Blood and synovial tissue metabolomics of rheumatoid arthritis rats treated with less polar ginsenosides / 中华风湿病学杂志

Objective:The therapeutic effect of less polar ginsenosides on rats with rheumatoid arthritis was studied, and the metabolic pathway that produce anti-inflammatory effect of less polar ginsenosides was identified.Methods:Rats were randomly divided into the control group, the model group, methotrexate treatment group, and high dose, medium dose, and low dose less polar ginsenosides groups. After 30 days of oral administration, less polar ginsenosides reduced the disease activity significantly in rats with rheumatoid arthritis. Blood and ankle synovial tissue metabolisms were measured by ultra performance liquid chromatography (UPLC) tandem mass spectrometry (MS) to explore the mechanism of less polar ginsenosides.The resulting data were subjected to principal component analysis and orthogonal partial least squares discriminant analysis(OPLS-DA).Results:Compared with the model group, erythrocyte sedimentation rate and RF decreased significantly in the high dose of less polar ginsenosides ( P<0.01). Metabolomics showed that R2X and R2Y of serum OPLS-DA were 0.626 and 0.904 respectively. The R2X and R2Y of synovial OPLS-DA were 0.429 and 0.689 respectively. Major differential metabolites were identified in the model group of rats, including arachidonic acid, valine, linoleic acid, and guanine nucleoside, etc. The main differential metabolites were identified in rats in the high dose group of less polar ginsenosides, including linoleic acid, betaine, eicosapentaenoic acid, alanine, methionine sulfoxide, isoleucine, etc. Conclusion:The metabolic spectrum has shown that inflammation is associated with linoleic acid metabolism, valine, leucine and isoleucine degradation, arachidonic acid metabolism. Less polar ginsenosides regulatethe linolenic acid metabolism, methionine metabolism and glucose alanine cycle.

78c can alleviate coliagen-induced arthritis and restore immune balance in rheumatoid arthritis / 中华风湿病学杂志

Objective:To explored the effect of 78c in treating collagen-induced arthritis (CIA) mice and to investigate its mechanism of effects.Methods:CIA mice model and CD38 +NK cells were treated with 78c. Cytokine concentrations and lymphocyte subtypes were measured in the mice peripheral blood and culture medium using flow cytometry. Mikenyi cell isolation kit was used to isolate CD4 + T cells and NK cells from peripheral blood mononuclear cells of healthy volunteers. CD38 + NK cells were enriched using the Miltenyi CD38 microbeads from the extracted NK cells. CD38 + NK cells with 78c pretreatment or not were cocultured with CD4 +T cells in transwells. The least significant difference (LSD) method was used for comparison between the two groups, and one-way analysis of variance was used for multi-group significance. Pearson correlation analysis was used for correlation analysis. Results:78c treatment significantly suppressed joint inflammation, inhibited the toe thickness of CIA mice, and reduced the number of while cell, neutrophils, platelets, and concentrations of IFN-γ, IL-6 and TNF-α ( t=6.10, P<0.001; t=4.00, P=0.002; t=3.09, P=0.012; t=2.31, P=0.043; t=3.58, P=0.005; t=2.68, P=0.002) in the CIA mice. The proportion of CD38 +NK cells decreased from (3.9±0.9)% to (2.4±0.3)% ( t=2.49, P=0.032), the proportion of regulatory T cell (Treg) increased from (0.81±0.33)% to (1.41±0.26)% ( t=2.74, P=0.021), and the concentration of IL-10 also increased from (99±37) pg/ml to (199±9) pg/ml( t=2.76 , P=0.020). The proportion of Treg in CD4 +T cells cocultured with 78c-pretreated CD38 +NK cells increased from (0.52±0.04)% to (0.69±0.08)% ( t=3.33, P=0.029) , the T helper cells (Th)17/Treg ratio decreased from (4.44±0.26) to (2.59±0.64) ( t=4.76 , P=0.009), and the Th1/Th2 ratio decreased from (14.8±1.6) to (8.1±1.3)( t=5.70 , P=0.005). Conclusion:78c can reduce the proportion of CD38 +NK cells, thereby reducing the inhibition of CD38 +NK cells on CD4 +T cell differentiation into Treg cells, leading to the restoration of immune balance. The results of this study suggest that 78c is a potential therapeutic agent for rheumatoid arthritis.

Study on the anti-inflammatory and analgesic pharmacodynamics of Zhonghua Dida Pill and its effect on inflammation in arthritis model rats / 国际中医中药杂志

Objective:To investigate the anti-inflammatory and analgesic effects of Zhonghua Dieda Pills; To preliminarily explore its mechanism on adjuvant arthritis model rats.Methods:Three inflammatory models and two pain models were used to investigate the anti-inflammatory and analgesic effects of Zhonghua Dieda Pills. After establishing the adjuvant arthritis rat model, the rats were divided into normal group, model group, dexamethasone group (0.8 mg/kg), and Zhonghua Dieda Pills (2.0, 1.0, 0.5 g/kg) groups according to random number table method. Each group was given corresponding drugs once a day for 5 weeks. The toe volume was measured at 1, 3 and 5 weeks after administration, and the swelling degree was calculated; the organ indices of rats were calculated and the histopathological changes of articular cartilage were observed by HE staining; the expressions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in joint tissues were detected by immunohistochemistry.Results:Zhonghua Dieda Pills (2.0 g/kg) group significantly reduced the swelling of foot and plantar of rats, reduced the swelling of ear of mice, and reduced the dry weight of granuloma of rats ( P<0.05); Zhonghua Dieda Pills (1.4 g/kg) group significantly reduced the number of twisting of rats, and the pain threshold after 3 h of administration was significantly higher than that of the control group ( P<0.05); Zhonghua Dieda Pills (2.0 g/kg) group significantly reduced the swelling of the foot and metatarsal of arthritic rats after 3-5 weeks of administration ( P<0.05), decreased the thymus index ( P<0.05), and reduced the expression levels of IL-1β, TNF-α and TGF-β in joint tissues ( P<0.05). Conclusion:Zhonghua Dieda Pills have confirmed anti-inflammatory and analgesic effects, which may play a therapeutic role in adjuvant arthritis model rats by reducing the levels of inflammatory factors such as IL-1β, TNF-α and TGF-β.

