Tat-Beclin1 Effectively Enhances The Chondroprotective Effect Of IL-1Ra In Rat Osteoarthritis Model

Introduction:The inflammatory cytokines,especially IL-1?,play important role in the development and progression of osteoarthritis(OA).Targeting inflammatory cytokine is an efficient treatment for OA.In this study,we aim to investigate the protective mechanism of IL-1 receptor antagonist(IL-1Ra)on OA chondrocytes,and to explore a method to promote IL-1Ra chondroprotection in OA rat model in vitro and vivo.Methods:In vitro:Chondrocytes were isolated from knee cartilage of neonatal male Sprague-Dawley rats(within 24 hours after birth).These cells were suspended in DMEM/F12 containing 10%fetal bovine serum(FBS),then cells were cultured with IL-1?(20ng/ml)stimulation for 36 hours,followed with the treatment of IL-1Ra(40ng/ml)for 36 hours:(1)total mRNA and proteins of Collagen ?,Aggrecan,Beclin1,and LC3B from chondrocytes were detected with real-time PCR and western blotting.Akt,P-Akt,mTOR,P-mTOR,ULK1,P-ULK1,NF-kB/p65,and P-NF-kB/p65 were detected with western blotting as well.Meanwhile,autophagic vacuoles were detected with immunofluorescence and transmission electron microscopy.(2)The protein level of LC3B in nucleus and cytoplasm was detected with western blotting analysis.(3)The acetylation level of LC3B protein in nucleus were detected with immunoprecipitation.(4)After cells were cultured with Tat-Beclin1(1O?M)for 8 hours,total proteins of Collagen II,Aggrecan,Beclinl,and LC3B were detected with western blotting.Meanwhile,autophagic vacuoles were detected with immunofluorescence and transmission electron microscopy.In vivo:45 Sprague-Dawley rats(about 4-5 weeks old male,120g)were used in inducing with anterior cruciate ligament transection combined with resection of medial menisci(ACLT + MMx).(1)Samples were divided into 7 groups:normal,sham,OA,OA + saline,OA+IL-1Ra,OA+Tat-Beclinl,and OA+IL-1Ra+Tat-Beclinl group.After surgery,these ingredients were injected into the knee joints(twice a week,)for two weeks.(2)After 3 weeks or 5 weeks,rats were sacrificed,and knee joints were acquired for H.E.staining,Safarinin-O staining,and immunohistochemistrical analysis(combining with CollagenII,Aggrecan,Beclin1 and LC3B antibody respectively),followed with morphological observation.Results:In vitro:(1)The results of real-time PCR and western blotting showed that IL-1Ra increased the mRNA and protein levels of Collagen?,Aggrecan,Beclinl and LC3B in 1L-1?-stimulated rat OA chondrocytes.(2)Meanwhile,the number of autophagic vacuoles was increased by the treatment of IL-1Ra.(3)The ratio of P-Akt/Akt,P-mTOR/mTOR,P-ULK1/ULK,and P-NF-KB/p65/NF-KB/p65 were increased by the treatment of IL-1Ra.Meanwhile,the acetylation level of LC3B in nuclear was decreased.(4)Tat-Beclinl not only could up-regulate the Collagenll and Aggrecan protein levels,but also could enhance the protective effect of IL-1Ra on OA chondrocyte(*P<0.05,**P<0.01,***P<0.001,****p<0.0001).In vivo:(1)The intra-articular injections with IL-1 Ra,Tat-Beclin 1,and IL-1 Ra+Tat-Beclin l could ameliorate cartilage impairment in rat OA models,compared with control group.Among them,the effect of IL-1Ra+TAT-beclina on cartilage is the best one.(2)The levels of Collagen?,Aggrecan,Beclinl,and LC3B were more increased in IL-1Ra,Tat-Beclin1,and IL-1 Ra+Tat-Beclin 1 groups,compared with control group.Among them,the effect of IL-1Ra+TAT-beclina on these protein expressions is the best one(*P<0.05,**P<0.01,***P<0.001,****p<0.0001)?Conclusion:In this study,IL-1Ra could promote the synthesis of extracellular matrix(Collagen? and Aggrecan),as well as the increased autophagy(Beclinl and LC3B)in IL-1?-stimulated rat OA chondrocytes and OA rat model.Akt/NF-KB/p65,Akt/mTOR/ULK1 and the deacetylation of LC3B were involved in regulating autophagy related to IL-1Ra.Tat-Beclinl could improve the protective effect of IL-IRa in early OA model.