis through the suppression of caspase-3 activation. Glycyrrhiza and Uncaria Hook may, at least in part, contribute to the neuroprotective effect of yokukansan. These mechanisms may underlie the improving effects of yokukansan on memory impairment and BPSD-like behaviors induced by Aβ oligomers.
和汉三才汤是一种传统的日本（汉方）药物，由七种草药组成，传统上用于治疗神经症、失眠和儿童夜间哭闹和烦躁。最近，这种药物已被报道可改善阿尔茨海默病（AD）患者常出现的行为和心理症状的痴呆（BPSD）。
淀粉样β（Aβ）低聚物对神经元具有极强的毒性，与 AD 中的神经退行性变有关。在动物中，和汉三才汤已被证明可改善过度表达淀粉样前体蛋白的转基因小鼠和经脑室内注射 Aβ 低聚物的小鼠的记忆障碍和 BPSD 样行为。这些结果表明，和汉三才汤有可能减轻 Aβ 低聚物的神经毒性。因此，本研究旨在探讨和汉三才汤（由七种草药组成）对体外 Aβ 低聚物诱导的神经毒性的改善作用，并确定和汉三才汤作用中的候选草药。
使用原代培养的大鼠皮质神经元。用 MTT 分析、DNA 片段分析和电子显微镜分析在 Aβ 低聚物和药物处理 48 小时后，评估 Aβ 低聚物（3µM）诱导的神经毒性和和汉三才汤（300-1000µg/mL）及其组成草药的改善作用。此外，还分析了与内质网（ER）应激相关的基因表达变化和与细胞凋亡密切相关的半胱天冬酶-3 活性的变化，以探讨潜在的机制。
和汉三才汤以剂量依赖性方式改善了 MTT 分析中的 Aβ 低聚物诱导的神经元损伤。该药物还抑制了 Aβ 低聚物引起的 DNA 片段化。电子显微镜分析表明，和汉三才汤减少了 Aβ 低聚物引起的核固缩和粗糙内质网的扩张。然而，Aβ 低聚物和和汉三才汤均未影响任何与 ER 应激相关的基因的 mRNA 表达，包括 CHOP 和 GRP78。另一方面，和汉三才汤剂量依赖性地抑制了 Aβ 低聚物诱导的半胱天冬酶-3 的激活。在和汉三才汤的七种成分中，甘草和钩藤（60-200µg/mL）抑制 Aβ 低聚物诱导的神经元损伤、DNA 片段化、核固缩和半胱天冬酶-3 激活的程度与和汉三才汤几乎相同。
本研究结果表明，和汉三才汤通过抑制半胱天冬酶-3 的激活，对 Aβ 低聚物诱导的神经元凋亡具有改善作用。甘草和钩藤可能至少部分参与了和汉三才汤的神经保护作用。这些机制可能是和汉三才汤改善 Aβ 低聚物诱导的记忆障碍和 BPSD 样行为的基础。
