Comparison of Three Experimental Models for Rat Osteoarthritis Induction

Background: Osteoarthritis is a slowly progressive and debilitating disease with high prevalence in adult population. Knee is one of the joints most affected by this disorder. There are several models for animals’ osteoarthritis induction, however it is not identified any paper that compares these techniques. The present study was aimed to define the most appropriate model for rats osteoarthritis induction. Material and Methods: 40 Wistar rats were distributed into 4 groups of 10 animals each: normality group (NG);meniscectomy group (MG);quinolone group (QG) and iodoacetate group (IG). Radiographic images of the rat’s knees were analyzed as well as the amount of chondrocytes in the epiphyseal and articular cartilage. Results: In the radiographic analysis, there was a low correlation between the raters. Regarding the amount of chondrocytes in the epiphyseal cartilage, it was noticed that the IG and QG groups had fewer chondrocytes than NG, in contrast to MG that reported similar results to normality (p > 0.05). There was no significant difference between IG and QG groups (p > 0.05). Regarding the amount of chondrocytes in articular cartilage, it was noticed that the IG group showed fewer chondrocytes than NG (p 0.05). There was no significant difference between QG and MG groups (p > 0.05). Conclusion: Intraarticular injection of iodoacetate in rats is the model with greatest effect on reduction of chondrocytes amount.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

The effect of triptolide （TP） on the expression of receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG） was explored in rat adjuvant induced arthritis （AA）. AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate （MTX） at the onset （day 9） of arthritis. On the peak of arthritis （day 24）, the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells （PBMC） was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group （.P〈0.01） ; The expression levels of RANKL in the synovium （P〈0.01） and bone （P〈0.05）, and OPG level in synovium （P〈0.05） were lower in TP group than in AA group （P〈0.05）. In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group （all P〈0.01）. It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

Effects of Moxibustion on the Paw Swelling and Pain Threshold in Rats with Experimental Arthritis

Objective： To observe and compare the effects of mild moxibustion and Chinese medicine on the paw edema and pain threshold in rats with experimental arthritis. Methods： Forty-six male Wistar rats were randomly divided into 5 groups, a control group （10 rats）, a model group （9 rats）, a Chinese medicine （CM） group （9 rats）, a moxibustion group （9 rats） and a Western medicine （WM） group （9 rats）. Except rats in the control group, rats in the other four groups were established to be arthritis model by cold stimulation and Freund＇s adjuvant. After modeling, rats in the control group and the model group were not treated. The remaining three groups were respectively treated with traditional Chinese medicine, moxibustion and Western medicine. Then the changes in the rat paw swelling and pain threshold were observed. Results： The paws of rats＇ swelled significantly after injection of adjuvant. After 7 d of treatment, the rats＇ paws of CM group were still markedly swollen, and compared with the CM group, the swelling of the paws in the WM group and the moxibustion group were statistically significant （P〈0.05）. The rats＇ mechanical pain threshold of the WM group, CM group and the moxibustion group were significantly higher than that of the model group with statistical significance （all P〈0.05）. In CM group, it was higher than in the WM group with statistical significance （P〈0.05）. That in moxibustion group was higher than that in the WM group without significance between the groups （P〉0.05）. Latent period of rat＇s paw withdrawal threshold of the thermal pain in western medicine group was significantly longer than that in the model group with statistically significant （P〈0.05）. But latent period of rat＇s paw withdrawal threshold of the thermal pain in the CM group and the moxibustion group extended without statistical significance （P〉0.05）. Conclusion： The mechanical pain threshold of rats with experimental arthritis can be significantly improved by mild moxibustion and Chinese medicine treatment, which have a certain analgesic effect on rats with experimental arthritis pain.

