AimTo explore the activation of necroptosis triggered by Enterococcus faecalis in human osteoblastic MG63 cells and provide new insights into the pathogenesis of refractory apical periodontitis.MethodologyThe viability of MG63 cells exposed to live E. faecalis was investigated using the cell counting kit‐8 assay. The relative expressions of specific markers for necroptosis, namely p‐RIPK3 and p‐MLKL, were determined by western blotting. Cells pretreated with necrosulfonamide and GSK’872, which are specific inhibitors for MLKL and RIPK3, respectively, were then subjected to lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry analysis and Hoechst 33342/PI double fluorescence staining. Lentiviral‐delivered short hairpin RNA (shRNA) targeting MLKL was employed to further confirm the activation of necroptosis in MG63 cells infected with E. faecalis. Transmission electron microscopy was additionally used to observe the morphological characteristics. Statistical analysis was conducted using Student’s t‐tests or one‐way ANOVA followed by the Student–Newman–Keuls test.ResultsThe infection with E. faecalis significantly inhibited the viability of MG63 cells in a multiplicity of infection‐ and infection time‐dependent manners (P < 0.05). In line with this, the expression levels of necroptosis‐related markers, p‐RIPK3 and p‐MLKL, were significantly increased postinfection (P < 0.05). Significant reductions in death rate were detected in the case of E. faecalis‐infected MG63 cells following pretreatment with the inhibitors of RIPK3 and MLKL (P < 0.01). Furthermore, silencing of MLKL by shRNA significantly decreased LDH release (P < 0.01) and resulted in less mitochondrial swelling and vacuole‐like changes, as well as reduced endoplasmic reticulum expansion.ConclusionsEnterococcus faecalis infection‐induced necroptosis of MG63 cells via the RIPK3/MLKL signalling pathway, which may exert a negative influence on the healing process of refractory apical periodontitis. This study may offer novel insights into the pathogenesis and potential therapeutic targets of refractory apical periodontitis.

The oral microbiome, one of the most complex and intensive microbial ecosystems in the human body, comprises bacteria, archaea, fungi, protozoa, and viruses. Dysbiosis of the oral microbiome is the initiating factor that leads to oral infectious diseases. Infection is a sophisticated biological process involving interplay between the pathogen and the host, which often leads to activation of programmed cell death. Studies suggest that pyroptosis, apoptosis, and necroptosis are involved in multiple oral infectious diseases. Further understanding of crosstalk between cell death pathways has led to pyroptosis, apoptosis, and necroptosis being integrated into a single term: PANoptosis. PANoptosis is a multifaceted agent of the immune response that has important pathophysiological relevance to infectious diseases, autoimmunity, and cancer. As such, it plays an important role in innate immune cells that detect and eliminate intracellular pathogens. In addition to the classical model of influenza virus-infected and Yersinia-infected macrophages, other studies have expanded the scope of PANoptosis to include other microorganisms, as well as potential roles in cell types other than macrophages. In this review, we will summarize the pathophysiological mechanisms underlying inflammation and tissue destruction caused by oral pathogens. We present an overview of different pathogens that may induce activation of PANoptosis, along with the functional consequences of PANoptosis in the context of oral infectious diseases. To advance our understanding of immunology, we also explore the strategies used by microbes that enable immune evasion and replication within host cells. Improved understanding of the interplay between the host and pathogen through PANoptosis will direct development of therapeutic strategies that target oral infectious diseases.

Oral infectious diseases represent a major neglected global population health challenge, imposing an increasing burden on public health and economy. Refractory apical periodontitis (RAP), mainly caused by Enterococcus faecalis , is a representative oral infectious disease with considerable therapeutic challenges.