Effect of hydroalcoholic leaf extract of Cassia fistula L.on typeⅡcollagen-induced arthritis in rats

Objective:To explore the effect of Cassia fistula on collagenⅡ-induced arthritis in rats.Methods:The effect of 250 and 500 mg/kg chloroform and hydroalcoholic extract of Cassia fistula leaf on collagenⅡ-induced arthritis was investigated by evaluating paw volume,arthritis index,spleen index,and biochemical parameters.Histopathological analysis and docking study were also performed.Results:A dose-dependent reduction in paw volume,arthritic index,and spleen index was observed following oral administration of the chloroform and hydroalcoholic extracts.Treatment with Cassia fistula extracts reduced tumor necrosis factor-α,interleukin(IL)-1β,IL-6,prostaglandin E_(2),aspartate aminotransferase,alanine aminotransferase,total leucocyte count,and erythrocyte sedimentation rate while increasing IL-10 level.In addition,Cassia fistula extracts improved joint architecture,and prevented cartilage and bone destruction.Docking analysis demonstrated that the physcion,1-octacosanol,5,3’,4’-trihydroxy-6-methoxy-7-O-α-Lrhamnopyranosyl-(1,2)-O-β-D-galactopyranoside and scopoletin may be responsible for the anti-arthritic effect of Cassia fistula.Conclusions:Cassia fistula suppresses the progression of collagenⅡ-induced arthritis by lowering the inflammatory factors,decreasing paw volume and arthritic index,and alleviating joint architecture.However,further studies are required to confirm the bioactive molecule responsible for the anti-arthritic potential of Cassia fistula.

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.

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.

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.

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.

Effect of Gui Zhi decoction on enteric mucosal immune in mice with collagen-induced arthritis

AIM： To explore the effect of Gui Zhi decoction on enteric mucosal immune in type Ⅱ collagen-induced arthritis （CIA） in DBA mice. METHODS： Eighty DBA/1, weighing 18-22 g, were randomly divided into four groups with 20 in each group： control group, CIA group, treatment groups at high dosage and low dosage （GZH and GZL）. CIA was induced by immunization with type Ⅱ collagen （CII） emulsified with equal complete adjuvant at 0.1 mg CII each mouse. Blood lymphocyte suspension was screened for CD4 and CD8 expression using a flow cytometry, the CD4 and CD8 and secretory IgA （sIgA）-positive cells in enteric lamina propria tested with immunohistochemical staining. Tumor necrosis factor-alpha （TNF-α）, interleukin-1 （IL-1）-β, and IL-6 concentrations in serum were assayed with RIA. RESULTS： Gui Zhi decoction can lower the arthritic scores and decrease the occurrence of arthritis. The CD4, CD8, and sIgA-positive cells in CIA mice are less than in control mice, and in Gui Zhi decoction at high dosage could restore the lowered CD4- and CD8-positive cells in lamina propria, and at both high and low dosages could increase the lowered sIgA-positive cells in lamina propria, even still lower than in normal mice. In periphery, the CD4 cells in periphery are higher in CIA mice than in control mice, and Gui Zhi decoction at high and low dosages could decrease the CD4 and CD8 cells. Also, Gui Zhi decoction at high dosage could decrease the IL-6 and TNF-α concentration in serum. CONCLUSION： Gui Zhi decoction can lower the arthritic scores and decrease the incidence of CIA in mice, and the mechanism is in part regulating enteric mucosal immune.

Expression of Peptidylarginine Deiminase 4 and Protein Tyrosine Phosphatase Nonreceptor Type 22 in the Synovium of Collagen-Induced Arthritis Rats

