Inhibition of Jaw Bone Marrow Mesenchymal Stem Cell Cuproptosis Attenuates Bone Loss in Apical Periodontitis via Suppressing the FoxO Signalling Pathway

AIM: Apical periodontitis (AP) is an infectious disorder characterized by periapical inflammation and alveolar bone destruction. Recent evidence suggests that cuproptosis, a distinctive form of copper-regulated cell death, has been implicated in the regulation of bone loss. This study aimed to investigate the involvement of cuproptosis and its mechanism in modulating bone loss in AP. METHODOLOGY: Periapical specimens from healthy subjects or AP patients were collected to evaluate copper content and the expression level of cuproptosis-associated proteins. Single-cell RNA sequencing (scRNA-seq) datasets (GSE171213, GSE181688, and GSE197680) were re-analysed to identify the specific cell types undergoing cuproptosis. An AP rat model was constructed and treated with the copper chelator tetrathiomolybdate (TTM). Alveolar bone loss was assessed using micro-CT, H&E staining, copper ion staining, and immunohistochemistry. In vitro, jaw bone marrow mesenchymal stem cells (JBMMSCs) were stimulated with lipopolysaccharide (LPS) or cuproptosis inducer (Elesclomol-Cu) to mimic the AP microenvironment. The effects of TTM on osteogenic differentiation were examined via qRT-PCR, Western blotting, ALP staining, and ARS staining. LC-MS/MS proteomics was utilized to explore the mechanism by which TTM attenuates bone loss. Moreover, cuproptotic JBMMSCs were treated with JY-2 (FoxO inhibitor) to determine whether cuproptosis aggravates osteogenic dysfunction via the FoxO/β-catenin axis. RESULTS: Copper concentrations were significantly elevated in human and rat AP tissues compared with healthy tissues. Analysis of scRNA-seq data revealed that cuproptosis predominantly occurred in JBMMSCs. In the rat model, TTM administration significantly alleviated alveolar bone loss by reducing cuproptosis in JBMMSCs. Furthermore, JBMMSCs stimulated with LPS or Elesclomol-Cu exhibited characteristics of cuproptosis and impaired osteogenic function. TTM treatment markedly reduced cuproptosis and restored osteogenic capacity. Mechanistically, cuproptosis in JBMMSCs led to the dephosphorylation of FoxOs. Conversely, TTM reversed these changes both in vivo and in vitro. Furthermore, inhibition of FoxO signalling with JY-2 disrupted FoxO3/β-catenin interaction, enhanced β-catenin expression and rescued the osteogenic dysfunction induced by cuproptosis. CONCLUSIONS: Cuproptosis acts as a novel driver of alveolar bone loss in AP by impairing the osteogenic potential of JBMMSCs. TTM targeting cuproptosis represents a promising strategy to attenuate bone resorption in AP by the cuproptosis/FoxO/β-catenin pathway.