The Effect Of High Glucose On Human Periodontal Ligament Fibroblasts After Inhibiting The Receptor For Advanced Glycation End Products

Part 1.Primary culture of human periodontal ligament fibroblasts and the expression of the receptor for advanced glycation end products under high glucose condition Objective: To isolate and culture human periodontal ligament fibroblasts(h PDLFs)in vitro culture system,then observe the effect of high glucose on expression of the receptor for advanced glycation end products(RAGE).Methods: The periodontal tissues were collected from the healthy premolars of young patients(12-15 years of age),and the cells were cultured with the method combined with enzyme digestion culture and tissue pieces culture.To identify sources of the cells,we adopted method of immunohistochemistry.To measure the effect of FPS-ZM1 on RAGE expression,cells were exposed to either DMEM/low glucose(control)or DMEM/high glucose,and protein expression was measured by Western blot analysis.Results: We obtain cells presented in long spindle or stellate shape congruously,expressed the mesoblastic cell marker vimentin but not the ectodermal cell marker cytokeratin.And the expression of RAGE increased while culturing in high glucose medium.Conclusion: The isolation and culture cells presented the characteristics of human periodontal ligament cells both in morphology and immunology,and high glucose would lead to the high expression of RAGE.Part 2.The inhibition of the receptor for advanced glycation end products promotes proliferation and repair of human periodontal ligament fibroblasts in response to high glucose Objective: To observe if inhibition of RAGE promotes proliferation and repair of h PDLFs stimulated by high glucose and discuss the signal pathway in relation to this process.Methods: Cells were cultured in media of high glucose with different concentrations of inhibitor and cell proliferation were measured by using Cell Counting Kit-8.Expression of collagen type 1(COL-1)and fibronectin(FN)were detected by real-time PCR and enzyme-linked immunosorbent assay method.The relative protein expression levels of NF-κB p65 and phosphorylated p65 were measured by Western blot.Results: RAGE expression increased in high glucose,and the growth of h PDLFs would be inhibited,as well as the expression of repair-related factors.The damaged ability of proliferation would recover after the inhibition of RAGE,and repair-related factors would recover in some extent,with a decline in the phosphorylation level of NF-κB p65.Conclusion: FPS-ZM1 rescued the proliferative capacity and repair capability of h PDLFs in response to high glucose,and the NF-κB signaling pathway might play a part role in this process.Part 3.The inhibition of the receptor for advanced glycation end products promotes proliferation and repair of human periodontal ligament fibroblasts in response to high glucose Objective: To observe if inhibition of RAGE effect the expression of IL-6 and TNF-α of h PDLFs stimulated by high glucose.Methods: Cells were cultured in high glucose with different concentrations of FPS-ZM1.We measured the expression of Interleukin 6(IL-6)and tumor necrosis factor-α(TNF-α)by real-time PCR and ELISA.Results: High glucose could led to the inflammatory response of h PDLFs,while FPS-ZM1 could reduce the expression of IL-6 and TNF-α both in gene expression level and protein level.Conclusion: FPS-ZM1 could rescue the inflammatory response of h PDLFs caused by high glucose.