Influence of Electroacupuncture on Tumor Necrosis Factor-αand Vascular Endothelial Growth Factor in Rats with Experimental Arthritis

Objective： To observe the effect of electroacupuncture （EA） on tumor necrosis factor-α （TNF-α） and vascular endothelial growth factor （VEGF） in peripheral blood and joint fluid of collagen-induced arthritis （CIA） rats. Methods： After CIA rat models were successfully established, the rats were given corresponding intervention treatments. The levels of TNF-α and VEGF in peripheral blood and joint fluid were measured. Results： The levels of TNF-α and VEGF levels in peripheral blood and joint fluid of CIA rats were higher than those of normal group （P〈0.05）; EA treatment significantly decreased the index levels, of significant difference compared with those before treatment （P〈0.05）, as well as the model group （P〈0.05）. Conclusion： EA can significantly decrease the TNF-α and VEGF levels in peripheral blood and joint fluid of CIA rats, and helps restrain further development of the disease.

Effects of Xinfeng capsule on pulmonary function based on treg-mediated notch pathway in a rat model of adjuvant arthritis

OBJECTIVE:To observe the impact of xinfeng xapsule(XFC) on pulmonary function in a rat model of adjuvant arthritis(AA) and to investigate the mechanism of action.METHODS:Forty rats were randomly divided into four groups of ten:normal control(NC);model control(MC);tripterygium glycosides tablet(TPT);and xinfeng capsule(XFC).Except for the NC group,AA was induced in all rats by intracutaneous injection of 0.1 mL Freund's complete adjuvant in the right paw on the 19th day.NC and MC groups were given(0.9%) physiological saline.The TPT and XFC groups were given TPT(10 mg/kg) and XFC(1.2 g/kg),respectively.Thirty days after administration,changes in paw edema(E),the arthritis index(AI),pulmonary function,levels of regulatory T-cells(Treg),ultrastructure of lung tissue,and expression of Notch receptors and ligands in lung tissue were observed.RESULTS:In the MC group,E and the AI were increased and pulmonary function significantly decreased;the structure of alveolar type-II cells was damaged;ratios of Treg in peripheral blood were reduced;and expression of Notch receptors such as Notch3 and Notch4 and ligands such as Delta1 in lung tissue were significantly increased whereas expression of Notch1,Jagged1 and Jagged2 were significantly decreased.After intervention with XFC,E and the AI were decreased;pulmonary function was enhanced;the structure of alveolar type-II cells was improved;and expression of Treg,Notch1,Jagged1,Jagged2 was elevated,whereas that of Notch3,Notch4 and Delta1 was reduced.CONCLUSION:XFC can not only inhibit E and the AI and improve joint symptoms,it can also improve pulmonary function and reduce inflammation in lung tissue.These actions could be carried out through increases in the expression of Treg,Notch receptors(Notch1) and ligands(Jagged1,Jagged2),and reductions in the expression of Notch3,Notch4 and Delta1.These phenomena would reduce the deposition of immune complexes and the inflammatory response in lung tissue,thereby improving joint symptoms and pulmonary function.

Effect of Xinfeng capsule on nuclear factor kappa B/tumor necrosis factor alpha and transforming growth factor beta 1/Smads pathways in rats with cardiac injuries induced by adjuvant arthritis

OBJECTIVE: To investigate effects of Xinfeng capsule(XFC) on cardiac function in rats with adjuvant arthritis(AA) and explore the mechanism of these effects.METHODS: Forty-eight rats were randomly divided into normal control(NC), model control(MC), methotrexate(MTX) and XFC groups of equal size. In all groups except for the NC group, 0.1 m L Freund's complete adjuvant(FCA) was intracutaneously injected in the right rear vola pedis to induce inflammation. Drugs were applied beginning 19 days after induction of inflammation. Normal saline was administered to the NC and MC groups and 1 mg/100 g MTX(weekly) and 0.12 g/100 g XFC(daily) to the MTX and XFC groups, respectively. Rats were sacrificed after 30 day of treatment. Toe swelling degree(TSD), arthritis index(AI), cardiac function and expression of nuclear factor kappa B(NF-κB) / tumor necrosis factor alpha(TNF-α) and transforming growth factor beta 1(TGF-β1)/Smads pathway proteins were measured.RESULTS: In the MC group, TSD and AI were greatly increased, while parameters of cardiac function were decreased and morphological analysis showed myocardial cell damage. Expression of TNF-α, NF-κB, Smad2, P-Smad2, Smad4 and TGF-β1 proteins were elevated in cardiac tissue, while Smad7 expression was decreased.TSD and AI values closely correlated to parameters of cardiac function and to levels of proteins in the NF-κB/TNF-α and TGF-β1/Smads pathways. Certain correlations were identified among TGF-β1 and NF-κB, Smad2, P-Smad2 and Smad4.With XFC intervention, both TSD and AI were decreased and parameters of cardiac function and ultrastructure of myocardial cells improved.Expressions of NF-κB, Smad2, and Smad4 proteins were greatly decreased and Smad7 expression was elevated, as compared with levels in the MC and MTX groups.CONCLUSION: XFC regulates expression of proteins in the NF-κB/TNF-α and TGF-β1/Smads pathways, decreases immune complex deposition in cardiac tissue and improves cardiac function in AA rats via upregulation of Smad7.

