LIPUS Regulates FOXO1 Phosphorylation to Promote Osteogenic Differentiation of Oxidatively Damaged Human Periodontal Stem Cells

TANG Li¹, PANG Hua², YANG Ke³, WANG Dong⁴, YAO Huan¹*

( ¹Department of Ultrasound, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; ²Department of Nuclear Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; ³Pediatric Research Institute, Children’s Hospital of Chongqing Medical University, National Clinical Research Center for ChildHealth and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science andTechnology Cooperation Base of Child Development and Critical Disorders, Chongqing Engineering Research Center of Stem CellTherapy, Chongqing 400014, China; ⁴Department of Ultrasound, Sichuan Provincial People’s Hospital (Affiliated Hospital ofUniversity of Electronic Science and Technology), Sichuan Medical College, Chengdu 610072, China)

Abstract:

This study was to investigate the protective effect and mechanism of LIPUS (low-intensity pulsed ultrasound) on osteogenic differentiation of oxidatively damaged hPDLSCs (human periodontal ligament stem cells). Thecellular oxidative damage model was constructed by using H2O2 at an action concentration of 300 µmol/L, whichwas divided into the control, H2O2, LIPUS and LIPUS+H2O2 groups. Cellular ROS (reactive oxygen species) andMDA (malondialdehyde) levels were detected by DCFH-DA probe and TBA colorimetric assay in each group, respectively. ALP (alkaline phosphatase) and alizarin red staining were used to assess the early and late osteogenicdifferentiation capacities of each group, respectively. WB (Western blot) evaluation of antioxidant enzymes [CAT(catalase) and SOD-2 (superoxide dismutase 2)], osteogenesis-related protein [Runx2 (runt-related transcriptionfactor-2), ALP and OPN (osteopontin)], FOXO1 (forkhead box protein O1) and p-FOXO1 (phospho-FOXO1).Subsequently, the cells were treated with FOXO1 inhibitor AS1842856 (100 nmol/L), and the experiments weredivided into control, H2O2+DMSO, LIPUS+H2O2+DMSO, and LIPUS+H2O2+AS1842856 groups. CCK-8 (cellcounting kit-8) assay detected cell viability, DCFH-DA probe detected the level of cellular ROS in each group, andWB detected the protein expression of RUNX2, ALP, and OPN in each group. Compared with the control group,both ROS and MDA levels were elevated and antioxidant enzyme protein expression was down-regulated in theH2O2 group (P<0.000 1); ALP and alizarin red positive staining as well as expression of osteogenesis-related proteinwere significantly reduced (P<0.000 1); and the p-FOXO1/FOXO1 protein ratio rose (P<0.000 1). Compared with theH2O2 group, the LIPUS+H2O2 group showed decreased ROS and MDA levels and upregulated expression of antioxidantenzyme proteins (P<0.000 1); ALP and alizarin red positive staining as well as protein expression of osteogenesis-relatedwere significantly increased (P<0.000 1); and p-FOXO1/FOXO1 protein ratio decreased (P<0.000 1). Compared withthe LIPUS+H2O2+DMSO group, the LIPUS+H2O2+AS1842856 group showed decreased cell viability, up-regulation ofp-FOXO1/FOXO1 protein expression, elevated ROS levels and significantly decreased protein expression of osteogenicdifferentiation (P<0.000 1). The results revealed that LIPUS exerted antioxidant defense and improved osteogenic differentiation of oxidatively damaged hPDLSCs by regulating the phosphorylation level of FOXO1.

CSTR: 32200.14.cjcb.2024.03.0005