Effects Of IL-1? On MMP-9 Expression In Cementoblast And Cell-mediated Degradation Of Collagen

In the local inflammation of periodontal tissues,host cells can secrete a variety of proteolytic enzymes to directly destroy the periodontal tissue structure,leading to destruction of connective tissue,cementum and alveolar bone resorption,and even tooth loss.Among these proteolytic enzymes,matrix metalloproteinases(MMPs)play an important role in the progression of destruction of periodontal tissues.Matrix metalloproteinases are a group of highly homologous zinc-dependent extracellular proteolytic enzymes with unique structural and catalytic properties that mediate tissue remodeling and degradation of extracellular matrix.Derived from the plaque biofilm in supporting periodontal tissues,lipopolysaccharides and/or other molecules recruit host immune cells to the site,and these cells respond by releasing proinflammatory cytokines,such as IL-1? and TNF-?.These cytokines further trigger a series of inflammatory responses.Interestingly,orthodontic treatment can also cause periodontal tissue-related cells to secrete proinflammatory cytokines such as IL-1? and IL-6.Cementum is a mineralized connective tissue covering the root surface,and it anchors the tooth root to the surrounding alveolar bone by the periodontal ligament.Therefore,it is important to ensure the integrity of cementum for tooth stability.Type I collagen is the main organic component of cementum.Its collagen fiber not only provides a depositional support for inorganic mineral salts,but also participates in the regulation of nucleation of hydroxyapatite.Cementoblasts are functional cells located on the surface of the cementum,which secrete collagen and non-collagen proteins,participate in the formation,repair and mineralization of cementum,and play an important role in maintaining the metabolism of cementum.This study was to investigate the effect of IL-1? on the expression of MMP-9 in cementoblasts and on cementoblasts-mediated collagen degradation,which provides understanding and sight in destruction of cementum tissues in inflammatory condition.Part 1Effect of IL-1? on the expression of MMP-9 in cementoblastsObjective: To investigate the effect of IL-1? on the expression of MMP-9 in cementoblasts.Methods:(1)OCCM-30 cell line was seeded into a 6-well cell culture plate at a cell density of 5×105/well,and treated with different concentrations of IL-1?(0,5,10,20,40 ng/ml)for 24 h.The expression of MMP-9 was detected by gelatin zymography or real-time PCR.(2)Treatment with 20 ng/ml IL-1? for different time(0,1,3,6,12,24 h),and then using gelatin zymography or real-time PCR to detect the expression of MMP-9.(3)In addition,OCCM-30 cells were treated with IL-1Ra and IL-1?,and the expression of MMP-9 was detected by gelatin zymography to further clarify the effect of IL-1? on MMP-9 expression.(4)Treatment of OCCM-30 cells with TNF-?or IL-6/IL-6 R alpha for different time(0,1,3,6,12,24 h),and then real-time PCR to detect the expression of MMP-9..Results:(1)Upon the treatment of IL-1?,the expression of pro-MMP-9(92KDa)secreted by cementoblasts was significantly up-regulated;activated-MMP-9 was also observed,but was not significantly increased.(2)IL-1? also increased MMP-9 expression at m RNA level in cementoblasts.(3)In addition,IL-1Ra(1000 ng/ml)blocked IL-1?-induced MMP-9 expression in cementoblasts.(4)TNF-?or IL-6/IL-6 R alpha also increased MMP-9 expression at m RNA level in cementoblastsConclusion: IL-1? can up-regulate the expression of pro-MMP-9 in cementoblasts in a time dependent manner.Part 2The mechanism of IL-1?-induced MMP-9 expression in cementoblastsObjective: To study the mechanism of IL-1?-induced MMP-9 expression in cementoblasts.Methods:(1)OCCM-30 cells was treated with IL-1? at a concentration of 20 ng/ml,and ERK1/2,JNK,P38,c-Fos,c-Jun,ATF were detected by western blot,EMSA or luciferase reporter gene;(2)pretreatment of OCCM-30 cells with specific chemical inhibotors U0126,SP600125,SB203580 and tanshinone IIA,and then treated with IL-1? for a certain period of time,gelatin zymography and real-time PCR were used to detect the expression of MMP-9;(3)OCCM-30 cells were pretreated with specific chemical inhibitors U0126 and SP600125,and then treated with IL-1? for a certain period of time,the luciferase reporter gene was used to detect the transcriptional activity of AP-1.The c-Fos and ATF-2 protein activities were detected by western blot,and the m RNA expression of c-Fos and c-Jun was detected by real-time fluorescent quantitative PCR.Results:(1)western blot showed that IL-1? activates ERK1/2,JNK,P38,c-Fos and ATF-2 proteins in a time-dependent manner in OCCM-30 cells.EMSA results showed that IL-1? enhanced AP-1 protein binding activity;luciferase reporter gene assay showed that IL-1? up-regulated AP-1 protein transcriptional activity;(2)gelatin zymogram and real-time PCR assay showed that specific chemical inhibitors U0126,SP600125 and tanshinone IIA attenuated IL-1?-induced MMP-9 expression in OCCM-30 cells;(3)the results of luciferase reporter gene assay show that specific chemical inhibitors U0126 and SP600125 inhibited IL-1?-induced AP-1 transcriptional activity.western blot and real-time PCR results show that the specific chemical inhibitors U0126 blocked IL-1? phosphorylated c-Fos protein and attenuated IL-1? up-regulated c-Fos m RNA expression;specific inhibitor SP600125 blocked IL-1? phosphorylated ATF-2 protein and attenuated IL-1? up-regulated c-Jun m RNA expression.Conclusion: IL-1? promotes the expression of MMP-9 by up-regulating the transcriptional activity of AP-1 by activating ERK1/2 and JNK signaling pathways.Part 3Effect of IL-1? on collagen degradation mediated by cementoblastsObjective: To study the effect of IL-1? on collagen degradation mediated by cementoblasts.Methods:(1)OCCM-30 cells was treated with IL-1? at a concentration of 20 ng/ml,MMP-13 expression was detected by western blot and real-time PCR.(2)Type I collagen was coated in 6-well culture plates.50 ul(about 60,000)OCCM-30 cells were seeded in the middle of the well plate and treated with IL-1? for 48 h,72 h and 96 h..(3)seeded 50 ul(about 60,000)OCCM-30 cells in a 6-well plate coated with type I collagen,then incubated OCCM-30 cell with plasmin and IL-1? for 72 h,Then the cells were digested with trypsin and the degradation of collagen was detected by Coomassie blue staining.(4)OCCM-30 cells was treated with IL-1? at a concentration of 20 ng/ml,the expression of TIMP-1 and TIMP-3 was detected by western blot and real-time PCR.Results:(1)western blot and real-time PCR showed that IL-1? up-regulated the expression of MMP-13 in OCCM-30 cells in a time-dependent manner;(2)Upon stimulation of IL-1? alone,OCCM-30 cell-mediated collagen degradation was not significantly enhanced;(3)OCCM-30 cell-mediated collagen degradation was significantly enhanced by plasmin and IL-1?;(4)Western blot and real-time PCR showed that IL-1? up-regulated the expression of TIMP-1 and TIMP-3 in OCCM-30 cells in a time-dependent manner.Conclusion: IL-1? enhances cementoblast-mediated collagen degradation by upregulating the expression of MMPs.IL-1? can up-regulate the expression of TIMP-1 and TIMP-3,which may negatively regulate IL-1?-induced collagen degradation to a certain extent.