Anti-inflammatory effect of Danhong injection through inhibition of GSDMD-mediated pyroptosis

Background: Pyroptosis is an inflammatory form of cell death that has been implicated in various infectious and non-infectious diseases. Gasdermin family proteins are the key executors of pyroptotic cell death, thus they are considered as novel therapeutic targets for inflammatory diseases. However, only limited gasdermin specific inhibitors have been identified to date. Traditional Chinese medicines have been applied in clinic for centuries and exhibit potential in anti-inflammation and anti-pyroptosis. We attempted to find candidate Chinese botanical drugs which specifically target gasdermin D (GSDMD) and inhibit pyroptosis.Methods: In this study, we performed high-throughput screening using a botanical drug library to identify pyroptosis specific inhibitors. The assay was based on a cell pyroptosis model induced by lipopolysaccharides (LPS) and nigericin. Cell pyroptosis levels were then evaluated by cell cytotoxicity assay, propidium iodide (PI) staining and immunoblotting. We then overexpressed GSDMD-N in cell lines to investigate the direct inhibitory effect of the drug to GSDMD-N oligomerization. Mass spectrometry studies were applied to identify the active components of the botanical drug. Finally, a mouse model of sepsis and a mouse model of diabetic myocardial infarction were constructed to verify the protective effect of the drug in disease models of inflammation.Results: High-throughput screening identified Danhong injection (DHI) as a pyroptosis inhibitor. DHI remarkably inhibited pyroptotic cell death in a murine macrophage cell line and bone marrow-derived macrophages. Mo-lecular assays demonstrated the direct blockade of GSDMD-N oligomerization and pore formation by DHI. Mass spectrometry studies identified the major active components of DHI, and further activity assays revealed sal-vianolic acid E (SAE) as the most potent molecule among these components, and SAE has a strong binding af-finity to mouse GSDMD Cys192. We further demonstrated the protective effects of DHI in mouse sepsis and mouse myocardial infarction with type 2 diabetes.Conclusion: These findings provide new insights for drug development from Chinese herbal medicine like DHI against diabetic myocardial injury and sepsis through blocking GSDMD-mediated macrophage pyroptosis.