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Objective: To observe the inhibitory effect of daphnetin which is active component from Daphne odora on collagenⅡ-induced arthritis (CIA) rats and investigate its immunoregulation effect on Th17 cells of CIA rats, and to provide the theoretical and experimental basis for the development of new drugs for RA.Methods: 1. Groups of experiments. 48 healthy female SPF Wistar rats which were at the weight of 170±10g were randomly divided into six groups: healthy group, CIA model group, daphnetin high, middle and low treatment group and the CIA positive control group; 2. The establishment of CIA models. The bovine-derived type II collagen was dissolved in certain volume of 0.5 mol / L acetic acid, and then added an equal volume of Freund’s incomplete adjuvant and was fully emulsified. The concentration of type II collagen emulsion was 0.25mg/mL. The CIA models of Wistar rats were established by intradermic injection of 0.1 mL of the type II collagen emulsion on the left foot and the same injections were repeated 21 days later; 3. The treatment with drugs. The Wistar rats were treated with drugs from the 20th day after the first immunization to the 40th day. Daphnetin high, middle and low treatment group were intraperitoneal injected with 4,2,1 mg / kg.d of daphnetin, CIA positive control group were intraperitoneal injected with 0.4mg/kg.d of triptolide, CIA model group and healthy group were intraperitoneal injected with the same volume of sterile saline for 21 days; 4. Tested the following indicators: (1) Body weight of the rats in each experimental group were measured; (2) The volume of the immunized foot was measured by YLS-7B plethysmometer and then was converted into swelling rate; (3) The X-ray photo of foot was photographed by PLANMECA TNTRA dental engine;(4) The pathological characteristic of the rats was assayed by histopathological HE staining techniques;(5) Flow cytometry was applied to detect the levels of RORγt, Foxp3, TGF-β, IL-6, IL-4, IFN-γand NF-κB in splenic T lymphocytes of each group.Results: 1. At the 21th day after treatment begining, the average body weight (g) of the healthy control group, CIA model group, CIA positive control group, and daphnetin high, middle and low dose group were respectively 191.00±6.78,189.38±6.12,193.5±10.10,187.11±6.09,194.50±10.92,195.00±7.59. The average body eight of all groups showed nificant difference (P>0.05); 2. The foot swelling rate of daphnetin high, middle, low dose group were significantly lower than CIA model group from the 24th day to 40th day after the first immunization (P<0.05). The foot swelling rate of daphnetin high, middle and low dose group and CIA positive control group were no significant difference (P>0.05); 3. X-ray film of daphnetin high, middle and low dose groups showed mild swelling of soft tissue on immunized foot, slightly thickened joint, marked joint space and clear edge of joints, and X-ray film of CIA model group showed marked swelling of soft tissue, thickened joints, narrow joint space and the ambiguous edge of joints; 4. H-E staining of daphnetin high, middle and low dose groups showed mild damage on synovial joint, joint space with mild stenosis, a more complete surface of articular cartilage, no significant proliferation of synovial cells, a certain amount of inflammatory cells infiltration, no fibrous tissue hyperplasia and pannus formation, while the CIA model group showed damage on synovial joint, narrow joint space, enlarged articular chondrocytes or destruction of cartilage, inflammatory cells infiltration, fibrous tissue hyperplasia and pannus erosion of articular cartilage and subchondral bone which lead to joint cartilage damage and subchondral bone dissolution; 5. The levels of RORγt and IL-6 of daphnetin high, middle and low dose groups were significantly lower than CIA model group (P<0.05) and were no significant difference compared with CIA positive control group (P>0.05); The level of Foxp3 of daphnetin high, middle and low dose groups were both significantly higher than CIA model group (P<0.05) and CIA positive control group(P<0.05); The level of TGF-βof daphnetin high, middle and low dose groups were significantly lower than CIA model group (P<0.05), the level of TGF-βof daphnetin high, middle and low dose groups were significantly lower than CIA positive control group (P<0.05), while the level of TGF-βof daphnetin high, middle and low dose group were no significant difference with healthy group(P> 0.05); The level of IL-4 of daphnetin high, middle and low dose groups were significantly lower than CIA model group (P<0.05) and CIA positive control group(P<0.05); The level of IFN-γof daphnetin high, middle and low dose groups were significantly lower than CIA model group (P<0.05), the level of IFN-γof daphnetin high dose group was no significant difference with CIA positive control group (P>0.05); The level ofNF-κB of any two groups were all no significant difference(P>0.05).Conclusion: 1. Daphnetin from Daphne odora had no obvious effect on body weight of Wistar rats. 2. Daphnetin from Daphne odora had obvious inhibitory effect on foot swelling of CIA rats within the dose of 1 ~ 4mg/kg.d. 3. Daphnetin from Daphne odora help to improve the severity of local joint in CIA rats; 4. Daphnetin from Daphne odora may regulate the Th17 cells to alleviate the symptoms of CIA arthritis by decreasing the levels of RORγt, TGF-β, IL-6, IL-4 and IFN-γand improving the level of Foxp3 in spleen T lymphocytes.
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