Protective Effects of an Octapeptide Identified from Riceberry™ (Oryza sativa) Protein Hydrolysate on Oxidative and Endoplasmic Reticulum (ER) Stress in L929 Cells

Abstract

Reactive oxygen species (ROS) play a critical role in oxidative stress and cellular damage, underscoring the importance of identifying potent antioxidants. This research focuses on the antioxidant capabilities of Riceberry™-derived peptides and their protective effects against oxidative and endoplasmic reticulum (ER) stress in L929 cells. By simulating human digestion, Riceberry™ protein hydrolysate was generated, from which antioxidant peptides were isolated using OFFGEL electrophoresis and LC-MS/MS. Notably, an octapeptide (VPAGVAHW) from the hydrolysate demonstrated significant antioxidant activity, particularly against oxidative stress induced by iodoacetic acid (IAA) or hydrogen peroxide (H₂O₂) and ER stress caused by tunicamycin (TM) in L929 cells. This peptide's effectiveness was evident in its dose-dependent ability to enhance cell viability and mitigate stress effects, although its efficiency varied with the stress inducer. Our study suggests that Riceberry™-derived peptides could serve as a promising natural antioxidant with potential benefits for health promotion and applications in the food industry, offering an environmentally friendly alternative to synthetic antioxidants.

Keywords: LC-MS/MS; Riceberry™; VPAGVAHW; antioxidant peptides; oxidative stress; rice.

Figure 1

Illustration of peptide fractionation via OFFGEL fractionation. (A) The OFFGEL experiments involved the separation of the 40% acetonitrile fraction following 3 h of electro-focusing using an electric field across a pH gradient ranging from 3 to 10. (B) The resulting 18 fractions are shown in individual microcentrifuge tubes after separation. Each tube corresponds to a specific pH range, displaying the distribution of peptides visually. The gradient of colors from red to yellow indicates the separation efficiency and the variation in peptide content across different pH values.

Figure 2

Effect of synthetic peptides on cell viability in L929 cells. The synthetic peptides at 50 µg/mL were tested by incubating cells with the peptides for 24 h, and the cytotoxicity was determined by the MTT assay. The lowercase letters above the bar graph indicates the significance of differences in cell viability between treatment groups. The symbol # followed by a number indicates the name of each peptide.

Figure 3

The impact of octapeptide #3 on L929 cells under oxidative stress induced by iodoacetic acid (IAA) and H₂O₂. (A) Illustrates the protective effect of octapeptide #3 on IAA-induced L929 cells, and (B) shows the protective effect of octapeptide #3 on H₂O₂-induced L929 cells. The percentage of cell viability is represented as the mean ± S.D. The lowercase letters above the bar graph indicates the significance of differences in cell viability between treatment groups.

Figure 4

The impact of octapeptide #3 on tunicamycin ™-induced ER stress in L929 cells. (A) Cell morphology visualized by confocal fluorescence microscopy. The staining includes ConA-FITC, emitting green fluorescence to highlight ER stress, and DAPI, emitting blue fluorescence to delineate the nuclear regions. ‘BF’ denotes the bright field images. (B) A comparative analysis of CTCF values among the NegCtrl, TM treated alone, and TM treated followed by octapeptide #3 treatment groups. The CTCF values are presented as the mean ± S.D. The lowercase letters above the bar graph indicates the significance of differences in cell viability between treatment groups.