IMMUNOLOGICAL STUDY ON ACUPUNCTURE TREATMENT OF COLLAGEN-INDUCED ARTHRITIS IN CX43-KNOCK-OUT RATS

Objective To observe the therapeutic effect of acupuncture for collagen-induced arthritis （CIA） in Cx43 knock-out mice and its underlying immunological mechanism.Methods Heterozygote （Cx43^＋/-） mice （n=64） and wild-type （Cx43^＋/＋） mice （n=46） were used in the present study.CIA model was es- tablished by intracutaneous injection of bovine collagenⅡ（immunonization）.Acupuncture of Zúsānlǐ（足三里ST36） was carried out from the 4^th week on after the initial immunization,once a day for 3 weeks.Arthritis score and days of onset of CIA were recorded.Intracellular contents of Th subgroups of splenic lymphocytes were detected with flow cytometry.Results The incidence rate of CIA and arthritis score in Cx43^＋/- mice were significantly lower than those in Cx43^＋/＋ mice （P〈0.05）.In Cx43^＋/＋ mice,3 weeks after acupuncture treatment,the score of arthritis symptoms and signs in acupuncture group was significantly lower than that in model group （P〈0.01）,while in Cx43^＋/- mice,no significant difference was found between model group and acupuncture group in arthritis score （P〉0.05）.In comparison with control group,percentages of both Thl and Th2 and Th1/Th2 increased significantly in 0x43^＋/＋ CIA model group （P〈0.05）,while compared with model group,the percentage of Th1,Th2 and Th1/Th2 in acupuncture group decreased evidently （P〈0.05） or moderately,showing that acupuncture can effectively suppress arthritis-induced increase of Thl in Cx43^＋/＋ mice.In 0x43^＋/- mice,Th1 level and Th1/Th2 of CIA model group increased considerably in comparison with control group （P〈0.05）,while compared with model group,the Th1,Th2 and Th1/Th2 of acupuncture group had no significant changes （P〉0.05）,indicating that the effect of acupuncture in suppressing increase of Th1 percent and Th1/Th2 was eliminated.Conclusion Acupuncture of Zúsānlǐ（足三里ST 36） can effectively relieve CIA symptoms and signs and inhibit increase of splenic Th1 in Cx43^＋/＋ mice but has no any effect on CIA score,Th1,Th2 and Th1/Th2 in Cx43^＋/- mice.It shows the effect of acupuncture in improving CIA is closely related to Cx43 gene.

Effectiveness of Huai Qi Huang Granules on Juvenile Collagen-induced Arthritis and Its Influence on Pyroptosis Pathway in Synovial Tissue

Summary:Huai Qi Huang(HQH)exerts great effects in clinic,such as anti-inflammation,immune-regulation,anti-cancer,and so on.However,the mechanism by which HQH protects juvenile idiopathic arthritis(JIA)is obscure.Thus,we explored deeply the protective mechanisms in juvenile collagen-induced arthritis(CIA)rat model.Pyroptosis is Gasdermin D(GSDMD)-dependent programmed cell death,involved in many diseases,such as sepsis.We investigated whether GSDMD-induced pyroptosis take part in mechanisms of juvenile CIA arthritis.Juvenile Wistar rats(3-4 weeks)were injected intradermally with fully emulsified bovine typeⅡcollagen and complete Freund's adjuvant to establish CIA rat models.Later,the CIA rats received oral administration of HQH(4.16 g/kg)once a day from the day 21 of modeling,with the treatment lasting for 28 days.Varieties of indicators were measured for evaluation of anti-inflammation effect of HQH,including hind paw swelling,arthritis scores,micro CT,and histopathological changes and the level of pro-inflammatory cytokines in the serum,including tumor necrosis factor alpha(TNF-α)and interleukin-18(IL-18).The expression of GSDMD and caspasein the joint synovial tissues was detected.The results demonstrated that the expression of the pyroptotic protein GSDMD and its upstream caspase-1 was significantly increased in the synovial tissues of CIA rats.The treatment of HQH ameliorated the symptoms in CIA rats,reduced levels of pro-inflammatory cytokines and hind paw swelling,down-regulated the expression of GDSMD and caspase-1.GSDMDinduced pyroptosis participated in the pathogenesis of CIA rats.The study supported that HQH can effectively improve joints inflammation of juvenile collagen-induced arthritis rats by inhibiting pyroptosis pathway in the joint synovial tissues.

