Sanguanphun T et al. (2022), Neuroprotective effects of a medium chain fatty...

ECB-ART-51086

Front Pharmacol 2022 Jan 01;13:1004568. doi: 10.3389/fphar.2022.1004568.

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Neuroprotective effects of a medium chain fatty acid, decanoic acid, isolated from H. leucospilota against Parkinsonism in C. elegans PD model.

Sanguanphun T , Sornkaew N , Malaiwong N , Chalorak P , Jattujan P , Niamnont N , Sobhon P , Meemon K .

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Sea cucumbers are marine organism that have long been used for food and traditional medicine in Asian countries. Recently, we have shown that ethyl acetate fraction (HLEA) of the crude extract of the black sea cucumber, Holothuria leucospilota, could alleviate Parkinsonism in Caenorhabditis elegans PD models. In this study, we found that the effective neuroprotective activity is attributed to HLEA-P1 compound chemically isolated and identified in H. leucospilota ethyl acetate. We reported here that HLEA-P1 could attenuate DAergic neurodegeneration, improve DAergic-dependent behaviors, reduce oxidative stress in 6-OHDA-induced C. elegans. In addition, HLEA-P1 reduced α-synuclein aggregation, improved behavior deficit and recovered lipid deposition in transgenic C. elegans overexpressing α-synuclein. We also found that HLEA-P1 activates nuclear localization of DAF-16 transcription factor of insulin/IGF-1 signaling (IIS) pathway. Treatment with 25 μg/ml of HLEA-P1 upregulated transcriptional activity of DAF-16 target genes including anti-oxidant genes (such as sod-3) and small heat shock proteins (such as hsp16.1, hsp16.2, and hsp12.6) in 6-OHDA-induced worms. In α-synuclein-overexpressed C. elegans strain, treatment with 5 μg/ml of HLEA-P1 significantly activated mRNA expression of sod-3 and hsp16.2. Chemical analysis demonstrated that HLEA-P1 compound is decanoic acid/capric acid. Taken together, our findings revealed that decanoic acid isolated from H. leucospilota exerts anti-Parkinson effect in C. elegans PD models by partly modulating IIS/DAF-16 pathway.

