Enteromorpha prolifera Extract Improves Memory in Scopolamine-Treated Mice via Downregulating Amyloid-β Expression and Upregulating BDNF/TrkB Pathway

Abstract

Enteromorpha prolifera, a green alga, has long been used in food diets as well as traditional remedies in East Asia. Our preliminary study demonstrated that an ethyl acetate extract of Enteromorpha prolifera (EAEP) exhibited the strongest antioxidant activity compared to ethanol or water extracts. Nonetheless, there has been no report on the effect of EAEP on memory impairment due to oxidative damage. This study investigated whether EAEP could attenuate memory deficits in an oxidative stress-induced mouse model. EAEP was orally administered (50 or 100 mg/kg body weight (b.w.)) to mice and then scopolamine was administered. The oral administration of EAEP at 100 mg/kg b.w. significantly restored memory impairments induced by scopolamine, as evaluated by the Morris water maze test, and the passive avoidance test. Further, EAEP upregulated the protein expression of BDNF, p-CREB, p-TrkB, and p-Akt. Moreover, EAEP downregulated the expression of amyloid-β, tau, and APP. The regulation of cholinergic marker enzyme activities and the protection of neuronal cells from oxidative stress-induced cell death in the brain of mice via the downregulation of amyloid-β and the upregulation of the BDNF/TrkB pathway by EAEP suggest its potential as a pharmaceutical candidate to prevent neurodegenerative diseases.

Keywords: BDNF/TrkB pathway; Enteromorpha prolifera; antioxidant defense system; cholinergic enzymes; scopolamine.

Figure 1

The schedule of the experiment.

Figure 2

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on memory-enhancing in scopolamine-induced mice. The escape latency time during the training days (a), escape latency time (b), and the number of platform area crossings (c) in Morris water maze test were measured in the mice. CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP 100: EAEP 100 mg/kg-treated group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 9/group). Statistical significance was indicated as * p < 0.05, or ** p < 0.01 compared to the control, and as # p < 0.05, or ## p < 0.01 compared to the scopolamine-induced group.

Figure 3

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on memory-enhancing in scopolamine-induced mice. Latency time in the passive avoidance test was measured in mice. CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP 100: EAEP 100 mg/kg-treated group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EAEP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 9/group). Statistical significance was indicated as *** p < 0.001 compared to the control, and as # p < 0.05, ## p < 0.01, or ### p < 0.001 compared to the scopolamine-induced group.

Figure 4

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on the cholinergic nervous system in scopolamine-induced mice. AChE (acethylcholine esterase) activity (a) and ChAT (choline acetyl transferase) activity (b) were measured in mice. CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP 100: EAEP 100 mg/kg-treated group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EAEP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 8/group). Statistical significance was indicated as * p < 0.05, or *** p < 0.001 compared to the control, and as ## p < 0.01, or ### p < 0.001 compared to the scopolamine-induced group.

Figure 5

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on antioxidant enzymes in scopolamine-induced mice. The MDA (malondialdehyde) level (a), GSH (glutathione) activity (b), GR (glutathione reductase) activity (c), and GPx (glutathione peroxidase) activity (d) were measured in mice. CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP 100: EAEP 100 mg/kg-treated group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EAEP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 8/group). Statistical significance was indicated as * p < 0.05, ** p < 0.01, or *** p < 0.001 compared to the control, and as # p < 0.05, ## p < 0.01, or ### p < 0.001 compared to the scopolamine-induced group.

Figure 6

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on hippocampal neurons in CA1 and CA3 region. Histological sections with hematoxylin and eosin (H&E) staining of the CA1 and CA3 regions in the hippocampi of mice (a), the number of surviving neurons in the CA1 region (b), and the number of surviving neurons in the CA3 region (c). CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP 100: EAEP 100 mg/kg treated-group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EAEP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 6/group). Statistical significance was indicated as *** p < 0.001 compared to the control, and as ## p < 0.01, or ### p < 0.001 compared to the scopolamine-induced group.

Figure 7

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on expression of p-Tau, APP, and Amyloid β in brain tissue from mice treated with scopolamine. The supernatant of brain homogenate was subjected to SDS-PAGE, and a western blot analysis was performed using each specific antibody against p-Tau, APP, Amyloid β, or β-actin. β-Actin was used as loading controls. The results are representative of three experiments conducted under each condition. CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP: EAEP 100 mg/kg-treated group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EAEP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 3/group). Statistical significance was indicated as *** p < 0.001 compared to the control, and as ### p < 0.001 compared to the scopolamine-induced group.

Figure 8

Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on expression of BDNF, p-CREB, p-TrkB, and p-Akt in brain tissue from mice treated with scopolamine. The supernatant of brain homogenate was subjected to SDS-PAGE, and a western blot analysis was performed using each specific antibody against BDNF, p-CREB, p-TrkB, and p-Akt or β-actin. β-Actin was used as loading controls. The results are representative of three experiments conducted under each condition. CON: Non-treated group; SCO: Scopolamine 2 mg/kg-treated group; TAC: Scopolamine 2 mg/kg + tacrine 10 mg/kg-treated group; EAEP 100: EAEP 100 mg/kg-treated group; SEAEP 50: Scopolamine 2 mg/kg + EAEP 50 mg/kg-treated group; SEAEP 100: Scopolamine 2 mg/kg + EAEP 100 mg/kg-treated group. All data are mean ± S.E.M. (n = 3/group). Statistical significance was indicated as ** p < 0.01, or *** p < 0.001 compared to the control, and as ## p < 0.01, or ### p < 0.001 compared to the scopolamine-induced group.