| Objective:to observe the effect of bone protective function with Artesunate (ART) and Methotrexate(MTX), Artesunate (ART) and Tripterygium Glucoside(TG)on adjuvant induced arthritis (Ajuvant Arthritis, AA) of rats.Methods: 1 Choose the Wistar rats weight from 170~200g , measure double paws volume, after sterilizing regularly on right rear foot ,then inject Freund's complete adjuvant (0.1ml/only) into the subcutis of every right rear paws to establish the AA model . 2 On the 21st day after modeling, divide randomly the rats presenting secondary reaction into the model group, the MTX group (1mg/kg.d), the ART group (80mg/kg.d), the TG group (8mg/kg.d), the MTX+ART group (1mg/kg.d+80mg/kg.d), the ART+TG group (80mg/kg.d+8mg/kg.d), each group comprises 8 samples, and randomly take 8 normal Wistar rats as the control group, measure the paw volumes of the rats in each group with the volume measuring instrument. Treats each therapeutic group to fill stomach continuously with the correspondent drugs for 14 days. 3 On the 15th day, measure the paw volumes of the rats, collect blood from the abdomen aorta, centrifugate it and takes the blood serum, the enzyme unites the immunity adsorption analytic method to determine the levels of TNF-α, IL-1β, RANKL, OPG in serum . After collecting the blood, select the ankle joints , decalcify, paraffin embed and fixate, HE dyes, observe the joint pathological change under the light microscope and assess pathological score, and use immunohistochemistry and the computer image analysis to detect on the expression of RANKL and OPG in synovial tissue. 4 All the data is analyzed by the SPSS13.0 statistics software.Results: 1 Effects on general condition and weight of AA rats:this experiment demonstrated that the condition of ART+TG group is best , the rats' state of mind,the diet and moving is good, the body weight increases unceasingly. In MTX group, MTX+ART group, the joint symptom reduce obviously on the later period compared to the model group, but weight gain is not obvious, in the MTX+ART group,2 rats appear diarrhea. The weight of rats after 14d post-administration: control group 258.37±15.33,ART group 251.50±10.32,TG group 249.62±10.08,ART+TG group 250.00±10.02 significantly higher than model group 232.50±16.33 , the difference is significant(P<0.05);The weight of AA rats in ART group,TG group,ART+TG group has no significant difference compared with control group (P>0.05).2 Effects on the left paw swelling degree and Arthiritis Index of AA rats: rat left paw swelling degree(ml)(% )and the AI in each therapeutic group after 14d post-administration:MTX group (0.95±0.36,37.76±5.21,4.75±1.28),ART group (1.46±0.19,50.38±8.04,5.37±0.51),TG group (1.65±0.14,48.95±7.96,5.50±0.75),MTX+ART group (1.2±0.46,35.59±6.83,3.87±0.64 ),ART+TG group ( 0.72±0.22,40.97±7.5,5.25±0.88)has the significant difference compared with the model group(2.46±0.17,83.72±11.97,12.00±2.20) (P<0.05); left paw swelling degree after 14d post-administration in ART group,TG group,MTX+ART group has the significant difference compared with MTX group (P<0.05);AI in ART group,TG group,ART+TG group has the significant difference compared with MTX +ART group (P<0.05).3 Effects on the level of the cell factors: IL-1β,TNF-α,RANKL and OPG in serum : IL-1β(pg/ml),TNF-α(pg/ml),RANKL (pg/ml),OPG (ng/ml) level and RANKL/OPG ratio in model group is (75.39±15.38,52.91±31.36,70.0±17.63,153.07±38.70,0.47±0.13), is obviously higher than normal control group ( 15.19±3.55,15.26±2.95,15.58±2.3,83.69±10.45,0.19±0.04), the difference is significant (P<0.05); IL-1β,TNF-α,RANKL level and RANKL/OPG ratio:MTX group(10.61±5.11,11.15±3.30,12.41±5.02,0.12±0.08),ART group ( 29.76±7.71,29.55±13.25,33.12±9.68,0.23±0.10),TG group (31.61±8.44,26.49±9.87,26.81±9.30,0.18±0.08),MTX+ART group(8.65±2.48,9.63±2.29,10.03±4.48,0.10±0.03),ART+TG group(18.61±6.81,20.91±9.98,23.39±7.1,0.15±0.04)reduce compared with model group(P<0.05), the difference in MTX+ART group is the most significant. IL-1β,TNF-αlevel in MTX group,MTX+ART group has no significance compared with control group (P>0.05); RANKL level in MTX+ART group has no significance compared with control group (P>0.05);OPG level in MTX group 123.11±10.27,ART group 147.56±35.13,TG group 152.57±22.69,MTX+ART group 152.47±21.87,ART+TG group 159.20±21.44 increase compared with control group(P<0.05),the difference with ART+TG group is the most significant.4 Effects on the pathological results: normal group: histology structure of ankle joint is normal; model group: the synovial membrane has the varying degree hyperemia, dropsy and the phlogocyte infiltration; the synovial membrane level increases (may reach 6), the arrangement disorder, some synovial membranes present the villose proliferation to the ticulare, articular cartilage obviously is destroyed, change to fibrosis; some also present the polynuclear giant cell infiltration. Pathology change of light microscope in each therapeutic group reduces compared with model group: proliferation of the synovial membrane reduces, the phlegmasia-cyte infiltration reduces, destruction of the cartilage reduces. The pathological score: model group 2.1±0.36, MTX group 1.15±0.30, ART group 1.55±0.42, TG group 1.69±0.67, MTX+ART group 0.83±0.29, ART+TG group 1.41±0.56 has the obvious difference compared with control group 0.12±0.23 (P<0.05). MTX group, ART group, TG group, MTX+ART group, ART+TG group has the obvious difference compared with model group (P<0.05). MTX+ART group has the obvious difference compared with MTX group (P<0.05).5 Effects on the immunohistochemistry result of OPG and RANKL expression in synovial membrane : rat synovial membrane tissue in normal control group RANKL,OPG positive cell and RANKL/OPG ratio is ( 0.06±0.01,0.05±0.01,1.2±0.20); the RANKL,OPG positive density of synovial membrane cellular layer and RANKL/OPG ratio in model group (0.39±0.06,0.19±0.02,2.05±0.60)is obviously higher than normal control group, the difference compared with normal group is significant (P<0.05); the RANKL,OPG positive density MTX group ( 0.18±0.01,0.12±0.02 ),MTX+ART group(0.15±0.02,0.11±0.01), ART+TG group (0.18±0.02,0.12±0.03)has the obvious difference compared with model group 0.39±0.06 (P<0.05). RANKL positive density in TG group is 0.23±0.04, it has significant difference compared with ART+TG group 0.18±0.02 (P<0.05). OPG positive density of the ART group 0.15±0.02 has significanct difference compared with ART+TG group 0.12±0.03 (P<0.05). RANKL/OPG ratio: MTX group 1.5±0.1, ART group 1.4±0.3,TG group 1.64±0.26,MTX+ART group 1.36±0.40,ART+TG group 1.5±0.13 is lower than model group ,the difference is obvious(P<0.05),MTX+ART group is the lowest. Conclusion:1 The combination of Artesunate (ART) and Tripterygium glucoside(TG),Artesunate (ART) and Methotrexate (MTX) can reduce the paw swelling degree,Arthiritis Index and the level of TNF-α,IL-1βin serum of the AA rats.2 The combination of Artesunate (ART) and Tripterygium glucoside ( TG ),Artesunate (ART) and Methotrexate (MTX) can change pathological damage of AA rats,reduce the level of RANKL and OPG in serum,the expression of RANKL/OPG ratio in the synovial ,maybe through RANKL/OPG rout to protect bone function .3 The effects of combination of the traditional Chinese medicine effective ingredient,combination of the traditional Chinese medicine with western medicine on inhibiting the bone damage and anti inflammation supplys a new idea and method to treating RA in the clinic.
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