Preventive effect of Ninjin-yoei-to, a Kampo medicine, on amyloid β_1-42-induced neurodegeneration via intracellular Zn²⁺ toxicity in the dentate gyrus

Abstract

Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer's disease, here we examined the effect of NYT on amyloid β1-42 (Aβ1-42)-induced neurodegeneration in the dentate gyrus.A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aβ staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aβ1-42induced increase in intracellular Zn 2+ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ1-42-induced Zn 2+ toxicity in the dentate gyrus. As a matter of fact, Aβ1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn 2+ -binding proteins, which can capture Zn 2+ from Zn-Aβ1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ1-42mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn 2+ toxicity induced by Aβ1-42.

Metallothionein synthesis increased by Ninjin-yoei-to, a Kampo medicine protects neuronal death and memory loss after exposure to amyloid β1-42

“…Male ddY mice (10 weeks of age) and Male Wistar rats (10 weeks of age), which were obtained from Japan SLC (Hamamatsu, Japan), freely access a control diet, a 3% NYT-containing diet, and water. NYT diet did not modify the body weight of mice 4 weeks after administration because of the almost the same intake between the control and NYT diets in the previous study [ 16 ]. Body weight of mice and rats was also almost the same between intakes of the control and NYT diets in the present experiments.…”

“…Thus, it is estimated that MTs can capture free Zn 2+ derived from Aβ 1-42 in the intracellular compartment. However, it was unclear whether the beneficial effect of NYT is directly linked with increased synthesis of MTs in the previous study [ 16 ]. When dexamethasone, an inducer of MT-I and MT-II, is intraperitoneally injected into rats once a day for 2 days, hippocampal MT level is significantly elevated 1 day after injection and reduces the increase in intracellular Zn 2+ derived from Aβ 1-42 , followed by rescuing the affected LTP [ 8 ].…”

“…Saline (vehicle) and Aβ 1-42 (ChinaPeptides, Shanghai, China) in saline (25 µM) was delivered into the lateral ventricle of mice at the rate of 0.5 µl/min for 40 min (500 pmol/mouse) via a microinjection canula as described previously [ 16 ].…”

“…The slides were rinsed with PBS for 5 min three times and incubated in blocking buffer containing Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific) in 3% BSA, 0.1% Triton X-100 in PBS (1:200 dilution) for 3 h at room temperature. Following three rinses in PBS for 5 min, the slides were bathed in 0.1% DAPI in PBS for 5 min, rinsed with PBS for 5 min three times, mounted with Prolong Gold antifade reagent, and placed at 4 °C for 24 h. Immunostaining images were measured in the dentate gyrus using a confocal laser-scanning microscopic system (Ex/Em: 495 nm/519 nm) (Nikon A1 confocal microscopes, Nikon Corp.) as described previously [ 16 ]. To obtain the best fluorescence images and measure the difference in fluorescence intensity among groups exactly, we first checked the relationship between the gain (fluorescence sensitivity) and fluorescence intensity and then carefully decided the best gain for measuring the exact changes in fluorescence intensity.…”

“…However, there is no report that NYT itself is effective on the AD pathophysiology. We have reported that neuronal death in the dentate gyrus induced by Aβ 1-42 is protected by pre-administration of NYT for 14 days [ 16 ]. In the present study, we presumed that NYT-induced synthesis of metallothioneins (MTs), Zn 2+ -binding proteins, which may reduce intracellular Zn 2+ toxicity by Aβ 1-42 , contributes to the protective effect.…”