Differentiation Of Human Periodontal Ligament Cells Stimulated By Pg-LPS And Its Mechanism

Objective: In recent years,liraglutide(LIRA)is a new type of drug used to treat diabetes mellitus.It is a glucagon-like peptide-1(GLP-1)analogue that binds and activates GLP-1 receptor(GLP-1R),which can be widely expressed in a variety of cells and tissues.Studies have shown that LIRA not only has hypoglycemic effect,but also has anti-inflammatory and bone protection effects.Therefore,we studied the effect of LIRA on osteogenic differentiation of human periodontal ligament cells(hPDLCs)after Porphyromonas gingivalis lipopolysaccharide(Pg-LPS)stimulation and further studied its mechanism.Methods: Preliminary identification of hPDLCs was assayed by the immunofluorescence staining and Alizarin red staining.the expression of GLP-1R on hPDLCs was detected by the immunofluorescence staining.The expression of inflammatory(IL-6 and TNF-?)Under different concentrations of Pg-LPS stimulation were measured by ELISA in hPDLCs.Effect of Pg-LPS on proliferation of hPDLCs determined by colony forming unit(CFU).ALP staining,Alizarin red staining,qRT-PCR,Western Blot and immunofluorescence staining were used to evaluate the effect of LIRA on osteogenesis of hPDLCs,The western Blot was performed to evaluate the Wnt/?-catenin signaling-related protein expression.Results: Immunofluorescence staining showed that hPDLCs were negative for pan-cytokeratin and positive for vimentin.The results showed that the primary cultured cells were derived from mesenchymal.Alizarin red staining was performed on day 21,indicated that hPDLCs had osteogenic differentiation potential.Immunofluorescence staining showed that GLP-1R was present on hPDLCs,and LIRA up-regulated the expression of GLP-1R.The expression of inflammatory factors(TNF-a,IL-6)in hPDLCs stimulated by Pg-LPS at 0.1,1,10,20 ug/mL for 24 hours was measured by ELISA.The results showed that differences in TNF-? and IL-6 content were statistically significant when the concentration of Pg-LPS was 1 ug/mL,compared with the control group(p<0.05),and the concentration of Pg-LPS had no effect on cell proliferation(p>0.05).ALP staining results showed that LIRA treatment group stained the deepest,LPS group stained the lightest,and LIRA+LPS group stained deeper than LPS group.Alizarin red staining showed that the area of mineralized nodules in LIRA group was large and deep stained under the same field of vision,the number of mineralized nodules in LPS group was the least,compared with LPS group,the number and area of calcified nodules in LIRA+LPS group increased.Quantitative analysis of calcium deposition showed that OD value in LIRA group was higher than that in control group(p<0.01),OD value in LPS group was lower than that in control group(p<0.01),OD value in LIRA+LPS group was higher than that in LPS group(p<0.01).Immunofluorescence staining showed that Runx2 was positive in all groups,and the fluorescence expression was strongest in LIRA group.The results of qRT-PCR showed that the expression of ALP and Runx2 mRNA was significantly up-regulated in LIRA group(p<0.01)and down-regulated in LPS group(p<0.01).Compared with LPS group,the expression of ALP and Runx2 in LPS+ LIRA group was significantly increased(p<0.01).The trend of protein expression of ALP and Runx2 was basically consistent with that of gene expression.Moreover,Pg-LPS strongly activated the Wnt/?-catenin signaling pathway(p<0.01)and reduced the osteogenesis of hPDLCs(p<0.01).which could be reversed by the LIRA.Conclusion: LIRA can enhance osteogenic differentiation of hPDLCs stimulated by Pg-LPS through inhibiting Wnt/?-catenin signaling pathway.