Anti-Oxidative Stress Activity Is Essential for Amanita caesarea Mediated Neuroprotection on Glutamate-Induced Apoptotic HT22 Cells and an Alzheimer's Disease Mouse Model

Abstract

Amanita caesarea, an edible mushroom found mainly in Asia and southern Europe, has been reported to show good antioxidative activities. In the present study, the neuroprotective effects of A. caesarea aqueous extract (AC) were determined in an l-glutamic acid (l-Glu) induced HT22 cell apoptosis model, and in a d-galactose (d-gal) and AlCl₃-developed experimental Alzheimer's disease (AD) mouse model. In 25 mM of l-Glu-damaged HT22 cells, a 3-h pretreatment with AC strongly improved cell viability, reduced the proportion of apoptotic cells, restored mitochondrial function, inhibited the over-production of intracellular reactive oxygen species (ROS) and Ca²⁺, and suppressed the high expression levels of cleaved-caspase-3, calpain 1, apoptosis-inducing factor (AIF) and Bax. Compared with HT22 exposed only to l-Glu cells, AC enhanced the phosphorylation activities of protein kinase B (Akt) and the mammalian target of rapamycin (mTOR), and suppressed the phosphorylation activities of phosphatase and tensin homolog deleted on chromosome ten (PTEN). In the experimental AD mouse, 28-day AC administration at doses of 250, 500, and 1000 mg/kg/day strongly enhanced vertical movements and locomotor activities, increased the endurance time in the rotarod test, and decreased the escape latency time in the Morris water maze test. AC also alleviated the deposition of amyloid beta (Aβ) in the brain and improved the central cholinergic system function, as indicated by an increase acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations and a reduction in acetylcholine esterase (AchE) levels. Moreover, AC reduced ROS levels and enhanced superoxide dismutase (SOD) levels in the brain of experimental AD mice. Taken together, our data provide experimental evidence that A. caesarea may serve as potential food for treating or preventing neurodegenerative diseases.

Keywords: Alzheimer’s disease; Amanita caesarea; apoptosis; cholinergic transmitters; oxidative stress.

Figure A1

Effect of AC on the over-load of intracellular Ca²⁺ caused by 12-h l-Glu. Quantitative analysis of Ca²⁺ values of fluorescence intensity obtained from Figure 2C (n = 6). ^## p < 0.01 vs. CTRL, * p < 0.05 vs. l-Glu-treated cells.

Figure 1

AC ameliorated l-Glu-induced cytotoxicity and apoptosis in HT22 cells. (A) AC has no significant influence on HT22 cell viability; (B) AC enhanced cell viability in l-Glu-damaged HT22 cells after 24 h co-incubation; (C) AC reduced proportion of the apoptotic cells in l-Glu-exposed HT22 cells detected by Annexin V-FITC/PI staining. Data are expressed as mean ± S.D. (n = 6). ^## p < 0.01 and ^### p < 0.001 vs. CTRL. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. l-Glu-treated cells.

Figure 2

AC ameliorated MMP loss, intracellular ROS and Ca²⁺ over-production, and the apoptotic alternations on the expression levels of proteins. (A) AC pretreatment restored the disruption of MMP caused by 12-h l-Glu exposure analyzing by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide staining (JC-1) (n = 6) (20×; Scale bar: 25 μm); (B) AC pretreatment inhibited the over-accumulation of intracellular ROS caused by 12-h l-Glu exposure detecting by DCFH–DA staining (n = 6) (20×; Scale bar: 25 μm); (C) AC ameliorated the over-load of intracellular Ca²⁺ caused by 12-h l-Glu exposure analyzing by Fluo 4-AM staining (n = 6); (D) AC reduced the expression levels of cleaved caspase-3, Bax, calpain 1 and apoptosis-inducing factor (AIF) in l-Glu-exposed HT22 cells after 24-h co-incubation. Quantification data was normalized by GAPDH, expressed as percentage of CTRL and mean ± S.D. (n = 6). ^# p < 0.05 and ^## p < 0.01 vs. CTRL, * p < 0.05 and ** p < 0.01 vs. l-Glu-treated cells.

Figure 3

3-h AC pre-exposure regulated the phosphorylation activities of PTEN, Akt and mTOR in l-Glu-exposed HT22 cells. Quantification data of the expressions of P-PTEN, P-Akt and P-mTOR were normalized by corresponding T-pTEN, T-Akt and T-mTOR. Data are expressed as mean ± SD (n = 6) and analyzed using one-way ANOVA. ^# p <0.05, ^## p < 0.01 and ^### p < 0.001 vs. CTRL, * p < 0.05 and ** p < 0.01 vs. l-Glu-treated cells.

Figure 4

AC improves AD-like behaviors in d-gal and AlCl₃ development AD mice. Compared with non-treated AD mice, 28-day AC administration enhanced horizontal movements(A) and vertical movements (B) in locomotor activity test; (C) enhanced the remaining time in rotating test; (D) reduced the escape latency time in WMT. Data expressed as mean ± S.E.M. (n = 12). ^# p < 0.05 and ^## p < 0.01 vs. control mice, * p < 0.05 and ** p < 0.01 vs. AD mice.

Figure 5

AC ameliorated oxidation stress in brain of AD mice. 28-day AC administration reduced the levels of ROS (A); and enhanced the levels of SOD (B) in brain of AD-like mice. Data expressed as mean ± S.E.M. (n = 12). ^## p < 0.01 vs. control mice, * p < 0.05 and ** p < 0.01 vs. AD mice.

Figure 6

The effect of AC on the levels of Aβ1-42 in serum and brain of AD-like mice. 28-day AC administration enhanced the levels of Aβ1-42 in serum (A); but reduced the levels of Aβ1-42 in brain (B) of AD-like mice. Data expressed as mean ± S.E.M. (n = 12). ^# p < 0.05 vs. control mice, * p < 0.05 vs. AD mice.

Figure 7

Schematic of experiments. The AD mouse model was established by given AlCl₃ (20 mg/kg, i.g.) and d-gal (120 mg/kg i.p.) daily for 56 days. From the 29th day, the mice were given AC at doses of 250,500 and 1000 mg/kg daily for 28 days. Behavioral tests were performed starting from the 54th day.