Experimental Study And Mechanism Of Aconitine Anti-Rheumatoid Arthritis

Objective: To investigate the therapeutic effect of aconitine on rheumatoid arthritis by establishing an animal model of adjuvant arthritis and collagen-induced arthritis.And investigated the effects of aconitine on the viability,cytokines and related proteins of RAW 264.7 cells so as to explore its mechanism.Methods 1.The therapeutic effect of aconitine on AA rat model.Fifty Wistar rats were randomly divided into control group,model group,methotrexate group,and aconitine groups of different dosages(0.025,0.050 mg/kg).Except the control group,each group was injection of intradermal Freund's complete adjuvant(0.1 mL)into right hindpaw of rats to establish adjuvant arthritis model.On the 10 th day after model onset,The aconitine were administered by gavage with AC(0.025,0.050 mg/kg)once daily,and the methotrexate group was administered with 0.5 mg/kg methotrexate twice per week,and all groups were treated for 19 days.After the last administration,the foot swelling was measured by toe volume meter,arthritis index was calculated by 5-grade scoring method,spleen and thymus index were calculated,and the pathological changes of right ankle joint were observed by HE staining.Results At 20,24 and 28 days after model establishment,the degree of swelling of the ankle in rats treated with aconitine(0.025,0.050 mg/kg).1.The therapeutic effect of aconitine on CIA rat model.the model of collagen-induced arthritis(CIA)was established: except for control group,the caudal root of rats were immunized subcutaneously with 0.2 mL of the emulsion containing 1 mg/mL of type II Collagen(CII)emulsion.One week later,a second injection of CII emulsion was administrated.After the rheumatoid arthritis model was successfully established.The rats with CIA were randomly divided into six groups: control group,CIA model group,(0.025,0.050 mg/kg)AC treatment group and 0.5 mg/kg methotrexate(MTX)positive control group.The drugs were administered by gavage in rats on the second day after arthritis onset.The body weights,arthritis scores and paw volumes were observedconsecutively.The ankle joints of rats were collected histological evaluation.Serum samples were collected to test the levels of TNF-?,IL-6.and indices of the thymus and spleen were determined.3.The anti-inflammatory mechanism of AC.RAW 264.7macrophage cells were cultured in vitro and treated with AC at various doses(0,0.002,0.003,0.005,0.011,0.021,0.042,0.083,0.165,0.330 mg/mL)for 24 h,the cell viability was detected by MTT assay.Lipopolysaccharides(LPS)group and LPS +AC groups(0.083,0.165,0.330 mg/mL)and control group were established.The level of TNF-? and IL-6 was detected by ELISA,the level of NO was detected by Griess.The protein expression levels of nuclear transcription factor ?B p65(NF-?B p65)was tested by Western blot.Results: Compared with the control group,the paw swelling,arthritis scores,Immune organ index(except AA rats' thymus index)and TNF-? and IL-6 levels of model group rats were significantly increased(P<0.01,P<0.05),AC could significantly reduce the degree of paw swelling in AIA/CIA rats,(P<0.01,P<0.05),decrease the arthritis score index(P<0.01),serum TNF-? and IL-6level of RA rats(P<0.01,P<0.05),but the level of TNF-? in AA rats were not significantly affected after AC treatments(P>0.05).Additionally,AC could reduce the spleen index of RA rats(P<0.01)and thymus index of CIA rats(P<0.05)and increase thymus index of AA rats.In addition,AC can significantly improve the pathological changes of ankle and decrease the pathological score on AA/CIA rats.(P<0.05,P<0.01).MTT results showed that the cells of each group of aconitine were able to maintain good viability,and there was no obvious cytotoxicity compared with the normal group,(P>0.05).The subsequent experiments showed aconitine concentrations of 0.083,0.165 and 0.330 mg/mL.AC induced a decrease in the level of nitric oxide(NO)and reduction of the levels of IL-6 and TNF-? in LPS-stimulated RAW 264.7 macrophages.Furthermore,AC decreased the phosphorylation of IKK?and I?B? and inhibited nuclear factor ? B?(I?B?)proteins,resulting in lower production of nuclear factor(NF)-?B transactivation.Conclusion: AC possesses an anti-arthritic effect in AA/CIA rats via inhibiting the release of pro-inflammatory cytokines and proteins that promote the NF-?B pathway.