LIPUS Regulates FOXO1 Phosphorylation to Promote Osteogenic Differentiation of Oxidatively Damaged Human Periodontal Stem Cells
This study was to investigate the protective effect and mechanism of LIPUS(low-intensity pulsed ul-trasound)on osteogenic differentiation of oxidatively damaged hPDLSCs(human periodontal ligament stem cells).The cellular oxidative damage model was constructed by using H2O2 at an action concentration of 300 μmol/L,which was divided into the control,H2O2,LIPUS and LIPUS+H2O2 groups.Cellular ROS(reactive oxygen species)and MDA(malondialdehyde)levels were detected by DCFH-DA probe and TBA colorimetric assay in each group,re-spectively.ALP(alkaline phosphatase)and alizarin red staining were used to assess the early and late osteogenic differentiation 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 transcription factor-2),ALP and OPN(osteopontin)],FOXO1(forkhead box protein O1)and p-FOXO1(phospho-FOXO1).Subsequently,the cells were treated with FOXO1 inhibitor AS 1842856(100 nmol/L),and the experiments were divided into control,H2O2+DMSO,LIPUS+H2O2+DMSO,and LIPUS+H2O2+AS1842856 groups.CCK-8(cell counting kit-8)assay detected cell viability,DCFH-DA probe detected the level of cellular ROS in each group,and WB 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 the H2O2 group(P＜0.000 1);ALP and alizarin red positive staining as well as expression of osteogenesis-related protein were significantly reduced(P＜0.000 1);and the p-FOXO1/FOXO1 protein ratio rose(P＜0.000 1).Compared with the H2O2 group,the LIPUS+H2O2 group showed decreased ROS and MDA levels and upregulated expression of antioxidant enzyme proteins(P＜0.000 1);ALP and alizarin red positive staining as well as protein expression of osteogenesis-related were significantly increased(P＜0.000 1);andp-FOXO1/FOXO1 protein ratio decreased(P＜0.000 1).Compared with the LIPUS+H2O2+DMSO group,the LIPUS+H2O2+AS1842856 group showed decreased cell viability,up-regulation of p-FOXO1/FOXO1 protein expression,elevated ROS levels and significantly decreased protein expression of osteogenic differentiation(P＜0.000 1).The results revealed that LIPUS exerted antioxidant defense and improved osteogenic differen-tiation of oxidatively damaged hPDLSCs by regulating the phosphorylation level of FOXO1.
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