Effects of Triptolide on the Expression and Activity of NF-κB in Synovium of Collagen-induced Arthritis Rats

Summary： The expression and activity of NF-kB in the synovium of collagen-induced arthritis （CIA） rats was detected in order to investigate the possible therapeutic effects of triptolide on rheumatoid arthritis （RA）. The experimental Wistar rat model of CIA was set up by intradermal injection of emulsion of bovine collagen 11 and the successful rate of setting-up models was evaluated by arthritis index （AI）. Rats were grouped randomly into three groups： normal, model and treatment group. The expression of TNF-α and IL-6 in synovial fluid was detected by ELISA, and the expression and activity of NF kB in synovium by immunohistochemistry method and by electrophoretic mobility shift assay （EMSA） respectively. As compared with normal group, the expression of TNF a and IL-6 in synovia （P〈0. 05）, and the expression and activity of NF-kB （P〈0.05） in synovium were increased in model group. There was statistical difference in above-mentioned indexes between model group and treatment group. Triptolide may play a protective role in IRA via downregulating the expression and activity of NF-kB in synovium.

Prediction of Response of Collagen-induced Arthritis Rats to Methotrexate： An ~1H-NMR-based Urine Metabolomic Analysis

Over one half the patients with rheumatoid arthritis （RA） are being treated with methotrexate （MTX）. Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis （CIA） and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M 1H-nuclear magnetic resonance （1H-NMR） for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats （n=20） was different from that from the non-responsive rats （n=11）. Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis （PLS-DA） （R2=0.812, Q2=0.604）. In targeted profiling, 13 endogenous metabolites （uric acid, taurine, histidine, methionine, glycine, etc.） were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that 1H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.

Therapeutic Actions of the Chinese Herbal Formulae with Cold and Heat Properties and Their Effects on Ultrastructures of Synoviocytes in Rats of the Collagen-Induced Arthritis

The therapeutic actions of Qing Luo Yin (QLY清络饮) with heat property and Wen Luo Yin (WLY温络饮) with cold property on pain, swelling of the ankle, arthritis index and ultrastructures of synoviocytes were compared in rats of type II collagen-induced arthritis (CIA), with tripterygium glycosidorum (TG) used as control. The results indicated that both QLY and WLY could reduce pain, swelling of the ankle and the arthritis index of CIA, and QLY had better effects in reducing the swelling of the ankle and controlling the secondary pathological lesions as compared with WLY. Investigation on the ultrastructures of synoviocytes indicated that both QLY and WLY could reduce the number of Golgi apparatus, rough surface endoplasmic reticulum, dense bodies, matrix filaments and vacuoles so as to suppress the excessive secretion of synoviocytes in rats of CIA.

Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance

Rheumatoid arthritis(RA)is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation,posing challenges in the development of effective treatments.Nuciferine,an alkaloid found in lotus leaf,has shown promising anti-inflammatory and anti-tumor effects,yet its efficacy in RA treatment remains unexplored.This study investigated the antiproliferative effects of nuciferine on the MH7A cell line,a human RA-derived fibroblast-like synoviocyte,revealing its ability to inhibit cell proliferation,promote apoptosis,induce apoptosis,and cause G1/S phase arrest.Additionally,nuciferine significantly reduced the migration and invasion capabilities of MH7A cells.The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis(CIA)rat model,where it markedly alleviated joint swelling,synovial hyperplasia,cartilage injury,and inflammatory infiltration.Nuciferine also improved collagen-induced bone erosion,decreased pro-inflammatory cytokines and serum immunoglobulins(IgG,IgG1,IgG2a),and restored the balance between T helper(Th)17 and regulatory T cells in the spleen of CIA rats.These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

