| ObjectiveThrough the observation of the effect of total saponins from rhizome of Anemone Flaccid on articular bone destruction of rats with collagen-induced arthritis(CIA),and on osteoclasts formation and bone resoroption induced by receptor activator of nuclear factor kB ligand (RANKL) in the murine macrophage cell line RAW264.7cells in vitro,to supply experimental evidence for clinical usage.MethodsThe healthy male SD rats of6weeks were randomly divided into6groups after weighing,named as Normal group, Vehicle group, MTX0.2mg/kg group,TS (15,30,60)mg/kg group, using II collagen (CII)emulsifier in zeroth and seventh days of the root of the tail subcutaneous scatter two injection induced,to established the CIA model. Groups of rats with Collagen II-induced Arthritis(CIA) were treated with methotrexate, TS according to the group from day l((the first immuing day) to the end of experiment(day28). The nomal rats and Vehicle rats without medicine intervention as the control groups. Observe and record joint swelling, arthritis incidence, degree of swelling, arthritis score, percentage of arthritis limb, chang in body weight of the rats of all groups every day. using X-ray film the same side of the ankle, observation of the joint clearance,bone erosion and bone destruction and adopting the double blind method for scoring, using mcro-CT to investigate bone distrution of knee from the same side, the left knee were then removed for histopathology, including HE staining, TRAP staining for OC.The serum and bone levels of IL-lβ、IL-6、IL-17, IL-23,TNF-a,RANKL,Osteocalcin,CTX-l,IgG,OPG were tested by ELISA; The protein leve of OPG, RANKL,RANK were tested by Western blot. Real-time PCR technique was used to determine the expressions of OPG, RANKL,RANK at transcription level.RAW264.7cells induced by RANKL were cultured with three variable concentration(0.1,0.5,2.5μg/ml)of TS、 W1、W2、W3、Saponin F separately in vitro for for24hours, the level of TNF-a in cell culture supernatant was examined by ELISA. RAW264.7cells induced by RANKL were cultured with three variable concentration of TS、W1、W2、W3、Saponin F in vitro for7days. The influence of TS、 W1、W2、W3、Saponin F on osteoclasts differentiation was assessed by osteoclast-like number and tartrate resistant acid phosphatase (TRAP) activity; resorption lacunae were stained by toluidine blue and were visualized by Philips XL30scanning electron microscope. The number of TRAP+cells and the percentage of resorption pits were counted. RAW264.7cell viability was assayed by MTT.Results1the effects of TS on disease severityfrom the model of success and disease severity index, the CIA model rats swelling, malformation clinical symptoms are manifested, TS (15、30、60) mg/kg groups could dose-dependent reduce joint swelling, arthritis incidence, degree of swelling, arthritis score, percentage of arthritis limb, the leve of IgG in serum.2The effects of TS on weightCompared with the normal group,vehicle group has slowly growth in bodyweight. Compared with the Vehicle group, TS (15、30、60) mg/kg groups could dose-dependent promote the growth of bodyweight, TS60mg/kg group’s body weight were close to the normal group,which indicated that there is no obvious side effects of TS.3The effects of TS on histopathologic and radiologic changesInflammation, pannus, cartilage and bone destruction were observed in the Knee and ankle joints of CIA rats. The results showed that TS (15、30、60) mg/kg groups could dose-dependent reduce inflammatory cell infiltration, synovial hyperplasia, pannus formation, cartilage, bone erosion.4The leve of CTX-1and Osteocalcin in serumThe levels of CTX-1in serum of Vehicle rats were higher compared with normal rats (P<0.05),TS (15、30、60) mg/kg groups could reduced the level of CTX-1, TS (30、60) mg/kg groups has the better effect (P<0.05, P<0.01). The levels of Osteocalcin in serum of Vehicle rats were lower compared with normal rats (P<0.01), TS60mg/kg group could increase OPG/RANKL obviously (P<0.05)5The TRAP staining of knee for OCThere is a lot of TRAP positive multinucleated cells in Vehicle group, TS (30,60) mg/kg group can decrease the number of TRAP+cells obviously (P<0.01,p<0.05).6The levels of cytokines in serum and boneThe levels of IL-1β. IL-6. IL-17, IL-23, TNF-a,RANKL in serum of Vehicle rats were higher compared with normal rats (P<0.01,or P<0.05). TS (15.30、60) mg/kg groups could reduced the levels of IL-17, IL-23, TNF-a,RANKL, in serum, TS (30.60) mg/kg groups has the better effect (P<0.01,or P<0.05).The levels of OPG/RANKL in serum of Vehicle rats were lower compared with normal rats (P<0.05), TS60mg/kg group could increase OPG/RANKL obviously (P<0.05)7The protein and mRNA levels of RANKL,RANK,OPG in ankle jointsThe protein and mRNA levels of RANKL,RANK, in ankle joints of Vehicle rats were higher compared with normal rats (P<0.01,or P<0.001),TS (15.30.60) mg/kg groups could dose-dependent reduced the level of RANKL,RANK (all P<0.05, P<0.01, P<0.001),and increase the level of OPG (P<0.05, P<0.01, P <0.001)8The effects of TS. W3on on osteoclasts differentiation and bone resoroptionRANKL could induce abnormally high expression of TNF-a in RAW264.7cells,three variable concentration(0.1,0.5,2.5μg/ml)of TS、Wi、W2、W3、 Saponin F could suppresse the expression of TNF-a, TS, W1, W3have better effect compared with W2, Saponin F. RANKL could induce RAW264.7cells to form TRAP positive multinucleated cells and form bone resorption pits on bone, three variable concentration(0.1,0.5,2.5μg/ml)of TS、 W3could decrease the number of TRAP+cells,as well as the area of bone resorption pits.Conclusion1TS can reduce the arthritis incidence,arthritis score and foot swelling and inhibit anti CII antibodies IgG levels in serum of CIA rats,and inhibit inflammation of the joints, inflammatory cells infiltration, synovial hyperplasia, pannus formation and the cartilage and bone destruction, and can dose dependent inhibit the expression of bone resorption marker CTX-1in serum, and to promote the expression of bone formation marker Osteocalcin, so as to reduce the CIA rats joint disease clinical symptoms, inflammation, to prevent bone destruction of CIA rats.2TS inhibit osteoclasts formation in CIA rats joints.3TS can reduce the expression of RANKL and RANK, and raise the expression of OPG and the ratio of OPG/RANKL, at both mRNA and protein levels.at the same time reduce the activities of the pro-inflammatory cytokines such as IL-1β\IL-6、 TNF-α、IL-17、IL-23in serum and joints.4RANKL induced osteoclastogenesis in these monocytic cells,and TS,W3inhibited RANKL-induced tumor necrosis factor-a production and osteoclast differentiation and bone resorption pit foemation.In conclusion, this study first proved by using the combined method that TS has the effect of preventing bone destruction in RA. its mechanism may be related to inhibit the expression of osteoclasts, regulate the balance of signaling pathways RANKL/OPG/RANK,and inhibit the expression of pro-inflammatory cytokines such as IL-1β、IL-6、TNF-α、IL-17、IL-23. |
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