Inhibition of receptor-interacting protein kinase-3 in the necroptosis pathway attenuates inflammatory bone loss in experimental apical periodontitis in Balb/c mice

Aim: To explore the role of Necroptosis in apical periodontitis (AP), this study investigated Necroptosis in a Fusobacterium nucleatum (Fn)-induced AP model of Balb/c mice and explored related intracellular signalling pathways in L929 cells affected by Fn.

Methodology: For the in vivo experiments, expression of receptor-interacting protein kinase-3 (RIP3) was inhibited using an adeno-associated virus and then the Balb/c mice model of AP was established by injecting Fn into the root canal of the first mandibular molars. Bone loss and number of osteoclasts were measured via micro-computed tomography and tartrate-resistant Acid Phosphatase staining, respectively; expression of RIP3 and phosphorylated Mixed Lineage Kinase domain-like protein (pMLKL) was detected by immunohistochemistry and western blotting; expression of mRNA of inflammatory cytokines was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). For the in vitro experiments, L929 cells transfected with RIP3-Mus-siRNA or negative control siRNA were co-cultured with Fn; thereafter, western blotting, detection of cell death and viability and qRT-PCR analyses were performed to assess the activation of Necroptosis pathway and expression of mRNA of inflammatory cytokines. Data were analysed with unpaired t-test and one-way analysis of variance with significance set at p < .05.

Results: The Fn-induced apical lesions were associated with apical bone loss, an increased number of osteoclasts, enhanced expression of pMLKL and increased mRNA levels of inflammatory cytokines(IL-1α and IL-1β); all these effects were alleviated by RIP3 inhibition (p < .05). L929 cells infected with Fn displayed increased expression of pMLKL and increased cell death (p < .05), together with decreased cell viability (p < .05), whilst transfection with RIP3-Mus-siRNA decreased the mRNA expression of inflammatory cytokines(TNF-α and IL-6, p < .05).

Conclusions: Necroptosis may be involved in AP progression. RIP3 inhibition ameliorated the expression of inflammatory cytokines and bone resorption in Fn-induced AP lesions in Balb/c mice.