The Role Of MAPKs Pathway In Modulation Of MMP-1/-13Expression By IL-1β In HPDLCs

The periodontal tissue remodeling is very actively in orthodontic treatment and isregulated by a series of systemic and local factors. This process is finally completed by thealveolar bone reconstruction, which is achieved by the balance activities between the boneresorption and formation[1]. It is regulated by complex regulating factors includingsystemic factors such as calcitonin, parathyroid hormone (PTH) and local factors such ascytokines (TNF, IL-1, IL-6), prostaglandin E2(PGE2) and growth factors through theinteraction of these factors in order to maintain bone metabolic balance[2].But theinflammatory cytokines often plays an important role in the pathological alveolar boneresorption and cementum resorption process[3].Inappropriate forces could induce the production of the sterile inflammatory factorsin periodontal tissue when orthodontic tooth movement[4], and this change may lead toincrease expression of matrix metalloproteinases (MMPs)[5].This mechanism research ofincreasing MMPs expression is also an important research subject in the stomatologyprofessional studies[6]. It has a broadly clinical application in the exploration of thealveolar bone and root resorption. This study aimed to investigate the following aspects:①Explore the effect ofinterleukin-1β (IL-1β) on the matrix metalloproteinase-1/-13(MMP-1/-13) expression inperiodontal ligament cells. and mechanism of the high expression of some factors relatedto the periodontal tissue remodeling during the reconstruction process in periodontaltissue.②Explore the effect of mitogen-activated protein kinases (MAPKs) on theexpression of matrix metalloproteinase-1/-13in human periodontal ligament cellsmediated by inflammatory cytokine IL-1β, and the possibility of inhibition of theexpression of related factors and resorption of alveolar bone and root via blockingMAPKS singaling pathways. The research includes two major parts:Research Contents and Methods1Effect of IL-1β on the MMP-1/-13expressions in hPDLCsHuman periodontal ligament cells were exposed to various concentrations of IL-1β(0.5ng/mL,5ng/mL,10ng/mL,20ng/mL) for24h, and then select the optimalconcentration. Human periodontal ligament cells were simulated at different times withthe optimal concentration of IL-1β (8h,24h,48h,72h). RT-PCR was used to detect themRNA expression of MMP-1and MMP-13in human periodontal ligament cells. Thenchoose the optimal concentration and action time that is10ng/mL IL-1β for24h inhPDLFs. Immunofluorescence staining and Western blot were used to observe expressionof MMP-1and MMP-13in hPDLFs.2Effect of MAPKs pathways on the expressions of the MMP-1/-13in hPDLCsThe effect of the MAPKs signaling pathway on the hPDLCs proliferation wasinvestigated by MTT method at different times (24h,48h,72h). RT-PCR and Western-blotwere used observe expression of MMP-1/-13in hPDLFs after inhibiton of IL-1β inducedMMP-1/-13expression by MAPKs inhibitors (ERK inhibitor PD98059, p38inhibitorSB203580, JNK inhibitor JNK inhibitorⅡ). Results1Effect of IL-1β on the MMP-1/-13expressions in hPDLCs1.1Expression of MMP-1/-13mRNA in different concentrationAccording to the result: lower concentrations of IL-1β (0.5ng/mL and5ng/mL) hadeffect on MMP-1and MMP-13mRNA expression in hPDLCs compared with the controlgroup (P<0.05); High concentrations of IL-1β (10ng/mL and20ng/mL) increased themRNA expression of MMP-1/-13in hPDLCs compared with the lower concentations ofIL-1β (0.5ng/mL and5ng/mL) groups (P<0.05); But the MMP-1and MMP-13expressionwas no significant difference between10ng/mL and20ng/mL groups (P>0.05). This resultimplied that IL-1β could promote the MMP-1and MMP-13expression in hPDLCs.10ng/mL group had the most significant promotion on the expression of MMP-1andMMP-13.1.2Expression of MMP-1/-13mRNA at various times with IL-1β stimulatedAccording to the above experimental results, we selected10ng/mL as a optimalconcentration and subsequently pretreated hPDLCs with this concentration at varioustimes (8h,24h,48h,72h). The result showed: MMP-1and MMP-13expression increase inthe10ng/mL IL-1β stimulated for8h group compared with the control group (P<0.05).But the expression of MMP-1and MMP-13in24h,48h and72h groups were higher thanthat in8h group (P<0.05), and there were no statistically significant differences betweenthe three groups (P>0.05).1.3Immunofluorescence staining resultsImmunofluorescence staining tests showed that the expression of MMP-1andMMP-13were weakly expression in control groups. But pretreated with10ng/mL IL-1β,the expression of MMP-1and MMP-13was strong positive in hPDLCs, and the MMP-1and MMP-13positive signals were located mainly in cytoplasm.1.4The expression of MMP-1/-13proteinAfter24h of pretreated,10ng/mL IL-1β increased the protein expression of MMP-1and MMP-13in hPDLCs compared with blank control group (P<0.05). 2Effect of MAPKs pathways on the expressions of the MMP-1/-13inhPDLCs2.1The effect of MAPKs pathways on cell proliferationAt the time of24hours, there was no significant difference between experimentalgroups and blank control groups (P>0.05). The value of OD in ERK inhibition group ismuch lower than that in IL-1β group in48h and it has statistically significant difference(P<0.05). In72h, the value of OD is lowest in ERK inhibition group and is highest inIL-1β group, it has a significant difference between the two groups (P<0.05).2.2Effect of MAPKs pathways on the expressions of the MMP-1/-13in hPDLCsBy RT-PCR and Western blot, the expression of MMP-1and MMP-13showed asignificant promote in the10ng/ml IL-1β group comparing with the control groups(P<0.05). These inhibitor group showed a significant inhibit the expression of MMP-1andMMP-13in hPDLCs (P<0.05).ConclusionsThe expression of MMP-1/-13could promoted by IL-1β in hPDLCs.The MAPKsinhibitor can inhibit the high expression of MMP-1/-13stimulated by IL-1β in hPDLCs.These results showed that MAPKs pathway is one of the signal transduction mechanismsof IL-1β modulate expression of MMP-1/-13in human periodontal ligament fibroblasts.