Ex Vivo Study Of The Change Of The Biocharacter Of HPDLFs Under The Stimulation Of High Glucose And AGEs

Aim: To preliminarily study the effect of AGEs on the periodontal tissue inpatients with diabetes and the mechanism of AGEs mediated destruction ofperiodontal tissue by studying the effects of high glucose and AGEs on thesecretion of inflammatory cytokines(IL-1β, IL-6, TNF-and IFN-γ), expressionof NF-κB, proliferation and apoptosis of human periodontal ligamentfibroblasts(hPDLFs). By comparing with high glucose, we try to verify thatthe AGEs is more important in mediating the destruction of periodontal tissue.In short, we try to provide solid basic evidence for clinical treatment of chronicperiodontitis in diabetes patients.Methods: The primary hPDLFs were isolated from extracted orthodonticpremolars. hPDLFs were divided into four groups: L (low glucose,5.5mmol/Lglucose), H(High glucose,25mmol/L glucose), L+A(low glucose and AGEs,5.5mmol/L glucose and100μg/ml AGEs) and H+A(high glucose and AGEs,25mmol/L glucose and100μg/ml AGEs). We set3time points (12h,24h and36h)to assess the proliferation and apoptosis of hPDLFs from different groups through cck-8and flow cytometry methods. Test the concentration of IL-1β、IL-6、TNF-and IFN-γ secreted by hPDLFs under the simulation of differentdose of AGEs(10μg/ml、50μg/ml and100μg/ml) during the same period byELISA. Assess the gene expression level of NF-κB of different groups. Theresults were analyzed by SPSS19.0. ANOVA analysis of Variance was used. Allvalues are expressed as mean±SD. p value<0.05was regarded as significant.Results:1. The effect on hPDLFs proliferation1) Cell growth curveThere are obvious differences among these4groups. Group H+A is the lowest.2) Cell cycleThe capacity of cell proliferation in four groups were statistically significantdifference (p<0.05), H+A group had the most inhibitive ability of cellproliferation.2.IL-1β、IL-6、TNF-、IFN-γ expression under different AGEs stimulationduration and IL-1β、IL-6、TNF-、IFN-γ expression under different AGEsstimulation dose.1) IL-1β expression (different duration)a in group：L: there is no statistical significanc（ep>0.05）；H: there is statisticalsignificance between24h,36h and12h（p<0.05）；L+A: there is statisticalsignificance among every time points（p<0.05）；H+A: there is statisticalsignificance between36h and12h、24h（p<0.05）.b among groups：12h: there is statistical significance，except for between Hand L+A（p<0.05）；24h: there is statistical significance except for between L+Aand H+A（p<0.05）；36: there is statistical significance among groups（p<0.05）.2) IL-6expression (different duration) a in group：L: there is no statistical significance（p>0.05）；H: there is nostatistical significance（p>0.05）；L+A: there is statistical significance amongevery time points（p<0.05）；H+A: there is statistical significance among everytime points（p<0.05）.b among groups：12h: there is statistical significance except for between L+Aand H+A（p<0.05）；24h: there is statistical significance except for between L+Aand H+A（p<0.05）；36h: there is statistical significance among group（sp<0.05）.3) TNF-expression (different duration)a in group：L: there is no statistical significanc（ep>0.05）；H: there is statisticalsignificance（p<0.05）, except for between12h and24h；L+A: there is statisticalsignificance except for between12h and24h（p<0.05）. H+A: there is statisticalsignificance among every time points（p<0.05）.b among groups：12h: there is statistical significance for between L and H,between L and L+A and between L and H+A（p<0.05）；24h: there is statisticalsignificance except for between L+A and H（p<0.05）；36: there is statisticalsignificance among groups（p<0.05）.4) IFN-γ expression (different duration)a in group：L: there is statistical significance between12h and36h（p<0.05）；There is statistical significance among every time points for the other groups（p<0.05）.b among groups：there is statistical significance among groups for every timepoints（p<0.05）.5) Cytokines expression for group H+A (different dose of AGEs)a IL-1β：there is statistical significance between100μg/ml and10μg/ml,50μg/ml (p<0.05).b IL-6： there is statistical significance between100μg/ml and10μg/ml (p<0.05).c TNF-: there is statistical significance between100μg/ml and10μg/ml,50μg/ml (p<0.05).d IFN-γ：there is statistical significance among different doses(p<0.05).3. NF-κB mRNA expression and apoptosis of hPDLCs under differentconditions.1) L: there is no statistical significance among different time points（p<0.05）.2)H: there is statistical significance between36h and12h,24h（p<0.05）,36h hasan obvious up-regulation of NF-κB mRNA.3)L+A, H+A: there is statistical significance among different time points（p<0.05）. NF-κB gene is up-regulated in a time dependent manner.4) there is a higher NF-κB gene expression at24h and36h in L+A and H+A（p<0.05）.5) There is a higher apoptosis rate in L+A and H+A.Conclusions:1. AGEs can suppress hPDLFs proliferation.2. AGEs can stimulate hPDLFs to secret IL-1β、IL-6、TNF-、IFN-γ in a timeand dose dependent manner.3. AGEs can up-regulate NF-κB gene expression in a time dependent manner.4. AGEs can promote the apoptosis of hPDLFs.