Holothuria scabra extracts confer neuroprotective effect in C. elegans model of Alzheimer's disease by attenuating amyloid-β aggregation and toxicity

Abstract

Background and aim: Alzheimer's disease (AD) is the most common aged-related neurodegenerative disorder that is associated with the toxic amyloid-β (Aβ) aggregation in the brain. While the efficacies of available drugs against AD are still limited, natural products have been shown to possess neuroprotective potential for prevention and therapy of AD. This study aimed to investigate the neuroprotective effects of H. scabra extracts against Aβ aggregation and proteotoxicity in C. elegans model of Alzheimer's diseases.

Experimental procedure: Whole bodies (WB) and body wall (BW) of H. scabra were extracted and fractionated into ethyl acetate (WBEA, BWEA), butanol (WBBU, BWBU), and ethanol (BWET). Then C. elegans AD models were treated with these fractions and investigated for Aβ aggregation and polymerization, biochemical and behavioral changes, and level of oxidative stress, as well as lifespan extension.

Results and conclusion: C. elegans AD model treated with H. scabra extracts, especially triterpene glycoside-rich ethyl acetate and butanol fractions, exhibited significant reduction of Aβ deposition. These H. scabra extracts also attenuated the paralysis behavior and improved the neurological defects in chemotaxis caused by Aβ aggregation. Immunoblot analysis revealed decreased level of Aβ oligomeric forms and the increased level of Aβ monomers after treatments with H. scabra extracts. In addition, H. scabra extracts reduced reactive oxygen species and increased the mean lifespan of the treated AD worms. In conclusion, this study demonstrated strong evidence of anti-Alzheimer effects by H. scabra extracts, implying that these extracts can potentially be applied as natural preventive and therapeutic agents for AD.

Taxonomy classification by evise: Alzheimer's disease, Neurodegenerative disorder, Traditional medicine, Experimental model systems, Molecular biology.

Keywords: Alzheimer's disease; Amyloid-β; Caenorhabditis elegans; Neuroprotection; Sea cucumber.

Graphical abstract

Fig. 1

Effects of H. scabra extracts on delaying paralysis in transgenic Aβ-expressing C. elegans, strain CL4176. Diagram illustrating the paralysis assay of CL4176 and CL802 (control) worms, showing the time at which the temperature was raised from 16 °C to 25 °C, when the H. scabra extracts were administered, and when the paralysis assay was done (A). Time duration of Aβ-induced paralysis in the transgenic CL4176 strain treated with 500 μg/ml of WBEA, BWEA, WBBU, BWBU fractions (B) and BWET fraction (D). Tetracycline treatment served as positive control, whereas 0.1% DMSO and ddH₂O were used as negative controls (B, D). Synchronous L1 worms were placed for 24 h at 16 °C on NGM plates fed with H. scabra extracts, tetracycline, or vehicle. The numbers of the paralyzed worms were counted at 26 h following temperature upshift to 25 °C then at 2 h intervals thereafter, and plotted as percent of unparalyzed worms from three independent experiments. CL802 which does not express Aβ transgene was used as transgenic control. Paralysis assays were quantified as PT₅₀ which is the duration at which 50% of worms were paralyzed after temperature upshift (C, E). An asterisk (∗∗) indicates the significant difference in paralysis curves at p < 0.01 compared with control.

Fig. 2

Effects of H. scabra extracts on neuronal functions of Aβ-expressed C. elegans CL2355 strain. Diagram illustrating the chemotaxis assay indicating the time of treatment in CL2355 and CL2122 (control) worms, when the H. scabra extracts were administered, and chemotaxis assay was done (A). Schematic illustration of chemotaxis assay (B). Chemotaxis assay was performed by using benzaldehyde as an attractant and 100% ethanol as a control odorant. Synchronized L1 worms were treated with either vehicle (DMSO or ddH₂O) or fractions of H. scabra extracts or TET (positive control). After treatments, worms were placed on the center of the assay plate and incubated at 25 °C for 1 h and CI was scored. The chemotaxis index in the control strain (CL2122) and the transgenic strain (CL2355) were scored (C, D). Data showed the mean ± SD value of CI from triplicated independent experiments. ∗∗ indicates statistically significant difference between treated and untreated groups at p < 0.01.

