| Rheumatoid arthritis (RA) is a chronic systemic inflammatory diseaseprimarily affecting the peripheral joints. Although the etiology of the diseaseremains unknown, cumulative findings suggest an important role for theimmune system in the inflammatory phase of the disease. Experimentalmodels of autoimmune arthritis are an invaluable resource for studying thepathogenesis of RA as well as for the testing of new products for theiranti-arthritic activity. The collagen-induced arthritis (CIA) is an autoimmunearthritis that resembles rheumatoid arthritis (RA) in many ways; therefore ithas been used most commonly as a model of RA.Collagen (CII)263-272peptide, an autoantigen in rheumatoid arthritis,is a specific human leukocyte antigen (HLA)-DR1/4-binding peptiderecognized by T-cell receptors (TCR). In the present experiment, CIAautoimmune arthritis model was used to evaluate the therapeutic effects ofNTAP on CIA rats and explored the possible mechanisms of action.Part1Therapeutic effects of NTAP on collagen-induced arthritis in ratsObjective: To investigate the therapeutic effects of NTAP on CIA in rats.Methods: The female Lewis rats (weigh180-200g) were immunizedintradermally at the base of the tail with0.1mg of freshly solubilized ofbovine type II collagen emulsified in incomplete Freund’s adjuvant (IFA) in a100μl injection volume to induce arthritis on day0, and boosted with the sameagent on day7. CIA rats were randomly divided into four groups (n≥8), themodel group, NTAP group, wild-type peptide (WP) group and irrelevantpeptide (IP) group. Meanwhile the eight normal rats were divided into thecontrol group. NTAP, wild-type peptide and irrelevant peptide (1mg,1.07mgand1mg/kg) were respectively administrated intravenously every other daybeginning at arthritis onset. The equal volume of PBS was administrated in the control and the model groups. Rats were sacrificed after administrating twoweeks. Clinical and arthritis scores were assessed. The pathological changes ofsynovial tissue in the knee joint were observed with light microscope by HEstaining. The expression of IL-17in the synovial tissue was detected byimmunohistochemisty method. The spleens and the synovial tissues wereisolated in rats.Results:1The arthritis score was significantly decreased in rats treated NTAPcompared with that in rats in the model (P<0.01). NTAP could surppress theseverity of CIA by decreasing the arthritis score and X ray score.2HE staining showed hyperplasia of synovial cell, pannus forming, andinfiltration with inflammatory cell in synovium tissue in the knee joint in themodel group. Compared with those of the model group, the pathologicalchanges of synovium tissue were significantly alleviated in NTAP group.3NTAP could selectively inhibit IL-17expression in local jointsynovium tissue.Conclusion: NTAP could surppress the severity of CIA and decreases thearthritis scores, and selectively inhibit IL-17expression in local jointsynovium tissue. It could be concluded that NTAP may play the therapeuticrole against CIA.Part2Effects of NTAP on T helper cells immunity in collagen-inducedarthritis ratsObjective: To observe the effects of NTAP on T cell population, thechanges of mRNA expression of cytokines and transcription factors related toT helper cells in the spleen in collagen-induced arthritis model rats andexplore the possible mechanisms.Methods: The percentage of Th1, Th2, Th17and Treg cells insplenocytes of CIA rats were determined by flow cytometry. Total RNA in thespleen was prepared, the T-bet, Gata-3, RORγt, Foxp3, and the IFN-γ, IL-4,IL-17and TGF-β mRNA expression were measured by quantitative RT-PCR. Results:1Compared with those of control group, the percentage of Th1and Th17cells and the ratio of Th1/Th2were significantly increased, and the percentageof Th2and Treg cells were significantly decreased in the splenocytes of modelgroup rats(P<0.01). Compared with those of model group, the percentage ofTh1and Th17cells and the ratio of Th1/Th2were significantly decreased andthe percentage of Treg cells were significantly increased in the splenocytes ofNTAP group rats (P<0.01). Compared with those of model group, thepercentage of Th2cells were increased and the percentage of Th17cell weredecreased in the splenocytes of wild type peptide group rats (P<0.05), and thepercentage of Th17cell were significantly decreased in the splenocytes ofirrelevant peptide group rats (P<0.01).2Compared with those of control group, IFN-γ、IL-17、IL-4mRNAexpression were significantly increased, and TGF-β mRNA expression wassignificantly decreased in the spleen