Huangqi Guizhi Wuwu Decoction suppresses inflammation and bone destruction in collagen-induced arthritis mice

Objective: Rheumatoid arthritis(RA) is a chronic inflammatory and destructive arthritis, characterized by inflammatory infiltration and bone destruction. Huangqi Guizhi Wuwu Decoction(HGWD) is traditional Chinese medicine, which has been applied in the treatment of RA in clinical. The aim of this study was to investigate the therapeutic effect of HGWD on collagen-induced arthritis(CIA) mouse model.Methods: DBA/1J female mice were used to establish the collagen-induced arthritis(CIA) model. HGWD was administered intragastrically once a day for four weeks starting on the 22nd day after the first immunization. The body weight, hind paw thickness and clinical score were measured every five days. Gait analysis, histopathological staining, enzyme-linked immunosorbent assay(ELISA), ultrasound imaging and micro-computed tomography imaging were performed to determine the effects of HGWD treatment on inflammation and bone structure in this model. Moreover, Real-time PCR and Western blot analysis were used to detect inflammatory factors m RNA and protein levels after HGWD intervention in RAW264.7 cells.Results: HGWD attenuated symptoms of arthritis, suppressed inflammatory synovium area and the serum levels of inflammatory factors, inhibited joint space enlargement in the knee and ankle joints,reduced numbers of osteoclasts, protected bone destruction, as well as improved motor function.HGWD decreased the expression of m RNA for inflammatory factors and the protein expression levels of p-NF-ΚB and IL-17.Conclusion: These results suggested that HGWD suppresses inflammation, attenuates bone erosion and maintains motor function in collagen-induced arthritis mice.

Attenuation of Collagen Induced Arthritis by Centella asiatica Methanol Fraction via Modulation of Cytokines and Oxidative Stress

Objective To investigate the anti-inflammatory, antioxidant and anti-arthritic effects of Centello asiatica methanolfraction （CAME） on collagen-induced arthritis （ClA）, an animal model of rheumatoid arthritis. Methods Arthritis was induced in female wistar rats by immunization with porcine type II collagen. The CIA rats were treated orally with CaME （50, 150, and 250 mg/kg/day） for 15 d （beginning on day 21 of the experimental period）. The clinical, histological, biochemical, and immunological parameters were assessed. Results CaME treatment （150 and 250 mg/kg） significantly attenuated the severity of CIA and reduced the synovial inflammation, cartilage erosion, and bone erosion as evident from both histological and radiographic data. The escalated plasma levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-12 alongwith nitric oxide in CIA rats decreased significantly on CaME treatment. The serum levels of type-Ⅱ collagen antibody were significantly lower in rats of CaME （150 and 250 mg/kg） treated group than those in the arthritic group. Furthermore, by inhibiting the above mediators, CaME also contributed towards the reversal of the disturbed antioxidant levels and peroxidative damage. Conclusion Our results clearly indicate that oral administration of CaME suppresses joint inflammation, cytokine expression as well as antioxidant imbalance, thereby contributing to an amelioration of arthritis severity in CIA rats.

Pharmacokinetic and pharmacodynamic study of triptolide-loaded liposome hydrogel patch under microneedles on rats with collagen-induced arthritis

Triptolide (TP), a major active component of Triptelygium wilfordii Hook.F. (TWHF), is used to treat rheumatoid arthritis (RA). However, it has a narrow therapeutic window due to its serious toxicities. To increase the therapeutic index, a new triptolide-loaded transdermal delivery system, named triptolide-loaded liposome hydrogel patch (TP-LHP), has been developed. In this paper, we used a micro-needle array to deliver TP-LHP to promote transdermal absorption and evaluated this treatment on the pharmacokinetics and pharmacodynamics of TP-LHP in a rat model of collagen-induced arthritis (CIA). The pharmacokinetic results showed that transdermal delivery of microneedle TP-LHP yielded plasma drug levels which fit a one compartment open model. The relationship equation between plasma concentration and time was C=303.59 x (e(-0.064t)-e(-0.287t)). The results of pharmacodynamic study demonstrated that TP-LHP treatment mitigated the degree of joint swelling and suppressed the expressions of fetal liver kinase-1, fetal liver tyrosine kinase-4 and hypoxia-inducible factor-1a in synovium. Other indicators were also reduced by TP-LHP, including hyperfunction of immune, interleukin-1/3 and interleukin-6 levels in serum. The therapeutic mechanism of TP-LHP might be regulation of the balance between Thl and Th2, as well as inhibition of the expression and biological effects of vascular endothelial growth factor. (C) 2015 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Metabolomics study of biochemical changes in the serum and articular synovium tissue of moxibustion in rats with collagen-induced arthritis