Objective To study the expression level of peptidylarginine deiminase 4(PADI4) and protein tyrosine phosphatase nonreceptor type 22(PTPN22) in the synovium of rat model of collagen-induced arthritis, and to explore their possible therapeutic role in rheumatoid arthritis. Methods Thirty-two female Wistar rats weighing 100±20 g were randomly assigned into 3-week collagen-induced arthritis(CIA) model group(n=8), 4-week CIA model group(n=8), 6-week CIA model group(n=8), and the control group(n=8). The body weight changes of each group were recorded. The expression levels of PADI4 and PTPN22 were detected and compared by the methods of immunohistochemical staining and Western blot. Results Arthritis of rat began to form 14 days after sensitization and the joint swelling reached peak at 28 days. The weights of the rats slowly grew both in CIA model groups and the control group. Immunohistochemical staining results showed that the positive expression of PADI4 and PTPN22 was mainly located in cartilage peripheral mononuclear cells, the cytoplasm of infiltrated cells, and bone marrow cavity. There were significant differences in the optical density of PADI4 and PTPN22 among CIA model groups and the control group(PADI4, 0.2898±0.012, 0.2982±0.022, 0.2974±0.031, 0.2530±0.013 in 3-week CIA model, 4-week CIA model, 6-week CIA model and control groups; PTPN22, 0.2723±0.004, 0.2781±0.010, 0.2767±0.008, 0.2422±0.019; all P <0.05). The expression bands of PADI4 were observed in Western blot 3 weeks after initial immunization, the thickest in the 4th week, and decreased in the 6th week. The expression bands of PTPN2 were observed at all the time points, with no obvious time-dependent trend. Conclusions PADI4 and PTPN22 are obviously correlated with CIA in rat model. PADI4 is expressed at early stage of the disease, while the expression of PTPN22 sustains throughout the course.

Effects of methotrexate on collagen-induced arthritis in male Wistar rats

To evaluate the effect of methotrexate on collagen-induced arthritis,micro-computed tomography(micro-CT)and histopathological analyses were used in male Wistar rats.Rats were divided randomly into three groups.Group 1 was treated with 0.9% saline,and groups 2 and 3 were boosted with type II collagen.From day 21 to 42,groups 1 and 2 were orally treated with 0.9% saline and group 3 was orally treated with 1.5 mg/kg methotrexate.All rats were sacrificed at day 42 after the first collagen treatment.Micro-CT analyses showed bony parameters,such as bone volume and trabecular number,were decreased in group 2 compared to group 1,and these parameters were recovered in group 3.Histopathological examination and pathological parameter scoring showed that the knee joints of rats in group 2 had severe joint destruction,showing cartilage and bone erosion,enlarged cavities with inflammatory cell infiltration and activation of synovial fibroblasts.By contrast,these changes were reduced in group 3.Taken together,methotrexate treatment showed therapeutic potential in male rat collageninduced arthritis model,and micro-CT analysis and histopathological tools could be integrated to assess the quantification/qualification of arthritic lesions.

The effect of alpha asarone, olive oil, and dexamethasone on collagen-induced arthritis (CIA) in the mouse

Aim of the Study: The primary aim of the study was to test the effect of 2,4,5-trimethoxy-1-propenylbenzene (alpha asarone), a hypocholes terolaemic drug, on the progression of collagen induced arthritis (CIA) in mice. Olive oil,the vehicle of alpha-asarone, and dexamethasonewere used as control treatments. Set-Up: Four groups of DBA/1 mice were immunised with chicken type II collagen (CII) via the intradermal route and either left untreated or were treated with alpha asarone, olive oil, or dexamethasone. A non-immunised group was an additional control. Follow-Up: The thicknesses of the rear and front footpads were continuously monitored, and the levels of anti-collagen antibodies were measured at the end of the experiment. The animals were then sacrificed, and their rear and front limbs were removed and processed forhistological examination. Results: Alpha asarone had no anti-inflammatory effect on CIA, and in one third of the animals, it showed a proinflammatory effect that was characterised by a marked accumulation of neutrophils. Olive oil did not show any obvious antiinflammatory effect on CIA, but it lowered the level of CII antibodies by 50%, suggesting a potential long-term antiinflammatory effect. As expected, dexamethasone had a clear anti-inflammatory effect on CIA. Con- clusion: Alpha asarone did not show any antiinflammatory effect on CIA in the mice under the above conditions;however, the accumulation of neutrophils in the CIA lesions of mice treated with alpha asarone and the effect of olive oil in downregulating the levels of anti-CII antibodies in CIA are two findings that warrant further investigation.

