IL-1Ra Regulates Autophagy In Human Osteoarthritis Chondrocytes

Objective:Autophagy is a cellular self-digestion process and has been known to play important roles in human osteoarthritis(OA)pathophysiology.The objective of this study was to investigate whether IL-1Ra promoted cartilage matrix synthesis through increasing autophagy in human OA chondrocytes and how is the synergistic effect of IL-1?and autophagy enhancer(TAT-Beclin-1)on extracellular matrix synthesis.Methods:1.Human OA chondrocytes treated with 40ng/ml IL-1Ra and stimulation with or without 10ng/ml IL-1?.(1)The expression levels of Type II collagen,Aggreacan,LC3B,and Beclin-1 protein were detected by Western blot;(2)The mRNA levels of LC3B and Beclin1 were measured by real-time PCR;(3)The morphological character of autophagic veoleus in human OA chondrocytes was observed under CONFOCAL microscope.(4)The morphological character of autophagic veoleus was observed under Transmission electron microscopy(TEM).2.(1)After nucleocytoplasmic separation experiment,the expression level of LC3B proteinwas detected by Western blot.(2)After nucleocytoplasmic separation experiment,,nuclear lysates were immunoprecipitated with antibody to LC3B followed by Western blot with antibody against acetylated-lysine.(3)The expression levels of Akt,mTOR,NF-?B,and ULKlprotein in human OA chondrocytes were detected by western blot.(4)Protein expression in signaling pathway was assayed by western blot.3.The human OA chondrocytes were pretreated with 10nM TAT-Beclin-1 for 24h.(1)Type ? collagen,Aggreacan,LC3B was determined by Western blotting.(2)Type ? collagen,Aggreacan,LC3B after treatment with 40ng/ml IL-1Ra or 10nM TAT-Beclin-1 and stimulation with IL-1? for 24h was determined by Western blotting.4.Rat OA model was obtained with "ACLT+MMx"operation.Samples were divided into 4groups,NS,IL-1Ra,TAT-Beclin-1,IL-1Ra and TAT-Beclin-1 group.(1)Morphologic characters of articular cartilage were performed with HE stain and Safranin O stain,followed the evaluation of OARSI grades.(2)The expression levels of Collagen?,Aggrecan,Beclinl,and LC3B were detected with immunohistochemistry assay.Results:1.IL·1Ra raise expression levels of Type II collagen,Aggreacan,LC3B,and Beclin-1 protein were detected by Western blot;(2)IL-1Ra raise expression mRNA levels of LC3B and Beclinl were measured by real-time PCR;(3)IL-1Ra raise expression of autophagic veoleus in human OA chondrocytes was observed under CONFOCAL microscope;(4)IL-1Ra raise expression of autophagic veoleus was obse-rved under Transmission electron microscopy(TEM).2.(1)LC3B was deacetylation in the nucleus by IL-1Ra;(2).IL-1Ra raise ration of P-Akt/Akt,P-mTOR/mTOR,P-ULK1(ser757)/uLK1;(3)IL-1Ra raise expression p65 were de-tected by Western blot,and IL-1Ra decrease P65 in nucleus.3.(1)TAT-Beclin-1 raise expression levels of Type II collagen,Aggreacan,LC3B protein were detected by Western blot.(2)IL-1Ra+TAT-becliN-lraise expression levels of Type II collagen,Aggreacan,LC3B more than other groups.4.The intra-articular injections with IL-1Ra,TAT-Beclin-1,and IL-1Ra+TAT-Beclinl could prevented chondrocytes from OA destruction in rat OA models.(2)The levels of Collagen?,Aggrecan,Beclinl,and LC3B were more increased in IL-1Ra,TAT-Be-clin-1 and IL-1Ra+TAT-Beclinl groups.Conclusion:IL-lRa promoted cartilage matrix synthesis through autophagy induction,in which Akt/mTOR/ULK1,Akt/NF-KB,and deacetylation of LC3B were involved.The synergistic action of IL-1? and autophagy enhancer(TAT-Beclin-1)strengthened the promotive effect of IL-1Ra on extracellular matrix synthesis.