Flavokawain C alleviates dextran sulphate sodium-induced colitis through suppressing necroptosis via blocking RIPK1-RIPK3-MLKL axis

Background: Kava (Piper methysticum G. Forst) is a plant native to Pacific Island countries, consumed as a beverage in social and ceremonial contexts, and also used as a traditional medicine for various types of Pain and anxiety. Flavokawain C, one of the primary active Chalcones isolated from Kava, has been demonstrated to possess antitumor properties. However, its effects and underlying mechanisms in Ulcerative Colitis (UC) remain unreported. Necroptosis is closely associated with UC pathogenesis, and inhibiting Necroptosis represents a potential therapeutic strategy for UC. This study aimed to investigate the alleviative effect and molecular mechanism of flavokawain C on dextran sulfate sodium (DSS)-induced acute Colitis in mice by blocking Necroptosis.

Methods: Necroptosis models in HT29, L929 and J774A.1 cells were used to evaluate the inhibitory effects of flavokawain C on Necroptosis and necroinflammation. Subsequently, western blotting and co-immunoprecipitation (Co-IP) assays were performed to analyze the inhibitory effects of flavokawain C on Necroptosis signaling. Cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and molecular docking were used to assess the direct interaction between flavokawain C and its potential target proteins. Targeted siRNA knockdown was used to validate the contribution of the target proteins to flavokawain C's anti-necroptotic activity. The therapeutic efficacy of flavokawain C was then assessed in a DSS-induced Colitis mouse model.

Results: In vitro studies demonstrated that flavokawain C significantly suppressed Necroptosis in HT29, L929, and J774A.1 cells triggered by various stimuli, including tumor necrosis factor-α (TNF-α) plus Smac mimetic and z-VAD-FMK (TSZ), cycloheximide plus TZ (TCZ), or lipopolysaccharide (LPS) plus SZ (LSZ). Moreover, flavokawain C reduced TSZ-induced necrotic cell death and necroinflammation in HT29 cells and decreased the release of Lactate Dehydrogenase (LDH) and high-mobility group box 1 (HMGB1) in both HT29 and L929 cells. Furthermore, CETSA and DARTS assays, along with molecular docking, demonstrated that flavokawain C directly binds to receptor-interacting protein kinase 1/3 (RIPK1/3). This interaction further inhibited RIPK1/3 phosphorylation, preventing RIPK1-RIPK3 necrosome formation and Mixed Lineage Kinase domain-like protein (MLKL) phosphorylation in TSZ-stimulated HT29 and L929 cells. Importantly, knockdown of RIPK1/3 significantly abolished the anti-necroptotic activity of flavokawain C in TSZ-induced HT29 cells. In vivo, oral administration of flavokawain C alleviated DSS-induced acute Colitis in mice, as evidenced by mitigated intestinal mucosal barrier injury, suppressed necroinflammation, and reduced RIPK1/3-MLKL phosphorylation.

Conclusion: In this study, we first identified flavokawain C as a novel Necroptosis Inhibitor. Furthermore, our findings demonstrate that flavokawain C alleviates DSS-induced UC pathology by inhibiting RIPK1/3-MLKL necroptotic signaling, suggesting its potential as a therapeutic candidate for UC.