Involvement of the Keap1-Nrf2-ARE pathway in the antioxidant activity of sinomenine

Sinomenine is a pure alkaloid isolated from Sinomenium acutum. This study is aimed to investigate the critical role of the nuclear factor erythroid 2-related factor 2 (Nrf2)-kelch-like ECH-associated protein-1(Keap1)-antioxidant response element (ARE) antioxidative signaling pathway in protecting sinomenine against H₂O₂-induced oxidative injury. Cytotoxicity and antioxidant experiments to initially determine the protective effects of sinomenine show that sinomenine has no effect on the decreased cell viability and presents similar potency in scavenging all three free radicals. The binding affinity between sinomenine and Keap1 was determined via fluorescence polarization assay, with IC₅₀ of 13.52 μM. Quantum chemical calculation and theoretical simulation illustrated that sinomenine located into the Nrf2-binding site of Keap1 via hydrophobic and hydrogen interactions, showing high stability and binding affinity. On the basis of the stable binding of sinomenine with Keap1, sinomenine efficiently induced nuclear translocation of Nrf2, and increased in ARE activity in a concentration-dependent manner. Quantitative polymerase chain reaction provided further evidences that sinomenine-induced protection upregulated ARE-dependent genes, such as NAD(P)H quinone oxidoreductase 1, hemeoxygenase-1, and glutamate-cysteine ligase modifier subunit. Western blot confirmed that sinomenine increased the expressions of these antioxidative enzymes. Taken together, in vitro and in silico evaluations demonstrate that sinomenine inhibits the binding of Keap1 to Nrf2, promotes the nuclear accumulation of Nrf2 and thus leads to the upregulated expressions of Nrf2-dependent antioxidative genes. Our findings also highlight the use of sinomenine for pharmacological or therapeutic regulation of the Nrf2-Keap1-ARE system, which is a novel strategy to prevent the progression of oxidative injury.