The phthalide compound tokinolide B from Angelica sinensis exerts anti-inflammatory effects through Nur77 binding

Background: Nur77, an orphan member of the nuclear receptor superfamily, regulates inflammatory diseases and is a therapeutic target for treating inflammation. Phthalides in Angelica sinensis exhibit anti-inflammatory activity. Purpose: This study aimed to screen compounds from A. sinensis phthalide extract that could exert antiinflammatory activity by targeting Nur77. To provide new theoretical support for better elucidation of Chinese medicine targeting mitochondria to achieve multiple clinical efficacies. Methods: The anti-inflammatory capacity of phthalides was assessed in tumor necrosis factor-alpha (TNF alpha)-stimulated HepG2 cells using western blotting. The interaction between phthalides and Nur77 was verified by molecular docking, surface plasmon resonance, and cellular thermal shift assay. Co-immunoprecipitation, western blotting, and immunostaining were performed to determine the molecular mechanisms. The in vivo anti-inflammatory activity of the phthalides was evaluated in a lipopolysaccharide (LPS)/d-galactosamine (dGalN)-induced acute hepatitis and liver injury mouse model of acute hepatitis and liver injury. Finally, the toxicity of phthalide toxicity was assessed in zebrafish experiments. Results: Among the 27 phthalide compounds isolated from A. sinensis, , tokinolide B (TB) showed the best Nur77 binding capacity and, the best anti-inflammatory activity, which was induced without apoptosis. In vivo and in vitro experiments showed that TB promoted Nur77 translocation from the nucleus to the mitochondria and interacted with tumor necrosis factor receptor-associated factor 2 (TRAF2) and sequestosome 1 (p62) to induce mitophagy for anti-inflammatory functions. TB substantially inhibited LPS/d-GalN-induced acute hepatitis and liver injury in mice. TB also exhibited significantly lower toxicity than celastrol in zebrafish experiments. Conclusion: These findings suggested that TB inhibits inflammation by promoting Nur77 interaction with TRAF2 and p62, thereby inducing mitophagy. These findings offer promising directions for developing novel antiinflammatory agents, enhance the understanding of phthalide compounds, and highlight the therapeutic potential of traditional Chinese herbs.