Therapeutical Effects Of NTAP On Rheumatoid Arthritis And Immunological Mechanism

Rheumatoid arthritis (RA) is a chronic, progressive, autoimmune disease,the characteristics are inflammatory cells infiltration in synovial joints, synovi-al hyperplasia, pannus formation, and continued destruction of articularcartilage and bone. The pathogeny of RA etiology is not clarified and therewere not effective drugs. Therefore it is necessary to explore the pathophysio-logical process of RA, and develop drug for the treatment of RA. NTAP wasobtained through screening a series of lead compounds and used in thetreatment of antigen mediated, gene related to systemic immune disease--rheumatoid arthritis. There are many common histological andimmunological features between Collagen-induced arthritis (CIA) rat modeland RA. As a classical model,CIA rat model is widely used to study thepathogenesis of RA and the effects of anti-inflammatory drugs.Therefore inpresent experiment, the animal model of collagen-induced arthritis isreconstructed, and the effects of NTAP on serum inflammatory cytokinesTNF-α、IL-6、IFN-γ and typeⅡcollagen antibody levels were observed, andthe peripheral blood T cells was determined, the immunological mechanismwas discussed.Part1Effects of NTAP on rheumatoid arthritisObjective: To observe the therapeutic effect of NTAP on rheumatoidarthritisMethods:6-7week-old female Lewis rats were randomly divided intocontrol group and the CIA model group. The emulsion of bovine collagen typeⅡa nd incomplete Freund’s adjuvantwas intradermally injected on the base ofthe tail in the CIA model rats, and the CIA model rats were strengthened theimmune with the same method after7days. Then the CIA model rats wererandomly divided into five groups: the model group, NTAP low dose group (0.25mg/kg), NTAP middle dose group (0.5mg/kg), NTAP high dose group(1.0mg/kg) and positive drug (methotrexate) group (0.5mg/kg). NTAPwere intravenously injected in NTAP low, medium and high dose groups rats.PBS was injected in the model group and the control group rats with the sameway and the volume. Methotrexate was injected in the abdominal cavity ofrats in the positive drug group twice a week.Treatment was continued for threeweeks.Rats were scored in each groups every other day during the experiment.The rats were anesthetized with20%urethane, blood was collected fromabdominal aortic for preparation of serum.The contents of TNF alpha, IL-6,IFN-γ and collagen type Ⅱ antibodyin serum were respectively determinedwith the enzyme-linked immunosorbent (ELISA) method. The ankle wasremoved, fixed, decalcified, dehydrated, embedded, sliced and stained byHE,then pathological changes of tissue were observed with the lightmicroscope.Results:1Compared with those of control group, the arthritis score wassignificantly increased in model group rats (P <0.05). Arthritis score in NTAPlow dose group rats were decreased in comparison with the model group rats,but there was not significant difference between the two groups. Comparedwith those of model group,arthritis score were significantly decreased inNTAP middle dose group and high dose group rats (P<0.01),and arthritis scorewas significantly decreased in positive group rats (P <0.01).2The contents of TNF-α, IL-6, IFN-γ, C Ⅱ antibodyin serum weresignificantly increased in the model group rats compared with those of thecontrol group(P<0.01).Compared with those of model group,the contents ofTNF-α, IL-6, IFN-γ, C Ⅱa ntibodyin serum were significantly decreased inNTAP high dose group rats(P <0.01or P <0.05), the contents of TNF-α, IL-6in serum were greatly decreased in positive group rats (P <0.05), and thecontents of IFN-γ in serum in NTAP medium dose group rats was markedlydecreased (P <0.05).3The synovial cells in the ankle were arranged in neat rows in the control group rats,and shape was regular. Synovial cells in the ankle wereproliferated,and there were a large number of inflammatory cell infiltration insynovial tissue,the synovial lining layer was thickened,bone and articularcartilage were significantly damaged in the model group rats.Compared withthose of model group rats, the pathological changes were significantlyalleviated in the NTAP groups.Conclusion: It could be concluded that the administration of NTAP couldsignificantly decrease the contents of TNF-α and IFN-γ in serum,alleviate thedamage of bone.Part2Effects of NTAP on the differentiation of T cell subsetsObjective: To observe the effects of NTAP in CIA rats peripheral blood Tcell subsets differentiation.Methods:6-7week-old female Lewis rats were randomly divided intocontrol group and CIA model group. The emulsion of bovine collagen typeⅡa nd incomplete Freund’s adjuvant was intradermally injected on the base ofthe tail in the CIA model rats, and the CIA model rats were strengthened theimmune with the same method after7days. Model rats were randomlydivided into two groups: the model group and NTAP group.The experimentwas divided into two parts:single dose and repeated doses. The blood wascollected at2h,48h,96h and144h after a single dose, then the proportion ofTh1, Th2, Th17and Treg in peripheral blood were analyzed with flowcytometry. The drugs were repeatedly administrated on the most seriouscondition once every other day for two weeks.The blood was collected at144hafter administration of drugs, then the proportion of Th1, Th2, Th17and Tregwere analyzed with flow cytometry.PBS were injected in rats of the modelgroup and control group in the same way and the volume.The arthritis scoresof rats in each groups were recorded every other day during the experiment.Results:1Changes of inflammation score: Compared with those of the controlgroup, the arthritis score were significantly increased in the model grouprats(P <0.01). Compared with thoes of model group, the arthritis score were significantly decreased in the NTAP in repeated doses group (P <0.01).2Changes of T cell subsets differentiation in the peripheral blood:Compared with those of control group, the percentage of Th1and Th17cellwere significantly increased, and the percentage of Th2and Treg cell weresignificantly decreased in the peripheral blood of model group rats(P <0.05orP <0.01). The percentage of Th1and Th17cell were significantly decreasedand the percentage of Th2and Treg cell were significantly increased in theperipheral blood of NTAP group rats compared with those of model group (P<0.01).Conclusion: The administration of NTAP could decrease the arthritisinduced by collagen,which may be related to change the percentage of Th1、Th2、Th17and Treg in the peripheral blood of CIA rat.Summary: It could be concluded that the NTAP has a beneficialtherapeutical effect on the arthritis induced by collagen in rats by decreasingthe contents of TNF-α and IFN-γ in serum, alleviating the damage of bone,decreasing the arthritis induced by collagen, and ameliorating the percentageof Th1、Th2、Th17and Treg in the peripheral blood in the CIA rats.