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Beneficial Effects of Cyclic Ether 2-Butoxytetrahydrofuran from Sea Cucumber Holothuria scabra against Aβ Aggregate Toxicity in Transgenic Caenorhabditis elegans and Potential Chemical Interaction.
Tangrodchanapong T
,
Sornkaew N
,
Yurasakpong L
,
Niamnont N
,
Nantasenamat C
,
Sobhon P
,
Meemon K
.
Abstract
The pathological finding of amyloid-β (Aβ) aggregates is thought to be a leading cause of untreated Alzheimer's disease (AD). In this study, we isolated 2-butoxytetrahydrofuran (2-BTHF), a small cyclic ether, from Holothuria scabra and demonstrated its therapeutic potential against AD through the attenuation of Aβ aggregation in a transgenic Caenorhabditis elegans model. Our results revealed that amongst the five H. scabra isolated compounds, 2-BTHF was shown to be the most effective in suppressing worm paralysis caused by Aβ toxicity and in expressing strong neuroprotection in CL4176 and CL2355 strains, respectively. An immunoblot analysis showed that CL4176 and CL2006 treated with 2-BTHF showed no effect on the level of Aβ monomers but significantly reduced the toxic oligomeric form and the amount of 1,4-bis(3-carboxy-hydroxy-phenylethenyl)-benzene (X-34)-positive fibril deposits. This concurrently occurred with a reduction of reactive oxygen species (ROS) in the treated CL4176 worms. Mechanistically, heat shock factor 1 (HSF-1) (at residues histidine 63 (HIS63) and glutamine 72 (GLN72)) was shown to be 2-BTHF's potential target that might contribute to an increased expression of autophagy-related genes required for the breakdown of the Aβ aggregate, thus attenuating its toxicity. In conclusion, 2-BTHF from H. scabra could protect C. elegans from Aβ toxicity by suppressing its aggregation via an HSF-1-regulated autophagic pathway and has been implicated as a potential drug for AD.
Figure 1. Chemical structure and HMBC and COSY correlations of Compound 1: 6-hydroxylabda-7(8),12(13)-dien-17-hydroxy-18-O-β-d-glucopyranosyl-pyran-15-one (Holothuria A) (A); Compound 2: 3-hydroxylabda-7(8),12(13)-dien-17-hydroxy-18-O-β-d-glucopyranosyl-pyran-15-one (Holothuria B) (B); Compound 3: palmitic acid (C); Compound 4: bis (2-ethylhexyl) phthalate (D); and Compound 5: 2-butoxytetrahydrofuran (2-BTHF) (E).
Figure 2. Effect of Holothuria scabra isolated compounds on amyloid-β (Aβ)-induced paralysis in Caenorhabditis elegans strain CL4176. (A) Diagram illustrating the paralysis assays showing the time at which the temperature was increased from 16 to 25 °C in CL4176 and its control stain CL802, as well as the feeding duration of worms with different treatment regiments. (B–D,H–J) Time course of paralysis progression caused by Aβ expression in worms treated with either 1% DMSO (Ctrl); different doses of H. scabra Compounds 1, 2, 3, 4, and 5 (1, 10, 25, and 50 µg/mL); or positive control frondoside A at an effective dose 1 µM throughout experimental time. The paralyzed worms were counted at 2 h intervals. Results are represented as percentages ± SD of unparalyzed worms from three independent experiments with at least 100 worms in each experiment. (E–G,K–M) The PT50 values were quantified as a mean of time at which 50% worms treated with or without compounds were paralyzed. Error bars indicate SD. * p < 0.05 and ** p < 0.01 vs. untreated control CL4176 worms.
Figure 3. Effect of the bioactive 2-BTHF (5) compound on Aβ species in transgenic C. elegans strains. (A,B) Representative Western blot of Aβ species in CL4176 and CL2006 worms treated with or without 2-BTHF (5) at 1 µg/mL and detected by anti-Aβ antibody (6E10) or anti-actin. The levels of the Aβ monomer band at around 4 kDa (C) and the Aβ oligomer band at 20 kDa (D) obtained from untreated and treated worm tissues were quantified by Image J. The arrows indicate actin (43 kDa) or counted Aβ species (4 and 20 kDa). Data are shown as mean ± SD of the indicated band density from three independent experiments (exp) with 1000 worms in each group. *** p < 0.001 vs. untreated control.
Figure 4. Effect of 2-BTHF (5) on Aβ-forming deposits in the CL2006 transgenic C. elegans strain. Representative images of C. elegans with 1,4-bis(3-carboxy-hydroxy-phenylethenyl)-benzene (X-34) staining in wild-type N2 (A) and transgenic strain CL2006 treated with either vehicle (B), 2-BTHF (5) at 1 µg/mL (C), or frondoside A at 1 µM (D). White arrows indicate X-34-positive spots of Aβ deposits in the worm’s head, which was separated from the body by a pharyngeal bulb region that is shown in a bright-field image. Scale bars indicate 20 µm. (E) Aβ deposits in each group were quantified and expressed as mean number of positive spots ± SEM of Aβ deposits/area in individual worm’s head. Data were obtained in triplicate for each experiment with 23 worms for each analysis (n = 69). *** p < 0.001 vs. untreated control CL2006.
Figure 5. Effects of 2-BTHF (5) on the expression levels of genes involved with autophagy, transcriptional activities in IIS pathway in C. elegans strains, and its potential target on heat shock factor 1 (HSF-1) molecule. mRNA levels of autophagy-related genes in CL4176 (A) or CL802 (B) treated with vehicle or 2-BTHF (5) at 1 µg/mL. mRNA levels of HSF-1 and dauer formation 16 (DAF-16) target genes in CL4176 (C) or CL802 (D) treated with or without the compound. All mRNA expression levels in worms were quantified by quantitative RT-PCR at 36 h after temperature increase and normalized with the internal control act-1. Data are expressed as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated control CL4176 worms. (E) Molecular docking model of 2-BTHF (5) binding to HSF-1. (F) 2-BTHF (5) molecule is bound to the DNA-binding domain (DBD) of HSF-1 at residues HIS63 and GLN72, shown in a magnified docking image.
Figure 6. Effect of 2-BTHF (5) on reactive oxygen species (ROS) in C. elegans strains. The transgenic CL4176 and CL802 C. elegans strains were cultured with or without the compounds starting from eggs at 16 °C for 36 h, followed by 25 °C for another 36 h. At the end of experiment, untreated and treated worms were subjected to a 2′,7′-dichlorodihydrofluorescein diacetate (DCF) assay for ROS detection. The ROS levels were expressed as percentage of fluorescence (%DCF) relative to untreated CL4176 control (Ctrl), which was set as 100%. Data were obtained in triplicate for each experiment with 60 worms for each analysis (n = 180). Error bars indicate SEM. * p < 0.05 vs. untreated CL4176.
Figure 7. Effect of 2-BTHF (5) on chemotaxis behavior in neuronal Aβ-expressing C. elegans CL2355 strain. (A) Schematic diagram of chemotaxis assay. CL2355 and its control strain CL2122 fed with vehicle or 2-BTHF (5) were placed on the center of the assay plate. After incubation for 1 h, migrated worms in each quadrant (A and D with chemical attractant; B and C for control without attractant) were scored and calculated for chemotactic index (CI). (B) Representative data are shown as CI ± SD and obtained in triplicate for each experiment with 60 worms for each analysis (n = 180) (n.s. p > 0.05, * p < 0.05, and *** p < 0.001).
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