Objective:To demonstrate the rheumatoid arthritis(RA)mechanisms by determining the biochemical changes.To investigate the therapeutic mechanism of moxibustion in RA-model rats using a gas chromatography-mass spectrometry(GC-MS)metabolomics approach.Methods:A total of 24 rats were divided into three groups as follows:normal control group,model group and moxibustion group.Rats in model group and moxibustion group were set up collagen-induced arthritis(CIA)model.Rats in moxibustion group were treated by moxibustion.After 3 weeks of intervention,right ankle joint,serum and articular synovium samples were collected.Right ankle joint samples were used for histopathological evaluation between 3 groups to get the pathological changes of tissues and cells.Serum and articular synovium samples were used to analyze the changed metabolites of moxibustion on RA rats by the GC-MS based metabolomics.Results:Treatment of moxibustion not only significantly increased the weight of CIA rats,reduced the swelling of hind paw,arthritic scores,IL-1β,TNF-αbut also improved histopathological evaluation in right ankle joint samples.Sixteen significantly altered metabolites were found in RA rats as potential biomarkers of arthritis.Thirteen metabolites,significantly adjusted by moxibustion to help relieve arthritis,were selected out as biomarkers of antiarthritic mechanism of moxibustion,which were mainly involved in phenylalanine,tyrosine and tryptophan biosynthesis,glycine,serine and threonine metabolism,phenylalanine metabolism,alanine,aspartate and glutamate metabolism,glyoxylate and dicarboxylate metabolism and aminoacyl-tRNA biosynthesis.Conclusions:We have indicated moxibustion treatment is able to resist inflammation in CIA rats effectively.Using GC-MS metabolomics technique,we detect novel metabolites in the moxibustion antiarthritic process,which may aid in advanced understanding of arthritis and therapeutic mechanism of moxibustion.

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats

Objective： To observe the effect of norcantharidin（NCTD） on collagen-induced arthritis（CIA） rats. Methods： Sixty Sprague-Dawley（SD） rats were randomly divided into 6 groups（n=10）： normal group, CIA model group（model group）, NCTD low-dose group [1.35 mg/（kg·d）], NCTD middle-dose group [2.7 mg/（kg·d）], NCTD high-dose group [5.4 mg/（kg·d）] and methotrexate（MTX） group [1.8 mg/（kg/w）]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin（H＆E） staining. The serum levels of interleukin（IL） 1β, IL-6, tumor necrosis factor（TNF）-α, vascular endothelial growth factor（VEGF）, IL-17 and transform growth factor（TGF） β were detected by enzyme linked immunosorbent assay（ELISA）. The mRNA expression of retinoid-related orphan nuclear receptor γ t（ROR γ t） and forkhead box P3（Foxp3） in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. Results： MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats＇ ankle joints compared with the model group（P〈0.05 or P〈0.01）. All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats（P〈0.05）. Only middle-and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats（P〈0.05）. However, NCTD has no effect on vascular endothelial growth factor（VEGF） level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group（P〈0.05）. The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group（P〈0.05）. Conclusion： NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

Protective Effects of Overexpression TCR Vβ5.2-HSP70 and TCR Vβ8.2-HSP70 against Collagen-Induced Arthritis in Rats

