Alleviating Effect of Lipid Phytochemicals in Seed Oil (Brassica napus L.) on Oxidative Stress Injury Induced by H2O2 in HepG2 Cells via Keap1/Nrf2/ARE Signaling Pathway

α-tocopherol (α-T), β-sitosterol (β-S), canolol (CA), and sinapic acid (SA) are the four main endogenous lipid phytochemicals (LP) found in <i>Brassica napus</i> L. seed oil, which possess the bioactivity to prevent the risk of several chronic diseases via antioxidant-associated mechanisms. Discovering the enhancer effects or synergies between LP is valuable for resisting oxidative stress and improving health benefits. The objectives of this study were to identify a potentially efficacious LP combination by central composite design (CCD) and cellular antioxidant activity (CAA) and to investigate its protective effect and potential mechanisms against H₂O₂-induced oxidative damage in HepG2 cells. Our results indicated that the optimal concentration of LP combination was α-T 10 μM, β-S 20 μM, SA 125 μM, and CA 125 μM, respectively, and its CAA value at the optimal condition was 10.782 μmol QE/100 g. At this concentration, LP combination exerted a greater amelioration effect on H₂O₂-induced HepG2 cell injury than either antioxidant (tea polyphenols or magnolol) alone. LP combination could reduce the cell apoptosis rate induced by H₂O₂, lowered to 10.06%, and could alleviate the degree of oxidative damage to cells (ROS↓), lipids (MDA↓), proteins (PC↓), and DNA (8-OHdG↓). Additionally, LP combination enhanced the antioxidant enzyme activities (SOD, CAT, GPX, and HO-1), as well as the T-AOC, and increased the GSH level in HepG2 cells. Furthermore, LP combination markedly upregulated the expression of Nrf2 and its associated antioxidant proteins. It also increased the expression levels of Nrf2 downstream antioxidant target gene (<i>HO-1</i>, <i>SOD-1</i>, <i>MnSOD</i>, <i>CAT</i>, <i>GPX-1</i>, and <i>GPX-4</i>) and downregulated the mRNA expression levels of Keap1. The oxidative-stress-induced formation of the Keap1/Nrf2 complex in the cytoplasm was significantly blocked by LP treatment. These results indicate that LP combination protected HepG2 cells from oxidative stress through a mechanism involving the activation of the Keap1/Nrf2/ARE signaling pathways.