| OBJECTIVE: To investigate the role of total flavonoids in Bidens pilosa L.by RANKL/RANK/OPG signaling in relieving bone destruction in rheumatoid arthritis.METHODS: Ten of the 60 SPF SD male rats（180-200 g）were randomly selected as the normal group（Control,n=10）,and another 50 rats were used to construct the rheumatoid arthritis（RA）model.The animal model of RA was established by subcutaneous injection emulsifier formed by bovine type II collagen and Freund’s incomplete adjuvant,and the successfully modeled rats（45 in total）were randomly divided into 5 groups: model group（Model,n=9）;Low dose TFB（l-TFB,n=9）,40 mg/kg.d,intragastric administration;medium dose TFB（m-TFB,n=9）80mg/kg.d,intragastric administration;high dose TFB,h-TFB,n=9,160mg/kg.d,intragastric administration;methotrexate group（MTX,n=9）,1mg/kg/w,administered intraperitoneally;normal group（Control,n=10）was administered with an equal volume of normal saline.All the tested rats received booster immunization after primary immunization,and the two immunization intervals were 21 days.Subsequent drug interventions were performed for 28 days.During the intervention period,the rats in each group were observed for the whole state and body weight every 3 days.Weighing,joint swelling measurement and arthritis index score;after drug intervention,the blood of each group was collected,and the expression of RANKL,OPG,TNF-α,IL-1β and IL-17 in serum was analyzed by ELISA.The level of the ankle joint was fixed and the tissue was sectioned.The synovial hyperplasia,vasospasm formation,cartilage erosion and subchondral bone destruction of the ankle joints were observed by HE staining.The imaging results of the joints of the rats in each group were observed by X-ray.The expression levels of RANKL,OPG,TNF-α,IL-1β and IL-17 in the joints of rats in each group were analyzed by immunohistochemistry.Results: 1.Successfully established a CIA rat model.2.The body weight of rats in each group changed:（1）The weight of Model group and each drug-administered group were lower than that of Control group,the weight of Model group was lower than that of each drug-administered group.（2）Joint swelling and arthritis index in rats: The joint swelling degree and arthritis index of the Model group were higher than those of the other groups（P<0.05）.The joint swelling degree and arthritis index of the h-TFB group were lower than other drug-administered groups.（P<0.05）.3.Changes in serum RANKL,OPG,TNF-α,IL-1β,IL-17 expression levels: RANKL,TNF-α,IL-1β,IL in serum of rats in each administration group compared with Model group The expression level of-17 was decreased（P<0.05）;the expression level of the above indicators was decreased in h-TFB group compared with MTX group（P<0.05）;the expression level of OPG in serum of each administration group was higher than that of Model group（P<0.05）.P<0.05),in which the h-TFB group was significantly more elevated than the l-TFB group（P<0.05）.4.The results of X-ray examination of the rats in each group: The control group had regular joints and joints,the joints were tight,the joint space was clear,the bones at both ends of the joint cavity were dense,and no bone destruction was observed.The joints of the Model group were deformed and jointed.The gap was narrowed,the bone destruction at both ends of the joint cavity was severe,and the osteoporosis was loose.The rats in each administration group had different degrees of reduction in the joint deformity,the narrowing of the joint space and the degree of bone destruction.Among them,the h-TFB group was the most obvious.X-ray imaging scores of the rats showed that the imaging scores of CIA rats were alleviated by TFB intervention,which showed a dose-effect relationship.The imaging scores of the rats in the l-TFB group were not compared with those in the Model group.Statistical difference（P>0.05）.The imaging scores of the m-TFB group,the h-TFB group and the MTX group were significantly different from those of the Model group（P<0.05）,and between the m-TFB group,the h-TFB group and the MTX group.There was no significant difference in imaging scores（P>0.05）.5.HE staining results of rat ankle joint: Synovial hyperplasia of the joints in the model group,intermittent destruction of the cartilage layer,more inflammatory cell infiltration and vasospasm invasion of the subchondral bone;no synovial membrane in the ankle joint of the Control group And vasospasm hyperplasia,articular cartilage bone surface is smooth and continuous;in each group,the synovial membrane and vasospasm of the ankle joint hyperplasia,the degree of cartilage destruction was significantly reduced compared with the model group,the h-TFB group was the most obvious.6.Immunohistochemical results of rat ankle joint: Compared with Model group,the expressions of RANKL,TNF-α,IL-1β and IL-17 were decreased in all groups,and OPG expression was increased.The expression of OPG was the most obvious in h-TFB group.Conclusion: 1.TFB can significantly improve arthritis symptoms,joint pathomorphological changes,synovial hyperplasia,and bone destruction in CIA rats.2.TFB can effectively alleviate the progression of RA disease,and its mechanism may be through lowering the expression of RANKL,TNF-α,IL-1β,IL-17,up-regulating the expression of OPG,and regulating the ratio of RANKL/OPG. |
