Lu M et al. (2021), Sea Cucumber-Derived Peptides Alleviate Oxidati...

ECB-ART-49195

Oxid Med Cell Longev 2021 Jan 01;2021:8842926. doi: 10.1155/2021/8842926.

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Sea Cucumber-Derived Peptides Alleviate Oxidative Stress in Neuroblastoma Cells and Improve Survival in C. elegans Exposed to Neurotoxic Paraquat.

Lu M , Mishra A , Boschetti C , Lin J , Liu Y , Huang H , Kaminski CF , Huang Z , Tunnacliffe A , Kaminski Schierle GS .

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Oxidative stress results when the production of oxidants outweighs the capacity of the antioxidant defence mechanisms. This can lead to pathological conditions including cancer and neurodegeneration. Consequently, there is considerable interest in compounds with antioxidant activity, including those from natural sources. Here, we characterise the antioxidant activity of three novel peptides identified in protein hydrolysates from the sea cucumber Apostichopus japonicus. Under oxidative stress conditions, synthetic versions of the sea cucumber peptides significantly compensate for glutathione depletion, decrease mitochondrial superoxide levels, and alleviate mitophagy in human neuroblastoma cells. Moreover, orally supplied peptides improve survival of the Caenorhabditis elegans after treatment with paraquat, the latter of which leads to the production of excessive oxidative stress. Thus, the sea cucumber peptides exhibit antioxidant activity at both the cellular and organism levels and might prove attractive as nutritional supplements for healthy ageing.

