Guo Ling, Li Liang
Stomatology Clinic, Meizhou People's Hospital, Meizhou Academy of Medical Sciences, Meizhou, Guangdong 514000, P.R. China.
Department of Stomatology, Xiangfang General Hospital, Heilongjiang Provincial Hospital, Harbin Institute of Technology, Harbin, Heilongjiang 150000, P.R. China.
Exp Ther Med. 2022 Jun;23(6):411. doi: 10.3892/etm.2022.11338. Epub 2022 Apr 27.
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.
牙周炎是一种具有多种不同病因的复杂牙科疾病。先前有报道称，Lin-28同源物A（LIN28A）可调节炎症，且其表达水平在牙周炎后的牙周组织中有所降低。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）检测人牙周活检组织中LIN28A和 runt相关转录因子2（RUNX2）的表达。RT-qPCR和蛋白质印迹分析还用于检测脂多糖（LPS）诱导后人牙周膜干细胞（hPDLSCs）中LIN28A和RUNX2的表达。构建LIN28A过表达质粒后，使用RT-qPCR和蛋白质印迹法检测LIN28A、RUNX2、骨桥蛋白、osterix和骨钙素的表达。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）的表达。使用蛋白质印迹分析评估核内和细胞质NF-κB p65的蛋白质表达以及NF-κB p65磷酸化。使用相应的商业检测试剂盒测定包括超氧化物歧化酶（SOD）和谷胱甘肽（GSH）在内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，牙周炎患者牙周活检组织中的LIN28A表达降低，与未处理的hPDLSCs相比，LPS诱导的hPDLSCs中的LIN28A表达降低，这与RUNX2表达呈正相关。结果显示，LIN28A过表达可减轻炎症损伤和氧化应激，同时改善LPS诱导的hPDLSCs中的ALP活性损伤，恢复RUNX2表达和成骨细胞矿化。总之，本研究表明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中的炎症损伤和氧化应激。
