IL-17 Induces The Expression Of RANKL And OPG In Human Periodontal Ligament Fibroblasts By P38MAPK Pathway

Objective:The objective is to explore whether p38 MAPK signaling pathway participates process of IL-17 and HPDLF mediateing osteoclast differentiation by investigating the effect of inhibitor of p38 MAPK signaling pathway(SB203580),on the expression of RANKL and OPG in human periodontal ligament fibroblasts(HPDLF),to investigate the possible signal transduction pathways of IL-17-induced human periodontal ligament cells to promote bone resorption and to explore the mechanism of IL-17 involved in orthodontic associated inflammatory root resorption.Methods:1.Primary HPDLF cells were cultured through the tissue culture method in vitro and purified by passage.The cells of the 4th generation cultured were identified by immunofluorescence cytochemistry.The effect of p38 MAPK inhibitor with six different concerntraions(0?2.5?5?10?20?40?mol/L)was detected on the proliferative activity of HPDLF by MTT assay,respectively.2.The expression of p38 MAPK phosphorylated protein was detected by Western blotting with 20ng/ml IL-17 stimulated HPDLF for 0,20,40,60,80 min.The experiment were divided into six groups: group A(blank control),groupB(DMSO control),group C(inhibitor),group D(IL-17),group E(IL-17+DMSO),group F(IL-17+inhibitor).Cells were treated with 10?mol/L of SB203580 and 20 ng/ml of IL-17 according to the grouping.3.The mRNA expression levels of RANKL and OPG in HPDLF were detected by RT-PCR and the protein expression of RANKL and OPG was detected by ELISA and Western blot.Results:1.The cultured cells were long spindle-shaped and grew radially.After passage,the cells were evenly distributed and the growth state was stable.Identified cells derived from mesenchymal cells and non-epithelial-derived cells.The cells could be identified as HPDLF combined with the site.The results of MTT assay showed that the activity of HPDLF increased gradually with the increase of concentration in 0-10?mol/L after HPDLF was stimulated by SB203580 at 0-40?mol/L.In 10?mol/L,the OD value was the highest and the proliferative activity of HPDLF was the strongest.In the 10-40?mol/L,the OD value decreased gradually with the increase of the concentration,and the proliferative activity of HPDLF decreased gradually.2.The relative expression of phosphorylated p38MAPK(p-p38MAPK)increased with time at stimulation of 20ng/ml IL-17.It reached the highest level at 60 min and began to decline at 60 min.3.Compared with the blank control group,the mRNA and protein expression of RANKL increased after IL-17 stimulation,the expression of OPG mRNA decreased,and the mRNA ratio of RANKL/OPG increased.IL-17 stimulated the mRNA and protein expression of RANKL,the expression of OPG mRNA increased,and the mRNA ratio of RANKL/OPG decreased.The function of IL-17 regulating the expression of RANKL and OPGmRNA,was blocked by P38 MAPK inhibitor.Conclusion:1.Human periodontal ligament fibroblast cell line was successfully established by tissue culture method.The cells were purificated and its biological characters were stable by passage.The suitable concentration of SB203580 could promote the proliferation of HPDLF,but the excessive concentration of SB203580 would inhibit the proliferation of it.Among them,the optimal concentration of HPDLF is 10?mol/L.2.IL-17 can activate p38 MAPK protein and it is time-dependent.IL-17 promotes the expression of RANKL in HPDLF,inhibits the mRNA expression of OPG and Increases the mRNA ratio of RANKL/OPG through p38 MAPK pathway.