| Objective: Temporomandibular Joint Osteoarthritis(TMJOA)is a prevalent disease in the oral and maxillofacial region,characterized by synovial inflammation,articular cartilage destruction,and subchondral bone remodeling.Synovial inflammation has been thought to initiate TMJOA.Synovitis is the earliest manifestation of TMJOA and the main source of pain.The synovial inflammation persists throughout the whole course of the disease.Synovial tissue consists of synovial lining layer and the connective sublining layer which contains connective tissue and blood vessels.Fibroblast-like synoviocytes(FLS),one of the main cells that constitute the synovial lining layer,secrete the proinflammatory cytokine Interleukin-1β(IL-1β)into joint cavity.IL-1β is a known cause of cartilage degeneration and often companied by vascular endothelial growth factor(VEGF)because angiogenesis and inflammation are closely integrated processes in TMJOA.As an anti-inflammatory cytokine,IL-36 receptor antagonist(IL-36Ra)blocks the activation of the downstream signaling pathway and the expression of inflammatory genes through antagonism.IL-36 Ra and IL-1β belong to the same IL-1superfamily.IL-36 Ra is an anti-inflammatory factor which is widely used in many diseases but has not been demonstrated in the temporomandibular joint.One of the objectives of this study was to detect the expressing and anti-inflammatory effect of IL-36 Ra in TMJ tissues.If IL-36 Ra can control early synovitis,it may prevent irreversible articular cartilage destruction and subchondral bone remodeling,and thereby alleviate the progression of TMJOA.Pyroptosis is a form of programmed cell death closely related to inflammation.Pyroptosis plays a key role in TMJOA.The canonical pyroptosis depends on the activation of Caspase 1 by inflammasomes.The most investigated inflammasome is NOD-like receptor family pyrin domain containing 3(NLRP3).After Caspase 1 is activated by the NLRP3 inflammasome,on the one hand,it promotes the maturation of pro IL-1β and pro IL-18;on the other hand,it cleaves Gasdermin D(GSDMD)to form pores in the cell membrane,causing cell rupture,and releasing mature IL-1β and IL-18 and other cytokines to the outside of the cell to cause a violent inflammatory response.Another objective of this study was to investigate whether IL-36 Ra can play an antiinflammatory role by inhibiting FLS pyroptosis mediated by NLRP3/Caspase 1 and reducing IL-1β production.Therefore,this study explored the expression and distribution of IL-36α,IL-36 Ra and pyroptosis pathway related molecules(NLRP3、Caspase 1、GSDMD 、 IL-1β)in temporomandibular joint tissues by testing TMJOA patients’ synovial tissues.Then animal experiments were displayed to confirm the therapeutic effect of IL-36 Ra on TMJOA mice.Last but not least,invitro experiments was undertook on FLS to investigate the molecular mechanisms of IL-36 Ra on TMJOA.We hope to provide scientific references and theoretical foundations for the treatment of TMJOA.Methods: 1.Testing of clinical patients’ samples: Patients were selected from the Department of Oral and maxillofacial Surgery,Affiliated Stomatological Hospital of China Medical University.Patients were diagnosed as TMJ osteoarthropathy by specialists strictly according to the diagnostic criteria for temporomandibular joint Disorders(RDC/TMD)and required temporomandibular joint disc repositioning surgery.Synovial tissues from TMJOA patients were collected during clinical surgery.Samples were divided into slight synovitis group and moderate/pronounced synovitis group by preoperative MRI analysis and HE staining of synovial tissues.Immunohistochemical staining was used to analyze the distribution and differential expression of IL-36α,IL-36 Ra,pyroptosis pathway related molecules(NLRP3,Caspase 1,GSDMD),pyroptosis product IL-1β and pro-angiogenic factor VEGF in both groups.Fibroblast-like synoviocytes were marked by Vimentin and the immunofluorescence co-localization test was used to determine whether the target proteins(IL-36α 、 IL-36 Ra 、 NLRP3 