Liu Chang, Lei Wanzhen, Zhang Lili, Zhang Chen, Gao Runtao, Jin Luyuan
Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China.
Department of General Dentistry and Integrated Emergency Dental Care, Beijing Stomatological Hospital, Capital Medical University, Beijing, China.
J Oral Rehabil. 2025 Mar;52(3):391-400. doi: 10.1111/joor.13918. Epub 2024 Dec 12.
Dental pulp stem cells (DPSCs) are widely used in research on dental tissue regeneration and systemic disease treatment. However, the oxidative microenvironment often causes cellular senescence, leading to decreased function. Our previous study demonstrated that pleiotrophin (PTN), a secreted extracellular matrix-associated protein, could rescue the proliferative capacity and osteogenic differentiation of replicative senescent DPSCs.
This study aimed to explore the influence and mechanism of PTN on dental pulp stem cells under HO-induced oxidative microenvironment.
DPSCs isolated from human third molars were treated with 100 μm HO for 4 h, mimicking the oxidative microenvironment. To investigate the influence of PTN on DPSC under HO-induced oxidative microenvironment, 50 pg/mL PTN was added in the culture medium for 48 h. RT-qPCR, western blotting, SA-β-gal staining, intracellular ROS production and immunofluorescence staining assays were used to analyse the cellular senescence, osteogenic differentiation capacity, oxidative stress conditions and possible mechanism.
HO treatment increased the ratio of SA-β-gal-positive DPSCs and upregulated the senescence-related gene expression, including P53, P21 and P16. PTN pretreatment downregulated the ratio of SA-β-gal-positive DPSCs and the expression of these genes. Besides, PTN pretreatment partially reversed the HO-induced decreased osteogenic differentiation potential of DPSCs, total antioxidant capacity and Nrf2 and HO-1 mRNA expression in DPSCs. Western blotting and immunofluorescent staining results indicated that PTN pretreatment enhanced the Nrf2 nuclear translocation under oxidative stress conditions and observable higher fluorescence signals in the nucleus denoted PTN and Nrf2 colocalisation. Western blotting results showed that PTN reversed the decreased expression of p-AKT in the HOinduced oxidative environment. However, the PI3K inhibitor LY294002 blocked the upregulated levels of total Nrf2. Immunofluorescence staining displayed that LY294002 also inhibited the nuclear translocation of Nrf2 which was enhanced under PTN pretreatment.
This study demonstrated that PTN could prevent senescent damage induced by HO on DPSCs, mainly by combining with Nrf2 and enhancing its nuclear translocation.
牙髓干细胞（DPSCs）广泛应用于牙组织再生研究及全身疾病治疗。然而，氧化微环境常导致细胞衰老，致使功能下降。我们之前的研究表明，多效生长因子（PTN），一种分泌型细胞外基质相关蛋白，可挽救复制性衰老DPSCs的增殖能力和成骨分化能力。
本研究旨在探讨PTN在过氧化氢（HO）诱导的氧化微环境下对牙髓干细胞的影响及其机制。
从人第三磨牙分离的DPSCs用100μM HO处理4小时，模拟氧化微环境。为研究PTN在HO诱导的氧化微环境下对DPSC的影响，在培养基中添加50 pg/mL PTN处理48小时。采用逆转录-定量聚合酶链反应（RT-qPCR）、蛋白质印迹法、衰老相关β-半乳糖苷酶（SA-β-gal）染色、细胞内活性氧（ROS）生成及免疫荧光染色分析细胞衰老、成骨分化能力、氧化应激状况及可能机制。
HO处理增加了SA-β-gal阳性DPSCs的比例，并上调了衰老相关基因表达，包括P53、P21和P16。PTN预处理下调了SA-β-gal阳性DPSCs的比例及这些基因的表达。此外，PTN预处理部分逆转了HO诱导的DPSCs成骨分化潜能降低以及DPSCs中总抗氧化能力、核因子E2相关因子2（Nrf2）和血红素加氧酶-1（HO-1）mRNA表达的降低。蛋白质印迹法和免疫荧光染色结果表明，PTN预处理增强了氧化应激条件下Nrf2的核转位，且细胞核中可观察到更高的荧光信号表明PTN与Nrf2共定位。蛋白质印迹法结果显示，PTN逆转了HO诱导的氧化环境中磷酸化蛋白激酶B（p-AKT）表达的降低。然而，磷脂酰肌醇-3激酶（PI3K）抑制剂LY294002阻断了总Nrf2上调水平。免疫荧光染色显示，LY294002也抑制了PTN预处理增强的Nrf2核转位。
本研究表明，PTN可预防HO对DPSCs诱导的衰老损伤，主要是通过与Nrf2结合并增强其核转位来实现。
