| Objective:Temporomandibular Joint Osteoarthritis(TMJOA)is a high-incidence disease of the maxillofacial region,which seriously affects the quality of life of patients.The synovial membrane of the temporomandibular joint is swollen and thickened,and cartilage damage increases the refractory disease.Pyroptosis is a form of cellular inflammatory injury that releases the inflammatory cytokines IL-1β,IL-18 after the assembly of the NLRP3inflammasome.As a pleiotropic factor,MIF is involved in the occurrence and development of inflammation,and the activated NF-κB pathway is the most directional in this process.This study aims to explore the pyroptosis of MIF/CXCR4-NF-κB-NLRP3 under the stimulation of abnormal fluid shear stress.The expression of pathway-related molecules in joint cells provides the basis for the pathological mechanism and targeted therapy of TMJOA.Methods:Part 1,(1)A retrospective study analyzed the clinical baseline data and synovial fluid of the TMJOA group and the temporomandibular joint internal derangement(TMJID)group collected from the temporomandibular joint disease clinic of the Stomatological Hospital Affiliated to Xinjiang Medical University;(2)The expression levels of MIF,IL-1β,IL-18,NLRP3 and Caspase-1 in synovial fluid of the two groups were detected by ELISA,and the differences were analyzed.And the clinical baseline data were sorted out by descriptive analysis.Part 2,(1)TMJO animal models of SD rats were established by Unilateral anterior crossbite(UAC)intervention for 4,8 and 12 weeks.Joint synovial membrane and cartilage tissue damage were observed by gross morphology,BV/TV,BS/TV,Tb.Th and Tb.N were analyzed by micro-CT,and cartilage degeneration was evaluated by OARSI score.(2)Serum and synovial fluid of temporomandibular joint were collected from rats in control group and UAC group,and THE expression level of MIF was detected by ELISA kit.(3)The distribution and expression of NLRP3,ASC,Caspase-1,GSDMD and inflammatory factors MIF,IL-1β,IL-18 and MIF receptor protein CXCR4 in synovial tissues and cartilage tissues of different treatment groups were detected by immunohistochemistry.(4)Western blot analysis of the above proteins and NF-κB pathway protein expression in UAC intervention group and control group.Part 3:(1)Fibroblast-like synovial cells(FLSs)and chondrocytes of SD rats were isolated and cultured,and different Fluid Flow Shear Stress(FFSS)was applied to the two cells,respectively,to detect the pyroptosis related molecules NLRP3,ASC,Caspase-1 and GSDMD.Expression levels of inflammatory factors MIF,IL-1β,IL-18 and MIF receptor CXCR4,and NF-κB pathway protein.(2)Mechanism level:MIF knockdown and MIF overexpressed lentivirus were transfected into both cells to detect the protein expression level of the above molecules under abnormal FFSS.After the use of site inhibitors/antagonists,the protein expression levels of the above molecules were detected under abnormal fluid shear force.AO/EB staining was used to detect the outcome of cell membrane rupture and death.Results:Part 1,(1)There were far more females in TMJOA patients and TMJID patients than males,and patients in both groups had periarticular pain and limited mouth opening.(2)The expression levels of MIF,IL-1β,IL-18,NLRP3 and Caspase-1 in synovial fluid of two group patients were detected and the above molecules were found to be highly expressed in synovial fluid of TMJOA patients(P<0.05).Part 2,(1)SD rats were treated with UAC for the degeneration and defect of the condylar cartilage of the TEMPORomandibular joint,and the synovial tissue was also observed to be swollen,congested and thickened.OARSI score gradually increased with the extension of UAC intervention time,indicating the occurrence and development of TMJOA.Compared with the control group,BV/TV decreased at 4W,8W and 12W after UAC intervention(P<0.01),while BS/BV increased(only significant difference was found in UAC-4W group).Compared with the control group,Tb.N was significantly decreased at UAC-4W(P<0.05)and UAC-8w(P<0.01).Compared with the control group,TB.Th decreased in all UAC intervention groups,but only in UAC-12W group,there was a statistical difference(P<0.01).(2)ELISA results showed that compared with the control group,the expression level of MIF in UAC group was significantly increased,and the expression level was mainly in synovial fluid.(3)Immunohistochemical results showed that MIF,IL-1β,IL-18,NLRP3,ASC,Caspase-1,GSDMD and CXCR4 positive cells were distributed in synovial tissues and cartilage tissues in UAC group,and the expression levels of these proteins were higher than those in control group after UAC intervention.Western blot showed that compared with the control group,the expression levels of MIF,IL-1β,IL-18,NLRP3,ASC,Caspase-1,GSDMD,CXCR4,p-P65/P65,p-IKBα/IKBαand IKKβin UAC intervention group were basically increased.Part 3,(1)In the experiment of FFSS loaded by fibroblast-like synovial cells(FLSs).Compared with 0 and 1dyn/cm~2 groups,the expression levels of NF-κB pathway(p-P65/P65,p-IKBα/IKBαand IKKβ),pyroptosis correlation(NLRP3,GSDMD,Caspase-1 and ASC)and inflammatory factors(MIF,IL-1β,IL-18)and CXCR4increased gradually(P<0.05).(2)In the experiment of FFSS loaded by chondrocytes.Compared with the 0 and 4dyn/cm~2 groups,the expression levels of the above molecules increased gradually with the increase of the loading force(8,12 and 16dyn/cm~2 groups)(P<0.05).(3)Under the action of FFSS,the cell shape of FLSs and chondrocytes changed significantly,such as cell swelling and rupture and the number of FLSs and chondrocytes was reduced.(4)After MIF knockdown,FLSs and chondrocytes expressed pyrophor-related molecules(NLRP3,GSDMD,Caspase-1 and ASC),inflammatory factors(MIF,IL-1β,Il-18)and MIF receptor protein CXCR4 were significantly decreased(P<0.05),and the cell membrane rupture death was delayed.After overexpression of MIF,the expression levels of above proteins were significantly increased(P<0.05),which accelerated cell membrane rupture death.(5)After the pretreatment of FLSs and chondrocytes with some site inhibitors and antagonists,the expression levels of these proteins were significantly decreased after the abnormal FFSS treatment(P<0.05),and the result of cell membrane rupture and death was reversed.Conclusion:1.High expression of inflammatory factors MIF,IL-1βand IL-18 and scorchnose-related mediator NLRP3 and Caspase-1 in TMJOA articular fluid.2.In THE UAC-induced TMJOA model samples,it was found that the degree of synovial inflammation and cartilage degeneration increased with the extension of UAC induction time,and with the extension of UAC induction time,the NF-κB pathway(-P65/P65,p-IKBα/IKBαand IKKβ),scorchdeath correlation(NLRP3,GSDMD,Caspase-1 and ASC),inflammatory factors(MIF,IL-1β,IL-18)and CXCR4 were increased.3.Abnormal fluid shear force induced scortosis in FLSs and chondrocytes.After MIF knockdown,the expression of scortose-related inflammatory factors and MIF receptor protein CXCR4 was significantly reduced under the action of abnormal fluid shear force,and the cell membrane rupture death was delayed.4.Abnormal fluid shear stress upregulates MIF-CXCR4,activates NF-κB pathway and then activates NLRP3-mediated pyrotopia.After site inhibitor combined with antagonist,NF-κB pathway and MIF/CXCR4-NLRP3 pyroptosis related pathway in FLSs and chondrocytes were inhibited.And the occurrence of cell permeation death was slowed or even reversed using above methods. |
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