Efecto protector del aceite de Sacha inchi ( Plukenetía volubilis linneo ) en ratas Holtzman con inducción de artritis experimental / Protective effect of Sacha inchi oil ( Plukenetía volubilis linneo ) in Holtzman rats with induction of experimental arthritis

RESUMEN Objetivo. Demostrar cual es el resultado de la protección del aceite de Sacha inchi (SI) al realizar una inducción artificial de artritis al inyectar carragenina a ratas Holtzman. Métodos. Estudio cuantitativo, experimental y correlacional; se usaron 30 ratas macho: divididos en cinco grupos aleatorios : 1) Solución salina fisiológica (SSF) 2 mL/kg; 2) Carragenina (C) 0,1 mL solución 2% vía intraarticular, en la zona de la articulación del fémur con la tibia izquierda; 3) C y SI 250 mg/kg; 4) C y SI 1125 mg/kg; y 5) C y SI 2250 mg/ kg; determinándose tiempo (segundos), tipo de prensión (normal, pobre, regular, moderada, intensa), e inflamación pannus, fibrosis pannus, mediante estudio histopatológico. Se aplicó análisis de varianza, y los test de Tukey y Fisher. Resultados. Hubo mayor porcentaje de efecto antiinflamatorio dosis dependiente y tiempo de prensión a 2250 mg/Kg, seguido por 1125 mg/Kg. El estudio histopatológico mostró un pannus leve y fibrosis ausente con la dosis más alta; a dosis de 1125 mg/Kg de aceite SI hubo pannus moderado, y fibrosis leve. Conclusiones. Se demostró el resultado protector del aceite de Sacha Inchi (SI) aumentando y mejorando el tiempo tipo de prensión y reducción del pannus en la artritis inducida por carragenina en ratas Holtzman.

ABSTRACT Objective. To demonstrate the protective effect of Sacha inchi oil (SI) in arthritis induced by carrageenan in Holtzman rats. Methods. Quantitative, experimental and correlational study; 30 rats, males, randomly distributed in 5 groups were used: 1) SSF 2 mL/kg; 2) Carrageenan; 3) 4) and 5) Sacha inchi. Except for the control, they received 0.1 mL 2% carrageenan intra-articularly (the area of the femur joint with the left tibia); sacha inchi oil orally 225, 1125 and 2250 mg/kg correspondingly; determining time (seconds), type of grip (normal, poor, regular, moderate, intense), and pannus inflammation, pannus fibrosis by means of histopathological study. Applying analysis of variance, Tukey and Fisher test. Results. There was a higher percentage of dose-dependent anti-inflammatory effect and grasp time at 2250 mg/Kg, followed by 1125 mg/Kg; and the histopathological study showed mild pannus and absent fibrosis with the highest dose, in contrast to doses of 1125 mg/Kg of oil there was moderate pannus, and mild fibrosis. Conclusions. The protective effect of Sacha inchi oil (SI) has demonstrated by increasing the time and improving the type of grasp and reducing the pannus in arthritis induced by carrageenan in Holtzman rats.

Study on the mechanism of moxibustion for rheumatoid arthritis based on liquid chromatography-mass spectrometry / 针灸推拿医学（英文版）

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.

Establishment and ultrasonic evaluation of hemophilic arthritis animal models / 中国医学影像技术

Objective:To establish rabbit models of hemophilic arthritis (HA) through injecting blood and iron dextran into articular cavity, and to monitor the changes of joint structures and evaluate the effect of modeling with ultrasound. Methods: A total of 25 New Zealand white rabbits were divided into Group A, B (each n=5) and C (n=15). In group A and group B, 1 ml rabbit arterial blood was injected into the articular cavity with a total of 36 times (3 times a week) in group A and 24 times (twice a week) in group B. In group C, 1 ml of iron dextran was injected into the articular cavity of rabbits, and then the rabbits were averagely divided into 4 weeks group (C1 group), 8 weeks group (C2 group) and 12 weeks group (C3 group). The changes of synovium and cartilage of the articular cavity were observed with ultrasound. Pathological examination was performed after modeling, and the pathological changes of articular cartilage and synovium were observed. Results: After modeling, the synovium in group A ([4.46±0.47]mm) and C ([4.08±0.44]mm) measured with ultrasound were both thicker than in group B ([2.43±0.39]mm, both P<0.05). In group A and B, the thickness and the grade of blood flow signal of synovial membrane increased gradually during modeling. In group C, the thickness and blood flow signal of synovial membrane increased gradually in the early stage of modeling but gradually thinned or weakened. At the end of modeling, damage of articular cartilage could be observed with ultrasound in group A and C. Synovial hyperplasia, infiltration of inflammatory cells and destruction of cartilage similar to HA were observed in each group under light microscope. The pathological changes in groups A and C1 were more significant than those in other groups. Conclusion: Rabbit models of HA could be established through injecting blood or iron dextran into the articular cavity, both could reflect the characteristic manifestations of HA via symptoms, ultrasonic and pathological features. Iron induced arthritis models had advantages of short modeling period, simple operation and easy to be monitored with ultrasound.