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	Figure 1. Sea cucumber-derived peptides are nontoxic and protect cells against hydrogen peroxide. (a) A protein hydrolysate from A. japonicus was subjected to centrifugal ultrafiltration to separate peptides less than 3000 Da. The purified pool was further subjected to size exclusion chromatography and LC-MS/MS analysis to identify peptide sequences. The identified peptide sequences were chemically synthesised and subjected to functional screening to identify antioxidant peptides. (b) Sequences and biophysical properties of peptides. (c) SH-SY5Y cells were plated at a density of 1 × 104 cells per well in 96-well plates and grown to approximately 70%-80% confluency. Cells were then treated with different concentrations of peptides for 24 h at 37°C before being washed once with PBS. Next, the cells were incubated with 600 μM H2O2 for 4 h without peptides at 37°C. The metabolic activity of cells was measured by the MTT assay using a plate reader to record absorbance at 570 nm. Metabolic activity is plotted as percentages relative to the untreated control group, which is denoted as 100%. Statistical significance was determined by Tukey one-way ANOVA. Error bars show SD (n = 3). Experiments were repeated three times. (d) SH-SY5Y cells were grown in 96-well cell culture dishes until they reached 60-70% confluency. Cells were then treated with different concentrations of peptides as shown in the figure. After treatment, cells were incubated for 72 h at 37°C. Using a colorimetric MTS assay, absorbance at 506 nm was recorded using a plate reader to quantify cell proliferation.
	Figure 2. Rescue of depleted GSH levels in stressed cells by sea cucumber-derived peptides and GSH. SH-SY5Y cells were grown in 96-well cell culture dishes until they reached ~80-90% confluency. Cells were then treated with peptides or GSH at 500 μM or 50 μM for 24 h at 37°C, then washed once with PBS before incubation with 40 μM monochlorobimane dye for 1 h in the absence of peptides and GSH at 37°C. Monochlorobimane fluorescence intensity in SH-SY5Y cells was measured using the Envision Multilabel plate reader at Ex/Em 405/486 nm wavelength 1 h after ROS induction with ABAP. “No peptide” represents cells treated with PBS and ABAP whereas “control” means no oxidative stress was induced. For each treatment, four replicates were performed (n = 4). Two-way ANOVA and Dunnett's multiple comparison test were applied to measure the statistical significance of each treatment relative to the no-peptide control as well as to compare differences between pairs of peptide concentrations. Statistical significance was determined by Tukey one-way ANOVA. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, and ∗∗P < 0.01. Error bars show SD (n = 4). Experiments were repeated three times.
	Figure 3. Sea cucumber-derived peptides reduce superoxide levels in mitochondria. (a) Detection of mitochondrial ROS by MitoSOX using confocal microscopy: SH-SY5Y cells were grown in glass-bottomed MatTek dishes until they reached partial confluency (50-70%). Cells were then treated with 50 μM peptides and were incubated for 24 h at 37°C. As a positive control, cells were also treated with 50 μM glutathione (GSH). After incubation, cellular oxidative stress was induced by treating cells with 2 mM H2O2 for 3 h at 37°C. Cells were then washed three times with PBS before labelling them with 2 μM MitoSOX Red dye for 30 min at 37°C. MitoSOX Red fluorescence intensity was then detected by confocal microscopy. (b) Detection of mitochondrial ROS by flow cytometry: cells were grown and treated as described in (a) except that the cells were cultured in 24-well plates until they reach ~80% confluency. After incubation with MitoSOX Red, cells were washed with PBS and harvested for fluorescence measurements by flow cytometry. In the bar diagram, “control” refers to the negative control where ROS are not induced, and “no peptide” represents the positive control where cells were treated with H2O2 in culture medium without any peptide treatment. Samples were compared with “no peptide,” and statistical significance was determined by Tukey one-way ANOVA. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05. Error bars show SD (n = 4). Experiments were repeated 3 times.
	Figure 4. Sea cucumber-derived peptides reduce mitophagy induced by oxidative stress. (a) SH-SY5Y cells were grown in glass-bottom MatTek dishes until they reached partial confluency (50-70%). Next, cells were treated with 50 μM peptides or 50 μM GSH. Peptides were added together with SiR-Lysosome, a lysosomal marker. 24 hours after incubation, cellular oxidative stress was induced by treating cells with 1 mM H2O2 for 2 h at 37°C. Cells were then washed three times with PBS before the addition of rhodamine B (a mitochondrial stain) for 30 min at 37°C. After a further wash, SiR-Lysosome and rhodamine B fluorescence were detected by confocal microscopy. White arrows point to the mitochondria colocalised with lysosomes. (b) Quantification of the colocalisation of mitochondria and lysosomes was done by measuring Manders' coefficient. Samples were compared with “no peptide,” and statistical significance was determined by Tukey one-way ANOVA. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05; NS: not significant. Error bars show SD (n = 4). Experiments were repeated three times.
	Figure 5. Cellular uptake of rhodamine B-labelled peptides. (a) Confocal imaging of SH-SY5Y cells incubated with either 2 μM or 50 μM rhodamine B-labelled peptide TP-WW-623 for 5 to 30 min at 37°C to show the cellular uptake mechanism. The ROI (region of interest) panel represents zoomed images from the marked squares in the panels labelled “rhodamine B.” (b) SH-SY5Y cells were incubated at 4°C for 15 min to block endocytosis before incubating them with either rhodamine B-labelled TP-WW-623 or free rhodamine B for a further 10 min at room temperature. These cells were then washed three times with PBS before imaging. Experiments were repeated twice.
	Figure 6. Rhodamine B-labelled peptide TP-WW-623 colocalises with lysosomes. (a) Lysosomes in SH-SY5Y cells were labelled with LAMP1-GFP before incubation with 50 μM rhodamine B-labelled peptide TP-WW-623 at 37°C. Cell images were recorded on three consecutive days to observe the colocalisation of peptides and lysosomes (see also Figure S2). (b) Images of cells recorded by SIM at day 3 to illustrate the colocalisation of peptides and lysosomes. White dashed lines indicate the cell boundary. Enlarged views of the white boxed regions are shown in the bottom panel (see also Figure S2 and Video 1).
	Figure 7. Sea cucumber-derived peptides improve the survival of C. elegans exposed to paraquat. Synchronized wild-type L1 nematodes were fed with E. coli NA22 and incubated for 42 h. The L4 nematodes were then washed and transferred to 96-well plates (15-20 nematodes per well; >100 nematodes for each treatment) containing E. coli NA22 (OD570 nm = 0.5) and peptide samples. After 24 h, the nematodes were exposed to 100 mM paraquat and the numbers of live nematodes were scored under a light microscope every 12 h. Representative Kaplan-Meier survival curves are shown for the nematodes treated with or without indicated concentrations of peptide samples TP-WW-620 (a), TP-WW-621 (b), and TP-WW-623 (c). Tables on the right panel show the quantification of the survival time of paraquat-intoxicated C. elegans at different conditions.

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