Pleiotrophin Prevents H2O2-Induced Senescence of Dental Pulp Stem Cells.

Abstract

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 H2O2-induced oxidative microenvironment. DPSCs isolated from human third molars were treated with 100 μm H2O2 for 4 h, mimicking the oxidative microenvironment. To investigate the influence of PTN on DPSC under H2O2-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. H2O2 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 H2O2-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 H2O2-induced 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 H2O2 on DPSCs, mainly by combining with Nrf2 and enhancing its nuclear translocation.