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	FIGURE 1. Effect of HLEA compounds 1–6 against 6-OHDA-induced DAergic neurodegeneration in C. elegans PD model. (A) Representative GFP expression pattern in CEP neurons (white arrows) of normal BY250, 6-OHDA-induced BY250, and 6-OHDA-induced worms with HLEA-P1 treatment at doses of 1, 5, and 25 μg/ml. (B–G) Graphical representation for relative fluorescent intensity of GFP expression in CEP neurons of normal BY250, BY250 exposed to 6-OHDA, BY250 exposed to 6-OHDA/DMSO, and BY250 exposed to 6-OHDA with HLEA compound 1–6 at doses of 1, 5, and 25 μg/ml. The data are presented as mean ± SD (n = 30, number of animals). The hash (#) indicates a significant difference between normal and 6-OHDA-induced worms (p < .05). The asterisk (*) indicates significant differences between untreated group (6-OHDA/DMSO) and HLEAs-treated groups at p < .05. Scale bar is 100 μm.
	FIGURE 2. Effect of HLEA-P1 on dopamine dependent behaviors in 6-OHDA-induced C. elegans PD model. (A) Graphical representation for relative basal slowing rate. The data are presented as mean ± SEM (with three independent replicates, n = 30 number of animals per replicate). (B) Graphical representation for ethanol avoidance index. The data are presented as mean ± SEM (three independent replicates, n ≥ 50 number of animals per replicate). The hash (#) indicates a significant difference between normal and 6-OHDA-induced worms (p < .05). The asterisk () indicates significant differences between untreated group (6-OHDA/DMSO) and HLEA-P1-treated groups, **p < .001.
	FIGURE 3. Effect of HLEA-P1 on reactive oxygen species in 6-OHDA-induced C. elegans PD model. Graphical representation for DCF fluorescence (%) in normal, 6-OHDA-, 6-OHDA/DMSO-, and 6-OHDA/DMSO plus HLEA-P1-treated worms compared to the untreated group. The data are presented as mean ± SEM (three independent replicates, n = 30 number of animals per replicate). The hash (#) indicates a significant difference between untreated and 6-OHDA-, 6-OHDA/DMSO-treated worms. The asterisk (*) indicates significant differences between 6-OHDA-, 6-OHDA/DMSO- and HLEA-P1-treated groups at p < .05.
	FIGURE 4. Effect of HLEA-P1 against α-synuclein aggregation and movement deficit in NL5901 C. elegans expressing α-synuclein. (A) Representative of YFP-tagged α-synuclein expression in body wall muscle cells of NL5901 strain. (B) Graphical representation for relative fluorescent intensity of YFP expression. (C) Graphical representation of the rate of movement of C. elegans with HLEA-P1 treatment. The data are presented as mean ± SD (n = 30, number of animals). The hash (#) indicates a significant difference between N2 wild type and NL5901 worms (p < .05). The asterisk () indicates significant differences between untreated group (DMSO) and HLEA-P1-treated group, p < .05, p < .01, *p < .001. Scale bar is 200 μm.
	FIGURE 5. Effect of HLEA-P1 on lipid restoration in transgenic NL5901 worms expressing α-synuclein. (A) Representative Nile red fluorescent images of wild-type N2, C. elegans NL5901 and C. elegans NL5901 treated with HLEA-P1 at 5 and 25 μg/ml. (B) Graphical representation for Nile red fluorescent signal. The data are presented as mean ± SEM (with three independent replicates, n ≥ 30 number of animals per replicate). The hash (#) indicates a significant difference between wild-type N2 and NL5901 worms (p < .05). The asterisk () indicates significant difference between untreated group (NL5901/DMSO) and HLEA-P1-treated groups, **p < .001. Scale bar is 200 μm.
	FIGURE 6. Effect of HLEA-P1 on nuclear localization of transcription factor DAF-16 in 6-OHDA-induced C. elegans. (A) Representative fluorescent images of different subcellular localization of DAF-16. (B) Graphical representation of subcellular distribution of daf-16. The data are presented as mean ± SEM (with three independent replicates, n ≥ 30 number of animals per replicate). The asterisk (*) indicated significant differences of DAF-16 nuclear localization between untreated group (6-OHDA/DMSO) and HLEA-P1-treated groups at p < .05.
	FIGURE 7. Effect of HLEA-P1 on SOD-3 and GST-4 in C. elegans PD model. (A,B) Representative SOD-3:GFP expression and mean fluorescent intensity of SOD-3:GFP in transgenic CF1553 in different conditions (normal CF1553, 6-OHDA-induced CF1553, 6-OHDA/DMSO-treated CF1553, and 6-OHDA-induced CF1553 treated with HLEA-P1 at 5 and 25 μg/ml). Scale bar is 100 μm. (C,D) Representative GST-4:GFP expression and mean fluorescent intensity of GST-4:GFP in transgenic CL2166 in different conditions (normal CL2166, 6-OHDA-induced CL2166, 6-OHDA/DMSO-treated CL2166 and 6-OHDA-induced CL2166 treated with HLEA-P1 at 5 and 25 μg/ml). Scale bar is 200 μm. The hash (#) indicates a significant difference between wild type N2 and transgenic worms (p < .05). The asterisk () indicates significant difference between untreated group (6-OHDA/DMSO) and HLEA-P1 treated groups, **p < .001.
	FIGURE 8. Effect of HLEA-P1 on expressions of mRNAs of DAF-16 target genes in C. elegans PD models. Graphical representation for fold change of gene expression level (2−(ΔΔCq)) of DAF-16 target genes in 6-OHDA-induced worms (A) and NL5901 worms overexpressing α-synuclein (B). The relative mRNA expressions were normalized with internal control act-1. The data represented as a mean ± SEM (three independent replicates, n = 800–1,000 number of animals per replicate). The asterisk () indicates significant differences between untreated group (DMSO) and HLEA-P1-treated group, p < .05, p < .01, *p < .001.
	FIGURE 9. (A) Chemical structure of HLEA-P1. (B) 1H-NMR spectrum of HLEA-P1, decanoic acid or capric acid in CDCl3.