Terapia fotodinâmica na artrite experimental induzida por Paracoccidioides brasiliensis / Photodynamic therapy in experimental arthritis induced by Paracoccidioides brasiliensis

OBJETIVOS: Avaliar a eficácia da terapia fotodinâmica com Brilliant Blue G no tratamento de um modelo experimental de artrite por Paracoccidioides brasiliensis (P. brasiliensis). MÉTODOS: Após a indução de artrite experimental com isolado de P. brasiliensis da linhagem Pb18 nos joelhos de ratos Wistar, os animais foram divididos em grupos e submetidos a terapia fotodinâmica com fotossensibilizador Brilliant Blue G intra-articular e a laserterapia apenas, sem o Brilliant Blue G. Todos os grupos receberam seus respectivos tratamentos do sétimo ao 11º dia. Para análise do edema foi mensurado o diâmetro latero-lateral do joelho de cada animal diariamente e após o período de tratamento os animais foram sacrificados para dissecação do joelho experimental e coleta de sangue para análise por ELISA, a fim de quantificar os níveis de anticorpos anti-P. brasiliensis. RESULTADOS: A aplicação da terapia fotodinâmica foi capaz de impedir a formação de edema quando comparado ao controle (p>0,005), bem como a produção de anticorpos anti-Gp-43 de P. brasiliensis (p=0,001). No exame anatomopatológico foi possível observar maior grau de sinovite e maior presença de granulomas com o fungo em seu interior no grupo que não recebeu tratamento quando comparado aos grupos que receberam a terapia fotodinâmica. CONCLUSÕES: A terapia fotodinâmica foi eficaz para atenuar a artrite experimental induzida por P. brasiliensis no modelo articular proposto.

AIMS: To evaluate the effectiveness of photodynamic therapy with Brilliant Blue G in the treatment of an experimental model of arthritis by Paracoccidioides brasiliensis (P. brasiliensis). METHODS: After the induction of experimental arthritis with isolated from P. brasiliensis of lineage Pb18 in the knees of Wistar rats, the animals were divided into groups and submitted to photodynamic therapy with intra-articular Brilliant Blue G photosensitizer and laser therapy only, without Brilliant Blue G. All groups received their respective treatments from the seventh to the 11th day. For edema analysis, the knee lateral-lateral diameter of each animal was measured daily and after the treatment period the animals were sacrificed for experimental knee dissection and blood collection for analysis by ELISA, in order to quantify levels of anti-P. brasiliensis antibodies. RESULTS: The results showed that the application of photodynamic therapy was able to prevent the formation of edema when compared to the control (p>0.005), as well as the production of anti-Gp-43 antibodies from P. brasiliensis (p=0.001). In the anatomopathological examination it was possible to observe a higher degree of synovitis and a greater presence of granulomas with the fungus inside the group that did not receive treatment when compared to the groups that received the photodynamic therapy. CONCLUSIONS: Photodynamic therapy was effective in attenuating the experimental arthritis induced by P. brasiliensis in the proposed joint model.

Study on pharmacodynamics of aconitine anti-adjuvant arthritis / 国际中医中药杂志

Objective To study the pharmacological function of the aconitine in treating adjuvant arthritis(AA).Methods Fifty rats were randomly divided into the control group, AA model group, methotrexate group(0.5 mg/kg), and aconitine groups of different dosages (25, 100μg/kg). Except the control group, each group was injection of intradermal Freund's complete adjuvant (0.1 ml) into right hindpaw of rats to establish adjuvant arthritis model. On the 10th day after model onset, the aconitine were administered by gavage with 25, 100μg/kg once daily, and the methotrexate group was administered with 0.5 mg/kg methotrexate twice per week, and all groups were treated for 19 days. After the last administration, the foot swelling was measured by toe volume meter, arthritis index was calculated by 5-grade scoring method, spleen and thymus index were calculated, and the pathological changes of right ankle joint were observed by HE staining.Results After the model establishment, compared with the model group, the degree of swelling of the ankle at 20 days (668.7 ± 144.5μl, 566.9 ± 179.3μl vs. 912.1 ± 200.5μl), 24 days (833.1 ± 144.0μl, 803.9 ± 213.4μlvs.1069.5 ± 164.6μl) and 28 days (736.4 ± 115.0μl, 835.7 ± 170.1μlvs. 1107.2 ± 215.8μl) in the aconitine groups significantly decreased (P<0.05 orP<0.01). After the model establishment, compared with the model group, arthritic index scores at 18 days (3.1 ± 0.7, 3.2 ± 0.4vs. 3.8 ± 0.6), 24 days (3.1 ± 0.5, 3.4 ± 0.5vs.3.9 ± 0.3), 28 days (2.7 ± 0.6, 3.2 ± 0.9 vs. 4.0 ± 0.0) in the aconitine groups significantly decreased (P<0.05). Compared with the model group, the spleen index (3.5 ± 0.4, 3.3 ± 0.4, 4.0 ± 0.6vs.4.9 ± 0.5) respectively in the low and high dose group of aconitine and methotrexate group (P<0.01).Conclusion Aconitine has a certain degree therapeutic effect on AA rats.

