| Rheumatoid arthritis (RA ) is a kind of chronic autoimmune disease charactered by astrosynovitis and destructive astropathy. There has been no specific therapeutics. The drug of geneticallyengineered antibody researched by the staff of biotechnology college in Northeast Agricultural University will make up a deficiency in the two-potency antibody drug treatment for the RA in China and reduce the difference in biotechnology drug between China and internation. The preclinical pharmacology research will provide the guarantee for the safety, effect and quality control of the biotechnology drug. The aim of this research is to prepare the animal disease models which reflect the features of RA and further to study its pathogenesis. This research provides the foundation for the preclinical pharmacological research of the orininal new drug.The action target of drug was the IL-1βand IL-17 which are the main proinflammatory factor in RA. The mechanism of IL-1βin RA has been clearly explained, that is mainly induced the synoviocyte and chondrocyte expression the collagenase and other protease and inhibition the synthesis of proteoglycan. IL-17, recent years finding in the synovium supernatant of RA patient, can significantly promote the secretion of TNF-αand IL-1β. The aggrecanase(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS) ADAMTS-4 can degradate the extracell matrix in cartilage. This research will culture the chondrocyte, and induce them with IL-17, then determine the ADAMTS-4 protein expression and mRAN change level. The aim of this research is to investigate the action mechanism of IL-17 in a local joint and further analyze the pathogeny of rheumatoid arthritis on the molecular level. This will supply a solid base for preclinical pharmacology research of the orinal drug.Firstly, the whole animl desease model was established. 45 male SD rat were divided into 3 groups: simply immune-induced group; immune-induced and cold wetness-stimulated group; control group. Collagen-induced arthritic rat were intradermally immunized at the base of the tail with 0.25 mL of the emulsion containing chicken typeⅡcollagen and Freund's complete adjuvant. On day 7 after the initial collagen immunization, the rats were intraperitoneally boosted with 0.1mL emulsion. Rat in immune-induced and cold wetness-stimulated group put in ice-water 1h for 10 days based on the immune with emulsion.The same volume of vehicle (glacial acetic acid)was administered as a control.Weight body and engorgement of foot and calw measured each week. Serum collected on 21d and 42d after initial collagen immunization for the determination of antibody for collagenⅡ; TNF-α; IL-1βand IL-17. Rats were euthanized by cervical dislocation on day 42 after initial collagen immunization. Histopathological features of peripheral ankles were assessed in hematoxylin-stained formalin-fixed paraffin-embedded sections. 5 rats in each group on day 42 after immune were anestetized by ethylether for radiological examination.Chondrocytes were isolated from articular cartilage of rats and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum. Chondrocytes were serum-starved in DMEM for 24h and treated with IL-17for another 24h or 48h. RNA was extracted by the guanidinium isothiocyanate procedure and reverse-transcribed. The resulting cDNA was used as a template for qRT-PCR for primers designed using ADAMTS-4 gene sequence. Expression of ADAMTS-4 was analysed, as well as the house keeping gene GAPDH to normalize expresⅡon between different samples relative mRNA levels of ADAMTS-4 were determined using the fomula 2-△ΔCt. The conditioned media were concentrated to SDS–PAGE under reduction and immunoblotted with anti-ADAMTS-4 antibody.the development of the blots was carried out using the ECL plus chemiluminescence substrate.After initial immune, the behind paws and joint were redness and swelling on the afterday, and then fadeaway gradually. After the boosted immune the whole paws and joint were swere inflammation. The disease incidence in simply immune-induced group was 80%; in immune-induced and cold wetness-stimulated group 85%. The solvent control group had no sequence inflammation.the levels of anticollagen antibody TNF-α; IL-1βand IL-17 in rat serum increased, and the difference were significece compared with control group (P<0.01).Rat ankle joint cavity of tissue sections in control group can be seen clearly; proliferation of synovial cell and chondrocyte in treated group can be seen. Inflammatory cell infiltrated into the joint cavity and lymphocyte infiltrated into the synovium tissue. Radiographs demonstrate marginal erosion, blurred joint and cartilage damage.After primary chondrocytes inoculation, under microscope, a large number of round cells in suspension, and had strong refraction, adherent cell Adherent cell body were flat, triangle or polygon, monolayer and mixed together gradually.the passage chondrocyte adherented completely within 24h. After freeze and recovery, the rate of living chondrocyte was 87%.All chondrocyte was found to express mRNA for ADAMTS-4. ADAMTS-4 in 20h and 48h group showed a statistically significant increase (P<0.01) in mRNA expression levels following induction of IL-17, respectively, 6.37 and 8.62 folds. The protein of ADAMTS-4 in chondrocyte showed the positive result using the western blotting.This research has prepared succeedfully the rheumatoid arthritis disease model with the following evaluation target: clinical symptom; arthritis index; the level of anticollagen antibody; TNF-α; IL-1βand IL-17; histopathology and radiological examination. The further research indicated that the IL-17 can induce the expression increase of the destructive enzyme-ADAMTS-4 and play an important role in the approaching destruction of cartilage matrix in rheumatoid arthritis. IL-17 could be the theruapeutic target.In the animal desease model, the level of IL-1β,IL-17 increased significantly compared to the control group. Consideration the environment was complex in vivo desease model, chondrocytes were cultured and induced with IL-17 in vitro in order to avoid the interferers of many factor. The results indicated directly that IL-17 can induce the chondrocyte expression the ADAMTS-4, which degradate the extra cell matrix in cartilage of local joint and resultly the abnormity of joint and even crippledom happened. This illustrate that blocking the action of Il-17 in RA promptly will treat effectly the symptom of rheumatoid arthritis. |
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