Sanguanphun T et al. (2023), Anti-Parkinson Effects of Holothuria leucospilo...

ECB-ART-51522

Mar Drugs 2023 Feb 23;213:. doi: 10.3390/md21030141.

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Anti-Parkinson Effects of Holothuria leucospilota-Derived Palmitic Acid in Caenorhabditis elegans Model of Parkinson's Disease.

Sanguanphun T , Promtang S , Sornkaew N , Niamnont N , Sobhon P , Meemon K .

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Parkinson's disease (PD) is the second most common neurodegenerative disease which is still incurable. Sea cucumber-derived compounds have been reported to be promising candidate drugs for treating age-related neurological disorders. The present study evaluated the beneficial effects of the Holothuria leucospilota (H. leucospilota)-derived compound 3 isolated from ethyl acetate fraction (HLEA-P3) using Caenorhabditis elegans PD models. HLEA-P3 (1 to 50 µg/mL) restored the viability of dopaminergic neurons. Surprisingly, 5 and 25 µg/mL HLEA-P3 improved dopamine-dependent behaviors, reduced oxidative stress and prolonged lifespan of PD worms induced by neurotoxin 6-hydroxydopamine (6-OHDA). Additionally, HLEA-P3 (5 to 50 µg/mL) decreased α-synuclein aggregation. Particularly, 5 and 25 µg/mL HLEA-P3 improved locomotion, reduced lipid accumulation and extended lifespan of transgenic C. elegans strain NL5901. Gene expression analysis revealed that treatment with 5 and 25 µg/mL HLEA-P3 could upregulate the genes encoding antioxidant enzymes (gst-4, gst-10 and gcs-1) and autophagic mediators (bec-1 and atg-7) and downregulate the fatty acid desaturase gene (fat-5). These findings explained the molecular mechanism of HLEA-P3-mediated protection against PD-like pathologies. The chemical characterization elucidated that HLEA-P3 is palmitic acid. Taken together, these findings revealed the anti-Parkinson effects of H. leucospilota-derived palmitic acid in 6-OHDA induced- and α-synuclein-based models of PD which might be useful in nutritional therapy for treating PD.

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	Figure 1. HLEA-P3 attenuated DAergic neurodegeneration induced by 6-OHDA in C. elegans model. (A) Representative fluorescence images of GFP-tagged CEP neurons (arrows) of normal BY250, 6-OHDA/DMSO-treated BY250 and BY250 exposed to 6-OHDA and treated with HLEA-P3 at doses of 1, 5, 25 and 50 μg/mL. (B) Graphical representations for the relative fluorescence intensity of GFP-tagged CEP neurons. The data are presented as a mean ± SEM (n = 30, number of animals). The hash (#) indicates a significant difference between normal and 6-OHDA-treated groups (p < 0.05). The asterisk (*) indicates significant differences between the untreated group (6-OHDA/DMSO) and HLEA-P3-treated groups at p < 0.05. Scale bar is 100 μm.
	Figure 2. HLEA-P3 recovered dopamine-dependent behaviors in 6-OHDA-treated C. elegans. Graphical representations of relative basal slowing rate (A) ethanol avoidance index (B) of normal, 6-OHDA-induced worms and 6-OHDA-induced worms and those treated with HLEA-P3. The data are presented as mean ± SEM. The hash (#) indicates a significant difference between normal and 6-OHDA-treated groups (p < 0.05). The asterisk () indicates significant differences between the untreated group (6-OHDA/DMSO) and HLEA-P3-treated groups, * p < 0.01, **** p < 0.0001.
	Figure 3. HLEA-P3 reduced oxidative stress in 6-OHDA-treated C. elegans. Graphical representations for relative changes in intracellular ROS levels using H2DCF-DA. The data are presented as mean ± SEM (three independent replicates, n = 30 number of animals per replicate). The hash (#) indicates a significant difference between normal and 6-OHDA-treated groups. The asterisk () indicates significant differences between the untreated group (6-OHDA/DMSO) and HLEA-P3-treated groups, p < 0.05.
	Figure 4. HLEA-P3 reduced α-synuclein aggregation and improved motor deficit in transgenic C. elegans NL5901 strain. (A) Representative fluorescence image of YFP-tagged α-synuclein expression in body wall muscle of NL5901 strain, NL5901 treated with DMSO, and NL5901 treated with various doses of HLEA-P3. (B) Graphical representations for relative fluorescence intensity of YFP expression. The data are presented as mean ± SEM (n = 30, number of animals). (C) Graphical representations for thrashing rates of wild-type worms, NL5901 and NL5901 treated with HLEA-P3. The data are presented as mean ± SEM (three independent replicates, n = 30 number of animals per replicate). The hash (#) indicates a significant difference between wild-type N2 and NL5901 worms (p < 0.05). The asterisk () indicated significant differences between the untreated group (DMSO) and HLEA-P3-treated group at p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001.
	Figure 5. HLEA-P3 modulated lipid deposition in transgenic C. elegans NL5901 strain. (A) Representative Nile red fluorescence images of wild-type N2, C. elegans NL5901 and C. elegans NL5901 treated with HLEA-P3 at 5 and 25 μg/mL. (B) Graphical representation for Nile red fluorescence signal. The data are presented as mean ± SEM (with three independent replicates, n ≥ 30 number of animals per replicate). (C) Graphical representation for fold changes of fatty acid desaturase genes: fat-5, fat-6 and fat-7. The data are presented as mean ± SEM (with three independent replicates, n = 800–1000 number of animals per replicate). The hash (#) indicates a significant difference between wild-type N2 and NL5901 worms (p < 0.05). The asterisk () indicates significant difference between the untreated group (DMSO) and HLEA-P3-treated groups, p < 0.05, **** p < 0.0001. Scale bar is 200 μm.
	Figure 6. HLEA-P3 improved lifespan of C. elegans PD models. (A) Kaplan–Meier survival curves of normal worms, 6-OHDA-induced worms and 6-OHDA-induced worms treated with 5 and 25 μg/mL HLEA-P3. (B) Kaplan–Meier survival curves of normal worms, NL5901, and NL5901 treated with 5 and 25 μg/mL HLEA-P3.
	Figure 7. (A) Graphical representations of fold changes of mRNA expression levels of antioxidant genes in 6-OHDA-treated worms treated with HLEA-P3. (B) Graphical representations of fold changes of mRNA expression levels of autophagic genes in NL5901 worms treated with HLEA-P3. The data are presented as mean ± SEM (three independent replicates, n = 800–1000 number of animals per replicate). The asterisk () indicates significant differences between the untreated group (DMSO) and HLEA-P3-treated group, p < 0.05, *** p < 0.001.

