Gongjin-Dan Enhances Hippocampal Memory in a Mouse Model of Scopolamine-Induced Amnesia

Abstract

We evaluated the neuropharmacological effects of Gongjin-Dan (GJD) on the memory impairment caused by scopolamine injection. BALB/c mice were orally treated with GJD (100, 200, or 400 mg/kg, daily) or tacrine (THA, 10 mg/kg) for 10 days, and scopolamine (2 mg/kg) was injected intraperitoneally. The radial arm maze and passive avoidance tests were performed to evaluate the animal's learning and memory. Scopolamine increased the task completing time, the number of total errors (reference and working memory error) in the radial arm maze task, and the latency time in the passive avoidance test, which were significantly ameliorated by treatment with GJD. The GJD treatment also attenuated the scopolamine-induced hyperactivation of acetylcholinesterase activity, and suppression of the expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and their receptors in the hippocampus. These effects of GJD were supported by both the doublecortin (DCX)-positive staining and Nissl staining, which were used to measure hippocampal neurogenesis and atrophy, respectively. These findings strongly suggest that GJD exerts a potent anti-amnesic effect, and its underlying mechanism might involve the modulation of cholinergic activity.

Conflict of interest statement

Competing Interests: The authors declare that no conflicts of interest exist. All the authors have approved the final article.

Figures

Fig 1. Fingerprint of GJD and experimental scheme.

The GJD and its 6 main components were subjected to HPLC. All chromatograms were obtained at a wavelengths of 203 Ginsenoside Rg1 and Rb1), 240 (morroniside and loganin), and 330 nm (nodakenin and decrusin). The detected peaks were merged (A). The L-muscone in Muschus was detected by gas chromatography (B). Two experimental schemes were shown (C).

Fig 2. Behavioral tests for the memory.

In the radial arm maze task, reference memory errors (A) and working memory errors (B) were recorded for 7 days (day 14–20 of the experiment). Time to complete the task (C) was recorded on experimental day 20. In the passive avoidance test, latency time was recorded on experimental day 22. The data are expressed as the means ± SD (n = 10). #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the vehicle group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Fig 3. Hippocampal levels of the AChE, CREB and neurotrophic proteins.

AChE activity (A) in the hippocampus was measured with an ELISA kit. The western blotting assay for the phosphorylated CREB, BDNF, and NGF levels in hippocampal tissues (B) and BDNF levels in HT22 cell (C), and their relative intensities (D) were shown. The data are expressed as the means ± SD (n = 7 or 3). #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with the vehicle group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Fig 4. Hippocampal expression levels of the mAChR1, TrkA, TrkB and synaptophysin mRNA.

Alterations in the expression levels of the mAChR1, TrkA, TrkB and synaptophysin mRNA were determined by real-time PCR. Gene expression was normalized to that of β-actin. The data are expressed as the means ± SD (n = 3). #P < 0.05 compared with the vehicle group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Fig 5. Immunohistochemistry of the DCX-positive cells in the hippocampus.

DCX-positive neural progenitor cells were stained in the subgranular zone of the hippocampus. Representative photomicrographs were taken at magnifications of 100 and 400× (A). The intensity of the DCX-positive staining was quantified (B). The data are expressed as the means ± SD (n = 3). ##P < 0.01 compared with the vehicle group; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Fig 6. Histochemical findings of the Nissl staining in the hippocampus.

The neuronal cell morphology in the hippocampus was detected using Nissl staining. Representative photomicrographs were taken at magnifications of 40 and 100× (A). The intensity of the labeled hippocampal neurons was quantified (B). The data are expressed as the means ± SD (n = 3). #P < 0.05 compared with the vehicle group; *P < 0.05, **P < 0.01 compared with the control group.