Effect Of Adenosine A3 Receptor On Rat Cartilage Damage Through Regulation Of NLRP3/caspase-1 Signaling

Osteoarthritis（OA）,a common joint disease in aged animals,which caused chronic pain and motor dysfunction.The pathogenesis of OA has not yet been fully elucidated.Irreversible articular cartilage damage caused by an imbalance between extracellular mat rix synthesis and degradation is the main feature of OA.At present,the drug treatment methods for OA are limited,and it is urgent to find drugs that can relieve the symptoms of the disease and delay the disease process.Adenosine A3 receptor（A3AR）has been studied as anti-inflammatory and pain-relief target in various diseases,and activation of A3AR can inhibit inflammation and alleviate chronic neuropathic pain.However,the role of A3AR activation in OA has not been clarified.Inflammation caused ex tracellular matrix degradation and pain in OA,and a variety of pro-inflammatory cytokines and inflammatory signaling pathways play an important role in the development of OA disease.Studies have shown that the pyroptosis pathway induced by the NLRP3 inflammasome is an important source of pro-inflammatory factors.Inhibiting the activation of the NLRP3 inflammasome may reduce joint inflammation and delay the development of diseases,such as rheumatoid arthritis,gout,and psoriasis.However,the role of the NLRP3 inflammasome-mediated pyroptosis pathway in cartilage degeneration in OA remains unknown.Based on the above research background,our study investigated whether the NLRP3 inflammasome-mediated pyroptosis pathway is involved in OA cartilage lesions in the rat OA model,and clarified whether A3AR activation plays a protective role in cartilage by inhibiting NLRP3/caspase-1/GSDMD pathway.Totally 60 SD rats were randomly divided into 5 groups in vivo,including Sham group（Control）;ACLT model group（O A）,ACLT+CF101 treatment group（100μg/kg）,ACLT+MRS1523 group（100μg/kg）,and ACLT+MRS1523+CF101 group（MRS1523 was given first,and CF101 was given 30 minutes later）.Histopathological analysis was performed to detecting the cartilage damage,and the ex tracellular matrix synthesis and degradation factors,the activation of NLRP3 inflammasome pathway,and the pro-inflammatory factors were examined to explore whether A3AR activation protects articular cartilage damage.In order to clarify the role of NLRP3/caspase-1/GSDMD signaling pathway in OA cartilage destruction,primary rat chondrocytes were cultured and stimulated with H ₂O₂ in vitro.The chondrocytes were divided into 6 groups,including control group,H ₂O₂ group（300μM）,MCC950 group（10μM）,MCC950+H₂O₂ group（10μM+300μM）,VX765 group（50μM）and VX765+H₂O₂ group（50μM+300μM）.After stimulation,the expression of biomarkers of articular cartilage injury and proteins associated with NLRP3 pathway were detected.In order to further clarify whether the activation of adenosine A3 receptors plays a role in regulating the signaling pathway mediated by the NLRP3 inflammasome,the in vitro experiments were divided into another 6 groups,including control group,H ₂O₂（300μM）group,CF101 group,CF101+H ₂O₂（1μM+300μM）group,MRS1523+H₂O₂（2μM+300μM）group,MRS1523+CF101+H₂O₂（2μM+1μM+300μM）group.Furthermore,the activation of NLRP3 signaling pathway,the expression of pro-inflammatory cytokines,and levels of upstream moleculars of NLRP3 activation(such as intracellular K⁺,Ca²⁺and ROS)were examined.The results showed as follows:（1）The mechanical pain threshold（PWT）of rats after ACLT decreased continuously,and the level of COX-2 in serum and synovial fluid increased significantly.After CF101（adenosine A3 receptor agonists）intervention,PWT gradually increased and the level of COX-2 decreased,indicating that CF101 relieved OA pain.