The Role And Mechanism Of Protease Activated Receptor 2 In Osteoarthritis

Objective 1.To investigate the expression of Protease activated receptor 2(PAR2)in osteoarthritis(OA)cartilage tissue and chondrocytes.2.To investigate the role and mechanism of Protease-activated receptor 2 antagonist AZ3451 in chondrocytes inflammation,autophagy,apoptosis and senescence in vitro.3.To investigate the effect of PAR2 antagonist AZ3451 on the progression of OA in vivo.Method 1.Protease activated receptor 2 expression detection: Anterior cruciate ligament resection surgery was used to prepare rat post-traumatic OA model(PTOA).To investigate the change of PAR2 level in OA development,we detected the difference of PAR2 expression between normal and PTOA rat cartilage by immunofluorescence staining.We also detected the change of PAR2,Collagen Ⅱ and Aggrecan in IL-1β stimulated rat chondrocytes.2.Inflammation and metabolism change detection: We first detected the inhibitory effect of AZ3451 on PAR2 expression in chondrocytes.We next detected the effect of AZ3451 in IL-1β induced expression of inflammatory cytokines and catabolic genes(i NOS,COX2,MMP1,MMP13 and ADAMTS5),as well as the degradation of cartilage matrix(Collagen Ⅱ,Aggrecan)in rat chondrocytes.3.Chondrocytes senescence change detection: Western-blot was used to detect the expression of senescence marker p16INK4 a in IL-1β induced chondrocytes.SA-β-gal staining was used to detect the SA-β-gal activity.4.Chondrocytes autophagy change detection: Western-blot was used to detect the expression of autophagy markers(Atg5,Atg7,Atg12,Beclin1 and LC3)in IL-1β-induced chondrocytes.The tandem GFP-RFP-LC3 adenovirus was utilized to confirm the change of autophagy flux and autophagy level in chondrocytes.5.Apoptosis change detection: Annexin V-FITC / PI staining and flow cytometry analysis was used to detect the apoptosis rates of chondrocytes.Western-blot was used to detect the change of the apoptosis related genes such as Bcl-2,BAX,Cyto C and Cleaved caspase 3 in chondrocytes.Mitochondrial membrane potential was used to detect the change of early apoptosis.TUNEL staining was used to detect the change of late apoptosis.6.Interactions between autophagy,apoptosis and inflammation: Western-blot was used to detect the change of Cleaved caspase 3 and LC3 in chondrocytes under the treatment of chloroquine(CQ).TUNEL staining was used to detect the change of chondrocyte apoptosis.Western-blot was used to detect the change of inflammation related proteins,such as Aggrecan,Collagen Ⅱ,ADAMTS5,MMP13 and COX2 in chondrocytes.7.Signal pathway change detection: Western-blot was used to detect the change of MAPK,PI3 K / AKT / mTOR,and NF-κB signaling pathways.Western-blot was used to detect the change of Aggrecan and Collagen Ⅱ after treatment with signaling pathways inhibitors.Western-blot was used to detect the changes of nuclear and plasma P65 expression.Immunofluorescence was used to detect the nuclear translocation of p65 subunits.8.In vivo experiments: To investigate the effect of AZ3451 on the progression of OA in vivo,AZ3451 was injected into the knee joints of the rat OA model.H&E,Toluidine blue and Safranin O/Fast Green staining of cartilage were used to detect the degree of cartilage damage.Immunohistochemical staining of MMP13,Cleaved-caspase3 and Beclin1 in the rat cartilage,and TUNEL staining were used to detect effects of AZ3451 in vivo.Result 1.We observed the percentage of PAR2 positive chondrocytes was significantly increased in rat OA cartilage,in comparison to normal cartilage.IL-1β lead to the degradation of collagen Ⅱ as well as aggrecan,and increased PAR2 expression in a time-and dose-dependent manner in rat