Two turtle-derived tripeptides as ARE-luciferase inducers suppress H2O2-induced oxidative stress

Activation of the Antioxidant response element (ARE) pathway contributes to protecting cells against oxidative stress. In the previous study, Asn-Cys-Ala and Cys-Thr-Ala derived from soft-shelled turtles demonstrated potent ARE-luciferase inducer activity in the HepG2 cell model. In this study, the effects of Asn-Cys-Ala and Cys-Thr-Ala on the Nuclear factor (erythroid-derived 2)-like 2/ Kelch-like ECH-associated protein 1 (Nrf2/Keap1) system in HepG2 cells were studied. The results showed that Asn-Cys-Ala and Cys-Thr-Ala regulated the Nrf2/ARE pathway by stabilizing the Nrf2 levels, which was achieved by decreasing the levels of Keap1. The EC 50 values for the cellular antioxidant activity of Asn-Cys-Ala and Cys-Thr-Ala were 81.53 and 58.60 μM, respectively. And the results also demonstrated that treatment with Asn-Cys-Ala and Cys-Thr-Ala could reduce the H 2 O 2 -induced changes in intracellular Reactive Oxygen Species (ROS), Malondialdehyde (MDA) levels, and antioxidant enzyme activity (Superoxide dismutase, SOD; Glutathione peroxidase, GSH-Px; Catalase, CAT) ( p < 0.05). • Asn-Cys-Ala and Cys-Thr-Ala, two turtle-derived tripeptides modulated the Nrf2/ARE pathway by enhancing Nrf2 level through the stabilization of Nrf2, which was accomplished by a decrease in the level of Keap1. • Asn-Cys-Ala and Cys-Thr-Ala have cellular antioxidant activity, with the superior antioxidant activity of Cys-Thr-Ala possibly attributed to its terminal cysteine residue. • Treatment with Asn-Cys-Ala and Cys-Thr-Ala could reduce the H 2 O 2 -induced changes on intracellular ROS, MDA levels, and antioxidant enzyme activity (SOD, GSH-Px, CAT).