Collagen-induced arthritis （CIA） is an animal model, which closely resembles human rheumatoid arthritis （RA） in pathogenesis and pathology. Evidence suggests that the inhibition of T lymphocytes or their functions can alleviate the progression of arthritis. So the administration of arthritogenic T cell receptor （TCR） variable region peptide or DNA vaccines encoding pathogenic TCR Vβ variable region may provide useful information for designing specific immunotherapies against autoimmune diseases. Heat shock proteins （HSPs） have the function of raising antigenic immunogenicity and HSP70 has a protective effect against arthritis. We previously demonstrated the presence of pathogenic predominant T cell receptor Vβ5.2 and Vβ8.2 clonotypes in the joints of CIA rats. In this study, we constructed the recombinant eukaryotic expression vectors pTARGET-TCR Vβ5.2/8.2-HSP70, and evaluated their protective effects on CIA rats. Protective effects were observed in CIA rats by injecting these recombinant DNA vaccines, which could alleviate arthritis index, decrease the levels of IFN-~ and anti-CII antibody in serum, and increase the levels of IL-4. Pathological changes were not as serious as those observed in control CIA rats. The rat injected with two combined vaccines showed better protective effects than CIA rats administered with individual vaccine. These results showed that recombinant DNA vaccines pTARGET-TCR Vβ5.2-HSP70 and pTARGET-TCR Vβ8.2-HSP70 could significantly alleviate the arthritic symptoms of CIA rats, and better protective effects could be achieved if these two vaccines were used in combination. Cellular ＆ Molecular Immunology.

Inhibitory Effect of the Extract of Phellodendron amurense Ruprecht Root on Collagen-Induced Arthritis in Mice

Objective： To investigate whether the dried root of Phellodendron amurense Ruprecht（Phellodendri cortex; PC） extract improves arthritic symptoms through anti-inflammatory and immune-modulatory effects in collagen-induced arthritis in mice. Methods： Rheumatoid arthritis（RA） was induced in male DBA/1 mice by immunization with type Ⅱ collagen（ColⅡ）. CIA mice were divided into 5 groups（n=10 per a group） with normal, CIA control, PC extract（50 mg/kg and 100 mg/kg）-treated, and meloxicam（50 mg/kg）-treated as the reference drug. The PC extract or meloxicam were administered orally in CIA mice once a day for 14 days after arthritis induction. Arthritic score, levels of anti-ColⅡ IgG2a antibody, prostaglandin E2（PGE2）, tumor necrosis factor（TNF）-α, and interleukin（IL）-17 in the sera of CIA mice were measured. Histopathological changes in the ankle joints of CIA mice were also analyzed by staining with hematoxylin and eosin（H and E）, safranin-O and immunohistochemistry using anti-TNF-α and anti-IL-17 antibodies. Results： The arthritic score was increased in CIA mice in a time-dependent manner, as were the serum levels of anti-ColⅡ IgG2a antibody, PGE2, TNF-α, and IL-17. However, the oral administration of PC extract at 50 and 100 mg/kg in CIA mice significantly decreased the arthritic scores, and the serum levels of anti-ColⅡ IgG2a, PGE2, TNF-α, and IL-17 compared with those in the CIA group（P〈0.05 or P〈0.01）. Furthermore, histopathological improvement of the joint architecture in CIA mice was observed after administration of PC extract. PC extract also significantly inhibited the expression of TNF-α and IL-17 in the joints of CIA mice by suppressing the expression of their m RNA and proteins. Conclusion： PC extract may improve the pathological progression of RA through the inhibition of joint destruction by synovial inflammation and immune-stimulation, therefore, it would be a potential anti-arthritic agent in RA.

