Antioxidant Peptides from the Fruit Source of the Oil Crop Litsea cubeba Ameliorate FFA-Induced Oxidative Stress Injury: Based on Nrf2/Keap1 Pathway and Molecular Dynamics Simulations

Abstract

In this study, we systematically investigated the mechanisms of the antioxidation and anti-lipid accumulation effects of antioxidant peptides from Litsea cubeba on a free fatty acid (FFA)-induced NAFLD model of HepG2 cells. The NAFLD cell model was constructed by inducing the HepG2 hepatocellular carcinoma cell line with 0.5 mmol/L FFAs, and AQRDAGLL, QEGPFVR, and DVPPPRGPL were given to the culture to study their lipid-lowering and antioxidant activities on NAFLD cells. The lipid-lowering activities of the three antioxidant peptides were evaluated by Oil Red O staining and TG and TC content assays, and the results showed that all three peptides had strong ameliorating effects on FFA-induced lipid accumulation in NAFLD cells. The intracellular antioxidant protease (CAT, GSH, and SOD) activity levels and lipid peroxidation (MDA) content were measured and intracellular ROS levels were detected. The results showed that after intervention with the antioxidant peptides, the intracellular ROS levels in the NAFLD model cells were significantly reduced, the SOD and CAT activities were increased, the GSH content was elevated, and the MDA content was reduced, which indicated that AQRDAGLL, QEGPFVR, and DVPPPRGPL were able to inhibit the oxidative stress of the cells effectively and to achieve the effect of intervening in NAFLD. JC-1 fluorescence staining experiments showed that the mitochondrial membrane potential function of NAFLD cells was restored under the effect of the antioxidant peptides. Molecular dynamics simulations revealed that the main driving force between QEGPFVR and Keap1 protein was van der Waals forces, ΔG = -62.11 kcal/mol, which indicated that QEGPFVR was capable of spontaneously binding to Keap1 protein.

Keywords: Keap1/Nrf2 pathway; Litsea cubeba antioxidant peptides; ROS; molecular docking; molecular dynamics simulations.

Figure 1

Comparison of essential amino acid contents of Litsea cubeba cake meal and IV2. (A) Specific values of essential amino acid contents. (B) Analysis of increased content of essential amino acids in IV2.

Figure 2

Effect of concentration of FFAs and peptides on the activity of HepG2 cells. (A) Effect of FFA concentration on the activity in HepG2 cells; (B) effect of antioxidant peptides on the activity in HepG2 cells; (C) effect of FFAs on intracellular lipid accumulation in HepG2 cells; (D) change in lipid droplet area; and (E) change in absorbance. * p < 0.05, *** p < 0.001 vs. Control, n = 3.

Figure 3

(A) Effect of antioxidant peptides on intracellular lipid; (B) change in absorbance; (C) change in lipid droplet area; and (D,E) TC and TG contents. *** p < 0.001 vs. Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. FFA, n = 3.

Figure 4

Effect of antioxidant peptides on the levels of (A) CAT; (B) SOD; (C) GSH-Px; and (D) MDA in HepG2 cells. *** p < 0.001 vs. Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. FFA, n = 3.

Figure 5

(A) Effect on intracellular ROS (100 µm); (B) change in absorbance; and (C–E) effect on the mRNA expression levels of Nrf2, HO-1, and NQO1 in cells under oxidative stress. * p < 0.05, *** p < 0.001 vs. Control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. FFA, n = 3.

Figure 6

(A) Detection of mitochondrial membrane potential by JC-1 staining (50 µm); (B) fluorescence intensity change; and (C–E) effect on the mRNA expression levels of MCJ, PPAR-α, and CPT-1a in HepG2 cells. ** p < 0.01, *** p < 0.001 vs. Control; ^# p < 0.05, ^## p < 0.01. vs. FFA, n = 3.

Figure 7

Molecular docking modeling of QEGPFVR with Keap1 (kelch). (A) Six-axis spider web graph of the properties of the antioxidant peptide; (B) structure of peptide QEGPFVR; (C) color band diagram of Keap1 protein docking complex with peptide QEGPFVR; and (D) waveform.

Figure 8

Localized pocket details of the Keap1 protein docking complex with peptide QEGPFVR. (A) Waveform; (B) QEGPFVR–Keap1 protein complex localized pocket details color band diagram; (C) waveform; and (D) QEGPFVR–Keap1 protein complex 3D interaction force demonstration. Yellow dashed lines are hydrogen bonds, and blue dashed lines are pi–pi conjugation.

Figure 9

Molecular dynamics simulations of QEGPFVR–Keap1 protein complex. (A) RMSD (blue lines indicate Keap1 protein, red lines indicate QEGPFVR–Keap1 protein complex); (B) RMSF (the blue background highlights the beta chain, which persists throughout the simulation for more than 70% of the time); (C) Interaction of QEGPFVR with Keap1 protein (green for hydrogen bonding; purple for water transport; pink for ionic bonding; blue for water bridges); (D) A schematic of detailed QEGPFVR interactions with Keap1 protein residues; (E) Calculation of the binding free energy of QEGPFVR to Keap1 protein; (F) Contribution of individual amino acids to the binding energy of QEGPFVR to Keap1 protein.

Figure 10

Overview of the roles of Nrf2, HO-1, NQO1, PPAR-α, CPT-1a, and MCJ in lipid accumulation and oxidative stress in HepG2 cells.