The Neuroprotective Properties of Hericium erinaceus in Glutamate-Damaged Differentiated PC12 Cells and an Alzheimer's Disease Mouse Model

Abstract

Hericium erinaceus, an edible and medicinal mushroom, displays various pharmacological activities in the prevention of dementia in conditions such as Parkinson's and Alzheimer's disease. The present study explored the neuroprotective effects of H. erinaceus mycelium polysaccharide-enriched aqueous extract (HE) on an l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cellular apoptosis model and an AlCl₃ combined with d-galactose-induced Alzheimer's disease mouse model. The data revealed that HE successfully induced PC12 cell differentiation. A 3 h HE incubation at doses of 50 and 100 µg/mL before 25 mM of l-Glu effectively reversed the reduction of cell viability and the enhancement of the nuclear apoptosis rate in DPC12 cells. Compared with l-Glu-damaged cells, in PC12 cells, HE suppressed intracellular reactive oxygen species accumulation, blocked Ca²⁺ overload and prevented mitochondrial membrane potential (MMP) depolarization. In the Alzheimer's disease mouse model, HE administration enhanced the horizontal and vertical movements in the autonomic activity test, improved the endurance time in the rotarod test, and decreased the escape latency time in the water maze test. It also improved the central cholinergic system function in the Alzheimer's mice, demonstrated by the fact that it dose-dependently enhanced the acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations in both the serum and the hypothalamus. Our findings provide experimental evidence that HE may provide neuroprotective candidates for treating or preventing neurodegenerative diseases.

Keywords: Alzheimer‘s disease mouse; Hericium erinaceus; glutamate; mitochondria; neuro-protection.

Figure 1

(A) HE and NGF induced PC12 cell differentiation determined by inverted microscope. Scale bar: 100 µm. (n = 6); (B) HE and NGF enhanced the expressions of β-tubulin III in PC12 cells. Data are expressed as mean ± SD (n = 6). * p < 0.05 and ** p < 0.01 vs. CTRL, HE: Hericium erinaceus aqueous extract; NGF: Nerve growth factor; CTRL: Non-treated cells.

Figure 2

(A) HE showed no effects on cell proliferation in normal DPC12 cells; (B) but strongly enhanced cell viability in l-Glu-induced DPC12 cellular apoptosis model; (C) HE restored l-Glu-induced nucleus morphological apoptotic alterations analyzed via Hoechst 33342 staining (n = 6). Scale bar: 100 µm. HE: Hericium erinaceus aqueous extract. Data are expressed as mean ± SD (n = 6). ^### p < 0.001 vs. CTRL, ** p < 0.01 and *** p < 0.001 vs. l-Glu-exposed cells. HE: Hericium erinaceus aqueous extract; CTRL: Non-treated cells.

Figure 3

(A) The disruption of mitochondrial membrane potential (MMP) caused by 12 h l-Glu exposure was strongly restored by 3 h HE pretreatment analyzed via JC-1 staining (n = 6). Scale bar: 100 µm; (B) the overaccumulation of reactive oxygen species (ROS) caused by 12 h l-Glu exposure was significantly reduced by 3 h HE pretreatment analyzed by DCFH-DA (2’,7’-dichlorofluorescein diacetate) staining (n = 6). Scale bar: 100 µm; (C) intracellular Ca²⁺ overload caused by l-Glu was strongly reversed by 3 h HE pretreatment analyzed via Fluo-4-AM staining (n = 6). Scale bar: 100 µm. Data are expressed as mean ± SD (n = 6). ^### p < 0.001 vs. CTRL, *** p < 0.001 vs. l-Glu-exposed cells. HE: Hericium erinaceus aqueous extract; CTRL: Non-treated cells.

Figure 3

(A) The disruption of mitochondrial membrane potential (MMP) caused by 12 h l-Glu exposure was strongly restored by 3 h HE pretreatment analyzed via JC-1 staining (n = 6). Scale bar: 100 µm; (B) the overaccumulation of reactive oxygen species (ROS) caused by 12 h l-Glu exposure was significantly reduced by 3 h HE pretreatment analyzed by DCFH-DA (2’,7’-dichlorofluorescein diacetate) staining (n = 6). Scale bar: 100 µm; (C) intracellular Ca²⁺ overload caused by l-Glu was strongly reversed by 3 h HE pretreatment analyzed via Fluo-4-AM staining (n = 6). Scale bar: 100 µm. Data are expressed as mean ± SD (n = 6). ^### p < 0.001 vs. CTRL, *** p < 0.001 vs. l-Glu-exposed cells. HE: Hericium erinaceus aqueous extract; CTRL: Non-treated cells.

Figure 4

In AlCl₃- and d-gal-induced AD mice, four-week HE treatment strongly enhanced (A) horizontal movements and (B) vertical movements in locomotor activity test; (C) increased the endurance time in rotating test; and (D) reduced the escape latency time in water-maze test compared with nontreated model mice. Data are expressed as mean ± SD (n = 10). ^### p < 0.001 vs. normal mice (CTRL), ** p < 0.01 and *** p < 0.001 vs. AD mice. HE: Hericium erinaceus aqueous extract.

Figure 5

HE treated AlCl₃- and d-gal-induced AD mice for four weeks, and blood and hypothalamus were collected. The levels of (A) acetylcholine (Ach) and (C) choline acetyltransferase (ChAT) in hypothalamus, and the levels of (B) Ach and (D) ChAT in serum were detected via ELISA method. Data are expressed as the percentage to controls and mean ± SD (n = 10). ^### p < 0.001 vs. normal mice (CTRL), * p <0.05 and ** p < 0.01 vs. AD mice. HE: Hericium erinaceus aqueous extract.

Figure 6

The experimental protocol for AlCl₃- and d-gal-induced Alzheimer’s disease mouse model establishment and drug administration.