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. 2024 Oct 27;16(21):3654.
doi: 10.3390/nu16213654.

Cytoprotective Effects of Antioxidant Peptides from Red Californian Worm (Eisenia foetida) Hydrolysate on Differentiated Caco-2 Cells

Yhoan S Gaviria  1 , José E Zapata  1 , Diego Miedes  2 , Amparo Alegría  2 , Antonio Cilla  2
Affiliations

Cytoprotective Effects of Antioxidant Peptides from Red Californian Worm (Eisenia foetida) Hydrolysate on Differentiated Caco-2 Cells

Yhoan S Gaviria et al. Nutrients. .

Abstract

Background/objectives: When prooxidants outweigh antioxidants, oxidative stress can occur, causing an accumulation of reactive oxygen species (ROS). This process can lead to cellular damage and plays a role in the development of numerous health conditions. This study aimed to investigate the cytoprotective effects on differentiated Caco-2 cells of hydrolysates derived from the red Californian worm (WH) and their fractions, identify the peptides responsible for this effect, and elucidate the mechanisms involved.

Methods: The WH was obtained through hydrolysis with Alcalase 2.4 L and subsequently fractionated to two fractions (F > 3 kDa and F < 3 kDa) using a ceramic membrane with a molecular weight cutoff of 3 kDa. The peptides found in the F < 3 kDa fraction, demonstrating the highest cytoprotective activity, were then sequenced via liquid chromatography-mass spectrometry analysis (LC-MS/MS), and molecular docking was conducted to elucidate the underlying antioxidant mechanisms.

Results: The hydrolysate of Eisenia foetida and its F < 3 kDa fraction exhibited no cytotoxicity, protected the cells from H2O2-induced oxidative stress (50% increase viability), preserved cell viability by restoring their redox status (ROS: 20% decrease, and glutathione (GSH): recovered to basal control levels) and cell cycle distribution, and decreased apoptosis (16%). Twenty-eight peptides were identified, with five showing antioxidant activity through stable interactions with myeloperoxidase (MPO) and Kelch-like ECH-associated protein 1 (Keap-1), KPEDWDDR being the peptide that presented the highest affinity with both molecules (-7.9 and -8.8 kCal/mol, respectively).

Conclusions: These results highlight the WH as a potential source of bioactive peptides for the management of oxidative stress.

Keywords: antioxidant peptide; cytoprotective effect; molecular docking; oxidative stress; red Californian worm (Eisenia foetida).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Cytoprotective activity of WH and its fractions (F) on differentiated Caco-2 cells. Results are presented as the mean ± standard deviation, derived from four independent experiments (n = 5). Significant differences (p < 0.05) are denoted by different letters (a–f).
Figure 2
Figure 2
Evaluation of the cytoprotective effect of worm hydrolysate (WH) and its fractions (F) on differentiated Caco-2 cells: (a) intracellular ROS; (b) intracellular glutathione (GSH); (c) intracellular calcium. Results are expressed as the mean ± standard deviation of four independent experiments (n = 5). Significant differences (p < 0.05) are denoted by different letters (a–e). AU: Arbitrary units.
Figure 3
Figure 3
Cell cycle distribution of differentiated Caco-2 cells treated with worm hydrolysate (WH) and its fractions (F). Results are presented as the mean ± standard deviation, derived from four independent experiments (n = 5). Different letters (a–d) for the treatments in the same cell cycle phase indicate significant differences (p < 0.05).
Figure 4
Figure 4
Effect of incubation with WH and its fractions (F) in differentiated Caco-2 cells. Results are presented as the mean ± standard deviation from four independent experiments (n = 5). Significant differences (p < 0.05) between treatments within the same cell state are denoted by different letters (a–e).
Figure 5
Figure 5
Molecular interaction of the KPEDWDDR peptide-Keap1: (a) molecule in quaternary representation; (b) surface hydrophobicity diagram; (c) charge interactions (pink color: Keap1 molecule; green color: KPEDWDDR peptide); (d) 2D interaction diagram.
Figure 6
Figure 6
Molecular interaction of the KPEDWDDR peptide-MPO: (a) molecule in quaternary representation; (b) surface hydrophobicity diagram; (c) charge interactions (white color: MPO molecule; red color: KPEDWDDR peptide; green color: hydrogen bonds); (d) 2D interaction diagram.
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