| Background:Condylar cartilage degeneration is one of the most important pathological features of TMJOA.Condylar cartilage is composed of both chondrocytes and cartilage extracellular matrix,and the interaction between chondrocytes and extracellular matrix plays a key role in the maintenance of cartilage homeostasis.Heparan sulphate proteoglycan（HSPG）are a group of transmembrane proteoglycans that directly connects chondrocytes and the extracellular matrixas an important link.Syndecan 4（SDC4）is highly expressed in the cartilage of knee osteoarthritis（OA）,but its role in TMJOA has not been reported.Methods:In the first part,the cyclic tensile stress（CTS）,or recombinant rat interleukin-1β（rrIL-1β）,was used to simulate rat condylar chnodrocytes.Western blotting and real-time quantitative polymerase chain reaction（RT-qPCR）were performed to detect the expression of SDC4,inflammatory factors（TNF-αand IL-1β）,degradation factors（matrix metalloproteinases,MMPs）,a disintegrin and metalloproteinase with a thrombospondin motifs 5（ADAMTS5）,and cleaved cysteinyl aspartate specific proteinase 3（cleaved-CASP3）.Twenty-four female C57BL/6J mice aged 6 weeks were randomly divided into control group（Con）and unilateral anterior crossbite group（UAC）,and were sacrificed at 3 weeks,6 weeks respectively（n=6）.Hematoxylin&eosin（HE）staining and Safranin O staining were performed to observe the degenerative changes in mandibular condylar cartilage.Immunohistochemical（IHC）staining and Western blot assays were performed to observe the changes of expression of SDC4,TNF-α,IL-1β,MMPs and cleaved-CASP3.In the second part,in the experiment in vitro,anti-SDC4 extracellular segment specific antibody was added to the supernatant of abnormal mechanical force-stimulated chondrocytes.Then the expression of SDC4,TNF-α,IL-1β,MMPs and cleaved-CASP3 was detected by RT-qPCR and Western blot.In the experiment in vivo,the anti-SDC4 antibody was injected into the joint cavity of MIA-induced TMJOA model.Then the expression of degradation factors and related molecules was detected by HE,SO,IHC staining and Western blot.In addition,condylar chondrocytes treated by SDC4 si RNA were stimulated with 10ng/m L rrIL-1β.The expression of cartilage extracellular matrix and matrix degradation factors was detected by RT-qPCR,Western blot and cellular immunofluorescence staining.In the experiment in vivo,SDC4 was specific knocked out in mouse condylar cartilage by the Cre-loxP system,and then TMJOA was induced by the monosodium iodoacetate（MIA）accordingly.The degenerative changes in mandibular condylar cartilage was observed by HE staining,safranin O staining and collagen type II（Col-II）IHC staining.The expression of SDC4,TNF-α,MMPs and cell-death numbers were observed by immunohistochemistry and TUNEL staining.In the last part,condylar chondrocytes were stimulated with 10 ng/m L rrIL-1βfor 0,15 min,30 min,1 h,2 h,and 4 h,the phosphorylation levels of p38、ERK、NFκB signal pathway were detected by western blot assays.In the experiment in vivo,the rats were treated with anti-SDC4 antibody in OA-condylar cartilage,and the changes of OA-related signaling pathway were detected by Western blot.Main results:1.SDC4 was upregulated in degenerative condylar cartilage and chondrocytes both in vivo and in vitro.After30%,0.5 Hz cyclic tensile stress loaded on rat condylar chondrocytes for 6 h and 12 h,the protein levels of SDC4,IL1β,TNF-α,and cleaved-CASP3 and mRNA expression of Sdc4,Mmp3,Mmp13,and Adamts5 increased significantly,while Agg mRNA expression decreased significantly.After stimulation of rat condylar chondrocytes with 0,1,10 and 100 ng/m L,the protein levels of IL-1β,MMP 3 and ADAMTS5 and mRNA expression of Sdc4,Mmp3,Mmp9,Adamts5 and Tnf-αmRNA increased significantly,while the protein level of Col-II and the mRNA expression of Agg decreased significantly.Compared with control group,the UAC model at 3 and 6 weeks could lead to decreased thickness of condylar cartilage,degradation of cartilage matrix（Col-II and AGG）and increased expression of SDC4 and IL-1β,TNF-α,MMP13,and cleaved-CASP3,while the expression of Col-II decreased significantly.2.Knockout or knockdown of SDC4 in condylar cartilage or chondrocytes alleviated TMJOA.SDC4 knockdown suppressed the MMP3 and ADAMTS5 upregulation,and enhanced the Col-II expression in rrIL-1β-induced condylar chondrocytes.In MIA-induced Sdc4flox/floxmouse,the number of chondrocytes in the condyle decreased sharply and the distribution was uneven,showing cell clusters and a large cell-free area,AGG and Col-II only distributing in the deep layer of cartilage,and the degeneration degree of the condylar cartilage was more serious.Tamoxifen injection deleted Sdc4 in the temporomandibular joint of the COL2+Sdc4flox/flox mice.Sdc4 knockout rescued MIA-induced degradation of cartilage matrix（Col-II and AGG）and the upregulation of SDC4,TNF-α,MMP13 and TUNEL.The addition of Anti-SDC4 antibody to the supernatant of condylar chondrocytes in vitro significantly reversed the mRNA expression of Sdc4 and Adamts5,Mmp3 and Casp3,as well as the protein levels of ADAMTS5,TNF-αand cleaved-CASP3.In vivo treatment with Anti-SDC4 effectively decreased the OARSI score of MIA-induced rat OA-condylar cartilage,inhibit the death of condylar chondrocytes and the loss of cartilage matrix（Col-II and AGG）,moreover decreased the proportion of SDC4,MMP13,cleaved-CASP3 positive cells,increased the protein levels of Aggrecan and Ki67.3.MAPK pathway may be involved in the biological effects of SDC4 intervention on the prognosis of TMJOAStimulated with different concentrations（0,1,10,100 ng/m L）rrIL-1β,the phosphorylation of ERK,p38 decreased significantly in the Condylar chondrocytes,while the phosphorylation of NFκB increased significantly.After 10 ng/m L rrIL-1βstimulation of condylar chondrocytes for 0,15 min,30 min,1 h,2 h,and 4 h,NFκB phosphorylation did not significantly change at each time point,ERK phosphorylation decreased at 1 h,2 h,4 h,p38 phosphorylation was significantly increased at 15 min,but decreased at 1 h,2 h,and 4 h.Rat-Ig G-treatment to the temporomandibular joint cavity of MIA model rats significantly reduced p38 and ERK phosphorylation in the condylar cartilage,while Anti-SDC4 extracellular segment-specific antibody treatment partially restored p38 and ERK phosphorylation.Conclusions:1.SDC4 is involved in the degeneration of the condylar cartilage induced by abnormal mechanical stress or elevated pro-inflammatory cytokines.2.The inhibition of SDC4 significantly alleviate condylar cartilage degeneration during TMJOA induced by abnormal mechanical stress or elevated pro-inflammatory cytokines.MAPK pathway may be involved in the biological effects of SDC4 intervention on the prognosis of TMJOA. |
