硫氧还蛋白相互作用蛋白介导的炎症稳态失衡在牙周膜干细胞骨向分化调控中的作用

doi:10.3969/j.issn.1006-7795.2025.06.007

首都医科大学学报 ›› 2025, Vol. 46 ›› Issue (6): 1000-1010.doi: 10.3969/j.issn.1006-7795.2025.06.007

硫氧还蛋白相互作用蛋白介导的炎症稳态失衡在牙周膜干细胞骨向分化调控中的作用

崔雨¹ ，张萍¹，孙毓徽¹，伍爵妃¹，昝佳琦¹，丁晓玲^2*，丁刚^1*

1.山东第二医科大学口腔医学院，山东潍坊 261053；2. 山东第二医科大学临床能力培训中心，山东潍坊 261053

收稿日期:2025-08-13 修回日期:2025-09-20 出版日期:2025-12-21 发布日期:2025-12-19
通讯作者: 丁晓玲, 丁刚 E-mail:dinggang@sdsmu.edu.cn，wfyxydxl@163.com
基金资助:
山东省自然科学基金项目(ZR2024MH147, ZR2023QH506)，潍坊市鸢都学者项目(ydxz2023002)。

Effects of thioredoxin-interacting protein-mediated inflammatory homeostatic imbalance in regulating the osteogenic differentiation of periodontal ligament stem cells

Cui Yu¹, Zhang Ping¹, Sun Yuhui¹, Wu Juefei¹, Zan Jiaqi¹, Ding Xiaoling^2*, Ding Gang^1*

1.School of Stomatology, Shandong Second Medical University, Weifang 261053, Shandong Province, China; 2. Clinical Competency Training Center, Shandong Second Medical University, Weifang 261053, Shandong Province, China

Received:2025-08-13 Revised:2025-09-20 Online:2025-12-21 Published:2025-12-19
Supported by:
This study was supported by Natural Science Foundation of Shandong Province (ZR2024MH147, ZR2023QH506), Weifang Kite Capital Scholars Program (ydxz2023002).

摘要/Abstract

摘要： 目的探讨硫氧还蛋白相互作用蛋白 (thioredoxin-interacting protein, TXNIP)在脂多糖(lipopolysaccharide, LPS)诱导的炎症稳态失衡条件下对牙周膜干细胞(periodontal ligament stem cells, PDLSCs)骨向分化能力的影响及潜在稳态调控机制。方法分离培养人PDLSCs，并进行鉴定，使用细胞计数试剂盒(cell counting kit-8, CCK-8)和实时荧光定量反转录聚合酶链式反应(real time fluorescence quantitative reverse transcription polymerase chain reaction, RT-qPCR) 筛选TXNIP 抑制剂 SRI37330 的最适浓度，以PDLSCs 为研究对象，分为Blank组(正常培养)、SRI37330组(正常培养条件下加入 SRI37330)、LPS 组(正常培养条件下加入LPS)、LPS+SRI37330组(LPS+SRI37330)、LPS+成骨诱导液(osteogenic medium, OM) 组(LPS+OM)、LPS+OM+ SRI37330组。碱性磷酸酶(alkaline phosphatase, ALP)染色和活性检测各组早期矿化水平，茜素红S法(alizarin red S, ARS)染色检测各组矿化基质的形成情况，RT-qPCR和蛋白质印迹(Western blotting, WB)检测骨向分化相关基因和蛋白的表达水平。RNA转录组测序分析TXNIP调控PDLSCs炎症－成骨稳态转换这一过程的潜在信号通路。结果成功培养出生长状态良好的PDLSCs。流式细胞术证实PDLSCs表达间充质干细胞表面标志物，不表达造血谱系表面标志物。根据CCK8和RT-qPCR结果，选取浓度为0.6 μmol/L的SRI37330用于后续实验。ALP染色、ALP活性检测、ARS染色、RT-qPCR和Western boltting 结果表明，LPS诱导的炎症稳态失衡可显著抑制PDLSCs成骨向分化，而加入TXNIP抑制剂SRI37330后则能明显逆转PDLSCs的骨向分化能力。RNA转录组测序分析表明，TXNIP抑制剂SRI37330可能通过mTOR信号通路和AMPK信号通路，促进PDLSCs在LPS诱导的炎症刺激下的成骨分化稳态的重建。结论在炎症稳态失衡状态下，TXNIP可能通过mTOR 通路和AMPK通路来抑制 PDLSCs 的成骨分化能力，靶向调控TXNIP有助于恢复炎症微环境下的骨稳态，可为稳态医学视角下的牙周组织再生提供新的治疗策略。

