LPS-induced upregulation of the TLR4 signaling pathway inhibits osteogenic differentiation of human periodontal ligament stem cells under inflammatory conditions

Toll-like receptor 4 (TLR4) is a transmembrane receptor responsible for the activation of a number of signal transduction pathways. Despite its involvement in inflammatory processes, the regulation of TLR4 signaling in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions remains to be fully elucidated. The present study aimed to clarify the regulatory mechanisms of the TLR4 signaling pathway and its role in the differentiation of hPDLSCs under inflammatory conditions. hPDLSCs from the periodontal tissues of healthy subjects and patients with periodontitis were identified by analyzing their cell surface marker molecules, and their osteogenic and adipogenic differentiation abilities. To determine the effect of TLR4 signaling on osteogenic and adipogenic differentiation under inflammatory conditions, cells were challenged with TLR4 agonist and antagonist under pluripotent differentiation conditions. Cell proliferation, apoptosis and migration were then determined using appropriate methods. The alkaline phosphatase (ALP) activity, Alizarin Red staining, Oil red O staining and relative gene and protein levels expression were also determined. The results showed that lipopolysaccharide (LPS)-induced inflammation inhibited cell proliferation and migration, promoted cell apoptosis and affected the cell cycle. Under inflammatory conditions, the activation of TLR4 decreased the activity of ALP and the expression of osteogenic markers, including osteocalcin, Runt-related transcription factor 2 and collagen I, compared with the control group, but increased the expression of adipogenesis-related genes poly (ADP-ribose) polymerase γ and lipoprotein lipase. The activation of TLR4 also induced the expression of proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, nuclear factor-κBP65 and TLR4, compared with that in the control group and the TLR4 antagonist group. The findings showed that LPS-induced upregulation of the TLR4 signaling pathway inhibited osteogenic differentiation and induced adipogenesis of the hPDLSCs under inflammatory conditions. The present study provided a novel understanding of the physiopathology of periodontitis, and a novel avenue for targeted treatments based on stem cell regeneration.