Effects Of Adenovirus-mediated Knockdown Of IRAK4 On Synovitis In The Osteoarthritis Rabbit Model

ObjectiveTo investigate the effect of adenovirus expressing IRAK4 sh RNA(Ad-sh IRAK4)mediated knockdown of IRAK4 on synovitis,cartilage damage and subchondral bone abnormality in rabbits with knee osteoarthritis(OA),and to determine whether IRAK4 can be used as a new target to intervene synovitis and improve OA pathology.MethodSix New Zealand white rabbits were randomly divided into sham operation group(control,n=3)and osteoarthritis model group(OA,n=3).A rabbit model of knee osteoarthritis was established by anterior cruciate ligament resection of the right knee.Six weeks after surgery,the knee joint of rabbits was observed by general observation and X-ray scan,and the expression of IRAK4 in normal synovial tissue and OA synovial tissue was assessed by Western blot.Twenty-five New Zealand white rabbits were randomly divided into sham operation group(Control),osteoarthritis model group(OA),adenovirus empty vector group(Ad-C),adenovirus-sh IRAK4 low titer group(Ad-sh IRAK4-L),and adenovirus-sh IRAK4 high titer group(Ad-sh IRAK4-H),with 5 rabbits in each group.In the sham operation group,only the right knee cavity was incised,but the ACL segment was not cut,and the other groups received right knee ACL resection.Two weeks after surgery,Ad-sh IRAK4(1×10~8pfu)was injected into the joint cavity of Ad-sh IRAK4-L group and Ad-sh IRAK4(1×10~9pfu)was injected into Ad-sh IRAK4-H group.Ad-c group was injected with empty adenovirus vector.Control group and OA group were injected with 0.9%normal saline.Micro-CT was used to assess the overall condition of the knee joint and the microstructure of the subchondral bone 6weeks after injection into the joint cavity.The joint cavity was opened and the synovium and cartilage of the knee were evaluated in general.q RT-PCR was used to detect the m RNA expression of IRAK4,TRAF6 and IL-1βin synovial tissue.The protein expression of IRAK4,TRAF6,TAK1,P-IKK and P-NF-κB in synovial tissue was evaluated by Western blot.Immunohistochemistry was used to detect the positive expression of IRAK4,TRAF6 and TAK1 in synovial tissue.In addition,the positive expression of IRAK4 protein in articular cartilage was detected by immunohistochemistry.H&E staining was used to assess pathological changes in synovium and cartilage.The levels of IL-1β,TNF-αand MMP-13 in synovial fluid were determined by ELISA.Data were collected for statistical analysis.ResultSix weeks after anterior cruciate ligament resection,the surface of femoral condyle and tibial plateau cartilage in the Control group was smooth,without defect or osteophyte,synovial hyperplasia and articular cavity effusion.However,the knee joint in OA model group had rough surface,obvious defect and osteophyte,and increased joint effusion and synovial hyperplasia.IRAK4 protein could be expressed in synovial tissues of Control group and OA model group.However,compared with the Control group,the protein expression level of IRAK4 in synovial tissues of OA model group was significantly increased.H&E staining results showed that the severity of synovial hyperplasia,inflammatory cell infiltration and angiogenesis were significantly increased in OA model group and Ad-C group compared with the Control group.However,in the Ad-sh IRAK4-L and Ad-sh IRAK4-H groups,there was less inflammatory cell infiltration,synovial hyperplasia and angiogenesis,but they were still higher than normal levels.ELISA results showed that the level of IL-1βin synovial fluid of OA model group was significantly higher than that of Control group.Ad-sh IRAK4 significantly reduced the level of IL-1βin OA synovial fluid in a titer-dependent manner.Similarly,TNF-αlevels in synovial fluid were significantly increased in the OA model group compared with the Control group.The level of TNF-αin OA synovial fluid was significantly decreased in Ad-sh IRAK4-H group.Notably,Ad-sh IRAK4 inhibits TLR/IL-1R signal transduction in synovial tissue.Ad-shirak4significantly decreased m RNA expression of IRAK4,TRAF6,IL-1βand protein expression of IRAK4,TRAF6,TAK1,P-IKK,P-NFκB in synovial tissue.In addition,immunohistochemical results showed that the expression levels of IRAK4,TRAF6and TAK1 in OA model group were higher than those in Control group.Ad-sh IRAK4decreased the expression of IRAK4,TRAF6 and TAK1 in synovium of rabbit knee osteoarthritis.The levels of IL-1β,TNF-αand MMP-13 in synovial fluid in OA model group were significantly higher than those in Control group,and Ad-sh IRAK4 could decrease the levels of IL-1β,TNF-αand MMP-13 in synovial fluid in OA model group.The gross view of articular cartilage and H&E staining results showed that Ad-sh IRAK4 could alleviate cartilage destruction in rabbit knee osteoarthritis model.In addition,immunohistochemical results showed an increase in the number of IRAK4protein positive cells in articular cartilage in the OA model group and the Ad-C group compared with the Control group.After Ad-sh IRAK4 injection,the expression of IRAK4 protein in articular cartilage decreased significantly,and the difference was statistically significant.Micro-CT analysis showed that Ad-sh IRAK4 treatment significantly increased BV/TV and TB.N compared with OA model group.ConclusionIRAK4 is involved in anterior cruciate ligamentectomy induced OA lesions of rabbit knee joint.Ad-sh IRAK4 can alleviate synovitis,cartilage destruction and subchondral bone microstructure changes of knee OA in rabbits,thus limiting or delaying the progression of OA.Therefore,selective inhibition of IRAK4 may be a new therapeutic strategy for the clinical prevention and treatment of OA.