Effects Of Silencing AdipoR1 On Joint Inflammation And Bone Erosion In Mice With Collagen-induced Arthritis

Part 1 Effect of silencing Adiponectin receptor 1 on joint inflammation and bone destruction in collagen-induced arthritisObjective:Previous studies have shown that Adiponectin(AD)is involved in the pathogenesis of rheumatoid arthritis(RA)and the high expression of AD in collagen-induced arthritis(CIA)mice can promote the secretion of inflammatory cytokines and metalloproteinase,activte osteoclasts and aggravate bone erosion.AD plays a biological role mainly through its receptors.The predominant expression of Adiponectin receptor 1(AdipoR1)in RA synovial tissue and CIA mice articular tissue suggests that it may be the main effector receptor of AD in promoting proinflammatory action.This study investigate the effects of silencing AdipoRl on reducing inflammation and bone erosion of CIA mice in vivo experiments.Methods:Titer of lentiviral shNC and shAdipoR1(shAdipoR1-1,shAdipoR1-2,shAdipoR1-3)was verified with 293T cells.Effective interference sequence was screened in DBA/1J mice with lentivirus intra articularly injection.CIA mice model was induced by type Ⅱ collagen immunization in DBA/1J mice.ShAdipoR1 lentivirus was intraarticularly injected into the knee joints of CIA mice on 21st day post the first immunization.The mice were sacrificed on 45th day post the firstl immunization.The efficiency of lentivirus transfection was measured by real-time PCR and western-blot Intervention effects was evaluated by the incidence of arthritis development,the clinical scores of the severity,the symptoms of paws and histopathological changes of knee and ankle in CIA mice.Micro-CT and three-dimensional reconstruction of knee joints and paws were used to assess the bone destruction with the evaluation indicators including bone mineral density,bone mass and bone surface area ratio of the femur and tibia,and the changes of cancellous bone density,bone volume fraction,trabecular number,trabecular thickness and structural model index.The expression of IL-6,TNF-α,IL-1β,IL-17,RANKL and OPG in joint tissue were detected by real-time PCR and western-blot.Results:ShNC and shAdipoRl lentivirus were 5×108 TU/ml.ShAdipoR1-1 sequence group significantly decreased the expression level of AdipoR1(p<0.05)with the optimum efficiency.The results of this study are shown by the experimental results of shAdipoR1-1.Knockdown of AdipoR1 delayed the onset of arthritis,reduced the clinical symptoms and clinical score,decreased the synovial hyperplasia,inflammatory infiltration pannus formation,and pathologic score(p<0.05).It also resulted in a reduced expression of inflammatory cytokines such as IL-6(p<0.05),TNF-α,IL-1β(p<0.05)and IL-17(p<0.01).Micro-CT confirmed a significant decrease in bone damage in shAdipoR1 treated CIA mice.Local inhibition of AdipoR1 increased the cortical and trabecular bone mineral density(p<0.05),bone mass,bone volume fraction(p<0.05),trabecular number and trabecular thickness,while the bone surface area ratio and structural model index(p<0.05)decreased.It downregulated the expression of RANKL and OPG/RANKL ratio(p<0.05).Conclusions:Local silencing of AdipoR1 reduced joint inflammation,decreased expression of inflammatory factors,alleviated bone erosion and osteoporosis,inhibited RANKL expression and decreased RANKL/OPG ratio.Our results suggest that AdipoR1 plays a key role in preventing the development of the disease,and silencing AdipoR1 may be a new target for the clinical treatment of RA.Part 2 Effect of silencing AdipoR1 on proliferation,apoptosis and expression of inflammatory factors in synovial fibroblasts of rheumatoid arthritis(MH7A)Objectives:Synovial fibroblasts are the main cells involved in RA chronic synovitis,synovial hyperplasia,pannus formation and progressive bone destruction.We detected elevated expression of AdipoR1 in synovial tissues.In this study,shRNA was used to silence the expression of AdipoR1 at the cellular level,and to observe the effect of MH7A on proliferation,apoptosis and proinflammatory response in synovial fibroblasts of rheumatoid arthritis(RA).Methods:The shAdipoR1 transfection efficiency of MH7A was detected by western-blot.The IL-6 expression was measured by real-time PCR in MH7A at 24h,48h and 72h with different concentrations of LPS(0,10,100,1000ng/ml),respectively.CCK8 kit and flow cytometry were used to detect the cell proliferation and apoptosis in LPS-induced shAdipoR1 group and negative control group,respectively.Real-time PCR was used to examine the relative expression of Bcl-2,BCL-xl,BAK,BAX and inflammatory cytokines such as TNF-α,IL1-β and IL-6.Results:The expression of fluorescent protein in MH7A cells transfected with lentivirus was detected nearly 100%by fluorescence microscope with MOI=10.The AdipoR1 expression in shAdipoR1 group was significantly lower than that in shNC group(p<0.05),indicating that AdipoR1 silenced cells were successfully constructed.The expression of IL-6 mRNA elevated with the increased concentration of LPS at 24h,48h and 72h.The mRNA expression levels in MH7A stimulated by LPS at a dose of 100ng/ml at 48h of shAdipoR1 group had the most significant difference with that of negative control group(p<0.01).The cell proliferation rate of shAdipoRl group was significantly lower than that of shNC group after LPS stimulation for 24 and 48 hours(p<0.01).And the apoptotic cells rate of shAdipoRl group was significantly higher than that of shAdipoRl group(p<0.01).Compared with that in shNC group,the mRNA expression levels of BCL-2 and BCL-xl were significantly decreased while BAX and BAK were significantly increased in shAdipoRl group(p<0.01).The mRNA expression levels of TNF-α(p<0.05),IL-1β(p<0.01)and IL-6(p<0.001)in shAdipoR1 group were significantly lower than those in shNC group.Conclusion:Our research shows that silencing AdipoRl can effectively reduce LPS induced proliferation of MH7A cells,promote apoptosis,and reduce the expression of LPS induced inflammatory factors.It suggests that AD-AdipoRl related signaling pathway may have inflammatory signal amplification in RA,and blocking this pathway can effectively block the inflammatory response of LPS induced MH7A cells.Our research provides new targets and ideas for the prevention and treatment of rheumatoid arthritis...