柚皮素调控叉头转录因子1/β-连环蛋白通路对过氧化氢诱导的人牙周膜干细胞氧化损伤的保护作用研究
Investigating the protective effect of naringenin on hydrogen peroxide induced oxidative damage of human periodontal ligament stem cells by regulating the forkhead box protein O-1/β-catenin pathway
作者：张丽, 彭世元, 唐飞扬, 蹇静薇, 袁硕声, 徐晓梅
Author：Zhang Li, Peng Shiyuan, Tang Feiyang, Jian Jingwei, Yuan Shuosheng, Xu Xiaomei
收稿日期：2024-12-24 年卷（期）页码：2025,43(4):559-559-569
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：牙周膜干细胞,叉头转录因子1/β-连环蛋白信号通路,氧化应激,柚皮素,成骨分化,
Key words：periodontal ligament stem cells,forkhead box protein O-1/β-catenin signaling pathway,oxidative stress,naringenin,osteogenic differentiation,
基金项目：四川省科技计划项目(2023ZYD0112);大学生创新创业项目(2023463);泸州市科技计划项目(2024LZXNYDJ041)
中文摘要
目的 探究柚皮素（NAR）对氧化应激状态下人牙周膜干细胞（hPDLSCs）成骨潜能的保护作用及其相关机制。 方法 用过氧化氢（H2O2）建立hPDLSCs氧化损伤模型，然后加入不同浓度的NAR及0.5 μmol/L的叉头转录因子1（FOXO1）抑制剂AS1842856对其进行干预。利用细胞计数试剂盒法（CCK8）筛选出H2O2及NAR的最适作用浓度。通过碱性磷酸酶（ALP）染色和实时荧光定量反转录聚合酶链反应（qRT-PCR）检测各组hPDLSCs中ALP、Runt相关转录因子2（RUNX2）、骨钙素（OCN）的表达情况，通过酶联免疫吸附试验（ELISA）和活性氧荧光探针（DCFH-DA）染色检测各组hPDLSCs中活性氧（ROS）、丙二醛（MDA）以及乳酸脱氢酶（LDH）的表达情况。同时，使用qRT-PCR和蛋白印迹法（Western blot）分别检测各组FOXO1和β-连环蛋白（β-catenin）的基因与蛋白表达水平。 结果 H2O2暴露导致hPDLSCs氧化损伤增加，细胞内ROS水平上升，MDA、LDH表达升高（P<0.05）；成骨分化降低，ALP染色变浅和成骨分化相关基因ALP、RUNX2、OCN的信使核糖核酸（mRNA）表达水平降低（P<0.05）。NAR共处理可缓解H2O2所致的hPDLSCs氧化损伤并提升其抗氧化能力、恢复其成骨能力。FOXO1抑制剂AS1842856可下调β-catenin的表达（P<0.05），明显减弱NAR的抗氧化作用及恢复成骨的能力（P<0.05）。 结论 NAR可以通过激活hPDLSCs内的FOXO1/β-catenin信号通路从而提升hPDLSCs的抗氧化能力，减轻氧化应激损伤及其所致的成骨能力丧失。
英文摘要
ObjectiveInvestigating the protective effect of naringenin (NAR) on the osteogenic potential of human periodontal ligament stem cells (hPDLSCs) under oxidative stress and its related mechanisms.MethodsThe oxidative damage model of hPDLSCs was established using hydrogen peroxide (H2O2) andthe hPDLSCs were treated with different concentrations of NAR and 0.5 μmol/L forkhead box protein O-1 (FOXO1) inhibitor AS1842856. After that, the cell counting kit-8 (CCK8) was used to determine the optimal concentrations of H2O2and NAR. The alkaline phosphatase (ALP) staining and real time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to assess the expression of ALP, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) in hPDLSCs of each group. The enzyme-linked immunosorbent assay (ELISA) and 2’,7’-dichlorofluorescin diacetate (DCFH-DA) staining were utilized to evaluate the expression of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in hPDLSCs. Meanwhile, qRT-PCR and western blot were used to detect the expression levels of FOXO1 and β-catenin, both are pathway related genes and proteins.ResultsH2O2exposure led to an increase in oxidative damage in hPDLSCs, characterized by a rise in intracellular ROS levels and increased expression of MDA and LDH (P< 0.05). At the same time, the osteogenic differentiation ability of hPDLSCs decreased, as evidenced by lighter ALP staining and reduced expression levels of osteogenic differentiation-related genes ALP, RUNX2 and OCN (P< 0.05). Co-treatment with NAR alleviated the oxidative damage in hPDLSCs, enhanced their antioxidant capacity, and restored their osteogenic ability. The FOXO1 inhibitor AS1842856 downregulated the expression of β-catenin (P< 0.05) and significantly diminished both the antioxidant effect of NAR and its ability to restore osteogenesis (P< 0.05).ConclusionNAR can enhance the antioxidant capacity of hPDLSCs by activating the FOXO1/β-catenin signaling pathway within hPDLSCs, thereby mitigating oxidative stress damage and alleviating the loss of osteogenic capacity.
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