激活转录因子3(ATF3)已被确定为与成骨细胞分化相关的调节因子。然而,ATF3对牙周炎中人牙周干细胞(hPDLSCs)成骨分化和增殖的影响尚未见报道。为了建立牙周炎的体外模型,用脂多糖(LPS)攻击hPDLSC。细胞计数试剂盒-8测定用于评估细胞活力,同时采用逆转录定量PCR和蛋白质印迹法检测ATF3的表达。使用ELISA评估炎症释放,以及西方印迹。使用C11BODIPY581/591探针探索脂质过氧化,生化试剂盒,硫代巴比妥酸反应性物质(TBARS)测定和DCFH-DA染色。铁和Fe2+水平,使用相应的试剂盒和蛋白质印迹法测定铁凋亡相关蛋白的表达水平。使用碱性磷酸酶染色评估成骨分化能力,茜素红染色和蛋白质印迹。使用蛋白质印迹鉴定与Nrf2/HO-1信号转导相关的蛋白质的表达水平。结果表明,ATF3在LPS诱导的hPDLSCs中表达上调。ATF3的敲减减轻了LPS诱导的hPDLSCs的炎症反应,随着TNF-α水平的增加,IL-6,IL-1β,Cox-2和iNOS,IL-10水平降低。ATF3沉默也导致TBARS生产率降低,降低活性氧的水平,铁,Fe2+,ACSL4和TFR1,而它升高了SLC7A11和GPX4的水平。此外,ATF3沉默促进hPDLSC矿化和细胞分化,并升高OCN2、RUNX2和BMP2的水平。此外,ATF3耗竭上调与Nrf2/HO-1信号相关的蛋白质的表达水平。Nrf2抑制剂ML385部分抵消了ATF3干扰对LPS攻击的炎症反应的影响,脂质过氧化,hPDLSCs的铁细胞凋亡和成骨分化能力。总之,结果表明,ATF3沉默抑制炎症和铁性凋亡,通过调节Nrf2/HO-1信号促进LPS诱导的hPDLSCs成骨分化,这可能为牙周炎的治疗提供有希望的治疗靶点。
Activating transcription factor 3 (ATF3) has been identified as a regulator associated with osteoblast differentiation. However, the effects of ATF3 on the osteogenic differentiation and proliferation of human periodontal stem cells (hPDLSCs) in periodontitis have not been reported. With the purpose of establishing an in vitro model of periodontitis, hPDLSCs were challenged with lipopolysaccharide (LPS). The Cell Counting Kit-8 assay was applied to assess cell viability, while reverse transcription-quantitative PCR and western blotting were employed to detect ATF3 expression. Inflammatory release was assessed using ELISA, together with western blotting. Lipid peroxidation was explored using the C11 BODIPY 581/591 probe, biochemical kits, thiobarbituric acid reactive substances (TBARS) assay and DCFH-DA staining. Iron and Fe2+ levels, and the expression levels of ferroptosis-related proteins were measured using corresponding kits and western blotting. Osteogenic differentiative capability was evaluated using alkaline phosphatase staining, Alizarin red staining and western blotting. The expression levels of proteins associated with Nrf2/HO-1 signaling were identified using western blotting. The results indicated that ATF3 expression was upregulated in LPS-induced hPDLSCs. The knockdown of ATF3 alleviated the LPS-induced inflammatory response in hPDLSCs, together with increased levels of TNF-α, IL-6, IL-1β, Cox-2 and iNOS, and decreased levels of IL-10. ATF3 silencing also led to lower TBARS production rate, and reduced levels of reactive oxygen species, iron, Fe2+, ACSL4 and TFR1, whereas it elevated the levels of SLC7A11 and GPX4. In addition, ATF3 silencing promoted hPDLSC mineralization and cell differentiation, and elevated the levels of OCN2, RUNX2 and BMP2. Additionally, ATF3 depletion upregulated the expression levels of proteins related with Nrf2/HO-1 signaling. The Nrf2 inhibitor ML385 partially counteracted the effects of ATF3 interference on the LPS-challenged inflammatory response, lipid peroxidation, ferroptosis as well as osteogenic differentiative capability in hPDLSCs. In summary, the results revealed that ATF3 silencing suppressed inflammation and ferroptosis, while it improved osteogenic differentiation in LPS-induced hPDLSCs by regulating Nrf2/HO-1 signaling, which may provide promising therapeutic targets for the treatment of periodontitis.