Therapeutic Effect of Dimethyl Dimethoxy Biphenyl Dicarboxylate on Collagen-Induced Arthritis in Rats

Objective： To study the effect of oral administration of dimethyl dimethoxy biphenyl dicarboxylate （DDB） on adjusting angiogeneic/inflammatory mediators and ameliorating the pathology of bones in rats with collagen-induced arthritis （CIA）. Methods： Wistar rat model of CIA was set up using bovine collagen type H. Fifty rats were divided into five groups randomly： normal, CIA model, DDB treatment, methotrexate （MTX） treatment, and combined DDB＋MTX treatment. Ankle joints of rats were imaged with digital X-ray machine to show the destruction of joints. Fore and hind paw and knee joints were removed above the ankle joint then processed for haematoxylin and eosin staining. Plasma levels of vascular endothelial growth factor （VEGF）, platelet derived growth factor, interleukin-8 （IL-8）, IL-4, tumor necrosis factor α （TNF-α）, and cyclooxygenase-2 （COX-2） were quantified by enzyme-linked immunosorbent assay. Nitric oxide levels were detected by Griess reagent. Results： Compared with the CIA model group, a remarkable reduction in various angiogenic （VEGF and IL-8） and inflammatory mediators （TNF-α, IL-4 and COX-2） after treatment with DDB either alone or combined with MTX （P〈0.05 or P〈0.01）. Histopathological and X-ray findings were confirmatory to the observed DDB anti-arthritic effect. The DDB-treated group showed amelioration in signs of arthritis which appeared essentially similar to normal. Conclusion： Our data shed light on the therapeutic efficacy of DDB in experimental rheumatoid arthritis （RA） compared with a choice drug （MTX） and it may be offered as a second-line drug in the treatment of RA.

Original article Inhibition of collagen-induced arthritis by DNA vaccines encoding TCR Vβ5.2 and TCR Vβ8.2

Background Arthritogenic T lymphocytes with common T cell receptor （TCR） Vβ clonotypes, infiltrating in the articulars of rheumatoid arthritis （RA） patients, play a central role in the pathogenesis of RA. TCR Vβ5.2 and TCR Vβ8.2 are the main pathogenic T cell clonotypes in the course of collagen-induced arthritis （CIA） progression in Lewis rats. To investigate a TCR-based immunotherapy for RA, we constructed recombinant DNA vaccines encoding TCR Vβ5.2 and TCR Vβ8.2, and evaluated the inhibitive effects of the two vaccines on CIA rats.Methods Genes encoding TCR Vβ5.2 and TCR Vβ8.2 were amplified by RT-PCR from spleen lymphocytes of Lewis rats and cloned into the eukaryotic expression vector pTargeT. The expression of vaccines was confirmed by RT-PCR and immunohistochemistry. The inhibitive effects of the vaccines on articulars of CIA rats were assessed with arthritis index evaluation and histology. Interferon γ （IFN-γ） and interleukin （IL）-4 production by spleen lymphocytes were tested with enzyme-linked immunospot assay （ELISPOT） technique, the changes in peripheral CD4^＋ and CD8^＋ lymphocyte populations were tested by flow cytometry, and the level of anti-CII antibody in serum was assayed by enzyme-linked immunosorbent assay （ELISA）.Results Recombinant DNA vaccines pTargeT-TCR Vβ5.2 and pTargeT-pTCR Vβ8.2 were successfully constructed. Both vaccines inhibited CIA, which alleviated the arthritis index score （P 〈0.05）, decreased the level of IFN-γ （P 〈0.05）, and reduced the ratio of CD4^＋/CD8^＋ lymphocytes （P 〈0.05） and the anti-CII antibody in serum （P 〈0.05）. In addition, the histological change in DNA-vaccinated rats was less serious than CIA rats. Compared to pTCR Vβ 8.2 and pTCR Vβ 5.2 groups, the group that was injected with a combination of the two vaccines showed stronger inhibitive effects on CIA than either individual vaccine.Conclusion The recombinant plasmids pTargeT-TCR Vβ5.2 and pTargeT-TCR Vβ8.2 have obvious inhibatory effects on CIA rats and better effects could be achieved when the vaccines were used in combination.

