Effect Of Vaspin On Hyperglycemia Induced Periodontal Ligament Stem Cells Proliferation And Osteogenic Differentiation Via The TGF-β1/Smad Signaling Pathway

Objective: Periodontal disease is characterized by alveolar bone loss and progressive tooth loss,which is the sixth major complication of diabetes.Periodontal ligament stem cells(PDLSCs)have been considered as promising candidates for repairing alveolar bone defect and mediating regeneration of periodontium.Visceral adipose tissue derived serine protease inhibitor(Vaspin)participates in various pathological processes in the body,such as inflammatory reaction,bone metabolism and glucose metabolism.The expression level of vaspin in periodontal tissue is high in periodontitis patients,and effectively reduced after initial therapy of periodontal diseases.It may be involved in the process of inhibiting PDLSCs from repairing periodontal tissue under high glucose and inflammatory microenvironment.The purpose of this study was to investigate whether vaspin alleviates the inhibition of high glucose on the proliferation and osteogenic differentiation of PDLSCs,and to preliminarily explore the underlining mechanisms.Methods:1.PDLSCs were isolated and cultured by tissue cultivation method,then purified by limited dilution method.Cell morphology and colony-forming ability were observed by light microscope.After directional induction,osteogenic and adipogenic staining were detected to further confirm their stem cell properties.2.Cell-counting kit-8(CCK8)was used to determine cell proliferation of PDLSCs incubated in medium supplemented with of different concentrations of vaspin(0,10,20,50,100,200 ng /m L).The effects of vaspin on the proliferation of PDLSCs were assessed in medium under 5.5m M glucose(NG group),NG plus vaspin,25 m M glucose(HG group)and HG plus vaspin conditions respectively.Alkaline phosphatase(ALP)activity,ALP staining and alizarin red staining were detected to evaluate Osteogenesis.Quantitative Real-time PCR and western blot were used to detect the expression of osteogenic related genes and proteins.3.Quantitative Real-time PCR and western blot experiments were conducted to detect TGF-β1/SMAD signal pathway related gene and protein expression for the above four experiment groups.Then inhibitor of the transforming growth factor-beta1(TGF-β1)type I receptor(SB431542)was administered to PDLSCs,osteogenesis was detected by Quantitative Real-time PCR and western blot.Statistical analysis of experimental data was performed using SPSS 26.0 software,and P<0.05 was considered statistically significant.Results:1.The PDLSCs were typical fibroblast like spindle-shape.Crystal violet staining suggested that PDLSCs displayed certain colony-forming ability.After osteogenic and adipogenic induction for 3 weeks,obvious mineralized nodules and lipid droplets were formed.2.Different concentrations of vaspin promoted PDLSCs proliferation ability in a dose-dependent manner,and the concentration of 100 ng/m L vaspin exhibited the best effect.Notably,high glucose significantly suppressed the proliferation of PDLSCs(P<0.05),but 100 ng/m L vaspin can alleviate inhibitory effects.100 ng/m L vaspin dramatically enhanced the proliferation of PDLSCs compared with the other groups(P<0.05).Incubation of PDLSCs under high glucose led to fewer ALP staining-positive cells and lower cellular ALP activity.The results of Alizarin red staining further confirmed that less calcium deposits were formed by PDLSCs under high glucose compared to the control group.After vaspin addition,the number of ALP staining-positive PDLSCs was increased,and ALP activity in cells was also enhanced.Consistently,the results of Alizarin red staining indicated that more calcium deposits were formed by PDLSCs with vaspin addition than those without.The results of Quantitative Real-time PCR revealed that the osteoblast differentiation-related genes ALP,Runx2,Col-I were downregulated in PDLSCs under high glucose.Similarly,the expression of osteoblast differentiation-related proteins Runx2,Col-I and OPN were also decreased in PDLSCs under high glucose.After vaspin addition,the gene levels of ALP,Runx2 and Col-I were significantly increased in PDLSCs under high glucose.The protein levels of Runx2,Col-I and OPN were also elevated after vaspin addition.3.A high glucose level significantly decreased the gene expression of TGF-β1,Smad2 and Smad3,as well as protein expression of TGF-β1,Smad2 compared to the control cultures.However,pretreatment of cells with vapsin increased expression of these proteins under HG conditions.TGF-β1/Smad signaling related-genes were significantly enhanced by vaspin compared with control group.Western blot analysis demonstrated that vaspin increased the protein levels of TGF-β1/Smad signaling relatedprotein.The results of Quantitative Real-time PCR showed that the gene levels of ALP,Runx2 and Col-I were significantly increased after inhibition of TGF-β1/Smad pathway.Similarly,the results of Western blot analysis revealed that the protein levels of OPN,Runx2 and Col-I in TGF-β1-downregulated PDLSCs were also increased after inhibition of TGF-β1/Smad pathwayConclusion: High glucose induced suppression of the proliferation and osteogenic differentiation of PDLSCs could be partially reversed by vaspin.Vaspin promotes PDLSCs osteogenesis under high glucose conditions by activating the TGF-β1/Smad pathway.