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	FIGURE 1. Effect of HLEA compounds 1–6 against 6-OHDA-induced DAergic neurodegeneration in C. elegans PD model. (A) Representative GFP expression pattern in CEP neurons (white arrows) of normal BY250, 6-OHDA-induced BY250, and 6-OHDA-induced worms with HLEA-P1 treatment at doses of 1, 5, and 25 μg/ml. (B–G) Graphical representation for relative fluorescent intensity of GFP expression in CEP neurons of normal BY250, BY250 exposed to 6-OHDA, BY250 exposed to 6-OHDA/DMSO, and BY250 exposed to 6-OHDA with HLEA compound 1–6 at doses of 1, 5, and 25 μg/ml. The data are presented as mean ± SD (n = 30, number of animals). The hash (#) indicates a significant difference between normal and 6-OHDA-induced worms (p < .05). The asterisk (*) indicates significant differences between untreated group (6-OHDA/DMSO) and HLEAs-treated groups at p < .05. Scale bar is 100 μm.
	FIGURE 2. Effect of HLEA-P1 on dopamine dependent behaviors in 6-OHDA-induced C. elegans PD model. (A) Graphical representation for relative basal slowing rate. The data are presented as mean ± SEM (with three independent replicates, n = 30 number of animals per replicate). (B) Graphical representation for ethanol avoidance index. The data are presented as mean ± SEM (three independent replicates, n ≥ 50 number of animals per replicate). The hash (#) indicates a significant difference between normal and 6-OHDA-induced worms (p < .05). The asterisk () indicates significant differences between untreated group (6-OHDA/DMSO) and HLEA-P1-treated groups, **p < .001.
	FIGURE 3. Effect of HLEA-P1 on reactive oxygen species in 6-OHDA-induced C. elegans PD model. Graphical representation for DCF fluorescence (%) in normal, 6-OHDA-, 6-OHDA/DMSO-, and 6-OHDA/DMSO plus HLEA-P1-treated worms compared to the untreated group. The data are presented as mean ± SEM (three independent replicates, n = 30 number of animals per replicate). The hash (#) indicates a significant difference between untreated and 6-OHDA-, 6-OHDA/DMSO-treated worms. The asterisk (*) indicates significant differences between 6-OHDA-, 6-OHDA/DMSO- and HLEA-P1-treated groups at p < .05.
	FIGURE 4. Effect of HLEA-P1 against α-synuclein aggregation and movement deficit in NL5901 C. elegans expressing α-synuclein. (A) Representative of YFP-tagged α-synuclein expression in body wall muscle cells of NL5901 strain. (B) Graphical representation for relative fluorescent intensity of YFP expression. (C) Graphical representation of the rate of movement of C. elegans with HLEA-P1 treatment. The data are presented as mean ± SD (n = 30, number of animals). The hash (#) indicates a significant difference between N2 wild type and NL5901 worms (p < .05). The asterisk () indicates significant differences between untreated group (DMSO) and HLEA-P1-treated group, p < .05, p < .01, *p < .001. Scale bar is 200 μm.
	FIGURE 5. Effect of HLEA-P1 on lipid restoration in transgenic NL5901 worms expressing α-synuclein. (A) Representative Nile red fluorescent images of wild-type N2, C. elegans NL5901 and C. elegans NL5901 treated with HLEA-P1 at 5 and 25 μg/ml. (B) Graphical representation for Nile red fluorescent signal. The data are presented as mean ± SEM (with three independent replicates, n ≥ 30 number of animals per replicate). The hash (#) indicates a significant difference between wild-type N2 and NL5901 worms (p < .05). The asterisk () indicates significant difference between untreated group (NL5901/DMSO) and HLEA-P1-treated groups, **p < .001. Scale bar is 200 μm.
	FIGURE 6. Effect of HLEA-P1 on nuclear localization of transcription factor DAF-16 in 6-OHDA-induced C. elegans. (A) Representative fluorescent images of different subcellular localization of DAF-16. (B) Graphical representation of subcellular distribution of daf-16. The data are presented as mean ± SEM (with three independent replicates, n ≥ 30 number of animals per replicate). The asterisk (*) indicated significant differences of DAF-16 nuclear localization between untreated group (6-OHDA/DMSO) and HLEA-P1-treated groups at p < .05.
	FIGURE 7. Effect of HLEA-P1 on SOD-3 and GST-4 in C. elegans PD model. (A,B) Representative SOD-3:GFP expression and mean fluorescent intensity of SOD-3:GFP in transgenic CF1553 in different conditions (normal CF1553, 6-OHDA-induced CF1553, 6-OHDA/DMSO-treated CF1553, and 6-OHDA-induced CF1553 treated with HLEA-P1 at 5 and 25 μg/ml). Scale bar is 100 μm. (C,D) Representative GST-4:GFP expression and mean fluorescent intensity of GST-4:GFP in transgenic CL2166 in different conditions (normal CL2166, 6-OHDA-induced CL2166, 6-OHDA/DMSO-treated CL2166 and 6-OHDA-induced CL2166 treated with HLEA-P1 at 5 and 25 μg/ml). Scale bar is 200 μm. The hash (#) indicates a significant difference between wild type N2 and transgenic worms (p < .05). The asterisk () indicates significant difference between untreated group (6-OHDA/DMSO) and HLEA-P1 treated groups, **p < .001.
	FIGURE 8. Effect of HLEA-P1 on expressions of mRNAs of DAF-16 target genes in C. elegans PD models. Graphical representation for fold change of gene expression level (2−(ΔΔCq)) of DAF-16 target genes in 6-OHDA-induced worms (A) and NL5901 worms overexpressing α-synuclein (B). The relative mRNA expressions were normalized with internal control act-1. The data represented as a mean ± SEM (three independent replicates, n = 800–1,000 number of animals per replicate). The asterisk () indicates significant differences between untreated group (DMSO) and HLEA-P1-treated group, p < .05, p < .01, *p < .001.
	FIGURE 9. (A) Chemical structure of HLEA-P1. (B) 1H-NMR spectrum of HLEA-P1, decanoic acid or capric acid in CDCl3.