Yuping Qi, Yijun Luan, Limei Wang
Department of Oral Medicine, Qilu Hospital of Shandong University, Wenhua West Road 107, 250012, Jinan, China.
Institute of Stomatology, Shandong University, Jinan, China.
Mol Biol Rep. 2023 Apr;50(4):3329-3339. doi: 10.1007/s11033-022-08173-8. Epub 2023 Feb 1.
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 aim of the present study was to investigate whether autophagy participates in hPDLSCs differentiation after treated with TNF-α and explore the underlying mechanisms.
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.
Low concentrations of TNF-α promote hPDLSCs osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR signaling.
肿瘤坏死因子-α（TNF-α）是牙周炎过程中牙槽骨代谢的关键炎症因子之一。自噬对于成骨细胞的正常功能不可或缺。然而，自噬在炎症微环境中对人牙周膜干细胞（hPDLSCs）成骨分化的影响及其潜在机制仍有待阐明。本研究的目的是探讨自噬是否参与TNF-α处理后hPDLSCs的分化，并探索其潜在机制。
通过茜素红S染色、油红染色和流式细胞术评估hPDLSCs的特征。用不同浓度的TNF-α处理hPDLSCs。使用雷帕霉素或3-甲基腺嘌呤（3MA）来实现或抑制自噬激活。使用ARQ092抑制AKT信号传导。使用细胞计数试剂盒-8（CCK8）测定法评估细胞增殖。应用实时逆转录聚合酶链反应测定法（RT-PCR）、蛋白质免疫印迹法、碱性磷酸酶（ALP）染色和茜素红S染色来评估成骨分化和自噬水平。CCK8结果显示，低浓度的TNF-α对细胞增殖无影响，而高浓度的TNF-α抑制增殖。低浓度的TNF-α促进hPDLSCs的成骨分化和自噬，而高浓度的TNF-α抑制hPDLSCs的成骨分化和自噬。hPDLSCs与0.1 ng/mL TNF-α与3MA或雷帕霉素共同孵育后，其成骨分化水平受到部分影响。ARQ092增强了0.1 ng/mL TNF-α诱导的ALP表达和矿化结节形成。
低浓度的TNF-α通过抑制AKT/mTOR信号传导激活自噬，从而促进hPDLSCs的成骨分化。