of model group rats(P<0.01). Comparedwith those of model group, IFN-γ、IL-17mRNA expression were significantlydecreased, IL-4、TGF-β mRNA expression were significantly increased in thespleen of NTAP group rats (P <0.05, P <0.01). Compared with those of modelgroup, IL-17mRNA expression were significantly decreased in the spleen ofwild type peptide and irrelevant peptide group rats (P <0.01).3Compared with those of control group, T-bet、RORγt mRNA expressionwere significantly increased, and Gata-3、Foxp3mRNA expression weresignificantly decreased in the spleen of model group rats(P <0.01). Comparedwith those of model group, T-bet、RORγt mRNA expression were significantlydecreased, Gata-3、Foxp3mRNA expression were significantly increased inthe spleen of NTAP group rats (P<0.01). Compared with those of model group,T-bet、Foxp3mRNA expression were significantly decreased and Gata-3mRNA were significantly increased in the spleen of wild type peptide.Compared with those of model group, RORγt mRNA expression weresignificantly decreased in the spleen of irrelevant peptide group rats (P<0.01).Conclusion: Administration of NTAP could potently suppress theseverity of CIA by decreasing the percentage of Th1and Th17cell and increasing the percentage of Th2and Treg cells, suggesting that NTAP mightbe a promising candidate for treatment of rheumatoid arthritis, compared towild type peptide and irrelevant peptide.Part3Effects of NTAP on apoptosis in collagen-induced arthritis ratsObjective: To study the effects of NTAP on the synoviocyte apoptosisand on the splenic T lymphocytes apoptosis in CIA rats and explore thepossible mechanism.Methods: The apoptotic rate of synoviocyte was measured by TUNEL.The expressions of Bcl-2and Bax in the synovial tissue were respectivelydetected by immunohistochemisty SP method. The apoptotic rate of CD3+Tlymphocyte in spleen was measured by flow cytometry technique.Results:1The apoptotic rate of synoviocyte was significantly decreased in themodel group compared with that of the control group (P<0.01). Comparedwith that of the model group, the apoptotic rate of synoviocyte wassignificantly increased in the NTAP group (P<0.01).2The expression of Bcl-2was significantly increased, the expression ofBax was significantly decreased in the model group compared with those ofthe control group (P<0.01, P<0.05). The expression of Bcl-2was significantlydecreased, the expression of Bax was significantly increased in the NTAPgroups compared with those of the model group (P<0.01).3Compared with those of the control group, the apoptotic rate of splenicCD3+lymphocytes was significantly decreased in the model group (P<0.05).Compared with those of the model group, the apoptotic rate of splenic CD3+lymphocytes was significantly increased in the NTAP group (P<0.05).Compared with those of the model group, no changes in the apoptosis rate ofsplenic CD3+lymphocytes were observed in the wild-type peptide andirrelevant peptide groups.Conclusion: NTAP could enhance the apoptotic rate of synoviocyte,attenuate the expression of Bcl-2, and increase the expression of Bax insynovial tissue in CIA rats. NTAP could ameliorate the apoptotic rate of splenic CD3+lymphocytes. It could be concluded that NTAP could play aprotective role against CIA by promoting synoviocyte apoptosis andameliorating the apoptotic rate of splenic CD3+lymphocytes.Summary:1NTAP could surppress the severity of CIA and decreases the arthritisscores. NTAP ameliorates the histopathology of CIA rats. NTAP could alsoselectively inhibit IL-17expression in local joint synovium tissue. It could beconcluded that NTAP may play the therapeutic role against CIA.2Administration of NTAP could potently suppress the severity of CIAby decreasing the number of Th1and Th17cells and by increasing the numberof Th2and Treg cells. Moreover, the expression of Gata-3, Foxp3, IL-4andTGF-β mRNA were significantly increased, the expression of T-bet, IFN-γ,RORγt and IL-17mRNA were obviously decreased in NTAP treated ratscompared with the model rats. The results suggest that NTAP might be apromising candidate for treatment of rheumatoid arthritis.3NTAP could enhance the apoptotic rate of synoviocytes, attenuate theexpression of Bcl-2, and increase the expression of Bax in synovial tissues inCIA rats. NTAP could ameliorate the apoptotic rate of splenic CD3+lymphocytes. It could be concluded that NTAP could play a protective roleagainst CIA by promoting synoviocyte apoptosis and ameliorating theapoptotic rate of splenic CD3+lymphocytes. |
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