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-1ß); 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.

Inhibition of Cathepsin K Alleviates Autophagy-Related Inflammation in Periodontitis-Aggravating Arthritis.

Rheumatoid arthritis (RA) and periodontitis share many epidemiological and pathological features, with emerging studies reporting a relationship between the two diseases. Recently, RA and periodontitis have been associated with autophagy. In the present study, we investigated the effects of cathepsin K (CtsK) inhibition on RA with periodontitis in a mouse model and its immunological function affecting autophagy. To topically inhibit CtsK periodontitis with arthritis in the animal model, adeno-associated virus (AAV) transfection was performed in periodontal and knee joint regions. Transfection of small interfering RNA (siRNA) was performed to inhibit CtsK in RAW264.7 cells. The effects of CtsK inhibition on the autophagy pathway were then evaluated in both in vivo and in vitro experiments. RA and periodontitis aggravated destruction and inflammation in their respective lesion areas. Inhibition of CtsK had multiple effects: (i) reduced destruction of alveolar bone and articular tissue, (ii) decreased macrophage numbers and inflammatory cytokine expression in the synovium, and (iii) alleviated expression of the autophagy-related transcription factor EB (TFEB) and microtubule-associated protein 1A/1B-light chain 3 (LC3) at the protein level in knee joints. Inhibition of CtsK in vitro reduced the expression of autophagy-related proteins and related inflammatory factors. Our data revealed that the inhibition of CtsK resisted the destruction of articular tissues and relieved inflammation from RA with periodontitis. Furthermore, CtsK was implicated as an imperative regulator of the autophagy pathway in RA and macrophages.