Effect of electroacupuncture on c-IAP1 mRNA and C-IAP2 mRNA in synovial tissues of rats with adjuvant arthritis

Objective: Td observe the therapeutic effect of electroacupuncture (EA) at Zusanli (ST 36), Guanyuan (CV 4) and Ashi points on adjuvant arthritis rats, and explore the mechanism of EA treatment of rheumatoid arthritis (RA). Methods: Sixty male rats were randomly divided into a normal group, a model group, a methotrexate group and an EA group, with 15 rats in each group. Rats in the normal group and the model group were routinely raised and did not receive treatment;rats in the methotrexate group received methotrexate at a dose of 0.35 mg/(kg bw), twice a week for 3 weeks;rats in the EA group received acupuncture at Zusanli (ST 36), Guanyuan (CV 4) and Ashi points, and the bilateral Zusanli (ST 36) and Ashi points were connected to EA apparatus, once a day for 3 weeks. The general status, the swelling degree of the toe, the arthritis index (Al) score, the pathological morphology of the ankle joint, and the mRNA expressions of cellular inhibitor of apoptosis protein (c-lAP) 1 and C-IAP2 in joint synovial tissue cells of the rats in each group were observed. Results: The swelling degree of the toe, Al score and mRNA expressions of c-IAPl and C-IAP2 in the model group were significantly higher than those in the normal group (all P<0.05). Compared with the model group, the swelling degree of the toe, Al score and mRNA expressions of c-IAPl and C-IAP2 in the methotrexate group and the EA group improved (P<0.01 or P<0.05);the expressions of c-IAPl mRNA and C-IAP2 mRNA in rat synovial tissues in the EA group were significantly higher than those in the methotrexate group (P<0.01). Conclusion: EA alleviates joint swelling in rats with adjuvant arthritis. The mechanism may be related to suppress!ng mRNA expressions of c-IAPl and C-IAP2, thus to induce apoptosis of synoviocytes.

Total glucosides of paeony improve complete freund’s adjuvant-induced rheumatoid arthritis in rats by inhibiting toll-like receptor 2-mediated tumor necrosis factor receptor-associated factor 6/nuclear factor-kappa B pathway activation

OBJECTIVE: To investigate the mechanism underlying anti-inflammatory and immunoregulatory effect of total glucosides of paeony(TGP) based on toll-like receptor 2(TLR2) mediated tumor necrosis factor(TNF) receptor-associated factor 6(TRAF6)/nuclear factor-kappa B(NF-κB) pathway activation in rats with rheumatoid arthritis.METHODS: Adjuvant arthritis(AA) model was developed by complete freund’s adjuvant(CFA) immunization. TGP(100, 50, 25 mg/kg) and celecoxib(2.8 mg/kg) were administered by intragastric administration for 21 d. Right hind paw swelling was assessed every 2 d. After 21 d, synovial changes of the ankle were detected by histopathology. CD4+and CD8+ T cell amounts in peripheral blood were measured by flow-cytometrically. Gene and protein levels of toll-like receptor(TLR)2, TRAF6, tumor necrosis factor ligand superfamily member 6(FASLG)in the spleen were assessed by RT-qPCR and Western Bolt, respectively. Nuclear expression of NF-κB p65 was detected by NF-κB p65 Assay Kit.RESULTS: Paw swelling and synovium lesions were obviously aggravated in AA rats. These symptoms were significantly relieved by TGP.The ratio of CD4+/CD8+ T cell was increased in AA rats, while TGP reduced this increased ratio.Gene and protein levels of splenic TLR2, TFAR6 and FASLG, and nuclear NF-κB p65 in AA rats were significantly increased, but overtly inhibited by TGP.CONCLUSION: These findings suggest that TGP’s anti-inflammatory effect onRA in rats with CFA may be related to the downregulation of TLR2/TRAF6/NF-κB pathway and the regulation of T cell subsets.

