Ilex latifolia Prevents Amyloid β Protein (25-35)-Induced Memory Impairment by Inhibiting Apoptosis and Tau Phosphorylation in Mice

Abstract

Ilex latifolia Thunb. (Aquifoliaceae), a Chinese bitter tea called "kudingcha," has been widely consumed as a health beverage and found to possess antioxidant, antidiabetic, antihypertensive, anti-inflammatory, and anti-ischemic activities. The aim of the present study was to investigate the neuroprotective effects of an ethanol extract of I. latifolia against amyloid β protein (Aβ)-induced memory impairment in mice and neurotoxicity in cultured rat cortical neurons. Memory impairment in mice was induced by intracerebroventricular injection of 15 nmol Aβ (25-35) and measured by the passive avoidance test and Morris water maze test. Chronic administration of I. latifolia (25-100 mg/kg, p.o.) significantly prevented Aβ (25-35)-induced memory loss. I. latifolia also prevented the decrease of glutathione concentrations, increased lipid peroxidation, expression of phosphorylated tau (p-tau), and changes in apoptosis-associated proteins in the memory-impaired mouse brain. Exposure of cultured cortical neurons to 10 μM Aβ (25-35) for 36 h induced neuronal apoptotic death. The neuronal cell death, elevation of intracellular Ca(2+) concentration, generation of reactive oxygen species, and expression of proapoptotic proteins caused by Aβ (25-35) in the cultured neurons were inhibited by treatment with I. latifolia (1-50 μg/mL). These results suggest that I. latifolia may have a possible therapeutic role in managing cognitive impairment associated with Alzheimer's disease. The underlying mechanism might involve the antiapoptotic effects mediated by antioxidant activity and inhibition of p-tau formation.

Keywords: Alzheimer's disease; Ca2+ signaling; antioxidant; cognition; neuroprotection.

FIG. 1.

Protective effects of I. latifolia against amyloid β protein (Aβ) (25–35)-induced memory impairment in mice. Learning and memory performance of the mice were assessed by (A) a passive avoidance test (n = 11–13) and (B) a Morris water maze test (n = 8–10). Values are expressed as the mean ± standard error of the mean (SEM) ^##P < .01 versus the sham; *P < .05 and **P < .01 versus 15 nmol Aβ (25–35).

FIG. 2.

Representative photomicrographs of (A) the hippocampus (upper) and CA1 region (lower), and (B) histogram of CA1 pyramidal cell numbers illustrating the inhibitory effect of I. latifolia on Aβ (25–35)-induced hippocampal damage in mice. Brain sections were stained with hematoxylin and eosin. Pathological features of the brain sections 7 days after intracerebroventricular injection of 15 nmol Aβ (25–35) were observed using a brightfield microscope. ^##P < .01 versus sham; *P < .05 and **P < .01 versus 15 nmol Aβ (25–35). Color images available online at www.liebertpub.com/jmf

FIG. 3.

Inhibitory effect of I. latifolia on Aβ (25–35)-induced expression of Bax and c-caspase-3 in mice brains. (A) Representative Western blot of proteins in the brains. (B) Bar graphs showing the relative ratio of Bax and c-caspase-3 proteins compared to β-actin. ^##P < .01 versus the sham; **P < .01 versus 15 nmol Aβ (25–35).

FIG. 4.

Inhibitory effect of I. latifolia on Aβ (25–35)-induced expression of phosphorylated tau (p-tau) in mice brains. (A) Representative Western blot of proteins in the brains. (B) Bar graphs showing the relative ratio of p-tau protein compared to β-actin. ^##P < .01 versus the sham; **P < .01 versus 15 nmol Aβ (25–35).

FIG. 5.

Inhibitory effect of I. latifolia on Aβ (25–35)-induced death of cultured cortical neurons measured by (A) an MTT assay and (B) Hoechst 33342 staining. Values are expressed as the mean ± SEM of data obtained from at least six independent experiments. ^##P < .01 versus the control; *P < .05 and **P < .01 versus 10 μM Aβ (25–35).

FIG. 6.

Inhibitory effect of I. latifolia on Aβ (25–35)-induced expression of Bax and c-caspase-3 in cultured cortical neurons. (A) Representative Western blot of proteins in the cultured neurons. (B) Bar graphs showing the relative ratio of Bax and c-caspase-3 proteins compared to β-actin. ^##P < .01 versus the control;*P < .05 and **P < .01 versus 10 μM Aβ (25–35).

FIG. 7.

Inhibitory effect of I. latifolia on Aβ (25–35)-induced elevation of [Ca²⁺]_i in cultured cortical neurons. [Ca²⁺]_i was monitored using a laser scanning confocal microscope. All images were processed to analyze changes in [Ca²⁺]_i at the single-cell level. Values are expressed as the relative fluorescence intensity (RFI). Each trace is a single-cell representative of at least four independent experiments.

FIG. 8.

Inhibitory effect of I. latifolia on Aβ (25–35)-induced reactive oxygen species generation in cultured cortical neurons. RFI was monitored using a laser scanning confocal microscope. Values are expressed as the mean ± SEM of data obtained from at least four independent experiments. ^##P < .01 versus the control; *P < .05 and **P < .01 versus 10 μM Aβ (25–35).