Madecassoside alleviates Clostridioides difficile infection by targeting ASC to inhibit inflammasome activation

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea, leading to inflammation due to the action of large clostridial toxins. Madecassoside (MA), a pentacyclic triterpene extracted from Centella asiatica, has broad anti-inflammatory effects. However, its therapeutic impact and mechanisms in CDI have not been thoroughly investigated. Here, we initially performed a CDI mouse model to analyze the improvement of symptoms by MA in vivo. Subsequently, we isolated primary bone marrow-derived macrophages (BMDM) in vitro and constructed an inflammation model stimulated by C. difficile isolates or toxins in macrophages, and analyzed the effects of MA. This study found that the administration of low and high concentrations of MA via continuous intragastric administration alleviated the symptoms in CDI mice, including improvements in body weight, colon length, and histopathological damage. ELISA and immunohistochemical staining showed that MA reduced the secretion of IL-1β in a dose-dependent manner in vivo and in vitro. Furthermore, the hyperactive state of inflammasome-related signaling pathway from the stimulated by C. difficile and toxin B was inhibited. Co-immunoprecipitation and confocal microscopy results indicated that MA inhibited the formation of the inflammasome complex and ASC speck. Molecular docking and SPR studies revealed MA's strong binding affinity for ASC. In conclusion, our study suggests that MA can reduce the activation of inflammasomes triggered by C. difficile toxins, potentially through its specific binding to ASC, which may pave the way for new treatment strategies against CDI.