Effect of electroacupuncture on c-IAP1 mRNA and c-lAP2 mRNA in synovial tissues of rats with adjuvant arthritis / 针灸推拿医学（英文版）

Objective:To 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 (AI) score,the pathological morphology of the ankle joint,and the mRNA expressions of cellular inhibitor of apoptosis protein (c-IAP) 1 and c-IAP2 in joint synovial tissue cells of the rats in each group were observed.Results:The swelling degree of the toe,AI score and mRNA expressions of c-IAP1 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,AI score and mRNA expressions of c-IAP1 and c-IAP2 in the methotrexate group and the EA group improved (P＜0.01 or P＜0.05);the expressions of c-IAP1 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 suppressing mRNA expressions of c-IAP1 and c-IAP2,thus to induce apoptosis of synoviocytes.

Therapeutic and immunomodulatory effects of human umbilical cord -derived mesenchymal stem cells on adjuvant arthritis in rats / 中国基层医药

To investigate the therapeutic and immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells ( hUC-MSCs) on adjuvant arthritis (AA) rats.Methods Twenty -four male Sprague-Dawley( SD) rats were established with AA by complete Freund＇s adjuvant method.They were randomly divided into model group and hUC -MSCs group (2×106 cells／mL,5×106 cells／mL,tail vein injection),and the Yisaipu group (2.8mg／kg,subcutaneous injection ),6 rats in each group.Another 6 male SD rats were used as the control group.After the model was established ,the body weight and paw volume were recorded weekly ,the whole body score and the arthritis index score were calculated ,and the joint swelling number was calculated.The animals were sacrificed after d35,the weight of thymus and spleen were weighed ,and the corresponding index was calculated ,the histopathological changes of the ankle joint were observed by HE staining .The percentages of CD4 +CD44 +T cell and CD4 +CD62L+T cell were detected by flow cytometry.The levels of TNF-α,IL-1βin the serum of AA rats were detected by ELISA.Results hUC-MSCs relieved paw volume ,the whole body score and arthritis index score ,and the joint swelling number in AA rats (F＝20.573,89.092,14.161,10.914,all P＜0.01).hUC-MSCs reduced thymus index[(0.120 ±0.032),(0.120 ±0.031)] and spleen index[(0.250 ±0.070),(0.240 ±0.018)] ( F＝6.339,4.105,all P＜0.01),improved structural damage of ankle joint.hUC-MSCs could regulate the percentage of T cell subsets(CD4 +CD62L+)[(7.0 ±1.4)%,(7.9 ±2.2)%],( CD4 +CD44 +) [(15.0 ±3.6)%,(12.0 ± 1.9)%] in spleen (F＝6.331,12.719,all P＜0.01),and down -regulate the levels of TNF -α[(172.0 ± 13.0)ng／L,(150.0 ±12.0)ng／L] and IL-1β[(75.0 ±36.0)ng／L,(74.0 ±20.0)ng／L] in serum (F＝8.221, 3.581,all P＜0.05) of AA rats by tail vein injection.Conclusion hUC-MSCs can promote the treatment of AA rats by regulating the function of T cells.

Therapeutic and immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells on adjuvant arthritis in rats / 中国基层医药

Objective@#To investigate the therapeutic and immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on adjuvant arthritis(AA) rats.@*Methods@#Twenty-four male Sprague-Dawley(SD) rats were established with AA by complete Freund's adjuvant method.They were randomly divided into model group and hUC-MSCs group (2×106 cells/mL, 5×106 cells/mL, tail vein injection), and the Yisaipu group (2.8mg/kg, subcutaneous injection), 6 rats in each group.Another 6 male SD rats were used as the control group.After the model was established, the body weight and paw volume were recorded weekly, the whole body score and the arthritis index score were calculated, and the joint swelling number was calculated.The animals were sacrificed after d35, the weight of thymus and spleen were weighed, and the corresponding index was calculated, the histopathological changes of the ankle joint were observed by HE staining.The percentages of CD4+ CD44+ T cell and CD4+ CD62L+ T cell were detected by flow cytometry.The levels of TNF-α, IL-1β in the serum of AA rats were detected by ELISA.@*Results@#hUC-MSCs relieved paw volume, the whole body score and arthritis index score, and the joint swelling number in AA rats (F=20.573, 89.092, 14.161, 10.914, all P<0.01). hUC-MSCs reduced thymus index[(0.120±0.032), (0.120±0.031)] and spleen index[(0.250±0.070), (0.240±0.018)] (F=6.339, 4.105, all P<0.01), improved structural damage of ankle joint.hUC-MSCs could regulate the percentage of T cell subsets(CD4+ CD62L+ )[(7.0±1.4)%, (7.9±2.2)%], (CD4+ CD44+ )[(15.0±3.6)%, (12.0±1.9)%] in spleen (F=6.331, 12.719, all P<0.01), and down-regulate the levels of TNF-α [(172.0±13.0)ng/L, (150.0±12.0)ng/L] and IL-1β [(75.0±36.0)ng/L, (74.0±20.0)ng/L] in serum (F=8.221, 3.581, all P<0.05) of AA rats by tail vein injection.@*Conclusion@#hUC-MSCs can promote the treatment of AA rats by regulating the function of T cells.

