Water extract of Humulus japonicus improves age‑related cognitive decline by inhibiting acetylcholinesterase activity and the acetylcholine signaling pathway

Abstract

The aging process is associated with a decline in certain cognitive abilities, including learning and memory. This age‑related cognitive decline is associated with a reduction in neurogenesis and alterations in the cholinergic system. Humulus japonicus (HJ), an ornamental plant in the family Cannabaceae, has been reported to exert beneficial effects against neurodegenerative pathophysiologies in mouse models of disorders such as Alzheimer's and Parkinson's disease. Despite the increasingly aging populations of numerous societies, no study has yet investigated the effects of HJ on cognitive decline associated with normal aging. The present study therefore aimed to examine the protective potential of HJ water (HJW) extract against age‑related cognitive decline and scopolamine‑induced cognitive impairment. The analyses revealed that the oral administration of HJW markedly improved novel objective recognition and spatial learning in the novel object recognition and Morris water maze tests, respectively, in aged mice. The administration of 600 mg/kg HJW further increased neurogenesis and CA1 thickness in the hippocampi of aged mice. In scopolamine‑induced cognitive impairment, administration of 400 or 600 mg/kg HJW markedly increased novel object recognition performance in scopolamine‑treated mice. The inhibitory effect of HJW on acetylcholinesterase (AChE) and the activation effects of HJW on the calcium/calmodulin‑dependent kinase (CaMK)IIα‑cAMP response element‑binding protein (CREB) and AKT‑glycogen synthase kinase‑3 β (GSK3β) pathways were further demonstrated. Overall, these results indicate that HJW administration improves cognitive function through the regulation of AChE activity and CaMKIIα‑CREB and AKT‑GSK3β pathways.

Keywords: Humulus japonicus; acetylcholine; aging; cognitive function; scopolamine‑induced model.

Figure 1.

Total ion chromatogram results of the standardized HJW by UPLC-ESI-qTOF-MS/MS spectrometry in the negative ion mode. A total of seven previously isolated compounds underwent peak assignments for the chemical profiling of standard HJW: benzyl-α-L-arabinopyranosy l-(1′-6′)-β-D-glucopyranoside (3); phenylethyl-α-L-arabinopyranosyl-(1′→6′)-β-d-glucopyranoside (6), vitexin (7), luteolin-7-O-β-d-glucopyranoside (8), apigenin-7-O-β-d-glucopyranoside (9), luteolin (10) and apigenin (11). Additionally, four compounds (1, 2, 4 and 5) were predicted based on their molecular weights and MS fragment ion patterns, as follows: Caffeoylquinic acid (1), apigenin-6,8-C-dihexoside (2), apigenin-6-C-glucosyl-8-C-arabinoside (4) and apigenin-6-C-arabinosyl-8-C-glucoside (5). HJW, water extract of Humulus japonicus; UPLC-ESI-qTOF-MS/MS, ultra-performance liquid chromatography coupled to a quadrupole/time of flight system mass spectrometry².

Figure 2.

Effects of HJW on locomotor activity and recognition memory in aged C57BL/6J mice. (A) Exploratory locomotion in the open field test for the 30 min test of the total distance moved. (B) Percentage of time spent sniffing and touching the familiar or novel objective for 10 min in the novel object recognition test. (C) Percentage of the number of touches on the familiar or novel objects at 10 min in the novel object recognition test. Young/vehicle group (n=9), Aged/vehicle group (n=7), Aged/HJW200 group (n=5), Aged/HJW400 group (n=10), Aged/HJW600 group (n=10). *P<0.05 and **P<0.01 vs. Aged/vehicle group. Data are presented as the mean ± SEM. Statistical analysis was performed using Student's t-test. HJW, water extract of Humulus japonicus.

Figure 3.

Effects of HJW on spatial learning memory in aged C57BL/6J mice. Results of the Morris water maze test. (A) Swimming speed during the training trials. (B) Latency time to the target platform during the training trials. (C) Percentage of time spent in the target quadrant following removal of the platform (D) The number of target crossings in the platform area. Young/vehicle group (n=9), Aged/vehicle group (n=7), Aged/HJW200 group (n=5), Aged/HJW400 group (n=10), Aged/HJW600 group (n=9). *P<0.05 and **P<0.01 vs. Aged/vehicle group. Data are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and Student's t-test. HJW, water extract of Humulus japonicus.

