Identification of RIPK3 as a novel p-AMPK substrate reveals a mechanism by which AdipoAI suppresses necroptosis to attenuate periodontitis

Necroptosis is implicated in the pathogenesis of various inflammatory diseases, including periodontitis. This study aimed to investigate the molecular mechanisms underlying necroptosis in gingival fibroblasts (GFs) and to evaluate the therapeutic potential of AdipoAI, a novel adiponectin receptor agonist, administered locally, along with its regulatory effect on necroptosis. Analysis of single-cell RNA sequencing data using the AUCell scoring system demonstrated significant activation of necroptosis in human periodontal tissues, with GFs identified as the primary cellular target. Integrated approaches, including molecular docking, co-immunoprecipitation, and immunofluorescence, confirmed a direct interaction between p-AMPK and RIPK3. By establishing a mouse model of experimental periodontitis and an LAZ-induced necroptosis model in hGFs, combined with techniques such as Western blotting, flow cytometry, and transmission electron microscopy, we found that local administration of AdipoAI significantly alleviated gingival inflammation and alveolar bone resorption in mice. In hGFs, AdipoAI effectively suppressed the activation of p-RIPK3 and p-MLKL and reduced the production of inflammatory factors. Mechanistic investigations revealed that AdipoAI binds to AdipoR1/APPL1 to activate the AMPK signaling pathway, facilitating the association between p-AMPK and RIPK3, thereby inhibiting RIPK3/MLKL-mediated necroptosis. This study is the first to unveil a novel mechanism by which AdipoAI through the AMPK-RIPK3 axis to suppress GFs necroptosis, offering new strategic insights and experimental evidence for targeted periodontitis therapy.