YY-1224, a terpene trilactone-strengthened Ginkgo biloba, attenuates neurodegenerative changes induced by β-amyloid (1-42) or double transgenic overexpression of APP and PS1 via inhibition of cyclooxygenase-2

Abstract

Background: Ginkgo biloba has been reported to possess free radical-scavenging antioxidant activity and anti-inflammatory properties. In our pilot study, YY-1224, a terpene trilactone-strengthened extract of G. biloba, showed anti-inflammatory, neurotrophic, and antioxidant effects.

Results: We investigated the pharmacological potential of YY-1224 in β-amyloid (Aβ) (1-42)-induced memory impairment using cyclooxygenase-2 (COX-2) knockout (-/-) and APPswe/PS1dE9 transgenic (APP/PS1 Tg) mice. Repeated treatment with YY-1224 significantly attenuated Aβ (1-42)-induced memory impairment in COX-2 (+/+) mice, but not in COX-2 (-/-) mice. YY-1224 significantly attenuated Aβ (1-42)-induced upregulation of platelet-activating factor (PAF) receptor gene expression, reactive oxygen species, and pro-inflammatory factors. In addition, YY-1224 significantly inhibited Aβ (1-42)-induced downregulation of PAF-acetylhydrolase-1 (PAF-AH-1) and peroxisome proliferator-activated receptor γ (PPARγ) gene expression. These changes were more pronounced in COX-2 (+/+) mice than in COX-2 (-/-) mice. YY-1224 significantly attenuated learning impairment, Aβ deposition, and pro-inflammatory microglial activation in APP/PS1 Tg mice, whereas it significantly enhanced PAF-AH and PPARγ expression. A preferential COX-2 inhibitor, meloxicam, did not affect the pharmacological activity by YY-1224, suggesting that the COX-2 gene is a critical mediator of the neuroprotective effects of YY-1224. The protective activity of YY-1224 appeared to be more efficacious than a standard G. biloba extract (Gb) against Aβ insult.

Conclusions: Our results suggest that the protective effects of YY-1224 against Aβ toxicity may be associated with its PAF antagonistic- and PPARγ agonistic-potential as well as inhibition of the Aβ-mediated pro-inflammatory switch of microglia phenotypes through suppression of COX-2 expression.

Keywords: APPswe/PS1dE9 transgenic mice; Cyclooxygenase-2 knockout mice; Microglia; Peroxisome proliferators-activated receptor γ; Platelet-activating factor; Terpene trilactone-strengthened G. biloba.

Fig. 1

Experimental design for evaluating the effects of YY-1224 or Gb on learning impairments in mice. a Effects of YY-1224 or Gb on Aβ (1-42)-induced memory impairment in COX-2 (+/+) and COX-2 (−/−) mice (Figs. 2, 3, 4, 5, 6, 7, and 8). Mice received YY-1224 or Gb for 14 consecutive days [7 days before Aβ (1-42) i.c.v. infusion and 7 days period of memory assessment after Aβ (1-42) i.c.v. infusion]. b Effects of meloxicam on the pharmacological activity of YY-1224 or Gb in response to neurotoxic changes (Figs. 9, 10, 11, 12, and 13) in APP/PS1 Tg mice. Mice received YY-1224 or Gb with or without meloxicam 90 consecutive days and additional 5-day period of memory assessment

Fig. 2

Effects of YY-1224 (YY) or Gb on Aβ (1-42)-induced memory impairment in mice. a Novel object recognition test. b Y-maze test. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.05, ^## P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 3

Effects of YY-1224 (YY) or Gb on Aβ (1-42)-induced PAFR expression in the hippocampi of mice. a Effect of YY on PAFR-immunoreactivity. b Effect of YY on PAFR mRNA expression. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 4

Effects of YY-1224 (YY) or Gb on Aβ (1-42)-induced PAF-AH I-immunoreactivity in the hippocampi of mice. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 5

Effects of YY-1224 (YY) or Gb on Aβ (1-42)-induced PAF-AH mRNA levels in the hippocampus. a Changes in PAF-AH I α1 mRNA expression. b Changes in PAF-AH I α2 mRNA expression. c Changes in PAF-AH I LIS1 mRNA expression. d Changes in PAF-AH II mRNA expression. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42); &P < 0.05 vs. COX-2 (+/+) mice treated with Gb + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 6

Effects YY-1224 (YY) or Gb on Aβ (1-42)-induced oxidative burdens in the hippocampus of mice. a Aβ (1-42)-induced lipid peroxidation. b Aβ (1-42)-induced protein oxidation. Lipid peroxidation and protein oxidation were evaluated by quantitative biochemical analyses and slot blot analyses. c Aβ (1-42)-induced synaptosomal formation of reactive oxygen species. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.05, ^## P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42); &P < 0.05 vs. COX-2 (+/+) mice treated with Gb + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 7