Research on effect of the extract of Caulis Sinomenii on the expression of TNF-α, Cat-G and Cat-S in collagen induced arthritis rats / 国际中医中药杂志

Objective To explore the effect of sinomenine on the expression of tumor necrosis factor-α (TNF-α),cathepsin G (Cat-G) and cathepsin S (Cat-S) in rats with collagen induced arthritis (CIA).Methods The 60 SPF male Wistar rats were randomly divided into normal group,model group,high dose group,medium dose group,low dose group,and dexamethasone group (with 1 0 in each group).In the normal control group,the rats were given ordinary feed.For the other five groups,the rats were used to build a CIA model and give pharmacologic intervention in the following 20 days.After 20 days of inflammation,sinomenine would be divided into three different dose groups,with 120 mg/kg,90 mg/kg and 60 mg/kg,respectively,and each group was given a gavage daily.For the dexamethasone group,7.5 mg/kg dexamethasone was given for gavage once a day.In terms of the model group and normal group,the rats were perfused with the same volume of saline once daily.Then,taken the pictures of foot paw X-ray and foot paw pictures of rats in each group,measured the content of TNF-α,Cat-G,Cat-S in blood serum,observed the pathological changes in the synovial tissue of rats in each group by tissue section,measured the content of TNF-α,Cat-G,Cat-S in in spleen of rats by Immunohistochemical staining.Results The results of X-ray showed that there were obvious soft tissue swelling,joint deformity and osteolysis in the paws of the model group,and the above symptoms were alleviated in different degrees in each treatment group compared with the model group.Compared with the model group,the expression of TNF-α (376.48 ± 22.21 pg/ml,369.45 ± 82.68 pg/ml,425.17 ± 153.51 pg/ml vs.457.63 ± 152.67 pg/ml),Cat-G (1 398.05 ± 167.32 pg/ml,1 337.65 ± 209.34 pg/ml,1 412.78 ± 67.65 pg/ml vs.2 283.03 ± 185.21 pg/ml),and Cat-S (662.18 ± 169.66 pg/ml,406.80 ± 41.93 pg/ml,452.76 ± 50.49 pg/ml vs.838.11 ± 141.86 pg/ml) in blood serum of sinomenine high dose group,medium dose group and low dose group significantly decreased (P＜0.05).The expression of TNF-α (0.28 ± 0.05,0.21 ± 0.03,0.34 ± 0.04 vs.0.50 ± 0.04),Cat-G (0.28 ± 0.02,0.18 ± 0.06,0.27 ± 0.02 vs.0.37 ± 0.03),and Cat-S (0.22 ± 0.02,0.18 ± 0.03,0.27 ± 0.02 vs.0.35 ± 0.03) in spleen tissue significantly decreased (P＜0.05).The results of HE staining showed that the synovial tissue of normal rats was normal,the synovial tissue cells of model rats were damaged,the expression of inflammatory cells was significantly increased,and pannus hyperplasia was observed.Inflammatory cell infiltration and pannus hyperplasia were decreased in each group after administration.Conclusions Sinomenine has a sound and comprehensive intervention effect on rheumatoid arthritis,and the mechanism may be related to the inhibition of the expression ofTNF-α,Cat-G and Cat-S.

The role of B-cell scaffold protein with ankyrin repeats 1 in collagen-induced arthritis / 中华风湿病学杂志

Objective To analyze the roles of B-cell ccaffold protein with ankyrin repeats 1 (BANK1) in collagen-induced arthritis (CIA) murine model and the correlation with disease severity.Methods CIA murine model were established and evaluated.In different disease stages,the serum levels of anti-C Ⅱ auto-antibodies and BANK1 were detected by electrochemiluminescence immunoassay(ELISA).Moreover,the expression of BANK1 mRNA in peripheral blood cells were detected by real-time polymerase chain reaction (PCR) and its correlation with clinical scores was analyzed.Then the percentage of BANK1 expression in B cells in spleen and draining lymph nodes were detected by flow cytometry and the level of BANK1 protein in spleen was detected by Western blotting according to the results afore mentioned.The data was analyzed by Statistical Product and Service Solutions (SPSS) Stastistics 21.0 and figures were made with Graph Pad Prism 6.Repeated measure ANOVA was used to assess differences between the two groups.Correlations were analyzed by Spearman correlation analysis.Linear regression analysis was done when a correlation was identified.Results The incidence of CIA was over 90％.The clinical scores of arthritic mice was positively correlated with the serum levels of anti C Ⅱ total IgG antibody (r=0.717 5,P ＜0.01),anti C] IgG2a antibody (r=0.675 3,P＜0.01) and anti C Ⅱ IgG2b antibody (r=0.889 4,P＜0.01) respectively.The BANK1 level in the serum and the BANK1 mRNA expression were significantly decreased in different disease stages in CIA mice when compared with normal mice.The negative correlation between the BANK1 mRNA expression and clinical scores (r=-0.485 4,P＜0.01) was observed.The percentage of BANK1 +CD19+ cells in spleen and draining lymph nodes and the level of BANK1 protein in spleen were reduced in CIA as well.Conclusion Along with the disease progress in CIA,BNK1 expression is declined,which weakens the negative regulation of BANK1 on B cells.This change goes hand in hand with the severity of arthritis.

