| Background and Purpose:Human periodontal ligament stem cells(hPDLSCs)are the dominant seed cells for oral tissue regeneration.Stem cells undergo replicative senescence during long-term in vitro expansion.Intracellular accumulation of reactive oxygen species(ROS)would be promoted in this process,which can impair stem cell function;Exogenous ROS stimulation can also induce premature senescence under oxidative stress,which can seriously affect tissue engineering efficiency.Numerous studies have shown that the characteristics of oxidative stress-induced senescence are the same as those of replicative senescence.Melatonin(MT)is a powerful free radical scavenger that can play a role in delaying the aging of hPDLSCs,but its ability to maintain the stemness properties of stem cells under oxidative stress conditions is inconclusive.The aim of this experiment was to observe the effects of hydrogen peroxide(H2O2)induced oxidative stress on the biological properties of hPDLSCs such as apoptosis,senescence,and stemness.And to explore whether MT plays a role in reversing the effects above.Finally,we will investigate the possible molecular mechanisms.Methods:1.Isolation,culture and surface markers identification of hPDLSCs:Primary cells obtained by tissue block method,and expression of cell surface markers were identified by flow analysis;After osteogenic and lipogenic induction,the cells were detected to verify whether they have ability of multi-directional differentiation.2.Construction of cell model with oxidative stress:hPDLSCs were pretreated with gradient concentration of H2O2 and cell activity was examined by Cell Counting Kit-8(CCK-8);Changes in cell morphology of each subgroup were observed under microscope;Levels of apoptosis and intracellular reactive oxygen species(ROS)were detected by flow cytometry;The proportion of ROS-positive cells was observed under fluorescence microscope.3.Effects of oxidative stress on senescence and stemness of hPDLSCs:Detect changes in expression levels of genes related to cell senescence and stemness by qRT-PCR and Western blot;Detect exterior manifestation of cell senescence with senescenceassociated β-galactosidase(SA-β-Gal)staining;After adding osteogenic inducers,the osteogenic differentiation levels of cells in the experimental group were determined by alkaline phosphatase(ALP)and alizarin red staining(ARS)to identify whether the level of osteogenic differentiation was weakened;Finally,confirm the working concentration of H2O2.4.The protective effects of MT on the biological characteristics of hPDLSCs under oxidative stress:Observe cell morphology,CCK-8 was used to detect the proliferative activity of cells with gradient concentrations of MT;Use flow cytometry to detect the changes of distribution of cell cycle;Detect intracellular ROS content and changes in the level of cell senescence and stemness-related indicators.Determine whether osteodifferentiation ability of hPDLSCs is restored.5.YAP mediates the above regulatory role of MT:YAP expression levels were measured throughout the study.YAP protein inhibitor Verteporfin(VP)was added to the above study.Senescence and stemness-related indicators were measured after antagonizing YAP.Results:1.The primary cells of hPDLSCs were obtained by tissue block adherence method and expression of their surface makers was consistent with mesenchymal stem cells.Moreover,the cells had abilities of multi-directional differentiation as well as strong cloning formation capability.2.After pretreatment of hPDLSCs with H2O2 for 2 hours,the cell proliferation activity was inversely proportional to the concentration.Part of the cells were apoptotic at high concentration,and the remaining cells were larger and fibroblast-like.Flow cytometry assay showed that the apoptosis rate was proportional to the concentration of H2O2.3.The ROS content in hPDLSCs was proportional to the H2O2 concentration via flow cytometry analysis.Under fluorescence microscopy,the fluorescence intensity and proportion of ROS(+)cells increased simultaneously with the concentration,which proved that the oxidative stress cell model had been successfully constructed.4.The mRNA and protein expression levels of P21,a cellular senescence-related gene,increased with the enhanced level of oxidative stress,while p-RB and genes related to cell stemness properties(C-MYC、OCT-4、NANOG)showed the opposite trend.SAβ-Gal positive staining ratio corroborated that the cells underwent premature oxidative stress.ALP and ARS staining demonstrated that the level of osteogenic differentiation of hPDLSCs were sharply declined under oxidative stress.5.Gradient concentration of MT had a positive effect on the proliferation of hPDLSCs,Besides,flow cytometry analysis showed a decrease in intracellular ROS content and a simultaneous decrease in fluorescence intensity and percentage of ROS-positive cells.6.In the H2O2+MT group,the features of senescence mitigated,the stemness and the osteo-differentiation ability was restored.Flow cytometry assay showed that MT reduced the proportion of cell cycle arrest in GO/G1 phase under oxidative stress.7.YAP protein expression level decreased after 200 μM H2O2 treatment,while the inhibited YAP protein level was partially restored after the addition of MT co-treatment.8.Lower concentrations of VP had no significant effect on the proliferation of hPDLSCs,and the inhibitory effects of H2O2 and VP on YAP protein expression were superimposed.After VP antagonized YAP,H2O2+MT+VP group showed elevated levels of cellular senescence with a subsequent decrease in stemness compared to H2O2+MT group.The protective effects of MT on the biological properties of oxidativestress induced hPDLSCs were demonstrated to be mediated through YAP proteins.Conclusion:H2O2-induced oxidative stress caused premature senescence and reduced stemness of hPDLSCs.Moreover,it exerted negative regulatory effects on cell proliferation,cycle,and ability of osteogenic differentiation.However,MT can effectively alleviate or even reverse these effects,in which YAP proteins plays an important role. |
PDF Full Text Request