Effects of YY-1224 (YY) or Gb on Aβ (1-42)-induced proinflammatory genes in the hippocampi of mice. a Changes in TNF-α mRNA expression. b Changes in IL-1β mRNA expression. c Changes in IL-6 mRNA expression. d Changes in IFN-γ mRNA expression. e Changes in iNOS mRNA expression. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42); &P < 0.05, &&P < 0.05 vs. COX-2 (+/+) mice treated with Gb + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 8

Effects of YY-1224 (YY) or Gb on Aβ (1-42)-induced PPAR expressions in the hippocampi of mice. a Changes in PPARα mRNA expression. b Changes in PPARγ mRNA expression. c Changes in PPARγ protein expression. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of six animals. *P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (42-1); ^# P < 0.01 vs. COX-2 (+/+) mice treated with vehicle + Aβ (1-42); &P < 0.05 vs. COX-2 (+/+) mice treated with Gb + Aβ (1-42) (three-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 9

Activity of YY-1224 (YY) or Gb on memory impairment and Aβ deposition in APP/PS1 Tg mice. a Effects of meloxicam on the pharmacological activity of YY or Gb in response to Y-maze performance and b novel object recognition. c, d Effect of meloxicam on the pharmacological activity of YY or Gb in response to Aβ-immunoreactivity in the hippocampus (c) and cortex (d). Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of 10 (a, b) or 5 (c, d) animals. ^# P < 0.05, ^## P < 0.01 vs. vehicle + 0.5% Na-CMC; & P < 0.05 vs. Gb + 0.5% Na-CMC (two-way ANOVA was followed by Fisher’s LSD pairwise comparisons). Scale bar = 200 μm

Fig. 10

Activity of YY-1224 (YY) or Gb on PAF-AH level in the hippocampus of APP/PS1 Tg mice. a, b Effects of meloxicam on the pharmacological activity of YY or Gb in response to PAF-AH-immunoreactivity in the CA1 (a) and CA3 (b) regions of the hippocampus. c Effects of meloxicam on the pharmacological activity of YY or Gb in response to PAF-AH I α2 mRNA expression in the hippocampus. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of five animals. ^# P < 0.05, ^## P < 0.01 vs. vehicle + 0.5% Na-CMC; &P < 0.05 vs. Gb + 0.5% Na-CMC (two-way ANOVA was followed by Fisher’s LSD pairwise comparisons). Scale bar = 200 μm

Fig. 11

Activity of YY-1224 (YY) or Gb on PPAR expressions in the hippocampus of APP/PS1 Tg mice. a Effects of meloxicam on the pharmacological activity of YY or Gb in response to PPARα mRNA expression. b, c Effects of meloxicam on the pharmacological activity of YY or Gb in response to PPARγ mRNA (b) and protein (c) expression. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of five animals. ^# P < 0.05, ^## P < 0.01 vs. vehicle + 0.5% Na-CMC; &P < 0.05 vs. Gb + 0.5% Na-CMC (two-way ANOVA was followed by Fisher’s LSD pairwise comparisons)

Fig. 12

Activity of YY-1224 (YY) or Gb on Iba-1-immunoreactivity in the APP/PS1 Tg mice. a, b Effects of meloxicam on the pharmacological activity of YY or Gb in response to Iba-1-immunoreactivity in the hippocampus (a) and cortex (b). Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of five animals. ^# P < 0.01 vs. vehicle + 0.5% Na-CMC; &P < 0.05 vs. Gb + 0.5% Na-CMC (two-way ANOVA was followed by Fisher’s LSD pairwise comparisons). Scale bar = 200 μm

Fig. 13

Activity of YY-1224 (YY) or Gb on mRNA expressions of microglial phenotype in the hippocampus. a–c Effect of meloxicam on the pharmacological activity of YY or Gb in response to CD16 (a), CD32 (b), and CD86 (c) mRNA expressions of M1 phenotype microglia/macrophages. d, e Effect of meloxicam on the pharmacological activity of YY or Gb in response to YM1 (d) and CD206 (e) mRNA expressions of M2 phenotype microglia/macrophages. Veh vehicle for YY or Gb (10% tween-80 in sterile saline). Each value is the mean ± S.E.M of five animals. ^# P < 0.01 vs. vehicle + 0.5% Na-CMC; &P < 0.05, &&P < 0.01 vs. Gb + 0.5% Na-CMC (two-way ANOVA was followed by Fisher’s LSD pairwise comparisons)