Methysticin (DL-Methysticin)

Cell experiment:

The mouse hepatoma cell line Hepa1c1c7 and AhR-deficient cell line CRL-2710 are grown in α-MEM supplemented with 10% FBS and antibiotics (50 U/mL Penicillin and 50 µg/mL Streptomycin) and maintained at 37°C in a humidified atmosphere with 5% CO2. The cytotoxicity of kava extract and kavalactones (e.g., Methysticin; 0.195313, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM) is assessed using the tetrazolium reduction cell viability assay (MTS). After treatment for 24 h, the MTS assay is performed. The viability of the cells is calculated by comparing the absorbance of the treated cells with that of the DMSO controls[1].

Animal experiment:

Mice[2]Treatment of transgenic APP/Psen1 mice (n=6) is started at an age of 25 weeks and lasted for 27 weeks. The animals are treated once a week with 6 mg/kg bodyweight Methysticin (0.15 mg/25 g mouse, corresponding to 100 µL of working solution). Control groups consist of wild type mice (n=6) and APP/Psen1 mice (n=6) which are vehicle-treated with an identical treatment regimen. At 52 weeks, the animals undergo behavioral testing and are euthanized afterwards. The brain hemispheres are separated to obtain both formalin-fixed tissue for paraffin embedding and tissue for biochemical analysis from the same animal. The left hemisphere is further dissected to separate the hippocampus from the remaining brain tissue. The fresh tissue is snap-frozen and immediately stored at -80°C. To induce Nrf2/ARE in the ARE-luciferase reporter gene mice, the mice receive 6 mg/kg bodyweight of Methysticin once. The mice's hippocampus, cortex, midbrain, and cerebellum are prepared and immediately snap-frozen in liquid nitrogen 6 h after Methysticin treatment[2].

References:

[1]. Li Y, et al. Methysticin and 7,8-dihydromethysticin are two major kavalactones in kava extract to induce CYP1A1. Toxicol Sci. 2011 Dec;124(2):388-99.
[2]. Fragoulis A, et al. Oral administration of Methysticin improves cognitive deficits in a mouse model of Alzheimer's disease. Redox Biol. 2017 Aug;12:843-853.