Mito tempo

To investigate the origin of redox signals, various inhibitors such as sodium diethyldithiocarbamate, GKT136901 (Sigma‐Aldrich, MO, USA), Mito‐tempo (MedChemExpress, NJ, USA), FCCP, oligomycin, rotenone and antimycin A (Agilent, CA, USA), and Batimastat (Selleckchem, Seoul, South Korea) were used as inhibitors. All of the inhibitors were incubated for 24 h at 37 °C. Furthermore, the cells were treated with CPI‐613 (MedChemExpress, NJ, USA) for drug screening and incubated for 24 h at 37 °C. After treatment, the cells were washed with DPBS for EC detection within 30 s and cytotoxicity testing.

mDPC6T cells stimulated with LPS and ATP were utilized to simulate the pyroptosis during the pulpitis pathological process. In detail, mDPC6T cells were first primed with 1 μg/mL LPS (#LPS25, Sigma Aldrich) for 24 h, followed by treatment with 2 mM ATP (#HY‐B2176, MedChemExpress) for 24 h (referred to as LA‐mDPC6T). To inhibit mitochondrial OS, mDPC6T cells were pre‐treated with 5 μM MitoTEMPO (#HY‐112879, MedChemExpress) or 2 μM NAC (#HY‐B0215, MedChemExpress) for 4 h prior to stimulation with LPS and ATP. To inhibit mitochondrial transfer and tunnelling nanotube (TNT) formation, cells were pre‐treated with 1 μM cytochalasin B (HY‐16928, MedChemExpress) for 6 h to interfere polymerization and interaction of actin. For signalling pathway inhibition, cells were pre‐treated with 1 μM NF‐κB inhibitor Bay 11‐7082 (#HY‐13453, MedChemExpress) or 5 μM IKKβ inhibitor ML120B (#HY‐15473, MedChemExpress) for 4 h before experiments. To generate the mBMSCs without the mitochondrial function (ρ^omBMSCs), cells were cultured in a medium additioned with 1 μg/mL ethidium bromide (15585011, Invitrogen), 100 μg/mL pyruvic acid (HY‐Y0781, MedChemExpress) and 50 μg/mL uridine (HY‐B1449, MedChemExpress) for at least 1 month. For the TNF‐α treatment, the mBMSCs were treated with different concentrations of mouse TNF‐α (#HY‐P7090, MedChemExpress) for 24 h.