关键词: 牙周膜干细胞, 炎性微环境, 骨向分化, 硫氧还蛋白相互作用蛋白, 脂多糖, SRI37330, 稳态医学

Abstract: Objective To investigate the effects of thioredoxin-interacting protein (TXNIP) on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) under lipopolysaccharide (LPS)-induced inflammatory homeostatic imbalance, and to explore potential homeostasis regulatory mechanisms. Methods Human PDLSCs were isolated, cultured, and identified. The optimal concentration of the TXNIP inhibitor SRI37330 was determined using cell counting kit-8 (CCK-8) method and real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). PDLSCs were cultured under Blank group (normal culture), SRI37330 group (addition of SRI37330 to normal culture medium), LPS group (LPS added under conventional culture conditions), LPS + SRI37330 group (LPS-treated PDLSCs with SRI37330), LPS + osteogenic medium (OM) group (LPS-treated PDLSCs with OM), and LPS + OM + SRI37330 group (LPS-treated PDLSCs with both osteogenic medium and SRI37330). Alkaline phosphatase (ALP) staining and activity assays were used to assess early mineralization levels in each group, and alizarin red S (ARS) staining were employed to evaluate mineralized matrix formation. RT-qPCR and Western blotting (WB) were employed to analyses measure expression levels of osteogenic-related genes and proteins. The potential signaling pathways that TXNIP regulates the inflammatory-osteogenic homeostasis transition in PDLSCs were analyzed with RNA transcriptome sequencing. Results PDLSCs with favorable morphology and growth status were successfully cultured. Flow cytometry validated that these cells were positive for mesenchymal stem cell markers, and negative for hematopoietic markers. Based on CCK8 assays and RT-qPCR results, SRI37330 with a concentration of 0.6 μmol/L was selected to treat PDLSCs in subsequent experiments. ALP staining, ALP activity detection, ARS staining, RT-qPCR, and WB results showed that LPS-induced inflammatory homeostatic imbalance could inhibit the osteogenic differentiation of PDLSCs, while the addition of TXNIP inhibitor SRI37330 could significantly reverse the inhibition of osteogenic differentiation ability of PDLSCs. RNA transcriptome sequencing analysis showed that SRI37330 may promote the reconstruction of osteogenic homeostasis in PDLSCs under inflammatory stimulation by regulating the mTOR and AMPK signaling pathway. Conclusion Under inflammatory homeostatic imbalance, TXNIP may inhibit the osteogenic differentiation ability of PDLSCs through the mTOR and AMPK pathway. Targeted regulation of TXNIP contributes to the restoration of bone homeostasis in inflammatory microenvironments and provides a new therapeutic strategy for periodontal tissue regeneration from the perspective of homeostasis medicine.

Key words: periodontal ligament stem cells, inflammatory microenvironment, osteogenic differentiation, thioredoxin-interacting protein, lipopolysaccharide, SRI37330, homeostasis medicine

中图分类号:

崔雨, 张萍, 孙毓徽, 伍爵妃, 昝佳琦, 丁晓玲, 丁刚. 硫氧还蛋白相互作用蛋白介导的炎症稳态失衡在牙周膜干细胞骨向分化调控中的作用[J]. 首都医科大学学报, 2025, 46(6): 1000-1010.

Cui Yu, Zhang Ping, Sun Yuhui, Wu Juefei, Zan Jiaqi, Ding Xiaoling, Ding Gang. Effects of thioredoxin-interacting protein-mediated inflammatory homeostatic imbalance in regulating the osteogenic differentiation of periodontal ligament stem cells [J]. Journal of Capital Medical University, 2025, 46(6): 1000-1010.

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硫氧还蛋白相互作用蛋白介导的炎症稳态失衡在牙周膜干细胞骨向分化调控中的作用

Effects of thioredoxin-interacting protein-mediated inflammatory homeostatic imbalance in regulating the osteogenic differentiation of periodontal ligament stem cells

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