The Effect Of Ebselen On The Osteogenic Differentiation Of Periodontal Ligament Stem Cells And Osteoclast Differentiation In Peripheral Blood Under High Glucose Environment

Objective: Diabetes and periodontitis are diseases with high prevalence worldwide and a bidirectional relationship exists.Periodontal ligament stem cells(PDLSCs)play an important role in periodontal tissue regeneration.In this experiment,we investigated the effects of Ebselen on the osteogenic differentiation of periodontal stem cells and the differentiation of peripheral blood osteoblasts in a high glucose environment,with the aim of providing theoretical guidance and therapeutic targets for the application of Ebselen in bone and periodontal tissue regeneration.Methods: PDLSCs were cultured and purified by limited dilution method.Cell proliferation activity was detected by CCK-8 after adding different concentrations(0,1,5,10,20 μM)of Ebselen.25 mmol/L glucose was used to simulate a high glucose microenvironment,and PDLSCs were induced to construct an oxidative damage model,and the transmission electron microscope ultrastructure of mitochondria was observed,reactive oxygen species(ROS)levels were measured by fluorescent H2 DCFDA.Alkaline phosphatase(ALP)staining and alizarin red staining were used to detect the level of osteogenic differentiation.Detection of osteogenesis related gene expression level by real-time fluorescence quantitative PCR.Western blotting was used to detect the expression of osteogenesis-related proteins and Glutathione peroxidase glutathione peroxidase 4(GPX4).Peripheral blood mononuclear cell(PBMC)were isolated by Ficoll method and induced to differentiate into mature osteoclasts,which were identified by tartrate-resistant acid phosphatase(TRAP)Acid phosphatase.The effects of different concentrations of Ebselen on osteoclast formation were observed by TRAP staining,and the expression of osteoclast-related genes was detected by real-time quantitative PCR.Graph Pad Prism 9 and SPSS 25.0 were used for statistical analysis of the experimental data.Results: 1.The cultured human PDLSCs were long fusiform cells.High glucose could induce the changes of mitochondrial morphology and Ros level in PDLSCs,inhibit the osteogenic differentiation of PDLSCs and decrease the expression of GPX4 protein.Ebselen promoted the proliferation of PDLSCs,increased the intracellular ALP level and calcium deposition,and promoted the expression of osteogenic related genes and proteins COL1 and Runx2(P < 0.05),furthermore,Ebselen restored the expression of COL1,Runx2 and GPX4 in PDLSCs with high glucose(P<0.05).2.Peripheral blood mononuclear cells were isolated from peripheral blood and induced into osteoclasts.Tartrate-resistant acid phosphatase(TRAP)staining showed a large number of multinucleated cells.The results of TRAP staining showed that Ebselen inhibited the formation of osteoclasts.The results of PCR showed that Ebselen inhibited the expression of TRAP,CTSK and MMP-9 m RNA,and the inhibition was more obvious with the increase of Ebselen concentration(P<0.05).Conclusion: 1.Ebselen may protect PDLSCs from oxidative stress induced by high glucose by increasing the activity of GPX4,thus restoring the osteogenic function of cells.2.Ebselen can inhibit osteoclast differentiation by inhibiting the expression of osteoclast-related genes.