Targeting the Nod-like receptor protein 3 Inflammasome with inhibitor MCC950 rescues lipopolysaccharide-induced inhibition of osteogenesis in Human periodontal ligament cells

Abstract

ObjectiveWe aim to investigate whether lipopolysaccharide-stimulated activition of Nod-like receptor protein 3 (NLRP3) Inflammasome inhibits osteogenesis in Human periodontal ligament cells (HPDLCs). Futhermore, to study whether MCC950 (a inhibitor of NLRP3 Inflammasome) rescues lipopolysaccharide-induced inhibition of osteogenesis in HPDLCs as well as the underlying mechanisms. MethodsHPDLCs were isolated from periodontal ligament of healthy orthodontic teeth from teenagers, and cells surface marker protein were detected by flow cytometry. Cells viability were determined by Cell Counting kit 8 assay. Enzyme-linked immunosorbent assay was used to analyze the secretion of proinflammatory factors. Western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were measured assessing the expression of NLRP3 and Caspase-1. RT-qPCR, Alizarin red staining and Alkaline phosphatase staining were tested to determine the osteogenic differentiation capacity of HPDLCs. ResultsIt was found that lipopolysaccharide in the range of concentrations from 10 to 100 μg/ml significantly inhibited HPDLCs viability at 24 h and significantly improved proinflammatory cytokine expressions at 8 h and 24 h. MCC950 reversed lipopolysaccharide-stimulated proinflammatory cytokine expressions including interleukin-1β and interleukin-18, but not tumor necrosis factor-α. In addition, MCC950 rescued the lipopolysaccharide-inhibited osteogenic gene (Alkaline phosphatase, Runt-related transcription factor 2, and Osteocalcin). Moreover, MCC950 downregulated lipopolysaccharide-induced relative protein of NLRP3 Inflammasome signaling pathway, such as NLRP3 and Caspase-1. ConclusionMCC950 rescues lipopolysaccharide-induced inhibition of osteogenesis in HPDLCs via blocking NLRP3 Inflammasome signaling pathway, and it may be used as a promising therapeutic agent for periodontitis or periondontal regenerative related disease.