Bone Protective Effect Of Moxibustion On Rheumatoid Arthritis Rabbits Based On "Dvl2,OPG/RANKL/RANK System And NF-?B Signaling Pathway"

Objective:Rheumatoid arthritis(RA)is a chronic inflammatory autoimmune disease involving multiple joints and is characterized by bone destruction in form of erosion.Bone destruction can lead to irreversible joint damage and functional disability,affecting the work and life of RA patients.At present,researches of RA mainly focus on synovitis,and mechanism of bone destruction is limited.Therefore,this study aims to(1)explore whether moxibustion has bone protection effect in RA rabbits;(2)To investigate whether Dvl2,OPG/RANKL/RANK system and NF-?B signaling pathway mediate the bone protection effect of moxibustion in RA rabbits;(3)To investigate the effects of Dvl2 on OPG/RANKL/RANK system and NF-?B signaling pathway in bone destruction of RA rabbits treated with moxibustion,thus to provide theoretical basis for clinical treatment of RA.Methods:66 CCV New Zealand rabbits were randomly divided into blank group,model group,model+moxibustion group,Dvl2 overexpression negative group,Dvl2 overexpression group,Dvl2 interference negative group,Dvl2 interference group,model+moxibustion+Dvl2 overexpression negative group,model+moxibustion+Dvl2 overexpression group,model+Dvl2 interference negative group,and model+Dvl2 interference group,with six rabbits in each group.In the first week,RA model was prepared by injecting Complete Freund's Adjuvant(CFA)into the knee cavity,and the intervention measures were "BL 23" and "ST 36" moxibustion treatment for 6 weeks.After the 7th week,Safranin O-Fast Green staining and SMASH score of knee cartilage was detected;Inflammatory factor IL-10 in synovial lavage fluid was detected by ELISA;BGP,Collagen ?,Dvl2,OPG,RANKL,RANK,and NF-?B signaling pathway molecules(p50,p52,IKK?,IKK?,I?B?,IKBKG,RelB,c-Rel,TAB3 and MAP3K7)of knee cartilage/bone were detected by PCR Array.Dvl2 expression of knee cartilage/bone was regulated by overexpression and interference expression of Dvl2 with lentivirus.Independent sample T test was used for comparison between two groups;OneWay ANOVA was used for comparison between three groups and above,and LSD method and Games-Howell test was used for comparison between two groups when variance was homogeneous and heterogeneous,respectively.P?0.05 was considered statistically significant.Results:(1)The knee cartilage/bone Safranin O-Fast Green staining of New Zealand rabbits:in the model group,almost complete loss of the cartilage surface,only had blue reverse staining,obvious proliferation of chondrocytes,and partial shedding of the cartilage layer;in the model+moxibustion group,the surface layer of cartilage nodules was rough,the superficial layer staining was lost,the chondrocytes increased and the arrangement was more homogeneous than that in the model group.The knee cartilage/bone pathological manifestations of the model+moxibustion+Dvl2 overexpression group were similar to those of the model group.The knee cartilage/bone pathological manifestations of the model+Dvl2 interference group were similar to those of the model+moxibustion group.(2)The SMASH score of knee cartilage injury of New Zealand rabbits:the model group was higher than that of the blank group(P?0.05),and the model+moxibustion group was lower than that of the model group(P?0.05);overexpression of Dvl2 in the model+moxibustion group increased SMASH score and aggravated cartilage injury(P<0.05),and there was no significant difference in the degree of injury between the model group(P>0.05).Interfering the expression of Dvl2 in the model group could reduce SMASH score and reduce cartilage injury(P?0.05),and there was no significant difference in the reduction degree between the model group and the model+moxibustion group(P>0.05).(3)Compared with the blank group,the expressions of knee cartilage/bone Dvl2,OPG,RANK and RelB in the model group were decreased(P?0.05),while the expressions of p50,IKK? and c-Rel were increased(P<0.05).Compared with the model group,the expressions of knee cartilage/bone Dvl2,OPG,RANKL,P50,IKK? and c-Rel in the model+moxibustion group were decreased(P?0.05),and the expression of RelB was increased(P<0.05).(4)Compared with model+moxibustion+Dvl2 overexpression negative group,the expressions of knee cartilage/bone Dvl2,OPG,p50,p52,IKK? and I?B? in the model+moxibustion+Dvl2 overexpression group were increased(P?0.05);Compared with model+Dvl2 interference negative group,the expressions of knee cartilage/bone Dvl2,RANK and IKK? in the model+Dvl2 interference group were decreased(P?0.05),and the expressions of OPG,RANKL,p50 and p52 were increased(P?0.05).(5)Compared with the Dvl2 overexpression negative group,the expressions of Dvl2,OPG,c-Rel,IKK? and IKK? in knee cartilage/bone were increased in Dvl2 overexpression group(P?0.05),while the expressions of p50 and p52 were decreased(P?0.05).Compared with the Dvl2 interference negative group,the expressions of Dvl2,OPG,RANKL,RANK and IKK? in knee cartilage/bone in Dvl2 interference group were decreased(P<0.05),and the expressions of c-Rel and TAB3 were increased(P<0.05).(6)Compared with the blank group,the expression of synovial lavage fluid IL-10 in the model group was increased(P<0.05);Compared with model group,the expression of synovial lavage fluid IL-10 in the model+moxibustion group was decreased(P<0.05).(7)Compared with the blank group,the expressions of knee cartilage/bone BGP and Collagen ? in the model group were decreased(P<0.05);Compared with model group,BGP expression was decreased(P<0.05)and there was no difference in Collagen ? expression(P>0.05)in the model+moxibustion group.Conclusions:(1)Six weeks Moxibustion can improve bone destruction of RA rabbits and has bone protection effect.(2)The mechanism of bone protection effect of moxibustion on RA rabbits is considered as follows:? mediated by the down-regulation of knee cartilage/bone Dvl2;? through the involvement of OPG/RANKL/RANK system,and NF-?B signaling pathway involved p50,IKK?,RelB and c-Rel of knee cartilage/bone;? through the synovial fluid IL-10 in knee joint cavity.(3)Dvl2 of knee cartilage/bone may mediate the expression of p50 and IKK? in NF-?B signaling pathway,but Dvl2 may not mediate the expression of OPG/RANKL/RANK system in the bone protective effect of moxibustion on RA rabbits.