、Caspase 1、GSDMD、IL-1β)were produced by fibroblast-like synoviocytes.2.Animal experiments: A TMJOA animal model was established by injecting 20 μl complete Freund’s adjuvant(CFA 1:1 mixed well with 0.9% saline)into the upper chamber of temporomandibular joint in 6-8 week-old male C57BL/6 mice.Micro-CT analysis,HE staining,Safranin O-Fast Green staining,and immunohistochemical staining were applied to evaluate the condition of synovial inflammation,articular cartilage destruction,and subchondral bone remodeling after 2 weeks to confirm the TMJOA animal model was successful established.One week after establishing the model,low,medium,and high doses(10 ng,50 ng,250 ng)of IL-36 Ra were injected into the temporomandibular joint cavity to treat TMJOA.A week later,the therapeutic effect of IL-36 Ra on TMJOA and the expression of IL-36α and pyroptosis pathway related molecules were evaluated through Micro-CT,HE staining,Safranin O-Fast Green staining,and immunohistochemical staining.The changes of body weight and food intake were monitored regularly during the experiment.3.In vitro experiments,fibroblast-like synoviocyte cells line SW982 were stimulated with lipopolysaccharide(LPS)and adenosine triphosphate(ATP)to induce pyroptosis in cells.After adding different concentrations of IL-36 Ra to apply anti-inflammatory treatment,CCK-8 and LDH test were used to detect cell activity and membrane integrity,respectively.ELISA was used to detect IL-1β secretion.Real-time PCR and Western Blot were used to detect the gene and protein expression of pyroptosis related factors,respectively.Rescue experiments further explored whether IL-36 Ra exerts anti-inflammatory effects by inhibiting FLS pyroptosis through NLRP3/Caspase 1 pathway.FLS was transfected with plasmid to overexpress NLRP3,a key protein of pyroptosis,and whether the overexpression of NLRP3 could reverse the inhibitory effect of IL-36 Ra on FLS pyroptosis was observed.Finally,the NLRP3 inhibitor MCC950 was used to further clarify whether IL-36 Ra can inhibit pyroptosis in FLS and play an anti-inflammatory role through blocking the NLRP3/Caspase 1 pathway which is activated by IL-36α.All the in vivo experiments were applied on more than three mice,and in vitro experiments were repeated three times.Experimental results are presented as mean±SD,with P<0.05 indicating statistical significance.Results: 1.A total of 28 patients were included,and 39 synovial tissues were collected.By MRI image evaluation and HE staining evaluation,39 cases of tissue were divided into two groups,18 cases of slight synovitis group and 21 cases of moderate/ pronounced synovitis group.Immunohistochemical staining results showed that the synovial tissue of TMJOA patients could express IL-36α and IL-36 Ra.IL-36α and IL-36 Ra were weakly positive expressed in the lining layer of synovium tissue in the slight synovitis group,but strongly positive expressed in moderate/pronounced synovitis group,with statistical significance between two groups(P<0.05).At the same time,the synovial tissues of TMJOA patients could also express NLRP3,Caspase 1,GSDMD,and the expressions of these proteins were higher in moderate/pronounced synovitis group than in the slight synovitis group,with statistical significance between two groups(P<0.05).The expressions of pro-inflammatory factor IL-1β and pro-angiogenic factor VEGF were also differently expressed between two groups(P<0.05).Immunofluorescence co-localization results showed that Vimentin was highly co-localized with IL-36α,IL-36 Ra,NLRP3,Caspase 1,GSDMD and IL-1β.2.The average daily food intake and body weight of mice decreased after CFA was injected into the temporomandibular joint.The TMJOA model can be effectively established in mouse by injecting CFA into the joint cavity.The TMJOA mice showed proliferation of lining layer cells,angiogenesis and inflammatory cell infiltration in the sublining layer of synovial tissue by HE staining.Obvious changes in cartilage were also observed: degeneration of condylar cartilage,thinning of