Effects of Agkistrodon in Different Dosage Forms on Collagen-Induced Arthritis in Rats

Objectives： To determine the effective dosage and formulation of agkistrodon in collagen-induced arthritis（CIA） rats. Methods： CIA was induced by injection of collagen in complete/incomplete Freund＇s adjuvant. Agkistrodon decoction, agkistrodon powder, and agkistrodon wine were administered daily starting from the onset of arthritis. Paw swelling degree was measured by using a volume-measuring instrument every 7 days after primary immunization. Arthritis index was measured and calculated using the ＂five scoring method＂ every 7 days. The levels of serum interleukin-1β（IL-1β） and type Ⅱ collagen Ig G antibodies were detected by enzyme-linked immunosorbent assay. Finally, all ankles were removed, and X-ray radiography was performed with In-vivo Imaging System FX. Samples were counterstained with hematoxylin and eosin for analysis. Results： Among the various dosage formulations of agkistrodon, high-dose powder, which was equivalent to an amount of 6 g/day in adults, showed better effects on the inhibition of joint swelling and reduction of arthritis index score. The relatively low levels of serum IL-1 and anti-type Ⅱ collagen Ig G antibodies, as well as the X-ray radiography and pathology results, further proved the superiority of the high-dose powder over the other formulations. The effect of decoction on inhibiting joint swelling was inversely proportional to the dosage. Other effects, such as reduction of arthritis index score and the levels of serum IL-1 and anti-type Ⅱ collagen Ig G antibodies, were directly proportional to the dosage. While the use of large dose agkistrodon wine led to negative effects. Conclusions： These data highlight the potential function of high-dose agkistrodon powder, which was equivalent to an amount of 6 g/day in adults. The powder can quickly relieve the symptoms of rheumatoid arthritis and prevent aggravation of disease, especially during the early period.

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Expression of Receptor Activator of Nuclear Factor Kappa B Ligand,Osteoprotegerin,and Tumor Necrosis Factor Receptor Superfamily Member 14 in Rats with Collagen-Induced Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe（温化蠲痹方, WJR） on expression of receptor activator of nuclear factor kappa B ligand（RANKL）, osteoprotegerin（OPG）, and tumor necrosis factor receptor superfamily member 14（TNFRSF14, also known as LIGHT） in rats with collagen-induced arthritis（CIA）. Methods： CIA rats were generated by subcutaneous injection of bovine collagen type-Ⅱ at the tail base. Sixty CIA rats were randomly assigned（10 animals/group） to： model, methotrexate（MTX）-treated（0.78 mg/kg body weight）, and WJR-treated（22.9 g/kg） groups. Healthy normal rats（n=10） were used as the normal control. Treatments or saline were administered once daily by oral gavage. Rats were sacrificed at day 28 post-treatment and knee synovium and peripheral blood serum were collected. Toe swelling degree and expression of RANKL, OPG, and LIGHT were determined by Western blot and immunohistochemistry. Results： Compared with the normal group, toe swelling degree was significantly increased in the model group（P〈0.01）. After treatment, toe swelling degree decreased significantly in the WJR and MTX groups compared with the model group（P〈0.01）. Compared with the normal group, expression of RANKL and LIGHT were significantly increased and OPG significantly decreased in peripheral blood and synovium of the model group（P〈0.01）. Conversely, RANKL and LIGHT expression were significantly reduced and OPG increased in the WJR and MTX groups compared with the model group（P〈0.01）. No statistically significant difference existed between WJR and MTX groups. Conclusion： WJR likely acts by reducing RANKL expression and increasing OPG expression, thus inhibiting RANKL/RANK interaction and reducing LIGHT expression, thereby inhibiting osteoclast formation/activation to block bone erosion.

Polyherbal formula SC-E3 inhibits rheumatoid arthritis activity in a mouse model of type-Ⅱcollagen-induced arthritis

