| ObjectivesPeriodontitis,a chronic infectious inflammatory disease,occurs in periodontal support tissue(including gingiva,periodontal ligament,alveolar bone,and cementum),commonly triggered by multiple factors,such as microbial components,host immune response,environmental and genetic risks,leading to the future damage of the periodontal supporting tissues.Human gingival fibroblasts(HGFs)are the most abundant cells in gingival connective tissues and they may directly interact with bacteria or their metabolites,and affect the progression of periodontitis by maintaining the stability of periodontal tissues and regulating the immune inflammatory response of the host.Plaque biofilm is the initiating factor of periodontitis,but the complex interaction between biofilm and host inflammatory immune response is a major cause of periodontal diseases.The ’red complex’ in the oral flora is a known risk factor for periodontitis.One of its main members is Porphyromonas gingivalis(P.gingivalis),which is the predominant pathogen in chronic periodontitis.As the most important microbial virulence that causes periodontal tissue destruction,lipopolysaccharide(LPS)has been considered as a structural component of P.gingivalis cell wall and can induce an immune response in HGFs and activate nuclear factor-kappa B(NF-κB)signaling pathway,leading to the production of proinflammatory cytokines such as interleukin(IL)-1,IL-6 and IL-8 in the periodontal connective tissue.Sitagliptin is a new oral hypoglycemic drug commonly used in clinic that can be used alone or in combination with other drugs for the treatment of type 2 diabetes.In addition to significantly lowering blood sugar,sitagliptin has extensive application prospects because of its multiple effects such as anti-inflammation,anti-tumor,potential renal protection,cardiovascular protection,tissue regeneration activities.Related studies reported that sitagliptin significantly decreased proinflammatory cytokines in type 2 diabetic patients,sitagliptin also possesses anti-inflammatory effects on RINm cell lines and H9c2 cells.However,it is still unclear the effect of sitagliptin on the inflammatory response of periodontal tissue cells,which needs further study and discussion.Therefore,the purpose of our study was to investigate the potential effects of sitagliptin on LPS-stimulated inflammation of HGFs in vitro and further clarify the underlying molecular mechanisms.Our present study aims to provide a theoretical and experimental basis for the application of sitagliptin in the prevention and treatment of periodontitis.Methods1.Isolation,cultivation and identification of HGFs:Human gingival fibroblasts were obtained from human healthy gingival tissues by enzymatic digestion.The 3rd to 5th generations of HGFs were applied for subsequent experiments.The morphological characteristics of the cells were observed under an inverted microscope,and the cells were identified by immunofluorescence.2.Cell viability detection:HGFs were treated with sitagliptin at 0,0.1,0.25,0.5,5,10,100,200,500 and 1000 μM for 24 and 48 h,cell viability was determined by cell-counting kit-8(CCK8).The effect of co-incubation with sitagliptin(0.1,0.25,0.5 μM)and LPS(5 μg/mL)on HGFs viability was also determined at 24 h and 48 h.3.Expression of inflammatory factors:the experiment was divided into five groups to detect the effect of different concentrations of sitagliptin on inflammatory factors expression:control,LPS,LPS+0.1 μM sitagliptin(LPS+0.1),LPS+0.25 μM sitagliptin(LPS+0.25),LPS+0.5 μM sitagliptin(LPS+0.5).The mRNA levels of interleukin(IL)-6,IL-8,superoxide dismutase 2(SOD2)and C-C motif ligand 2(CCL2)were evaluated by quantity real-time polymerase chain reaction(qRT-PCR).The protein expression levels of IL-6,IL-8 and CCL2 in the supernatant were examined by Enzyme-linked immunosorbent assay(ELISA)4.Mechanism of anti-inflammatory:this experiment was divided into four groups:control group,LPS group,LPS+sitagliptin group,LPS+NF-κB inhibitor BAY11-7082 group.Western blot analysis was used to examine nuclear factor-kappa B(NF-κB)signaling pathway.The effect of BAY11-7082(NF-κB pathway inhibitor)on inflammatory cytokines at the gene level was verified by qRT-PCR.Results1.Isolation,cultivation and identification of HGFs:HGFs were successfully isolated and cultured by enzymatic digestion.The results of cell immunofluorescence assay showed that HGFs obtained by enzymatic digestion were positive for vimentin staining and negative for cytokeratin 17 staining.2.CCK8 results showed that incubation of HGFs with 0.1,0.25 and 0.5 μM sitagliptin had no significant effect on HGFs proliferation after treating for 24 and 48 h(P>0.05).Sitagliptin at 5～1000 μM showed significant inhibitory effects on cell proliferation(P<0.001).Stimulation with 5 μg/mL LPS in the absence or the presence of 0.1,0.25 and 0.5 μM sitagliptin for 24 and 48 h showed no effect on the viability of HGFs(P>0.05).3.Sitagliptin inhibited the expression of inflamatory factors:sitagliptin dose-dependently inhibited LPS-elevated gene levels of CCL2(P<0.001),IL-6(P<0.01),IL-8(P<0.01)and SOD2(P<0.05)expression at 12 and 24 h.ELISA results showed that CCL2,IL-6 and IL-8 protein were upregulated in varying degrees with LPS stimulation(P<0.01),and sitagliptin decreased the expression of the three proteins in HGFs.4.The expression of inflammatory factors could be downregulated by sitagliptin via NF-κB signaling pathway:the data from western blot analysis indicated that treatment with LPS significantly activated NF-κB.However,sitagliptin could block NF-κB signaling pathway by inhibiting p-p65 and p-IκBα phosphorylation(P<0.001,P<0.0001)and IκBα degradation(P<0.0001).To further confirm that sitagliptin inhibited the expression of inflammatory factors through NF-κB pathway,the mRNA levels of IL-6,IL-8,CCL2 and SOD2 were analyzed by qRT-PCR after addition of 5 μM NF-κB pathway inhibitor(BAY 11-7082).The results showed that 0.5 μM sitagliptin inhibited LPS-induced expression of CCL2(P<0.001),IL-6(P<0.001),IL-8(P<0.001)and SOD2(P<0.0001).The levels of CCL2,IL-6,IL-8 and SOD2 in 5 μM BAY11-7082 group were significantly decreased(P<0.0001).ConclusionsThe present study indicated that sitagliptin inhibited P.gingivalis LPS-induced production of the pro-inflammatory cytokine IL-6,IL-8,CCL2,SOD2 expression in HGFs by blocking the activation of the NF-κB signaling pathway,which provide a theoretical and experimental basis for the application of sitagliptin in the prevention and treatment of periodontitis. |
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