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	Figure 1. HLEA-P3 attenuated DAergic neurodegeneration induced by 6-OHDA in C. elegans model. (A) Representative fluorescence images of GFP-tagged CEP neurons (arrows) of normal BY250, 6-OHDA/DMSO-treated BY250 and BY250 exposed to 6-OHDA and treated with HLEA-P3 at doses of 1, 5, 25 and 50 μg/mL. (B) Graphical representations for the relative fluorescence intensity of GFP-tagged CEP neurons. The data are presented as a mean ± SEM (n = 30, number of animals). The hash (#) indicates a significant difference between normal and 6-OHDA-treated groups (p < 0.05). The asterisk (*) indicates significant differences between the untreated group (6-OHDA/DMSO) and HLEA-P3-treated groups at p < 0.05. Scale bar is 100 μm.
	Figure 2. HLEA-P3 recovered dopamine-dependent behaviors in 6-OHDA-treated C. elegans. Graphical representations of relative basal slowing rate (A) ethanol avoidance index (B) of normal, 6-OHDA-induced worms and 6-OHDA-induced worms and those treated with HLEA-P3. The data are presented as mean ± SEM. The hash (#) indicates a significant difference between normal and 6-OHDA-treated groups (p < 0.05). The asterisk () indicates significant differences between the untreated group (6-OHDA/DMSO) and HLEA-P3-treated groups, * p < 0.01, **** p < 0.0001.
	Figure 3. HLEA-P3 reduced oxidative stress in 6-OHDA-treated C. elegans. Graphical representations for relative changes in intracellular ROS levels using H2DCF-DA. The data are presented as mean ± SEM (three independent replicates, n = 30 number of animals per replicate). The hash (#) indicates a significant difference between normal and 6-OHDA-treated groups. The asterisk () indicates significant differences between the untreated group (6-OHDA/DMSO) and HLEA-P3-treated groups, p < 0.05.
	Figure 4. HLEA-P3 reduced α-synuclein aggregation and improved motor deficit in transgenic C. elegans NL5901 strain. (A) Representative fluorescence image of YFP-tagged α-synuclein expression in body wall muscle of NL5901 strain, NL5901 treated with DMSO, and NL5901 treated with various doses of HLEA-P3. (B) Graphical representations for relative fluorescence intensity of YFP expression. The data are presented as mean ± SEM (n = 30, number of animals). (C) Graphical representations for thrashing rates of wild-type worms, NL5901 and NL5901 treated with HLEA-P3. The data are presented as mean ± SEM (three independent replicates, n = 30 number of animals per replicate). The hash (#) indicates a significant difference between wild-type N2 and NL5901 worms (p < 0.05). The asterisk () indicated significant differences between the untreated group (DMSO) and HLEA-P3-treated group at p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001.
	Figure 5. HLEA-P3 modulated lipid deposition in transgenic C. elegans NL5901 strain. (A) Representative Nile red fluorescence images of wild-type N2, C. elegans NL5901 and C. elegans NL5901 treated with HLEA-P3 at 5 and 25 μg/mL. (B) Graphical representation for Nile red fluorescence signal. The data are presented as mean ± SEM (with three independent replicates, n ≥ 30 number of animals per replicate). (C) Graphical representation for fold changes of fatty acid desaturase genes: fat-5, fat-6 and fat-7. The data are presented as mean ± SEM (with three independent replicates, n = 800–1000 number of animals per replicate). The hash (#) indicates a significant difference between wild-type N2 and NL5901 worms (p < 0.05). The asterisk () indicates significant difference between the untreated group (DMSO) and HLEA-P3-treated groups, p < 0.05, **** p < 0.0001. Scale bar is 200 μm.
	Figure 6. HLEA-P3 improved lifespan of C. elegans PD models. (A) Kaplan–Meier survival curves of normal worms, 6-OHDA-induced worms and 6-OHDA-induced worms treated with 5 and 25 μg/mL HLEA-P3. (B) Kaplan–Meier survival curves of normal worms, NL5901, and NL5901 treated with 5 and 25 μg/mL HLEA-P3.
	Figure 7. (A) Graphical representations of fold changes of mRNA expression levels of antioxidant genes in 6-OHDA-treated worms treated with HLEA-P3. (B) Graphical representations of fold changes of mRNA expression levels of autophagic genes in NL5901 worms treated with HLEA-P3. The data are presented as mean ± SEM (three independent replicates, n = 800–1000 number of animals per replicate). The asterisk () indicates significant differences between the untreated group (DMSO) and HLEA-P3-treated group, p < 0.05, *** p < 0.001.