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Caspase-3/GSDME-mediated pyroptosis leads to osteogenic dysfunction of osteoblast-like cells
Zhixiao Wu1, Qian Ding1, Muxin Yue1
1Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology & Research Center of Engineering and Technology for Computerized Dentistry Ministry of Health & NMPA Key Laboratory for Dental Materials, Beijing, China.
Objective:
Cell pyroptosis is implicated in progressive bone loss in dental inflammatory diseases. We induced caspase-3/Gasdermin E (GSDME)-mediated pyroptosis in osteoblast-like cells and evaluated the effects on osteogenesis.
Materials And Methods:
Osteoblast-like cells were treated with various concentrations of sodium butyrate (NaB) to identify the most appropriate for inducing caspase-3/GSDME-mediated pyroptosis. Cells were divided into control, NaB and NaB+Ac-DEVD-CHO (specific caspase-3 inhibitor) groups. Pyroptosis level was evaluated by immunofluorescence, morphological observation, flow cytometry, lactate dehydrogenase (LDH) release assays, mRNA and protein levels of pyroptosis-related markers. Then, inflammation level, osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteogenic function were detected.
Results:
Treatment with 10 mM NaB increased caspase-3 expression, GSDME cleavage, LDH release and the number of pyroptotic cells, with morphologic changes, indicating GSDME-mediated pyroptosis induction. The pyroptosis-related changes were abolished by caspase-3 inhibition. Caspase-3/GSDME-mediated pyroptosis triggered the expression of inflammatory cytokines and RANKL, downregulated alkaline phosphatase (ALP) activity, mineralisation level, mRNA and protein levels of multiple osteogenic markers. These effects were partly reversed by Ac-DEVD-CHO.
Conclusion:
Caspase-3/GSDME-mediated pyroptosis induced by NaB activated the inflammatory response, reduced osteogenic differentiation and disturbed OPG/RANKL axis, leading to osteogenic dysfunction in osteoblast-like cells.
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