Effects Of Artesunate On The Levels Of TNF-α, IL-1β In The Serum Of Rats With Ajuvant Arthritis And Apoptosis Of Synovial Fibroblasts

Objecitive: To observe the therapeutic effect of Artesunate (ART) on Ajuvant Athritis(AA) of rat and its influences on the levels of TNF-αand L-1? in the serum of rats with AA and apoptosis of synovial cell. Methods: Choose the male Wistar rats weight from150-180g, determine the volumes of their rear paws, then inject Freund's complete adjuvant (FCA) into the subcutis of their right rear paws after sterilizing regularly to establish AA rat models. On the 19th day after modeling, divide randomly the rats presenting secondary response into Model group, ART group, Threewingnut root group, each group comprises 8 samples. Meanwhile, choose randomly 8 normal Wistar rats as control group. Measure the paw volumes of rats in each group, then IG the rats in each therapeutic group with correspondent drugs for successive 14 days. On the 15th day, measure the paw volumes of the rats. Collect blood from eye sockets, centrifugate it and get the serum, determine the levels of TNF-α, IL-1? by Radioimmunoassay. Cultivate the synovial fibroblasts regularly to the forth generation, use the 96-well plate to determine the best concentration and acting time of artesunate's effect on the apoptosis of synovial fibroblasts by MTT method. Put cover slips (1×1cm) into the wells of the 24-well plate, and drop the cell suspension (1×105)in lgarithm growth phase onto the cover slips, then add 1ml DMEM culture medium into the wells, put the plate into the CO₂ incubator to cultivate at 37℃for 24h, add the artesunate (200μg/ml)into some of the wells, do nothing about the control wells. Put the plate back into the incubator to cultivate at 37℃for 24h, take the samples out and fix them in 4% paraformaldehde for 20 min, dry them in the air, then to observe the apoptosis of synovial fibroblasts by TUNEL method and determine gene expression of the Bcl-2 and Bax by immunohistochemical method. Results: 1.The paw volumes of rats with AA have been determined on the 15th day after treating with ART. (1) The average swelling degrees(%)of FCA-injected paws of rats in each group are: Model group 177.00±37.5, ART group 68.81±28.19, Threewingnut root group 66.00±30.78, Control group 27.25±4.20. The inhibitory rate of ART on the swelling paw is 61.1% and that of Threewingnut root is 63%. There is significant difference ( p<0.05 ) compared ART and Threewingnut root group as well as control group with Model group. There is no significant difference (p>0.05) between the ART group and Threewingnut root group, but compared the two groups with Control group there is significant difference( p<0.05) . (2) The average swelling degrees ( % ) of non-FCA-injected paws of rats in each group on are: Modelgroup 83.25±21.47, ART group 28.00±7.76. The inhibitory rate of ART on the swelling paw is 58.6% and that of Threewingnut root is 56.8%. There is significant difference(p<0.05)compared ART and Threewingnut root group as well as control group with Model group, but there is no significant difference (p>0.05) between ART group and Threewingnut root group. There is also no significant difference (p>0.05) compared ART and Threewingnut root group with Control group.2. The levels of TNF-α, IL-1 ? in the serum of rats determined by RIA are(ˉx±S,ng/ml)Model group 8.70±2.34,0.10+0.03,Control group 5.40±1.15, 0.05±0.01,ART group 5.79±1.56, 0.06±0.01, Threewingnut root group 6.16±0.945, 0.058±0.015. There is significant difference ( p<0.05 ) compared ART and Threewingnut root group as well as control group with Model group, but there is no significant difference (p>0.05) between ART group and Threewingnut root group. There is also no significant difference (p>0.05) compared ART andThreewingnut root group with Control group. 3. The result of in vitro synovial fibroblasts culture shows that ART (200μg/ml)can obviously induce the apoptosis of synovial fibroblasts after acting for 24h. Its apotosis index(AI) (%) is 68.20±4.08,and that of control group is 5.00±1.26. There is significant difference(p<0.01) between the two groups. 4 . The expression of Bcl-2 and Bax protein in the synovial fibroblasts are determined by immunohistochemical method. The label index (LI) of Bcl-2 in Control group(%) is 61.60±4.48, and that...