Study on the effect of Pin1 novel depressor all-transretinoic acid in collagen-induced arthritis mice / 中华风湿病学杂志

Objective To investigate the effect of Pin1 novel depressor all-transretinoic acid (ATRA) in collagen-induced arthritis (CIA) mice and elucidate the underlying mechanisms.Methods CIA model mice were established,then the qualified ones were treated with ATRA and other medicines respectively.Paw thickness and volume were measured once a week in each group.The production of tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) was detected by enzyme-linked immunosorbent assay (ELISA) in serum.Pathological changes of joints tissues were observed by hematoxylin and eosin (HE) staining.Western blot was used to detect the expression of Pin1 and nuclear factor-κB (NF-κB) between different groups.The measurement date were compared with single factor analysis of variance and independent sample t test.Results Compared with healthy control group,the paw thickness,volume and arthritis scores were significantly increased in CIA mice,compared with disease control group,joints swelling and arthritic score index were reduced in both treatment groups;TNF-α and IL-6 expression were significantly increased in CIA mice [(50.1±1.3) pg/ml,(67.6±8.3)) pg/ml] when compare with health control group [(47.1±0.6) pg/ml,(43.0± 5.2) pg/ml] (t=-9.0,P＜0.01;t=-6.9,P＜0.01),and both were decreased in treatment groups compare with disease control group (TNF-α,F=35.5,P＜0.05;IL-6,F=17.15,P＜0.01);HE staining study demonstrated that ATRA and Dexamethasone could significantly suppressthe pathological changes of joints tissues;Western blot assay demonstrated that the higher arthritis scores was,the more synovial pin1 expressed in mice,moreover ATRA and Dexamethasone decreased Pin1 and NF-κB expression in CIA mice synovial.Conclusion ATRA can successfully decrease inflammation scores and joints pathology damage in CIA mice via weaken the synovial Pin1 expression.

Grape seed proanthocyanidin extract ameliorates murine autoimmune arthritis through regulation of TLR4/MyD88/NF-κB signaling pathway

BACKGROUND/AIMS: Grape seed proanthocyanidin extract (GSPE) has been reported to have a beneficial effect on regulating inf lammation. However, the anti-inflammatory mechanism of GSPE remains unclear. The aim of this study was to verify the influence of GSPE on the Toll-like receptor 4 (TLR4)-mediated signaling pathway in the regulation of murine autoimmune arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in dilute brown non-agouti (DBA)/1J mice. The mice were treated with GSPE (0 or 100 mg/kg) intraperitoneally. The severity of arthritis was assessed clinically, biochemically, and histologically. Immunostaining for TLR4 was performed. The expressions of TLR4 and downstream signaling molecules were analyzed by Western blot. The effect of GSPE on lipopolysaccharide (LPS)-induced TLR4 activation was also evaluated using RAW264.7 cells and fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis and from those with osteoarthritis. RESULTS: GSPE attenuated the clinical severity of arthritis and decreased histological damage. GSPE treatment reduced the number of TLR4-stained cells in the synovium of mice with CIA. GSPE also downregulated the expression of TLR4, myeloid differentiation factor 88 (MyD88) and phosphorylated IκBα synovial protein in CIA mice. Concurrently, GSPE inhibited the nuclear translocation of nuclear factor-κB (NF-κB) subunits (p65 and p50). LPS-induced TLR4 activation was suppressed by GSPE in human FLS as well as in murine macrophages in vitro. CONCLUSIONS: Our results demonstrated that GSPE ameliorated CIA by regulating the TLR4-MyD88-NF-κB signaling pathway.

Short term histopathological effects of GaAlAs laser on experimentally induced TMJ osteoarthritis in rabbits

Abstract The aim of this study was to evaluate the biostimulation (BS) effect of the gallium-aluminum-arsenide (GaAlAs) diode laser by histopathology with an experimental osteoarthritis (OA) model in the temporomandibular joints (TMJ) of rabbits, in the early period. GaAlAs diode laser is used for pain reduction in TMJ disorders. Twenty-four adult male New Zealand white rabbits were randomly divided into three equal groups: Control Group (CG), Study Group 1 (SG-1), and Study Group 2 (SG-2). Mono-iodoacetate (MIA) was administered to the right TMJs of all rabbits. The rabbits did not undergo any treatment for four weeks to allow the development of osteoarthritis. In SG-1, laser BS was applied to the rabbits at 940 nm, 5 W, and 15 J/cm2 in continuous wave mode at 48-hour intervals for 14 sessions; and in SG-2, laser BS was applied with the same parameters at 24-hour intervals for 28 sessions. Laser BS was not applied to the rabbits in CG. All rabbits were sacrificed simultaneously. The TMJ cartilage, osteochondral junction, chondrocyte appearance, and subchondral ossification were evaluated histopathologically. There was no statistically significant difference between the groups in terms of cartilage, osteochondral junction, chondrocyte appearance, and subchondral ossification values (p > 0.05). The laser BS protocol used in the study had no positive histopathological effects on TMJ OA in the early period.

Intra-articular injection of ozone on serum receptor activator for nuclear factor-κB ligand and osteo-protegerin level in collagen-induced arthritis rats / 中华风湿病学杂志