（2）According to histopathological examination,the articular cartilage surface was obviously defective in the ACLT model group,the chondrocytes were arranged disorderly,the red staining of matrix was reduced,and the OARSI score was significantly increased.CF101treatment group showed slight damage to the articular cartilage,and cell proliferation occured in the cartilage surface and OARSI score was significantly decreased.MRS1523（an adenosine A3 receptor antagonist）pretreatment and then given CF101 showed that the cartilage surface damage was significantly aggravated,and the matrix red staining was greatly reduced.It is indicated that CF101 activates A3AR and exerts a protective effect on cartilage,and MRS1523 counteracted the effect.（3）In the ACLT group,the expressions of Col-II and aggreacan were down-regulated,and the expressions of cartilage matrix degrading enzymes,including MMP-13 and ADAMTS-5 were up-regulated.When compared with ACLT group,CF101 treatment group showed increased expressions of Col-II and aggreacan,and decreased expressions of MMP-13 and ADAMTS-5.Rather,MRS1523+CF101 significantly reversed the effect of CF101 on matrix degradation.（4）The protein expressions of NLRP3,ASC,caspase-1 and GSDMD in the ACLT group were significantly increased,and the levels of IL-1β,IL-18 and TNF-αin serum and joint cavity lavage fluid were significantly increased.When compared with ACLT group,CF101inhibited the NLRP3 pathway protein and the expression of downstream pro-inflammatory cytokines.In contrast,MRS1523+CF101 antagonized the effect of CF101,indicating that A3AR activation exerts a chondroprotective effect by inhibiting NLRP3-mediated inflammatory signaling pathway in cartilage.（5）After H₂O₂ treatment of chondrocytes,the release of LDH was significantly increased,the protein expressions of NLRP3,caspase-1 p20 and GSDMD-N,and the expression levels of IL-1βand IL-18 in the cell culture supernatant were significantly up-regulated as compared with control.Moreover,the levels of collagen degradation biomarkers COMP and CTX-II were increased and the expression level of pro teoglycan synthesis biomarker GAG was significantly decreased.As compared with H ₂O₂ group,chondrocytes pre-treated with MCC950（a NLRP3-specific inhibitor）and VX765（a caspase-1-specific inhibitor）,significantly inhibited the release of LDH,reduced th e gene and protein expression of NLRP3/caspase-1/GSDMD-N,and the expression of IL-1βand IL-18,decreasd the expression levels of COMP and CTX-II and increased the expression of GAG.The results indicated that NLRP3/caspase-1 pathway was involved in the p rocess of chondrocyte injury,and inhibiting the NLRP3 pathway in chondrocytes could reduce cell damage and delay the degradation of OA matrix.（6）After H₂O₂ treatment of chondrocytes,the protein expressions of NLRP3,caspase-1p20,GSDMD-N and the expression levels of IL-1βand IL-18 increased,the double positive staining of caspase-1/PI increased,and the inflammation of NLRP3 was reduced as compared with control.Moreover,the upstream activator K⁺level was significantly reduced,the Ca²⁺level and ROS levels were significantly increased.When compared with H₂O₂ group,the expression of NLRP3 pathway-related proteins and pro-inflammatory cytokines was inh ibited,the double positive staining of caspase-1/PI was significantly reduced,and the ROS release was significantly reduced after CF101 treatment,while K⁺and Ca²⁺level had no significant change.MRS1523 antagonized the protective effect of CF101.Thes e results indicated that CF101 activates A3AR in chondrocytes,thereby inhibiting the activation of the NLRP3inflammasome-mediated pyroptosis pathway and the release of pro-inflammatory factors by reducing the level of intracellular ROS.In conclusion,MCC950 and VX765 inhibit NLRP3/caspase-1 signaling and relieve extracellular matrix degradation in chondrocytes.CF101 treatment activates adenosine A3receptors,thereby inhibits NLRP3/caspase-1/GSDMD pathway induced pyroptosis and inflammation through inhibiting the release of intracellular ROS in chondrocytes.Furthermore,CF101 treatment reduced the production of matrix-degrading enzymes,and relieved OA pain and articular cartilage damage,ultimately delay the development of OA disease.