The Effects Of Y-27632 On Proliferation,Apoptosis,Migration,Differentiation And Stemness Maintenance Of Human Periodontal Ligament Stem Cells

Background and Objectives:Y-27632 is related Rho kinase inhibitor and involves in various cellular functions that include actin cytoskeleton organization,cell proliferation,cell migration,cell apoptosis and cell differentiation.Since then,a number of studies indicate that Y-27632 promotes the proliferation,differentiation and pluripotency of embryonic stem cells,adipose-derived stem cells and prostate stem cells.And other studies have demonstrated that Y-27632 can promote the migration of dental pulp cells.These results may indicate the possibility of application for periodontal tissue regeneration in some degree.Periodontal ligament stem cells(PDLSCs)are adult mesenchymal stem cells isolated from human periodontal tissue,have multilineage differentiation potential and can differentiate into osteoblasts,cementoblast-like cells and fibroblasts,and play an important role in periodontal tissue regeneration and tissue repair.Thus they are regarded as candidates for periodontal tissue regeneration.The purpose of this study was to evaluate the effects of different concentrations of Y-27632 on biological characteristics of cell proliferation,apoptosis,migration,differentiation and pluripotency of PDLSCs in vitro,and to explore the mechanism of Y-27632 promoting PDLSCs proliferation and to provide a new strategy for periodontal therapy.Methods:(1)Cultivation of human PDLSCs:human PDLSCs were harvested from healthy periodontal ligament tissue of the premolars or the third molar extracted for orthodontic reasons by limiting dilution assay.PDLSCs were subcultured at confluence.PDLSCs from the third to fifth passage were used for subsequent experiments.(2)Cell viability:the effects of Y-27632 on the proliferation and cell viability of PDLSCs were detected by cell-counting kit-8(CCK8),5-ethynyl-2'-deoxyuridine(EdU)labeling assay and colony forming efficiency assay.(3)Cell apoptosis:the effect of Y-27632 on cell apoptosis was measured by flow cytometry after annexin V-FITC and propidium iodide(PI)staining.(4)Cell migration and wound-healing assays:the effects of Y-27632 on cell migration and wound-healing ability were assessed by transwell chemotaxis assay and wound-healing assay.(5)Osteogenic differentiation:ALP activity after 7,14 days with or without Y-27632 stimulation was detected by ALP activity assay kit.The relative amount of calcium nodules and calcium deposition after 28 days were evaluated by Alizarin Red S staining and cetylpyridinium chloride(CPC)colorimetry,respectively.Osteogenesis-related genes ALP and runt related transcription factor 2(Runx2)of PDLSCs with Y-27632 treatment were detected by qRT-PCR.(6)Adipogenic differentiation:the amount of lipid droplet after 21 days with Y-27632 treatment was assessed by Oil Red O staining and isopropyl alcohol colorimetry.Adipogenesis-related genes peroxisome proliferator-activated receptor y(PPARy)and lipoprotein lipase(LPL)of PDLSCs with Y-27632 treatment were detected by qRT-PCR.(7)Pluripotency:the stemness-ralated genes expression levels of c-Myc,Nanog,Klf4 and Oct4 with the treatment of Y-27632 were detected by qRT-PCR.(8)Mechanism of cells proliferation:the effect of extracellular signal-regulated kinase(ERK)inhibitor U0126 on Y-27632-induced proliferation of PDLSCs was detected.Afterwards,western blot analysis was performed to explore the activation of Y-27632 on ERK and p38 signal pathway.(9)Statistical analysis:data was analyzed using SPSS 19.0 with differences between groups assessed by one-way ANOVA.Statistical probability of P<0.05 was considered significant.Results:(1)PDLSCs were successfully harvested and cultured in a good condition.(2)CCK8 assay and EdU labeling assay showed that 10 ?M and 20 ?M Y-27632 promoted the proliferation of PDLSCs(P<0.05),and cell proliferation peaked at the concentration of 20 ?M(P<0.01).The result of colony-forming efficiency assay showed that there was no significant difference between control group and experimental groups(P>0.05).However,the sizes of single cell-derived colonies in experimental groups were larger than that in control group,and cells in experimental groups exhibited star-like morphology.(3)The effect of Y-27632 on PDLSCs apoptosis was analyzed using flow cytometry analysis.The result showed that Y-27632 had no significant effect on PDLSCs apoptosis.(4)The chemotactic capability of Y-27632 on PDLSCs was quantified with transwell migration assay and wound-healing assay.The results indicated that Y-27632 enhanced migration and wound-healing capability of PDLSCs(P<0.05).(5)The result of ALP activity assay showed that 10?M and 20?M Y-27632 significantly inhibited ALP activity compared with negative control group after 7 and 14 days(P<0.01).The results of Alizarin Red S staining and CPC colorimetry indicated that Y-27632 significantly decreased the amount of calcium nodules and inhibited calcium deposition of PDLSCs(P<0.01).The result of qRT-PCR showed that treatment of PDLSCs with Y-27632 significantly decreased the expression of ALP and Runx2 at day 14(P<0.01).(6)The results of Oil Red O staining and isopropyl alcohol colorimetry indicated that Y-27632 significantly promoted the formation of lipid droplets compared with control group(P<0.01).The result of qRT-PCR showed that treatment of PDLSCs with Y-27632 significantly increased the expression of PPARy and LPL at day 14 and 21(P<0.05)(7)The result of qRT-PCR showed that the gene expression levels of c-Myc,Nanog,Klf4 and Oct4 with the treatment of Y-27632 were significantly higher than that in control group at day 3 and 7(P<0.05).(8)ERK signal pathway involved in Y-27632-induced proliferation of PDLSCs.The result of CCK8 indicated that Y-27632-induced growth of PDLSCs was significantly inhibited by the addition of ERK inhibitor U0126(P<0.05).The result of western blot assay showed that Y-27632 up-regulated phosphorylation of ERK after stimulation.Moreover,phosphorylation level of ERK decreased by the addition of U0126.However,Y-27632 had no effect on p38 phosphorylation(P>0.05).These results indicated that ERK rather than p38 signaling pathway involved in Y-27632-induced proliferation of PDLSCs.Conclusions:In this study,we demonstrated that Y-27632 enhanced the proliferation and migration of PDLSCs.Moreover,Y-27632 maintained the sternness and enhanced the formation of fat droplets while significantly inhibited ALP activity and the formation of mineral deposition.Furthermore,we demonstrated that the basic molecular mechanism for Y-27632-induced cell proliferation was ERK pathway.Therefore,we speculated that Y-27632 might have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site,and then enhancing the proliferation of these cells and maintaining their pluripotency.