the cartilage layer,disordered arrangement of chondrocytes layer,and significant reduction in the number of cells in the proliferative layer and hypertrophic layer,the bone trabeculae in the subchondral bone layer were abnormal and disordered.Safranin O-Fast Green staining showed that the proteoglycan in the condylar cartilage of TMJOA mice were degraded and the cartilage matrix were lost.Micro-CT showed that condylar surfaces were destroyed and subchondral bone density decreased.However,after injecting different doses of IL-36 Ra into the joint cavities of TMJOA mice,the average daily food intake and body weight of the mice were higher than that of the mice in the TMJOA group.At the same time,injecting different doses of IL-36 Ra into the joint cavities of TMJOA mice can improve the pathological manifestations of TMJOA,reduce the proliferation of cells in the synovial lining layer,reduce the angiogenesis and inflammatory cell infiltration in the sublining layer of synovial tissue,and alleviate cartilage degeneration and abnormal remodeling of subchondral bone.Immunohistochemical staining results showed that the expressions of IL-36α and pyroptosis related factors(NLRP3、Caspase 1、GSDMD and IL-1β)in synovial tissue and condylar cartilage tissue were decreased after IL-36 Ra injection,with statistical significance(P<0.05).3.Applying 10 μg/ml LPS for 12 h and 5 m M ATP for 1 h could effectively induce pyroptosis in FLS.Compared with Control group,the activity of FLS was decreased,LDH release was increased,IL-1β in supernatant was increased in the pyroptosis group,and the expression of pyroptosis related proteins was increased by Western Blot test,with statistical significance(P<0.05).100 ng /ml IL-36 Ra was applied into FLS with pyroptosis condition,which improve cell activity,significantly reduce the release of LDH and reduce the secretion of IL-1β.Real-time PCR results showed that the gene expression levels of NLRP3,GSDMD and IL-1β were down-regulated.Western Blot results showed that the protein expression levels of activated Caspase p10,NLRP3,cleaved GSDMD-N and mature IL-1β were significantly decreased,with statistical significance(P<0.05).After transfecting FLS with plasmid to overexpress NLRP3,CCK8 results showed that the activity of cells that were originally up-regulated by IL-36 Ra,but decreased after overexpressing NLRP3.ELISA showed that IL-1β level in the supernatant could be down-regulated by IL-36 Ra,but increased after overexpressing of NLRP3.Real-time PCR and Western Blot results also showed that the down-regulated effects of IL-36 Ra on pyroptosis related factors in FLS were reversed after the overexpression of NLRP3 at gene level and protein level,respectively.Finally,the reverse experiments proved that 100 ng/ml of pro-inflammatory factor IL-36α could reduce cell activity and increase gene and protein expression levels of pyroptosis related factors(NLRP3 、 Caspase 1 、 GSDMD and IL-1β).After adding IL-36 Ra to play an antagonistic role,cell activity was increased and gene and protein expression levels of pyroptosis related factors(NLRP3 、 Caspase 1 、 GSDMD and IL-1β)were downregulated.The addition of NLRP3 inhibitor MCC950 can also increase the decrease of cell activity induced by IL-36α,and down-regulate the increase of gene and protein levels of pyroptosis related factors induced by IL-36α.Conclusion: 1.IL-36α,IL-36 Ra and pyroptosis related factors(NLRP3、Caspase 1、GSDMD、IL-1β)were all expressed in the synovial tissues of TMJOA patients,and the expression levels were higher in the synovial tissues with more severe inflammation.Besides,all these factors were mainly produced by fibroblast-like synovial cells.2.Injection of IL-36 Ra into temporomandibular joint can improve TMJOA synovial inflammation,cartilage degeneration and subchondral bone destruction in mice.3.As a receptor antagonist,IL-36 Ra may reduce the production of IL-1β through inhibiting NLRP3/Caspase 1 pathway mediated pyroptosis,and thus reduce the cascade inflammatory reaction and relieve TMJOA. |
