Pharmacological Basis for the Use of Evodiamine in Alzheimer's Disease: Antioxidation and Antiapoptosis

Abstract

Evodiamine (Evo), a major alkaloid compound isolated from the dry unripened fruit of Evodia fructus, has a wide range of pharmacological activities. The present study sought to explore the neuroprotective effects of Evo in l-glutamate (l-Glu)-induced apoptosis of HT22 cells, and in a d-galactose and aluminum trichloride-developed Alzheimer’s disease (AD) mouse model. Evo significantly enhanced cell viability, inhibited the accumulation of reactive oxygen species, ameliorated mitochondrial function, increased the B-cell lymphoma-2 protein content, and inhibited the high expression levels of Bax, Bad, and cleaved-caspase-3 and -8 in l-Glu-induced HT22 cells. Evo also enhanced the phosphorylation activities of protein kinase B and the mammalian target of rapamycin in the l-Glu-induced HT22 cells. In the AD mouse model, Evo reduced the aimless and chaotic movements, reduced the time spent in the central area in the open field test, and decreased the escape latency time in the Morris water maze test. Evo reduced the deposition of amyloid beta 42 (Aβ42) in the brain, and increased the serum level of Aβ42, but showed no significant effects on Aβ40. In addition, six weeks of Evo administration significantly suppressed oxidative stress by modulating the related enzyme levels. In the central cholinergic system of AD mice, Evo significantly increased the serum levels of acetylcholine and choline acetyltransferase and decreased the level of acetylcholinesterase in the serum, hypothalamus, and brain. Our results provide experimental evidence that Evo can serve as a neuroprotective candidate for the prevention and/or treatment of neurodegenerative diseases.

Keywords: Alzheimer’s disease; Evodiamine; apoptosis; cholinergic system; oxidative stress.

Figure 1

Chemical structure of Evodiamine.

Figure 2

Evo showed neuroprotective effects on HT22 cells against l-Glu-caused damage. (A) 3-h pre-treatment with Evo enhanced cell viability in HT22 cells exposed to 25 mM l-Glu for 24 h. ### p < 0.001 vs. CTRL. ** p < 0.01 vs. l-Glu-treated cells; (B) 3-h pre-treatment of Evo reduced the apoptosis rate in HT22 cells exposed to l-Glu for 24 h (n = 10); (C) 3-h pre-treatment of Evo ameliorated the dissipation of MMP in HT22 cells exposed to l-Glu for 12 h (Scale bar: 100 μm; n = 10); (D) 3-h pre-treatment of Evo inhibited the over-accumulation of ROS in HT22 cells exposed to l-Glu for 12 h (Scale bar: 100 μm; n = 10).

Figure 3

Evo regulated the expression levels of apoptosis-related proteins in HT22 cells exposed to l-Glu for 24 h. Evo increased the expression levels of Bcl-2, P-Akt, and P-mTOR, and reduced the expression levels of Bax, Bad, and cleaved-caspase-3 and -8. Quantification data were normalized by (A) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (B) corresponding total proteins and the average fold increase in band intensity compared with the CTRL group are marked, respectively (n = 6).

Figure 4

Evo improved the behavioral performances of AD mice. Compared with non-treated AD mice, 42-day Evo administration (A) decreased the time mice spent in the central area in the open field test and (B) reduced the escape latency time in the Morris water maze test. Data are expressed as mean ± S.E.M. (n = 18). ## p < 0.01 and ### p < 0.001 vs. healthy mice (CTRL), * p < 0.05 and ** p < 0.01 vs. AD mice (model).

Figure 5

Forty-two-day Evo administration (A) enhanced the levels of Aβ42 in serum and (B) reduced the levels of Aβ42 in the cerebral cortices, but failed to influence the levels of Aβ40 in the (C) serum and (D) cerebral cortices of AD mice. Data are expressed as mean ± S.E.M. (n = 9). # p < 0.05 and ## p <0.01 vs. healthy mice (CTRL), * p < 0.05, and ** p < 0.01 vs. AD mice (model).

Figure 6

No significant pathologic changes were noted in (A) the hippocampus (magnification 40×); (B) spleen (magnification 200×); and (C) kidney (magnification 200×) among all experimental groups (n = 9).

Figure 7

Schematic of experiments. The AD mouse model was established by administering AlCl₃ (20 mg/kg, i.g.) and d-gal (120 mg/kg i.p.) daily for 91 days. From the 50th day, the mice were given Evo at doses of 10 and 40 mg/kg daily for 42 days. Behavioral tests were performed starting from the 89th day.