chondrocytes.2.10 μM AZ3451 could effectively inhibit PAR2 expression in rat chondrocytes.PAR2 antagonist AZ3451 inhibited the IL-1β-induced expression of inflammation and metabolism indicators(COX2,iNOS,ADAMTS5,MMP1,MMP13)in rat chondrocytes.AZ3451 could also alleviate the IL-1β-induced cartilage matrix(Collagen Ⅱ,Aggrecan)degradation in rat chondrocytes.3.The IL-1β-treated chondrocytes exerted higher SA-β-gal activity and p16INK4 a protein expression compared with the control group,while PAR2 antagonist AZ3451 significantly prevented this process.4.AZ3451 treatment efficiently increased the levels of autophagy-related proteins such as,Atg5,Atg7,Atg12,Beclin1 and LC3,which were decreased by IL-1β stimulation.AZ3451 also enhanced autophagosome conversion to autophagolysosomes,which ultimately restored the IL-1β-induced disruption of autophagy flux.5.Annexin V-FITC/PI staining and flow cytometry analysis showed that AZ3451 treatment caused a marked decrease of apoptotic chondrocytes induced by IL-1β.AZ3451 downregulated the IL-1β-induced increased Cyto C and Cleaved caspase 3 expression,and reversed the BAX/Bcl-2 ratio in rat chondrocytes.IL-1β stimulation decreased the mitochondrial membrane potential,while AZ3451 treatment remarkably reversed this process.AZ3451 treatment downregulated the IL-1β-induced increasing levels of TUNEL positive cells.6.Western-blot results showed that the IL-1β-mediated upregulation of cleaved caspase 3 was significantly attenuated by AZ3451,while inhibition of autophagy with CQ reversed the anti-apoptotic effect of AZ3451.Similarly,the reduction of the percentage of TUNEL positive cells was also markedly attenuated by CQ.CQ treatment decreased the increased the level of COX2,MMP13 and ADAMTS5 in rat chondrocytes,and also attenuated the IL-1β-induced degradation of collagen Ⅱ and aggrecan.Combined treatment of AZ3451 and CQ couldn’t inhibit the protective effect of AZ3451 in IL-1β-induced catabolic factors(COX2,MMP13 and ADAMTS5).While we observed that the AZ3451 decreased degradation of collagen Ⅱ and aggrecan was partially weakened in the presence of CQ.7.Western-blot results showed that PAR2 antagonist AZ3451 could inhibit the activation of IL-1β-induced P38/MAPK,NF-κB,and PI3K/AKT/mTOR signaling pathways.Combined therapy of P38 / MAPK inhibitor SB203580、PI3K / AKT inhibitor LY294002 and NF-κB inhibitor JSH-23 with AZ3451 played a better protective role in inhibiting the degradation of cartilage matrix.AZ3451 also inhibited the nuclear translocation of p65 subunits induced by IL-1β in chondrocytes.8.We observed the significant superficial articular cartilage erosion as well as the loss of proteoglycan in the PTOA group.In contrast,AZ3451 treatment group exerted the less cartilage damage and the richer proteoglycan compared to the PTOA group.AZ3451 could increase the Beclin1 expression,and decrease MMP13 and Cleaved-Caspase 3 expression in rat OA cartilage in vivo.The TUNEL results showed that the PTOA group exerted higher proportion of chondrocytes apoptosis,while AZ3451 treatment reversed such pathological change.Conclusion 1.PAR2 is highly expressed in rat OA cartilage tissue and in IL-1β treated chondrocytes.2.PAR2 antagonist AZ3451 suppresses IL-1β-induced inflammation response,cartilage degradation,premature senescence and apoptosis in rat chondrocytes.AZ3451 also alleviates the IL-1β-induced autophagy downregulation in rat chondrocytes.The activation of the P38/MAPK,NF-κB and PI3K/AKT/mTOR pathways was blocked by AZ3451 treatment,indicating that these pathways were partially involved in the protective effects of PAR2 antagonist AZ3451 in OA.3.PAR2 antagonist AZ3451 rescues cartilage destruction in rat OA model in vivo.