BACKGROUND: NLRP3 inflammasome-mediated pyroptosis in gingival fibroblasts (GFs) plays a pivotal role in periodontitis pathogenesis. Mitochondrial dysfunction serves as a critical upstream trigger for NLRP3 inflammasome activation, while mitophagy acts as a key homeostatic mechanism. The regulatory mechanisms of mitophagy in modulating GF pyroptosis remain poorly defined.
METHODS: Human GFs were used in this study. An in-vitro inflammatory environment was created using 5 μg/mL lipopolysaccharide (LPS). Mitophagy was either activated using P62-mediated mitophagy inducer (PMI, 10 μM) or inhibited with Mdivi-1 (10 μM) in LPS-stimulated and healthy GFs, respectively. Mitophagy was visualized by immunofluorescence, alongside quantification by qRT-PCR/Western blot analysis of proteins associated with mitophagy (PINK1, Parkin, and Beclin-1). Mitochondrial integrity was comprehensively assessed by transmission electron microscopy (TEM), mitochondrial membrane potential (MMP) fluorescence assays, intracellular ROS/mtROS quantification via flow cytometry, and mitochondrial DNA (mtDNA) copy number analysis. NLRP3 inflammasome activation was analyzed by qRT-PCR and Western blot. Pyroptotic cell death was determined by propidium iodide (PI) staining, LDH release, and IL-1β/IL-18 secretion assays.
RESULTS: LPS stimulation suppressed mitophagy in GFs, which was effectively rescued by PMI treatment. In contrast, the mitophagy inhibitor decreased basal mitophagy in healthy GFs. PMI-mediated mitophagy enhancement restored mitochondrial function in LPS-exposed GFs, as demonstrated by improved MMP, reduced ROS/mtROS levels, and normalized mtDNA/nDNA ratios. Mechanistically, mitophagy activation attenuated LPS-induced NLRP3 inflammasome assembly, thereby reducing pyroptotic cell death and IL-1β/IL-18 secretion.
CONCLUSIONS: These findings revealed that mitophagy safeguarded against NLRP3 inflammasome-dependent pyroptosis in GFs by preserving mitochondrial homeostasis, highlighting its therapeutic potential for periodontitis management.
