Effects of nuclear factor-κB signaling pathway on periodontal ligament stem cells under lipopolysaccharide-induced inflammation

Lipopolysaccharide (LPS) induces inflammatory stress and apoptosis. This study focused on the effect of nuclear factor kappa B (NF-κB) signaling pathway on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) after LPS induction and its mechanism. We first isolated hPDLSCs from human tooth root samples in vitro. Then, flow cytometry detected positive expression of cell surface antigens CD146 and STRO-1 and negative expression of CD45, suggesting the hPDLSCs were successfully isolated. LPS significantly induced increased apoptosis and diminished proliferation of hPDLSCs. The NF-κB pathway agonist phorbol 12-myristate 13-acetate (PMA) or p65 overexpression inhibited the proliferation of LPS-treated hPDLSCs and promoted apoptosis. PMA also promoted LPS-induced up-regulation of the expression of inflammatory factors TNF-α and IL-6 and down-regulation of the expression of anti-inflammatory factor IL-10. Additionally, LPS was confirmed to lead to a reduction of alkaline phosphatase (ALP) activity, calcium nodules, and expression of osteogenic markers Runt-related transcription factor 2 (Runx2) and osteopontin. This reduction could be promoted by PMA. Western blotting further indicated that PMA could promote LPS-induced decrease of expression of p65 (cytoplasm), and total cellular proteins IKKα and IKKβ in hPDLSCs, while protein expression of p-IκBα (cytoplasm) and p65 (nucleus), and p-IκBα/IκBα ratio was elevated. By contrast, inhibition of the NF-κB pathway (PDTC) or small-interfering RNA targeting NF-κB/p65 (p65 siRNA) showed the opposite results. In conclusion, activation of NF-κB signaling in LPS-induced inflammatory environment can inhibit the proliferation and osteogenic differentiation of hPDLSCs. This study provides a theory foundation for the clinical treatment of periodontitis.