Fig. 1

Mortality curve and experimental workflow chart. (A) Larval zebrafish were exposed to different concentrations of EUMF (100, 200, 300, 400, 500, 600, 700, 800, and 1,600 μg/mL) from 3 to 6 dpf. The mortality was recorded within each group at 3, 4, 5, and 6 dpf. Dead larvae were judged using missing heartbeats. (B) Larvae at 3 dpf were co-exposed to AlCl3 and three different concentrations of EUMF from 3 to 6 dpf. At 6 dpf the zebrafish were subjected to a behavioral test. In addition, we also evaluated the AchE activity, Aβ deposition, and apoptosis in the brain and performed RT-qPCR.
Fig. 2

Effect of EUMF on AlCl3-induced locomotion impairments in zebrafish. (A) Dgital track map. The red, green, and black lines depict fast, medium, and slow movements, respectively (n = 10). (B) Total distance moved in the Ctl, AlCl3, and AlCl3 + EUMF groups (***P < 0.001 vs. Ctl; ###P < 0.001 vs. AlCl3; n = 10). (C) Speed change in the Ctl, AlCl3, and AlCl3 + EUMF groups (the speed change after light stimulus is demarcated by the frame; n = 10).
Fig. 3

Inhibition of EUMF on Aβ aggregation in zebrafish. (A) The Aβ plaques in the brain region were stained using thioflavin S in the Ctl, AlCl3, and AlCl3 + EUMF groups (Aβ is demarcated by arrows; scale bar = 100 μm). (B) Statistical analysis of the Aβ plaque count in each group (***P < 0.001 vs. Ctl; #P < 0.05; ###P < 0.001 vs. AlCl3; n = 10).
Fig. 4

Inhibition of EUMF on the AChE activity in zebrafish (***P < 0.001 vs. Ctl; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AlCl3; n = 10).
Fig. 5

Effect of EUMF on apoptosis in the brains of the AlCl3-modeled zebrafish. (A) The apoptotic cells were stained with TUNEL (scale bar = 100 μm). (B) Statistical analysis of the apoptotic cells count in the larvae heads (***P < 0.001 vs. Ctl; ###P < 0.001 vs. AlCl3; n = 10).
Fig. 6

Transcriptional alterations of genes. The amount of gene expression was exhibited as the relative expression (shown as fold) compared with the Ctl. (**P < 0.01, ***P < 0.001 vs. Ctl; #P < 0.05, ###P < 0.001 vs. AlCl3). (A–D) Expressions of genes involved in autophagy. (E) Transcript levels of ache. (F) Transcript levels of slc6a3.
Fig. 7

The proposed mechanism underlying the anti-AD effect of EUMF. EUMF inhibits the excessive autophagy and abnormal expressions of the ache and slc6a3 genes to exert the therapeutic effects against AD-like symptoms. Flavonoids in EUMF may contribute to this biological process.
Acknowledgments
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