Antioxidant Activity of Novel Casein-Derived Peptides with Microbial Proteases as Characterized via Keap1-Nrf2 Pathway in HepG2 Cells

Abstract

Casein-derived antioxidant peptides by using microbial proteases have gained increasing attention. Combination of two microbial proteases, Protin SD-NY10 and Protease A "Amano" 2SD, was employed to hydrolyze casein to obtain potential antioxidant peptides that were identified by LCMS/ MS, chemically synthesized and characterized in a oxidatively damaged HepG2 cell model. Four peptides, YQLD, FSDIPNPIGSEN, FSDIPNPIGSE, YFYP were found to possess high 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability. Evaluation with HepG2 cells showed that the 4 peptides at low concentrations (< 1.0 mg/ml) protected the cells against oxidative damage. The 4 peptides exhibited different levels of antioxidant activity by stimulating mRNA and protein expression of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as nuclear factor erythroid-2-related factor 2 (Nrf2), but decreasing the mRNA expression of Kelch-like ECH-associated protein 1 (Keap1). Furthermore, these peptides decreased production of reactive oxygen species (ROS) and malondialdehyde (MDA), but increased glutathione (GSH) production in HepG2 cells. Therefore, the 4 casein-derived peptides obtained by using microbial proteases exhibited different antioxidant activity by activating the Keap1-Nrf2 signaling pathway, and they could serve as potential antioxidant agents in functional foods or pharmaceutic preparation.

Keywords: HepG2 cells; Keap1-Nrf2 pathway; Microbial protease; antioxidant peptide; casein hydrolysate.

Fig. 1. DPPH scavenging ability and hydrolysis degrees (DH) of the casein hydrolysates by different ratios of Protin SD-NY10 and Protease A “Amano” 2SD.

Data are presented as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 2. DPPH scavenging ability of different peptide fractions.

Data are presented as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 3. DPPH scavenging ability of nine synthesized peptides at concentration of 5.0 mg/ml.

Data are presented as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 4. Effect of AAPH on the viability of HepG2 cells (A), and effects of Pep1(B), Pep3(C), Pep6(D), Pep7(E) on the viability of HepG2 cells.

Data are presented as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 5. The relative mRNA expression level of CAT, SOD, GSH-Px, Keap1, and Nrf2.

Each mRNA expression was normalized to that of the ribosomal protein GAPDH and expressed relative to the control level. Data are expressed as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 6. Protein expression related to antioxidant signaling pathway (A), and relative protein content in the presence of Pep1(B), Pep3(C), Pep6(D), and Pep7(E), when compared to the control.

Lanes 1, 4, 7, 10 represent samples with Pep1, Pep3, Pep6, Pep7 at concentration of 0.1 mg/ml, respectively; Lanes 2, 5, 8, 11 represent samples with Pep1, Pep3, Pep6, Pep7 at concentration of 0.5 mg/ml, respectively; Lanes 3, 6, 9, 12 represent samples with Pep1, Pep3, Pep6, Pep7 at concentration of 1.0 mg/ml, respectively. Data are expressed as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 7. Effect of four peptides on the activities of CAT (A), SOD (B), and GSH-Px (C).

Data are expressed as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).

Fig. 8. Effect of four peptides on the production of ROS (A), MDA (B), and GSH (C).

Data are expressed as mean ± SD, and results marked with the same letters are not significantly different (p > 0.05).