Phytosomal curcumin alleviates collagen-induced arthritis by downregulating Th17 and upregulating Treg cell responses in rats

Objective:To explore the effects of a nano-formulation of curcumin(phytosomal curcumin)on the clinical and pathological symptoms of collagen-induced arthritis(CIA)in rats.Methods:Forty male Wistar rats were immunized with an emulsion containing bovine typeⅡcollagen and incomplete Freund's adjuvant and then administered phytosomal curcumin post-immunization.Clinical symptoms and histological analysis of the synovial tissues were performed.The effect of phytosomal curcumin on Th17 and Treg parameters was also evaluated.Results:Phytosomal curcumin reduced the clinical severity and paw swelling in CIA-induced rats,which was accompanied by a reduction in the number of inflammatory cell infiltration in the synovial tissue.Additionally,treatment with phytosomal curcumin significantly inhibited CIA-associated mediators as well as increased the anti-inflammatory mediators in comparison to the control groups.Conclusions:Phytosomal curcumin could improve CIA autoimmune responses and can be considered a potential candidate for the treatment of rheumatoid arthritis.

Protective effects of Dioscin on TNF-α-induced collagen-induced arthritis rat fibroblast-like synoviocytes involves in regulating the LTB4/BLT pathway

Background and Objective:LTB4 has been shown to be involved in rheumatoid arthritis(RA)pathogenesis.The effect of Dioscin(Dio)on the LTB4 pathway of RA have not been reported yet.This study aimed at further exploring whether Dioscin’s effects on TNF-αinduced collagen-induced arthritis(CIA)rat fibroblast-like synoviocytes(FLS)connected with the LTB4 and its receptor pathway.Materials&Methods:In this experiment,control group,TNF-αgroup,and different concentrations of Dioscin groups were established.Cell viability was evaluated using MTT assay.The levels of LTB4 in the samples of above groups were measured using ELISA.The mRNA expression levels of LTA4H,BLT1,and BLT2 were detected by quantitative real time PCR,while the expression level of LTA4H proteins were detected using western blot.The distribution of LTA4H was assessed by immunofluorescence assay.Results:the LTB4 level of TNF-αgroup in sample supernatant was higher than both control group and Dioscin groups with decreased LTB4 levels(p<0.05).Compared with the control group,the expression of LTA4H was significantly increased in TNF-αgroup(p<0.05),whereas LTA4H expressions were significantly decreased in all Dioscin groups when compared to TNF-αgroup(p<0.05).The mRNA expressions of BLT1 and BLT2 were markedly higher in TNF-αgroup than those in control group while Dioscin treatment significantly inhibited the increased expressions of BLT1 and BLT2 induced by TNF-α(p<0.05).Conclusions:These results firstly demonstrate that the protective effect of Dioscin on TNF-αinduced FLS may involve in its reducing LTB4 production by down-regulating LTA4H expression,and may inhibit its downstream pathway by decreasing LTB4 receptors levels.This findings suggest that dioscin produces a potential therapeutic effects for RA via its influencing LTA4H/LTB4/BLT pathway.

Liposome-encapsulated dimethyl curcumin inhibits over-activation of rat lymphocyte to relieve collagen-induced arthritis by regulating intracellular proteolysis and PCNA/p21/caspase-3 pathway

Dimethyl curcumin,a synthetic derivative of curcumin,has good anti-cancer,anti-arthritis and anti-inflammatory properties,but underline mechanism was unclear.Activation,differentiation,proliferation and apoptosis of lymphocytes have important functions during arthritis development and immune homeostasis.Over-activation of lymphocyte causes inflammatory cytokines storm,leading to an immune imbalance and tissue damages.The aim of this study was to investigate the anti-inflammation mechanism of liposome encapsulated dimethyl curcumin(Lipo-DiMC)on over-activated rat spleen lymphocytes.In this study,Lipo-DiMC was produced to increase the solubility;the primary rat spleen lymphocytes were extracted and the Concanavalin A induced cell activation model was established to study the cellular responses to Lipo-DiMC treatment in-vitro.The results showed that Lipo-DiMC suppressed Concanavalin A-induced cell proliferation,inhibited cells entering G2/M phase,and reduced the ratio of necrosis/apoptosis by manipulating intracellular p53/p21/PCNA/JNK-1 pathways.Furthermore,Lipo-DiMC increased the accumulations of intracellular matrix metalloproteinase-9 and reduced matrix metalloproteinase-9 secretion of rat spleen lymphocytes.Also,Lipo-DiMC increased intracellular caspase-3 expression and reduced Cat-C activity in activated rat spleen lymphocytes,involving in intracellular proteolysis.Our findings suggest that dimethyl curcumin effectively alleviates Concanavalin A induced over-activation of rat spleen lymphocytes,thereby inhibiting inflammatory cytokines storm and restoring cell homeostasis.

