| Part 1.The effect of FPS-ZM1 on the proliferation of bone marrow mesenchymal stem cells and the expression of receptor for advanced glycation end products under high glucose conditionObjective: The objective is to investigate the regulation effect of specific inhibitor of receptor for advanced glycation end products(FPS-ZM1)on high glucose(HG)-induced proliferation in bone marrow mesenchymal stem cells(BMSCs),then to explore the optimal concentration and action time of FPS-ZM1 for use in the follow-up trial;Furthermore,to find out the effect of high glucose on expression of the receptor for advanced glycation end products(RAGE)in BMSCs.The broader objective is to find a new target to antagonize the inflammatory effect of high glucose on BMSCs,and to assess a new idea for prevention or treatment of diabetes related periodontitis or peri-implantitis.Methods: BMSCs were cultured in DMEM/low glucose(normal control)or DMEM/high glucose.The cells from the three passage were used in the following research.Cells were cultured in media of high glucose with the treatment of various concentrations of FPS-ZM1(250 n M,500 n M,750 n M),low glucose culture group without FPS-ZM1 was set as normal control,high glucose culture group without FPS-ZM1 was set as positive control.Cell Counting Kit-8(CCK-8)was served as detection of the cell proliferation,and the RAGE level was assessed using Western blot.Results: Compared with the normal control group,the proliferation ability of bone marrow mesenchymal stem cells under high glucose condition was promoted,and reached the peak at 48 h.The proliferation rate decreased significantly with the extension of time in high glucose environment.The co action of low concentration of FPS-ZM1 and high glucose had no significant effect on bone marrow mesenchymal stem cells.With the increase of the concentration of FPS-ZM1,it reversed the effect of high glucose on bone marrow mesenchymal stem cells,however,there was no significant difference between high glucose+500 n M FPS-ZM1 and high glucose+750 n M FPS-ZM1groups(P>0.05).In addition,the ability of bone marrow mesenchymal stem cells to express RAGE protein was induced by high glucose,while FPS-ZM1 could reduce the expression level of RAGE protein in bone marrow mesenchymal stem cells.Conclusion: 500 n M FPS-ZM1 was the best choose to be the next experimental concentration.In addition,high glucose can increase the expression of RAGE in BMSCs,and FPS-ZM1 can decrease the expression of RAGE in BMSCs.This suggests that RAGE-related intracellular signal transduction exists in BMSCs stimulated by high glucose,and FPS-ZM1,as a mature specific RAGE inhibitor,also has an effective RAGE blocking effect on BMSCs in high glucose environment.FPS-ZM1 may be a promising therapeutic agent for periodontal tissue or peri-implant inflammation induced by high glucose.Part 2.FPS-ZM1 reduces high glucose-induced inflammation in bone marrow mesenchymal stem cells and the research on related signaling pathwayObjective: The objective is to evaluate the regulation effect of specific inhibitor of receptor for advanced glycation end products(FPS-ZM1)on high glucose(HG)-induced inflammation in bone marrow mesenchymal stem cells(BMSCs).Furthermore,to find out how TXNIP/NLRP3(Thioredoxin-interacting protein/NLR family,pyrin domain containing protein 3)inflammasome signaling pathway participates in FPS-ZM1 regulating inflammation under high glucose.Methods: BMSCs were cultured in DMEM/low glucose(normal control)or DMEM/high glucose.The cells from the three-six passage were used in the following research.The cells were randomly divided into four groups,the normal control group,high glucose group,high glucose+FPS-ZM1 group and high glucose+resveratrol(Res)group.The inflammasome markers including interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured with enzyme linked immunosorbent assay(ELISA).The m RNA and protein expressions of TXNIP,caspase-1,Thioredoxin(TRX),ASC(apoptosis-related speck-like protein containing CARD),NLRP3 were respectively investigated using Real-time quantitative polymerase chain reaction(RT-q PCR)and Western-blotting(WB).Resveratrol(Res)was used to ulteriorly determine the role of TXNIP/NLRP3 inflammasome signaling pathway by inhibiting it.Results: FPS-ZM1 suppressed the protein activation levels of TNF-α,IL-1β,IL-6,which are highly induced by high glucose.In addition,FPS-ZM1 inhibited the m RNA and protein expressions of TXNIP,Caspase-1,NLRP3,ASC and promoted TRX expression.Res exerted the same effect as FPS-ZM1 by suppressing TXNIP/NLRP3 inflammasome signaling pathway.Conclusion: FPS-ZM1 attenuated HG-induced inflammation in BMSCs.Furthermore,the molecular mechanism for this effect is mediated by TXNIP/NLRP3 inflammasome signaling pathway.Thus,a new idea and feasibility for the prevention and treatment of diabetes-related periodontitis or peri-implant periodontitis is provided. |
