Effects And Mechanisms Of Carbon Monoxide Releasing Molecule-3 On Temporomandibular Joint Osteoarthrits Induced By Interleukin-1?

BackgroundOsteoarthritis is one of the most important types of temporomandibular joint(TMJ)disorder syndrome.In animals,it is usually caused by wear and degeneration of temporomandibular region.Articular cartilage injury is the main feature,which is characterized by apoptosis of chondrocytes,the destruction of extracellular matrix of cartilage,and absorption of subchondral bone.It was found that interleukin-1?(IL-1?)can induce the formation of TMJ arthritis,while carbon monoxide(CO)can inhibit apoptosis and reduce inflammatory response in vitro.Carbon monoxide molecule-3(CORM-3)can simulate the controlled slow release of CO in the physiological environment.However,the effects and mechanism of carbon monoxide molecule-3 on IL-1? induced TMJ arthritis are still unclear.ObjectiveIn order to explore the regulatory effect and mechanism of carbon monoxide releasing molecule-3 on osteoarthritis in in vivo experiment,we established the rat model of TMJ arthritis,the chondrocytes of the temporomandibular condyle were induced by IL-1? to explore the regulatory mechanism of carbon monoxide releasing molecule-3 on inflammatory chondrocytes in vitro.Methods(1)The isolation,culture and identification of primary rat condylar chondrocytes:The chondrocytes of rat condyle were isolated and cultured by the method of secondary enzyme digestion and identified as chondrocytes by the method of toluidine blue staining.(2)The HO-1 gene of condylar chondrocytes was silenced by Si-RNA transfection,and the efficiency of HO-1 silencing was detected by PCR.Group:Blank group:normal cells cultured in the normal medium;Inflammation group:chondrocytes were cultured in the medium containing IL-1?;iCORM-3 group:chondrocytes were cultured in the medium containing IL-1? and iCORM-3(CORM-3 solution was prepared before 24 hours in advance to inactivate it);CORM-3 group:chondrocytes were cultured in the medium containing IL-1? and CORM-3;Silence group:chondrocytes that the gene of HO-1 was silenced were cultured in the medium containing IL-1? and CORM-3.The concentration of IL-1? used in this step is 10 ng/ml,and the concentrations of CORM-3 and iCORM-3 are 200?M.RT-qPCR was used to detect the genes expression of Col-2,MMP-3,MMP-9 and MMP-13 in each group.The experiment was repeated at least three times,and the results were statistically analyzed with Graphpad Prism.If there are only two groups in the experiment,T test is used for comparison.If there are more than two groups in the experiment,the pairwise comparison between the groups is conducted by one-way analysis of variance and Turkey test.The difference of P<0.05 was considered to be statistically significant.(3)The establishment of rat model of TMJ arthritis:rats were induced by bilateral temporomandibular joint cavity injection of complete adjuvant(CFA,50 ?g)and IL-1?(10 ng/ml),and HE staining was used to identify whether TMJ arthritis model was established?(4)In the control group,50?1 saline was injected into the TMJ;in the inflammatory group,50 ?l CFA containing IL-1 ?(10 ng/ml)was injected into TMJ;In the iCORM-3 group,50?1 of CFA containing IL-1?(10 ng/ml)was injected into the TMJ and the pretreated iCORM(CORM-3 solution was prepared before 24 hours in advance to inactivate it)was injected intraperitoneally after 24 hours for successive 7 days;in CORM-3 group,50 ?l CFA containing IL-1?(10 ng/ml)was injected into TMJ,24 hours later,CORM-3 was injected intraperitoneally for successive 7 days.The concentrations of CORM-3 and iCORM-3 are 10 mg/kg/day.On the eighth day,the inflammatory lesions of condylar cartilage were extracted and then detected by HE staining,and the expressions of HO-1,Col-2,MMP-3,MMP-9 and MMP-13 were detected by immunohistochemistry.Results(1)After subculture,the cells extracted from the condylar cartilage of the rat were in good condition and were identified as chondrocytes by toluidine blue staining.(2)CORM-3 significantly inhibited the expression of MMP-3,MMP-9 and MMP-13 in the rat condylar chondrocytes stimulated with IL-1?,but significantly increased the expression of Col-2 and HO-1.The regulatory effect of CORM-3 on the expression of Col-2,MMP-3,MMP-9 and MMP-13 in the rat condylar chondrocytes was significantly weaker after HO-1 gene silencing,suggesting that the anti-inflammatory effect of CORM-3 on the condylar chondrocytes stimulated with IL-1? was partially mediated by HO-1 pathway.(3)After 50 ?l CFA containing IL-1?(10 ng/ml)was injected into bilateral TMJ of rats,TMJ region swelling,the decreased frequency of food intake and the blood and tears around the eyes was observed.One week later,HE staining showed that the space between the temporomandibular joint disc and the condylar cartilage was smaller than that in the normal condition,the condylar cartilage layer was thinner,and the articular disc was destroyed,the joint cavity was full of inflammatory secretions and fibrin,there were blood vessels and fat droplets near the articular disc.These results showed that establishment of the rat TMJ arthritis model was successful.(4)Immunohistochemical staining showed that CORM-3 increased the expression of HO-1 and Col-2,decreased the expression of MMP-3,MMP-9 and MMP-13,indicating that CORM-3 inhibited inflammatory response of temporomandibular arthritis in vivo,and this effect might be related to HO-1.ConclusionsCarbon monoxide releasing molecule-3 inhibits the inflammatory response in rat condylar chondrocytes stimulated with IL-1?,and TMJ-OA induced by interleukin-1?,which is evidenced by the data that CORM-3 significantly inhibits the expression of MMP-3?MMP-9 and MMP-13,but increases the expression of Col-2.The regulatory effects of CORM-3 might be via HO-1 pathway,evidenced by the decreased regulation of CORM-3 after HO-1 silence.The results of the study provide a new approchment for the treatment of TMJ arthritis.