Clustering human dental pulp fibroblasts spontaneously activate NLRP3 and AIM2 inflammasomes and induce IL-1ß secretion.

Objectives: The objective of the present study was to investigate whether NOD-like receptor family pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes pathways were involved in an experimental model of fibroblast activation named nemosis, which was used to mimic circumstances without bacteria stimulation. Methods: Nemosis of human dental pulp fibroblast (DPFs) was induced by three-dimensional culture in U-shaped 96-well plates and investigated by scanning electron microscopy (SEM). DPFs monolayers were used as control. Annexin V-FITC/7-AAD apoptosis assay was performed on the DPFs spheroids by flowcytometry. Caspase-1 activity detection assay was conducted on the DPFs spheroids. Quantitative real-time polymerase chain reaction (qRT-PCR), cytokine measurements, Western blot and the effect of COX-2 inhibitor on spheroids was studied. Results: SEM study observed human dental pulp fibroblast clusters and cell membranes damage on the surface of DPFs spheroids. The percentages of necrotic cells from DPFs spheroids gradually increased as the incubation time increased. A statistically significant increase in caspase-1 activity was observed after DPFs spheroids formation. DPFs spheroids displayed significant amounts of NLRP3, AIM2 mRNA and protein expression, caspase-1 mRNA expression and cleaved Caspase-1 protein expression and high IL-1ß concentrations (P < 0.05) than DPFs monolayers. Specific COX-2 inhibitor (NS-398) decreased NLRP3 mRNA and protein expression, cleaved Caspase-1 protein expression, Caspase-1 activity and IL-1ß mRNA expression and IL-1ß concentrations (P < 0.05). However, Specific COX-2 inhibitor had no impact on AIM2 mRNA and protein expression, caspase-1 mRNA expression and pro-Caspase-1 protein expression. Conclusions: In conclusion, clustering human DPFs spontaneously activated NLRP3 and AIM2 inflammasomes and induced IL-1ß secretion which could be partially attenuated by COX-2 inhibitor. Thus, nemosis could become a powerful model for studying mechanisms underlying aseptic pulpitis.

Glucagon-like peptide-1 receptor promotes osteoblast differentiation of dental pulp stem cells and bone formation in a zebrafish scale regeneration model.

Bone cells express the glucagon-like peptide 1 receptor (GLP-1R). However, its presence and role in human dental pulp derived stem cells (hDPSCs) remains elusive. Hence, in the current study, we isolated hDPSCs and differentiated them into osteoblasts, where GLP-1R expression was found to be upregulated during osteoblast differentiation. GLP-1 receptor agonist, liraglutide peptide treatment, increased osteoblast differentiation in hDPSCs by increasing calcium deposition, ALP activity, and osteoblast marker genes, Runx2, type 1 col, osteonectin, and osteocalcin. Furthermore, activation of long non-coding RNA (LncRNA) LINC00968 and microRNA-3658 signalling increased Runx2 expression. Specifically, liraglutide increased LncRNA-LINC00968 expression while decreasing miR-3658 expression. LINC00968 targets miR-3658, and miR-3658 targets Runx2. Additionally, in an in-vivo study, zebrafish scale regeneration model, liraglutide promoted calcium deposition, osteoblastic cell count, collagen 1α, osteonectin, osteocalcin, runx2a MASNA isoform expression (transcribed from promoter P1), and Ca/P ratio in scales. Overall, GLP-1R activation promotes osteoblast differentiation via Runx2/LncRNA-LINC00968/miR-3658 signalling in hDPSCs and promotes bone formation in zebrafish scale regeneration.

Absent in melanoma 2 (AIM2) in rat dental pulp mediates the inflammatory response during pulpitis.

INTRODUCTION: In recent years, the inflammasome has been determined to play an important role in inflammatory diseases. However, the role of the inflammasome in pulpitis remains unclear. Absent in melanoma 2 (AIM2) is a type of inflammasome that recognizes cytosolic double stranded DNA and forms a caspase-1-activating inflammasome with apoptosis-associated speck-like protein containing a caspase activating recruiting domain. In this study, we determined whether AIM2 was expressed in pulp cells and defined the role of AIM2 in the initiation of inflammation within the dental pulp. METHODS: In the in vivo study, the right maxillary molars from male adult Sprague-Dawley rats (250-350 g) were exposed to the pulp. In the in vitro study, the pulp cells isolated from the mandibular incisors of the Sprague-Dawley rats (2 weeks) were conventionally cultured. Immunofluorescence staining was used to determine the expression and distribution of AIM2 in the rat dental pulp tissues and cells in the presence or absence of inflammatory stimulation. Western blotting and real-time polymerase chain reaction were performed to determine whether there was a correlation between AIM2 expression levels and inflammation both in vivo and in vitro. RESULTS: In healthy dental pulp tissues and cells, AIM2 was only detected in the odontoblast layer. Stimulation significantly increased AIM2 expression in both the dental pulp tissues and cultured cells. The mRNA and protein levels of AIM2 were significantly up-regulated in response to inflammatory stimulation in a dose-dependent manner. Moreover, we also found that AIM2 expression correlated with interleukin-1 levels. These results reveal a direct relationship between the AIM2 inflammasome and pulpitis. CONCLUSIONS: Our study demonstrates that AIM2 is expressed in dental pulp tissues and mediates the inflammatory response during pulpitis. Therapeutic interventions aimed at reducing AIM2 expression may be beneficial in the treatment of pulpitis.