Figure 4.

Effects of HJW on neurogenesis in aged C57BL/6J mice. (A) Representative Nissl staining images of the CA1, CA3 and DG hippocampus regions. Scale bar, 200 µm. (B) Quantification of hippocampus layer thickness. (C) Representative DCX-staining images of the hippocampus DG region. Scale bar, 100 µm. (D) Quantification of DCX-positive cells in the DG regions. Aged/vehicle group (n=7) and Aged/HJW600 group (n=9). *P<0.05 vs. Aged/vehicle group. Data are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA. HJW, water extract of Humulus japonicus.

Figure 5.

Inhibitory effects of HJW on AChE in C57BL/6J mice. (A) AChE inhibitory effects on HJW. (B) Inhibition of the AChE in the cortex of C57BL/6J mice. (C) Inhibition of the AChE in the hippocampus of C57BL/6J mice *P<0.05 and **P<0.01 vs. control group. Data are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA. HJW, water extract of Humulus japonicus; AChE, acetylcholinesterase; DP, donepezil.

Figure 6.

Effects of HJW on recognition memory in scopolamine-induced memory impaired mice. (A) Percentage of time spent sniffing and touching the familiar or novel objectives at 10 min in the novel objective recognition test. (B) Percentage of time spent sniffing and touching the familiar or novel objects for 10 min in the novel objective recognition test. Vehicle/vehicle group (n=12), Vehicle/scopolamine group (n=12), HJW200/scopolamine group (HJW200 mg/kg, n=12), HJW400/scopolamine group (HJW400 mg/kg, n=12) and HJW600/scopolamine group (HJW600 mg/kg, n=10). **P<0.01 vs. Vehicle/scopolamine group. Data are presented as the mean ± SEM. Statistical analysis was performed using Student's t-test. HJW, water extract of Humulus japonicus; DP, donepezil.

Figure 7.

Effects of HJW on the phosphorylation of NR2B, CaMKIIα and CREB in the hippocampus of scopolamine-induced mice. Western blot analysis of pNR2B (Tyr1472), NR2B, pCaMKIIα (Thr286), CaMKIIα, pCREB (Ser133) and CREB. The signal intensities of the bands normalized to actin are shown. (A) A representative western blot image. (B) Quantitative analysis of the NMDA receptor subunit pNR2B (Tyr1472), NR2B, pCaMKIIα (Tyr286), CaMKIIα, pCREB (Ser133) and CREB. *P<0.05 and **P<0.01 vs. Vehicle/vehicle group, ^#P<0.05 and ^##P<0.01 vs. Vehicle/scopolamine group. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. HJW, water extract of Humulus japonicus; NR2B, NMDA receptor subtype 2B; CaMK, calcium/calmodulin-dependent kinase; CREB, cAMP response element-binding protein; p, phosphorylated; NMDA, N-Methyl-d-aspartate.

Figure 8.

Effects of HJW on the phosphorylation of Akt, GSK3β and the synaptic marker PSD95, gephyrin, ChAT in the hippocampus of scopolamine-induced mice. Western blot analysis of pAkt (Tyr308), Akt, pGSK3β (Ser9), GSK3β, PSD95, gephyrin and ChAT. The signal intensities of the bands normalized to actin are shown. (A) Representative western blot image. (B) Quantitative analysis of pAkt (Tyr308), Akt, pGSK3β (Ser9) and GSK3β. (C) Representative western blot image. (D) Quantitative analysis of PSD95, gephyrin and ChAT. *P<0.05 and **P<0.01 vs. Vehicle/vehicle group, ^#P<0.05 and ^##P<0.01 vs. Vehicle/scopolamine group. Data are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA. HJW, water extract of Humulus japonicus; GSK3β, glycogen synthase kinase-3 beta; ChAT, choline acetyltransferase; p, phosphorylated.