Objective:SC-E3 is a polyherbal formula that contains five medicinal herbs used frequently in traditional herbal medicine.In our previous study,we demonstrated the antioxidant and anti-inflammatory effects of SC-E3.The present study examined the effects of SC-E3 in a mouse model of type-II collagen-induced arthritis(CIA).Methods:In vivo,male DBA/1 J mice were immunized by intradermal injection of bovine type-II collagen and complete or incomplete Freund’s adjuvant,to induce arthritis.SC-E3 was orally administered daily for 23 days.In vitro,bone marrow-derived macrophages(BMMs)were treated with macrophage colony-stimulating factor(M-CSF)and receptor activator of nuclear factor-j B ligand(RANKL)in the absence or presence of SC-E3.Results:Administrations of SC-E3 were found to have anti-arthritic effects in the joints of CIA mice,as evidenced by reduced paw swelling,bone erosion and deformation,inflammatory cell infiltration,and inflammation in synovial membrane.SC-E3 also reduced serum levels of tumor necrosis factor-a,interleukin-1 b,aspartate aminotransferase and alanine aminotransferase.Furthermore,tartrateresistant acid phosphatase-positive osteoclast numbers in the joints were significantly lower in SC-E3-treated CIA mice than in CIA mice.In addition,the differentiations of BMMs to multinucleated osteoclasts induced by M-CSF and RANKL stimulation were dose-dependently reduced by SC-E3.Conclusion:These results suggest that SC-E3 possesses substantial anti-arthritic activity because it inhibits pro-inflammatory cytokines and osteoclastogenesis,and that SC-E3 has potential therapeutic use for the treatment of rheumatoid arthritis.

Effect of Qubi Zhentong Recipe(祛痹镇痛方)on Synovial Gene Expression in Collagen-Induced Arthritis in Rats

Objective： To investigate the effect of the Chinese medical formula Qubi Zhentong Recipe （祛痹镇痛方 QZR） on the synovial gene expression profile in collagen-induced arthritis （CIA） rats. Methods： Ten rats were randomly chosen from 60 rats as the control group, and the other 50 rats were used for the CIA models. The CIA model group was constructed by bovine injection of type Ⅱ collagen through the rats＇ neck and tail. Twenty rats were randomly chosen from 34 successful CIA models and randomly assigned into two groups： the model group （n=10） and the QZR group （n=10）. The QZR group was fed intragastrically with QZR 22.9 g/（kg.d） （10 times the clinical adult dose）, and the CIA model group was given the same dose of normal saline. Both model and QZR groups were administered treatment once a day. Total RNA was collected from the knee joint synovium after 30 days. The change in gene expression profile was analyzed by a whole gene chip. Results： A total of 76 genes showed a difference in expression between CIA model group and the control group; 35 genes were down-regulated and 41 were up-regulated. A total of 67 genes showed a difference in expression between the model group and the QZR group; 48 genes were down-regulated and 19 were up- regulated. Conclusions： QZR may affect CIA by stimulating multiple genes and targets, which are related to oncogenes, apoptosis, metabolism, the immune system, ion channels, and transport proteins.

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Proliferation and Apoptosis of Synoviocytes in Rats with Collagen-Inducing Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe （温化蠲痹方, W JR） on proliferation and apoptosis of synoviocytes in rats with collagen-inducing arthritis （CIA）. Methods： A CIA model was induced by intradermal injection of bovine collagen type 1[ emulsion at the base of rat tails. Thirty modeled healthy Wistar rats were randomly assigned to one of three groups （10 per group）： the model group, the methotrexate （MTX）- treated group （0.78 mg/kg） and the WJR-treated group （22.9 g/kg）. A group of 10 healthy rats was used as normal control. Treatments or normal saline for the control group were administered by oral gavage once daily. Rats were sacrificed after 30-day treatment and subjected to the following examinations： arthritis index （AI） was estimated, inflammatory cell infiltration and proliferation in synovial membrane were evaluated by microscopy, the synoviocyte apoptosis was determined by TUNEL assay, and the cell apoptosis index was calculated. Results： AI was lowered significantly in the WJR group compared to the model group （P〈0.01）. The pathological findings observed in the model group were reversed in the WJR group, including increase in inflammatory cell infiltration and synoviocyte proliferation in synovial membrane and reduction in cell apoptosis index （all P〈0.01）. Conclusions： Synoviocyte proliferation and apoptosis reduction were present in CIA rats. WJR was effective in treating the rat model of CIA. The therapeutic effect might be exerted through inducing apoptosis and suppressing proliferation of synoviocytes.