Restoration and preservation effects of mung bean antioxidant peptides on H2O2-induced WRL-68 cells via Keap1-Nrf2 pathway

Mung bean antioxidant peptides (MBAPs) were prepared from mung bean protein hydrolysate, and four peptide sequences including Ser-Asp-Arg-Thr-Gln-Ala-Pro-His (similar to 953 Da), Ser-His-Pro-Gly-Asp-Phe-Thr-Pro-Val (similar to 956 Da), Ser-Asp-Arg-Trp-Phe (similar to 710 Da), and Leu-Asp-Arg-Gln-Leu (similar to 644 Da) were identified. The effects of MBAPs on the oxidation-induced normal human liver cell line WRL-68 were analyzed to determine the mechanism protecting the oxidation-induced injury. The results showed that the cells were subjected to certain oxidative damage by H2O2 induction, as evidenced by decreased cell number and viability, overproduction of intracellular ROS, and decreased mitochondrial membrane potential. Compared with the H2O2-induced group, the MBAP-treated oxidation-induced group exhibited significantly higher cell number and viability, and the intracellular ROS was similar to that of the control group, suggesting that MBAP scavenges excessive intracellular free radicals after acting on the oxidation-induced cells. Combined with Western blotting results, it was concluded that the MBAP-treated oxidation-induced group also significantly promoted the expression of proteins related to the kelch-like ech-related protein 1 (Keap1)/ nuclear factor e2-related factor 2 (Nrf2) signaling pathway, which resulted in an approximately 2-fold increase in antioxidant enzymes, and a decrease in malondialdehyde content of approximately 55% compared to oxidatively-induced cells, leading to the recovery of both cell morphology and viability. These results suggest that MBAPs scavenge intracellular free radicals and improve oxidative stress in hepatocytes through the expression of Keap1/Nrf2 pathway-related protein, thereby reducing oxidative attack on the liver. Therefore, MBAP is applied as a nutritional ingredient in the functional food field, and this study provides a theoretical basis for the high utilization of mung bean proteins.