Objective To evaluate the clinical efficacy of intra-articular injection of ozone in collagen-induced arthritis (CIA) rats and to assess its effects on serum receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) levels. Methods Forty weight age malched Wistar rats were randomly divided into normal control group (normal group), the CIA model group (CIA group), ozone (O 3 group), and methotrexate (MTX group). In addition to the normal control group, Freund's complete adjuvant and bovine type Ⅱ collagen were injected to establish the rat model of CIA. After the model was sucessfully developed, double ankle injection concentration ozone group of 40 μg/ml of O3 each 1 ml, 1 times a week, a total of injection for 3 weeks for the experimenal group. MTX group of 0.9 mg/kg was injected 1 times a week for 3 weeks for the MTX group. Degree of foot swelling was measured, and radiographic assessment of arthritis index (AI) score was performed. One week after treatment, angular vein blood was collected for the rats after the intervention, flow multi-factor detection technology was used to test each rat. T test or Wilcoxon rank test was used to compare the difference between groups. Results ① After 3 week administration with O3, dcgree of foot swelling, and AI of the O3 group was reduced significanly than the CIA group during the same period (foot swelling degree: O3 group: (4.21±0.14) ml, CIA group (9.12±0.17) ml, T=64.08, P=0.00; AI O3 group: [(2.97± 0.18) ml, CIA group: 5.76 ±0.13, T=37.24, P=0.00], and X-ray showed joint damage was alleviated. ② The serum level of RANKL in the CIA group was significantly higher than of the normal group [CIA model group 1890.70(797.03, 10571.94)], normal group [74.46(29.21, 95.37), T=43, P=0.005] during the same period; The serum level of RANKL in the O3 group was significantly lower than the CIA group [O3 group 28.09 (14.11, 207.30), CIA group 1890.70 (797.03, 10571.94), T=39, P0.05).③Serum RANKL/OPG of the CIA group was significantly higher than that of the normal group during this period, the difference was statistically significant [CIA group 250.68(42.33, 2959.78), normal group 4.32(3.16,5.30), T=36, P0.05). Conclusion Intra-articular injection of concentration of 40 μg/ml of O3 can reduce RA rat joint swelling degree, which may relate to the mechanism that O3 can lower levels of serum RANKL and RANKL/OPG ratio, reduce osteoclast formation and activation.

Metformin regulate the balance of Th17/regulatory T cells and expression of serum cytokines through AMPK-mTOR signaling pathway in collagen-induced arthritis / 中华风湿病学杂志

Objective To evaluate therapeutic effects of metformin on the expression of serum cytokines,balance of splenic Th17/regulatory T cells (Treg) and expression of splenic AMPK-mTOR in collagen-induced arthritis (CIA) rats,and the mechanism thereof.Methods The rat model of CIA was established by injecting bovine type Ⅱ collagen.Forty rats were randomly divided into five groups:the CIA model group(sterile water by gavage),Met-30 mg/kg group (metformin 30 mg ·kg-1 ·d-1 by gavage),Met100 mg/kg group(metformin 100 mg ·kg-1 ·d-1 by gavage),Met-300 mg/kg group(metformin 300 mg ·kg-1 ·d-1 by gavage) and control group (sterile water by gavage).The hind paw volume was recorded once a week.The serum level of cytokines,tumor necrosis factor (TNF)-α,interleukin (IL)-1β,IL-6,and IL-17,were measured by enzyme linked immunosorbent assay (ELISA) 35 days after the initial immunization.The quantity of Th17 and Treg cells were examined by flow cytometry.The expression of AMPK,p-AMPK,mTOR and p-mTOR were examined by western blotting.One-way analysis of variance was used to evaluate the experimental data.Results The values of hind paw volume were decreased on the 35 d of the initial immunization in the Met100 mg/kg [(2.43±0.37) ml,t=2.97,P＜0.05] and Met-300 mg/kg groups [(2.58±0.21) ml,t=2.96,P＜0.05] than those of CIA model group (2.97±0.23) ml.The serum levels of TNF-α,IL-1β,IL-6 and IL-17 on the 35-d after the initial immunization were significantly lower in the metformin therapeutic groups [Met-300 mg/kg group TNF-α (104±8) pg/ml,t=42.77,P＜0.05;IL-1β (183±24) pg/ml,t=60.457,P＜0.05;IL-6 (19.3±2.8) pg/ml,t=53.62,P＜0.05;IL-17 (64.5±6.7) pg/ml,t=47.92,P＜0.05] than those of the CIA model group [TNF-α (246±8) pg/ml;IL-1β (1 336±40) pg/ml;IL-6 (87.0±5.1) pg/ml;IL-17 (282.3±6.8) pg/ml].The quantity of Th17 and Treg cells were significantly different in the Met-300 mg/kg group than those of the CIA model group 35 d after the initial immunization [Th17(6.57±0.39) vs (9.89±0.53),t=8.74,P＜0.05;Treg (7.60±0.45) vs (3.94±0.61),t =8.37,P＜0.05].The expression of p-AMPK on the 35 d after the initial immunization in the metformin therapeutic groups was significantly higher than in the CIA model group(P＜0.05),and the expression of p-mTOR was significantly lower (P＜0.05).Conclusion Metformin can significantly inhibit the serum levels of TNF-α,IL-1β,IL-6 and IL-17 in CIA model rats,and regulate the balance of Th17/Treg through AMPK-mTOR signaling pathway.