Effects of Huatan Tongluo decoction on vascular endothelial growth factor receptor 2 expression in synovial tissues of rats with collagen-induced arthritis

OBJECTIVE: To determine the therapeutic effect and potential mechanism of Huatan Tongluo decoction on rats with collagen-induced arthritis.METHODS: Forty specific pathogen-free Wistar rats were selected, and 10 were randomly selected as the control(group 1). The remaining rats were injected intradermally with emulsified type Ⅱ bovine collagen at the tail base and back, followed by a booster 7 d post first immunization. After establishing collagen-induced arthritis(CIA), rats were randomly divided into three groups(n = 10). The rats were treated orally for 30 d as follows: group 1, saline; group 2, model(saline); group 3, tripterygium polyglycoside(TP; 7.81 mg/kg, positive control);group 4, Huatan Tongluo decoction(HTTL; 7.5 g/kg). Body weight, ankle swelling and arthritis index were measured over the course of the study. The rats were sacrificed 30 d after treatment. Morphological changes in the synovium were observed by hematoxylin and eosin staining. Pannus formation and synovial thickness in the left ankle were observed by color Doppler ultrasoundVascular endothelial growth factor(VEGF) and VEGFR2 protein levels were measured by immunohistochemistry.VEGF/VEGFR2 mRNA levels were measured by real-time quantitative polymerase chain reaction.RESULTS: Compared with the model group, a significantly lower arthritis index was observed in the positive control group(P < 0.05) and HTTL group(P < 0.01), after treatment. Both positive control and HTTL reduced intra-articular pannus formation and synovial thickening. Furthermore, VEGF mRNA,and VEGFR2 protein and mRNA levels were significantly downregulated(P < 0.05) in the treatment groups.CONCLUSION: Inhibition of the expression of VEGF and VEGFR2 in synovial tissues and the formation of pannus and synovial hyperplasia may be part of the mechanism of HTTL for relieving the symptoms of rheumatoid arthritis in CIA rats.

Study on the mechanism of moxibustion for rheumatoid arthritis based on liquid chromatography-mass spectrometry

Objective To explore the mechanism of moxibustion for rheumatoid arthritis(RA)by observing the metabolite changes in urine using liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis.Methods Twenty-four rats were randomly divided into a control group,a model group,and a moxibustion group.Rats in the model and moxibustion groups were established as collagen-induced arthritis(CIA)models.The control and model groups did not receive any intervention;rats in the moxibustion group received moxibustion at Shenshu(BL23)and Zusanli(ST36).After three weeks of intervention,ankle joint,serum,and urine samples were collected for pathological examinations and metabolomic tests.Results After moxibustion treatment,the CIA rats showed increased body mass,reduced swelling of the hind paws and arthritis score,decreased serum cytokine levels,and improved histopathological evaluation of the ankle joint.Twenty-four significantly altered metabolites were found,mainly involved in alanine metabolism,taurine and hypotaurine metabolism,tricarboxylic acid cycle,phenylalanine metabolism,tyrosine metabolism,and primary bile acid biosynthesis.These metabolites may serve as potential biomarkers for RA.Conclusion Moxibustion can effectively resist inflammation in CIA rats.The potential biomarkers and the abnormal metabolic pathways of RA can be identified by LC-MS-based metabolomics.Metabolomics may be an effective way to explain the mechanism of moxibustion in treating RA.