Glycyrrhiza and Uncaria Hook contribute to protective effect of traditional Japanese medicine yokukansan against amyloid β oligomer-induced neuronal death

Ethnopharmacological relevance: 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).

Aim of the study: 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.

Materials and methods: 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.

Results: 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.

Conclusions: 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.