Preventive effect of Ninjin-yoei-to, a Kampo medicine, on amyloid β1-42-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 β_1-42 (Aβ_1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ_1-42 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 Aβ_1-42-induced increase in intracellular Zn²⁺ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ_1-42-induced Zn²⁺ toxicity in the dentate gyrus. As a matter of fact, Aβ_1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn²⁺-binding proteins, which can capture Zn²⁺ from Zn-Aβ_1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ_1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn²⁺ toxicity induced by Aβ_1-42.

Keywords: Alzheimer’s disease; Ninjin-yoei-to; Zn2+ dysregulation; amyloid β1-42; metallothionein.

Fig. 1.

Aβ_1-42-induced neurodegeneration determined by propidium iodide (PI) staining is rescued after Ninjin-yoei-to (NYT) administration for 4 weeks. Four weeks after administration of the NYT-containing diet (upper), PI fluorescence was measured in the dentate granule cell layer surrounded by the dotted line (lower-left). Bar; 50 µm. Each bar and line (mean ± SEM) represent the rate (%) of PI fluorescence after Aβ_1-42 injection to that after saline (vehicle) injection, which was represented as 100% (lower-right). **P<0.01, vs. saline, ^##P<0.01, vs. Aβ (Tukey’s test). saline, n=18; Aβ, n=32; Aβ/NYT, n=18.

Fig. 2.

Aβ_1-42 uptake in the dentate gyrus is not modified after Ninjin-yoei-to (NYT) pre-administration for 2 weeks. The NYT-containing diet was administered to mice for 2 weeks and Aβ_1-42 was intracerebroventricularly injected into the mice (upper). One hour after the start of injection, Aβ immunostaining was determined in the dentate granule cell layer (GCL) (lower-left). Bar; 50 µm. Each bar and line (mean ± SEM) represent the rate (%) of Aβ staining after Aβ_1-42 injection to that after saline (vehicle) injection, which was represented as 100% (lower-right). *P<0.05, vs. saline (Tukey’s test). saline, n=6; Aβ, n=3; Aβ/NYT, n=3.

Fig. 3.

Aβ_1-42-induced increase in intracellular Zn²⁺ in the dentate gyrus is reduced after Ninjin-yoei-to (NYT) pre-administration for 2 weeks. The NYT-containing diet was administered to mice for 2 weeks and Aβ_1-42 was intracerebroventricularly injected into the mice (upper). One hour after the start of injection, intracellular ZnAF-2 fluorescence was determined in the dentate granule cell layer (lower-left). Bar; 50 µm. Each bar and line (mean ± SEM) represent the rate (%) of ZnAF-2 fluorescence after Aβ_1-42 injection to that after saline (vehicle) injection, which was represented as 100% (lower-right). *P<0.05, vs. saline: ^#P<0.05, vs. Aβ (Tukey’s test). saline, n=10; Aβ, n=28; Aβ/NYT, n=18.

Fig. 4.

Aβ_1-42-induced neurodegeneration determined by propidium iodide (PI) staining is rescued after Ninjin-yoei-to (NYT) pre-administration for 2 weeks. Two weeks after administration of the NYT-containing diet, Aβ_1-42 was intracerebroventricularly injected into mice (upper). Two weeks later, PI fluorescence was measured in the dentate granule cell layer surrounded by the dotted line (lower-left). Bar; 50 µm. Each bar and line (mean ± SEM) represent the rate (%) of PI fluorescence after Aβ_1-42 injection to that after saline (vehicle) injection, which was represented as 100% (lower-right). **P<0.01, vs. saline, ^##P<0.01, vs. Aβ (Tukey’s test). saline, n=18; Aβ, n=25; Aβ/NYT, n=9.

Fig. 5.

Aβ_1-42-induced neurodegeneration determined by Fluoro-Jade B (FJB) staining is also rescued after Ninjin-yoei-to (NYT) pre-administration for 2 weeks. Two weeks after administration of the NYT-containing diet, Aβ_1-42 was intracerebroventricularly injected into mice (upper). Two weeks later, FJB fluorescence was measured in the dentate granule cell layer surrounded by the dotted line (lower-left). Bar; 50 µm. Each bar and line (mean ± SEM) represent FJB-positive cells in the unit area after injection of vehicle and Aβ_1-42. (lower-right). **P<0.01, vs. saline, ^###P<0.001, vs. Aβ (Tukey’s test). saline, n=12; Aβ, n=32; Aβ/NYT, n=24.

Fig. 6.

Metallothionein (MT) level in the dentate gyrus is elevated after Ninjin-yoei-to (NYT) administration for 2 weeks. The NYT-containing diet was administered to mice for 2 weeks (left-upper). MT immunostaining was determined in the dentate granule cell layer (left-lower). Bar; 50 µm. Each bar and line (mean ± SEM) represent the rate (%) of MT staining after NYT administration to that after the control diet administration, which was represented as 100% (right). **P<0.01, vs. control (t-test). Control, n=8; NYT, n=12.

Fig. 7.

Proposed rescue mechanism of Ninjin-yoei-to (NYT) via induced synthesis of MTs on Aβ_1-42-induced neurodegeneration via intracellular Zn²⁺ toxicity. Aβ_1-42 uptake into dentate gyrus neurons play a key role for Aβ_1-42-mediated pathogenesis, in which Zn²⁺ toxicity via the uptake is crucial (upper) [9].