Study on the balance rebuilding of regulatory T cells/Th17 cells by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin for the treatment of rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the regulatory T cells (Treg) induced by 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin (TCDD) on the aryl hydrocarbon receptor activator from na?ve T cells, in collagen-induced arthritis (CIA)-rheumatoid arthritis (RA) mouse in the correction of rheumatoid arthritis pathological process on Th17/Treg cell imbalance. Methods ① Rat CIA model was established by bovine Ⅱ collagen injection. The rats were divided into 3 groups: the normal group (N), the CIA model group (C), and the TCDD group (T).② The percentage of Th17 and Treg from peripheral blood mononuclear cells (PBMCs) were analyzed with flow cytometry (FCM). Th17 and Treg cells related cytokine including interleukin (IL)-1β, IL-6, IL-17A, IL-23, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β were measured with enzyme linked immunosorbent assay (ELISA). ③ The changes of the arthritis parameters, and radiological of T/NT before and after treatment were observed. ④ After two weeks of treatment, the rats were killed. The swollen joints were used for pathological assessment and arthritic pathology injury score was evaluated. ⑤ All data were analyzed by one-way analysis of variance (ANOVA) test and t test. Results In group C:When the second immunization was compared with the first, the percentage of Th17 cells was significantly increased [(2.99 ±0.16)%, (4.02 ± 0.33)%, t=-6.82, P<0.01], while Treg cells were significantly reduced [(2.67 ±0.57)%, (1.79 ±0.39)%, t=3.11, P=0.000], Th17/Treg was significantly increased (1.14 ±0.14, 2.27 ±0.39, t=-4.53, P=0.039). In group T, the percentage of Th17 cells were significantly reduced [(2.69±0.24)%, (1.21±0.25)%, t=5.12, P<0.01] before and after treatment, while Treg cells were significantly increased [(2.76 ±0.36)%, (3.78 ±0.22)%, t=-6.24, P<0.01], Th17/Treg was significantly reduced (1.08±0.07, 0.21±0.05, t=-11.92, P=0.027). In group C:When the second immunization was compared with the first, the Th17 cells related cytokines were significantly increased inclu-ding IL-1 [(96±12) pg/ml, (153±18) pg/ml, t=-13.24, P<0.01], IL-6 [(246±14) pg/ml, (295±15) pg/ml, t=-9.41, P=0.000], IL-17 [(76 ±14) pg/ml, (99 ±16) pg/ml, t=-5.78, P<0.01], IL-23 [(85 ±9) pg/ml, (102 ±11 ( pg/ml, t=-11.71, P<0.01], TNF-α [(303 ±12) pg/ml, (345 ±17) pg/ml, t=-15.56, P=0.007], while Treg cells related cytokines TGF-β was significantly reduced [(42 ±7) ng/ml, (35 ±8) ng/ml, t=7.52, P=0.012]. Th17 cells related cytokines were significantly reduced including IL-1 [(93 ±10) pg/ml, (79 ±8) pg/ml, t=10.52, P<0.01], IL-6 [(245 ±11) pg/ml, (151 ±8) pg/ml, t=19.23, P<0.01], IL-17 [(76 ±10) pg/ml, (62 ±9) pg/ml, t=7.37, P=0.005], IL-23 [(84 ±11) pg/ml, (38 ±6) pg/ml, t=14.48, P=0.000], TNF-α [(294 ±8) ng/ml, (195 ±9) ng/ml, t=23.35, P<0.01], while Treg cells related cytokine TGF-β was significantly increased (41 ±8, 61 ±10, t=-10.61, P=0.000. Conclusion TCDD can increase Treg cells, reduce Th17 cells, so the Th17/Treg cell in RA patho-genesis could be re-balanced.

Magnetic resonance imaging of macrophages for the diagnosis of rat collagen-induced arthritis: a preliminary study / 中华风湿病学杂志

Objective To explore the optimal time and sequence for getting the best magnetic resonance (MR) imaging when MR image of synovium macrophages was used for the diagnosis of collageninduced arthritis (CIA) in a rat model,and whether it can be used to monitor the efficacy of drug treatment.Methods CIA was induced by subcutaneous injection of chicken type Ⅱ collagen and complete Freund's adjuvant (CFA).Arthritis rats were randomly divided into the model group,the leflunomide group and the control group.Knees of the model group rats were imaged before and 24 h,48 h,72 h after USPIO intravenous administration (300 μmol Fe/kg) on day 28,29,30,31,respectively.From day 28,the leflunomide group was given a gauge of drug at a dose of 8 mg/kg.Then they were imaged before and 24 hours after USPIO administration on day 42,43 respectively.MR sequences included SE T1WI,SE T2WI,GRE T2 * WI.After the completion of MR imaging,rats were sacrificed to obtain histopathologic samples of synovial membrane.LSD-t test and one-way analysis of variance (ANOVA) were used for statistical analysis.Results No distinct signal enhancements were observed on the 24 h,48 h,72 h post-contrast enhancement on T1WI.On T2WI,signal intensity ratio of synovium (SNR) pre-contrast and 24 h,48 h post-contrast were 24.13±1.96,17.09± 1.23,19.14±0.91,respectively.On T2 * WI,SNR pre-contrast and 24 h,48 h post-contrast were 22.28±0.92,11.40±0.53,17.18±0.63,respectively.Distinct signal changes were observed on 24 h,48 h post-contrast on T2WI and T2 * WI (P＜0.05).The changes between SNR at 24 h,48 h,72 h post-contrast and pre-contrast were-29.09±2.42,-20.83±2.90,-6.2±2.9 respectively on T2WI,which were-48.4±1.3,-22.9±0.8,-8.2±1.6 respectively on T2 * WI.Changes were more obvious at 24 h post-contrast than 48 h post-contrast on both T2WI and T2 * WI (P＜0.05).The quantitative analyses were coinci-dent with the visual differences in signal changes between pre-contrast and post-contrast images.Difference between △SNR of leflunomide group and the control group on T1WI was not significant,while that on T2WI and T2 * WI were significantly different (P＜ 0.01).Histological examination confirmed the uptake of iron in the macrophages of arthritic knees.Signal intensity changed more on GRE T2 * WI than SE T2WI in all arthritis rats.Conclusion GRE T2 * WI is more sensitive for the diagnose of rat CIA,and 24 h post-contrast is better than 48 h and 72h post-contrast to get better images.We successfully observed the effects of leflunomide through signal changes of synovium,and the technique maybe contribute to diagnosis and therapeutic monito-ring of rheumatoid arthritis.