Fig. 3

Effects of H. scabra extracts in decreasing Aβ deposits in transgenic CL2006 worms. An illustration indicating the time duration of treatment and Aβ deposit detection (A). A schematic diagram indicates position of pharyngeal bulbs (Ba) and representative images of X-34 staining in wild-type N2 (Bb), in transgenic CL2006 fed with DMSO (control) (Bc), and ddH₂O (control) (Bh), with 500 μg/ml of WBEA (Bd), BWEA (Be), WBBU (Bf), BWBU (Bg), BWET (Bi), and TET at 50 μM (Bj). White arrows indicate Aβ deposits in the worm head. Quantitative analysis of Aβ deposits in the transgenic CL2006 fed with H. scabra extracts compared with control (DMSO or ddH₂O) (C). The Aβ deposits was quantified by ImageJ software and expressed as mean number ± SD of Aβ deposits/anterior area of a worm (n = 30 for each analysis) (C). ∗∗ indicates statistically significant difference between treated and untreated groups at p < 0.01.

Fig. 4

Effects of H. scabra extracts on Aβ species in transgenic CL2006 strain detected by anti-Aβ 6E10 antibody. Representative images of Western blotting of Aβ species in worms fed with or without H. scabra extracts at dose 500 μg/ml (A, D), or 50 μM TET (positive control) (D), and detected by anti- Aβ (6E10) or anti-actin antibodies. Quantification of intensities of Aβ bands at 4 kDa an d 20 kDa were carried out with ImageJ software (B–C, E-F). Vertical line indicated Aβ oligomer band at MW higher than 20 kDa, whereas arrow heads indicated Aβ monomer band at MW 4 kDa and actin band (43 kDa). Data are expressed as mean ± SD of relative band intensity normalized against actin from three independent experiments. ∗, ∗∗ indicate statistically significant difference between treated and untreated groups at p < 0.05 and 0.01, respectively.

Fig. 5

Effects of H. scabra extracts on Aβ species in transgenic CL2006 strains as detected by anti- Aβ oligomer A11 antibody. Representative images of Western blotting of Aβ species in worms fed with or without H. scabra extracts at dose 500 μg/ml (A, D), or 50 μM TET (positive control) (D), and detected by anti-Aβ (A11) or anti-actin antibodies. Quantification of intensities of high molecular weight Aβ oligomer bands at 75 kDa (B, E) and 150 kDa (C, F) were carried out with ImageJ software. Vertical line indicated high molecular Aβ oligomer bands at 75–150 kDa, while arrow head indicated actin band (43 kDa). Data are expressed as mean ± SD of relative band intensity against actin from three independent experiments. ∗, ∗∗ indicate statistically significant difference between treated and untreated groups at p < 0.05 and 0.01, respectively.

Fig. 6

Effect of H. scabra extracts on lifespan of transgenic Aβ-expressing worms. The survival curves show the lifespans of transgenic worms CL2006 (A), CL4176 (B), and CL2355 (C) treated with BWET compared to the control; CL2006 (D), CL4176 (E), and CL2355 (F) treated with WBEA and BWEA compared to the control; and CL2006 (G), CL4176 (H), and CL2355 (I) treated with WBBU and BWBU compared to the control.

Fig. 7

Effect of H. scabra extracts on the reduction of ROS levels in the transgenic CL4176 (A–B) and CL2006 (C–D) worms fed with vehicle or H. scabra extracts at a dose 500 μg/ml or TET 50 μM (positive control). Results are expressed as percentage of fluorescence (%DCF) relative to vehicle-treated transgenic CL4176 and CL2006 (untreated control) worms, which is set as 100%. Bars represent the standard error (SD). ∗∗indicates statistical significance at p < 0.01 compared with control groups.