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.

Bone-derived MSCs encapsulated in alginate hydrogel prevent collagen-induced arthritis in mice through the activation of adenosine A_(2A/2B)receptors in tolerogenic dendritic cells

Tolerogenic dendritic cells(tol DCs)facilitate the suppression of autoimmune responses by differentiating regulatory T cells(Treg).The dysfunction of immunotolerance results in the development of autoimmune diseases,such as rheumatoid arthritis(RA).As multipotent progenitor cells,mesenchymal stem cells(MSCs),can regulate dendritic cells(DCs)to restore their immunosuppressive function and prevent disease development.However,the underlying mechanisms of MSCs in regulating DCs still need to be better defined.Simultaneously,the delivery system for MSCs also influences their function.Herein,MSCs are encapsulated in alginate hydrogel to improve cell survival and retention in situ,maximizing efficacy in vivo.The three-dimensional co-culture of encapsulated MSCs with DCs demonstrates that MSCs can inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines.In the collagen-induced arthritis(CIA)mice model,alginate hydrogel encapsulated MSCs induce a significantly higher expression of CD39^(+)CD73^(+)on MSCs.These enzymes hydrolyze ATP to adenosine and activate A_(2A/2B)receptors on immature DCs,further promoting the phenotypic transformation of DCs to tol DCs and regulating naive T cells to Tregs.Therefore,encapsulated MSCs obviously alleviate the inflammatory response and prevent CIA progression.This finding clarifies the mechanism of MSCs-DCs crosstalk in eliciting the immunosuppression effect and provides insights into hydrogel-promoted stem cell therapy for autoimmune diseases.

Electroacupuncture stimulating Zusanli(ST36),Sanyinjiao(SP6)in mice with collagen-induced arthritis leads to adenosine A2A receptor-mediated alteration of p38αmitogen-activated protein kinase signaling and inhibition of osteoclastogenesis

OBJECTIVE:To observe the effect of electroacupuncture(EA)stimulating Zusanli(ST36),Sanyinjiao(SP6)on inhibition of osteoclastogenesis and the role of the adenosine A2A receptor(A2AR)and the p38αMitogen-Activated Protein Kinase(MAPK)signaling pathway in mediating this effect.METHODS:Mice with collagen induced arthritis(CIA)received different treatments.Immunohistochemistry and western blotting were used to determine the levels of multiple signaling molecules in these joints[receptor activator of nuclear transcription factor-κB(NF-κB)ligand(RANKL),receptor activator of NF-κB(RANK),tumor necrosis factor receptor associated factor 6(TRAF6),p38α,NF-κB,and nuclear factor of activated T cells C1(NFATc1)].Osteoclasts were identified using tartrate-resistant acid phosphatase(TRAP)staining.RESULTS:The immunohistochemistry results indicated upregulation of p38α,NF-κB,and NFATc1 in the CIA-control and CIA-EA-SCH58261 groups,but reduced levels in the CIA-EA group.Western blotting indicated upregulation of RANKL,RANK,TRAF6,p38α,NF-κB,and NFATc1 in the CIA-control and CIA-EA-SCH58261 groups,but reduced expression in the CIA-EA group.Osteoclasts were more abundant in the CIA-control and CIA-EA-SCH58261 groups than in the CIA-EA group.CONCLUSIONS:EA treatment enhanced the A2AR activity and inhibited osteoclast formation by inhibition of RANKL,RANK,TRAF6,p38α,NF-κB,and NFATc1.SCH58261 reversed the effect of EA.These results suggest that EA regulated p38α-MAPK signaling by increasing A2AR activity,which inhibited osteoclastogenesis.

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.

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.

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 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.