Low concentrations of tumor necrosis factor-alpha promote human periodontal ligament stem cells osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR pathway

Tumor necrosis factor-alpha (TNF-α) is one of the crucial inflammatory factors in alveolar bone metabolism during the process of periodontitis. Autophagy is indispensable for proper osteoblast function. However, the effects of autophagy on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory microenvironment and the underlying mechanisms remain to be clarified. The

Low concentrations of TNF-α promote hPDLSCs osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR signaling.

Characterizations of hPDLSCs were evaluated by Alizarin-red S staining, Oil red staining and flow cytometry. hPDLSCs were treated with various concentrations of TNF-α. Rapamycin or 3MA was used to achieve or inhibit autophagy activation. AKT signaling was inhibited using ARQ092. Cell proliferation was evaluated using Cell Counting Kit-8 (CCK8) assay. Real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR), western blot, alkaline phosphatase (ALP) staining and Alizarin Red S staining were applied to evaluate levels of osteogenic differentiation and autophagy. CCK8 showed that low concentrations of TNF-α had no influence on cell proliferation, while high concentrations of TNF-α inhibited proliferation. Low concentrations of TNF-α promoted osteogenic differentiation and autophagy, while high concentrations of TNF-α inhibited osteogenic differentiation and autophagy in hPDLSCs. The levels of osteogenic differentiation in hPDLSCs were partly effected after co-incubation with 0.1 ng/mL TNF-α with 3MA or Rapamycin. ARQ092 enhanced 0.1 ng/mL TNF-α-induced ALP expression and mineral nodule formation.