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Abstract：
Periodontitis is a complex dental condition that has a number of different etiologies. Lin-28 homolog A (LIN28A) has been previously reported to regulate inflammation, where its expression levels have been indicated to be lower in periodontal tissues following periodontitis. However, there is a lack of evidence to indicate the precise role of LIN28A in periodontitis. In the present study, LIN28A and Runt-related transcription factor 2 (RUNX2) expression were measured in human periodontal biopsy tissues using reverse transcription-quantitative PCR (RT-qPCR). RT-qPCR and western blot analyses were also used to measure LIN28A and RUNX2 expression in human periodontal ligament stem cells (hPDLSCs) following lipopolysaccharide (LPS) induction. Following construction of the LIN28A overexpression plasmid, the expression of LIN28A, RUNX2, osteopontin, osterix and osteocalcin were detected using RT-qPCR and western blotting. Additionally, RT-qPCR was used for the detection of proinflammatory biomarkers (IL-8, IL-1β and IL-6) and alkaline phosphatase (ALP) expression. Protein expression of intranuclear and cytoplasmic NF-κB p65 and NF-κB p65 phosphorylation were assessed using western blot analysis. The expression of antioxidant factors including SOD and GSH were determined using corresponding commercial assay kits. ALP and the mineralization capacity of hPDLSCs were detected by ALP activity assay and Alizarin red staining. The expression of LIN28A was found to be decreased in periodontal biopsy tissues from periodontitis patients compared with normal tissues and LPS-induced hPDLSCs compared with untreated hPDLSCs, which was positively correlated with RUNX2 expression. LIN28A overexpression was revealed to attenuate inflammatory damage and oxidative stress whilst improving ALP active damage, restoring RUNX2 expression and osteoblastic mineralization in LPS-induced hPDLSCs. In conclusion, the present study suggests that LIN28A serves a key role as a mediator of osteoblast differentiation and mineralization. In addition, LIN28A was able to alleviate inflammatory injury and oxidative stress in LPS-induced hPDLSCs.
摘要：
牙周炎是一种复杂的牙科疾病，具有许多不同的病因。Lin-28同源物A（LIN28A）先前已被报道调节炎症，其中已表明其在牙周炎后的牙周组织中的表达水平较低。然而,缺乏证据表明LIN28A在牙周炎中的确切作用。在本研究中,使用逆转录定量PCR（RT-qPCR）在人牙周活检组织中测量LIN28A和Runt相关转录因子2（RUNX2）的表达。RT-qPCR和蛋白质印迹分析也用于测量脂多糖(LPS)诱导后人牙周膜干细胞(hPDLSC)中的LIN28A和RUNX2表达。在构建LIN28A过表达质粒后，LIN28A的表达，RUNX2骨桥蛋白,使用RT-qPCR和蛋白质印迹法检测osterix和骨钙蛋白。此外,RT-qPCR用于检测促炎生物标志物(IL-8、IL-1β和IL-6)和碱性磷酸酶(ALP)表达。使用蛋白质印迹分析评估核内和细胞质NF-κBp65的蛋白表达和NF-κBp65磷酸化。使用相应的商业测定试剂盒测定包括SOD和GSH的抗氧化因子的表达。通过ALP活性测定和茜素红染色检测hPDLSCs的ALP和矿化能力。与正常组织相比，在牙周炎患者的牙周活检组织中发现LIN28A的表达降低，与未治疗的hPDLSCs相比，LPS诱导的hPDLSCs的表达降低，与RUNX2表达呈正相关。LIN28A过表达可减轻炎症损伤和氧化应激，同时改善ALP活性损伤，在LPS诱导的hPDLSCs中恢复RUNX2表达和成骨细胞矿化。总之,本研究提示LIN28A作为成骨细胞分化和矿化的介质发挥关键作用.此外,LIN28A能够减轻LPS诱导的hPDLSCs的炎症损伤和氧化应激。
