The Effects Of Compressive Forces On The Activation Of P38 MAPK And Expression Of IL-17 And IL-6 In Human Periodontal Ligament Fibroblasts

Objective:The objectives of this study was to investigate the relationshipamong compressive force,cytokine,andsignal transduction.Weestablished a compressiveforces model for HPDLF in vitro,and we would identifywhetherthe p38 MAPK is involvedin the mechanotransduction process and to explore the effect of the p38 MAPK on down-stream cytokine expression.Methods:Human periodontal ligament fibroblasts cell line was established by sequential digestion from theroots of the premolars extracted from healthy youngvolunteersduringtheir orthodontic treatment.Immunocytofluorescensewas used to identify the source of the cultured cells.CCK8 assay was also used to explore the influences of different compressiveforces(0-5g/cm2)and differentconcentrations(0-16μmol/L)of SB203580 which is a specific inhibitor of p38 MAPK on HPDLF proliferation.Besides,the HPDLF was treated with different compressiveforces(0-5g/cm2).Thenthep38 MAPKactivationafterthecompressiveforces was detected by western-blot for the active kinase(tyrosine/threonine phosphorylated)in HPDLF.Furthermore,the role of p38 MAPK of HPDLF in response to compressiveforces is investigated by using a pyridinal imidazole compound(SB203580).HPDLF were subjected to SB203580 for 60 min,and under the stimulation of4g/cm2 compressive forces for 12 or24 h,real-time PCR and ELISA were respectivelydetected the mRNAexpression of IL-17 R and IL-6 and theprotein expression of IL-17A、IL-17 R and IL-6.Results:1.The cells were long strip,Cell immunofluorescence showed that Vimentin staining was positive and Keratinstaining was negative.Considering the material parts,the cells was HPDLF.2.CCK8 assay showed that compressiveforces(1-4g/cm2)could promote thegrowthofHPDLF.It reached its peak on the compressiveforcesof 2 g/cm2,and growed slowlyor graduallyreduce in 3-4 g/cm2 group.When the compressiveforces was increased,theproliferation activity was dramaticlly inhibited.And CCK8 assay revealed that the concentrations of 1-8μmol/L of SB203580 could accelerate the proliferation of HPDLF.However HPDLFproliferation activity was inhibited atthe concentrations of 16μmol/L of SB203580.3.Western-blot suggested that the activity of p38 MAPK was increased from 10 min,and it reached its peak value at 30 min.At last it declined at 60 min under the stimulation of 2-5g/cm2.However,p38 MAPK was not actived in the pressure of 0.5-1g/cm2.4.The mRNA of IL-17 R andIL-6 and the protein of IL-17A、IL-17 R and IL-6 increased in the group of 24 h than the 12 h.Besides,SB203580 significantly inhibited the mRNA and protein expression of IL-6 stimulated by compressiveforce in HPDLF.But it have no effect on the expression of IL-17 A and IL-17RConclusion:1.It was successfully to established the HPDLF cellby using tissue culture method.2.It could promote the proliferation of HPDLF when the forces and concentrations of SB203580 was suitable.However excessive compressive forces and concentrations of SB203580 wouldinhibit HPDLF’s activity.3.p38 MAPK was a signal transduction molecule under compressive force in HPDLF.The mechanotransduction that the p38 MAPK involved in was a dynamai process.4.p38 MAPK may be an important cooprative mechanism to regulate IL-6synthesis induced by compressive force,but our data could not prove the synthesis of IL-17 A and IL-17 R was relative to p38 MAPK under compressive force in HPDLF.