OBJECTIVETo show the involvement of lymphocyte-derived catecholamines in the pathogenesis of rheumatoid arthritis (RA), we investigated the change in expression of tyrosine hydroxylase (TH), a rate-limiting enzyme of catecholamine synthesis, by CD4+ T lymphocytes in lymphoid tissues of DBA/1 mice with collagen-induced arthritis (CIA).

METHODSCIA model was induced by chicken type II collagen in DBA/1 mice. The joints of the mice were observed for clinical score of swelling on and after the 22nd day of primary immunization. Pathological changes of ankles were examined by staining of tissue sections with hematoxylin and eosin on the 35th and 55th day following primary immunization. Immunofluorescent histochemistry was used to identify the number of TH-positive, CD4-positive, and double-labeled cells in the mesenteric lymph nodes and the spleen.

RESULTSPaw-swelling onset was on days 29 - 32 after the first immunization in DBA/1 mice. Clinical score for swelling of the paws reached peak on day 46 after the first immunization. Compared with the ankles of intact or vehicle mice, the joints of CIA mice had these characteristics: increased inflammatory cells in the synovial tissues, proliferated synoviocytes in the multilayers, narrowed articular space, and destructed articular cartilages. Simultaneously, the number of TH-positive, CD4-positive, and double-labeled cells in the mesenteric lymph nodes and the spleen was significantly increased on days 35 and 55 following the first immunization. Between day 35 and day 55 post-immunization, there was no significant difference in the number of these positive cells.

CONCLUSIONCD4+ T lymphocytes up-regulate TH expression in the process of CIA and therefore, it is suggested that endogenous catecholamines of lymphocytes involve in the pathogenesis of RA.

Animals ; Arthritis, Experimental ; metabolism ; Arthritis, Rheumatoid ; CD4-Positive T-Lymphocytes ; metabolism ; Lymphoid Tissue ; metabolism ; Male ; Mice ; Mice, Inbred DBA ; Tyrosine 3-Monooxygenase ; metabolism

AIMTo determine the exact roles of prolactin (PRL) in the pathogenesis of rheumatoid arthritis (RA) and supply experimental basis for clinical treatment of RA, and to investigate the expression of matrix metalloproteinase-9 (MMP-9) in the synovium of adjuvant arthritis rats.

METHODSForty rats were divided into four groups (n = 10): (1) Normal control group (group A); (2) Adjuvant arthritis control group (group B); (3) Hyperprolactinemic adjuvant arthritis group (group C); (4) Hypoprolactinemic adjuvant arthritis group (group D). The content of PRL in the serum was detected by radio-immunoassay method. The activity of MMP-9 was analyzed by gelatin zymography. The alteration of MMP-9 immunoreactivity were investigated by means of immunohistochemistry in the synovium of all groups. The expressions of MMP-9 were investigated by Western blot in the synovium of all groups.

RESULTSCompared with group A, the activity and expression of MMP-9 of group B in the synovium were highly increased. The activity and expression of MMP-9 in the synovium were the most distinctive in group C. Compared with group B, the activity and expression of MMP-9 in the synovium were decreased in group D, but still higher than group A.

CONCLUSIONThe present results indicated that PRL might involved in the pathogenesis of RA by regulating the secretion of MMP-9 in the synovium.

Animals ; Arthritis, Experimental ; metabolism ; Arthritis, Rheumatoid ; physiopathology ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Prolactin ; blood ; physiology ; Random Allocation ; Rats ; Rats, Wistar ; Synovial Membrane ; metabolism

OBJECTIVETo observe different effects of moxibustion on extracellular potassium ion in acupoint under physiological and pathological status and provide experimental evidence for exploring action mechanism of moxibustion on acupoint local.

METHODSForty female SD rats were randomly divided into a blank group, a blank-moxibustion group, a model group and a model-moxibustion group, 10 cases in each one. The complete Freund's adjuvant(CFA) was adopted to establish model of adjuvant arthritis (AA) in the model group and model-moxibustion group. No treatment was given in the blank group and model group while moxibustion was applied at "Zusan-li" (ST 36) for 30 min in the blank-moxibustion group and model-moxibustion group. The tissue fluid in "Zusanli" (ST 36) was collected with microdialysis and real-time analyzed by electrolytic analyzer. The change of concentration of potassium ion in "Zusanli" (ST 36) was observed.

RESULTS(1) Under physiological status, the concentration of extracellular potassium ion in the blank group was not changed within 150 min (P > 0.05); before the moxibustion, the concentration of extracellular potassium ion in the blank-moxibustion group was (1.21 +/- 0.31) mmol/L, and after treatment it was gradually increased and reached its peak at (2.38 +/- 0.42) mmol/L after 60 min (P < 0.05), then it was reduced. 150 min after the treatment, concentration of potassium ion was slightly higher than that before moxibustion as well as that in the blank group. The concentration in the blank-moxibustion group at 60 min was statistically significant compared with that in the blank group (P < 0.05). (2) Under pathological status, the concentration of extracellular potassium ion in the model group was not changed within 150 min, differences of which at each time point was not statistically significant (all P > 0.05). Before the moxibustion, the concentration of extracellular potassium ion was (1.09 +/- 0.12) mmol/L in the model-moxibustion group, and it was immediately increased to (1.96 +/- 0.18) mmol/L after moxibustion. 60 min and 90 min after the moxibustion, it still maintained a higher level, which was (1.87 +/- 0.29) mmol/L and (1.59 +/- 0.16) mmol/L respectively (both P < 0.05). The differences of each time point after moxibustion in the model-moxibustion group were statistically significant compared with those in the model group (all P < 0.05).

CONCLUSIONThe moxibustion could increase the concentration of potassium ion in rat's acupoint local under physiological status but time of effect is short; with moxibustion at "Zusanli" (ST 36) under pathological status, the concentration of local potassium ion is obviously increased and maintains for a long time.

Acupuncture Points ; Animals ; Arthritis, Experimental ; metabolism ; therapy ; Disease Models, Animal ; Female ; Humans ; Moxibustion ; Potassium ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study on the analgesic mechanism of electroacupuncture (EA) in the rat of adjuvant arthritis (AA).

METHODSForty-eight SD rats were randomly divided into a blank control group (n = 8), an inflammatory group (n = 10), an acupoint EA group (n = 10), a non-acupoint EA group (n = 10) and a contralateral acupoint EA group (n = 10). Complete Freund's adjuvant (CFA) 50 microL were injected into the left malleolus' articular cavity in the rats except the blank control group for preparing single local adjuvant arthritis (AA) model. EA was given every other day to the acupoint EA group, the non-acupoint EA group and the contralateral acupoint EA group. The improving effects of EA at "Huantiao" (GB 30) and "Yanglingquan" (GB 34) on the dorsal flexion pain score and the swell of dorsum of hind paw were investigated, and effects of EA on Glial cell line-derived neurotrophic factor (GDNF) positive cells immunoreactivity in the inflammatory tissue of the AA rats on the 14th day after injection of adjuvant were observed with immunohistochemical technique.

RESULTSHyperalgesia and local swell, and GDNF in the dermal and subcutaneous tissue around the inflammatory ankle joint in the inflammatory groups were significantly higher than those in the blank control group. EA on the ipsilateral and contralateral acupoints reduced the pain, promoted the recovery of swell, and decreased the positive area percentage and mean optical density of GDNF positive cells in the dermal and the subcutaneous tissues. However, the non-acupoint EA group did not have this action.

CONCLUSIONEA can regulate expression of GDNF in local dermal tissue of the inflammatory focus in the AA rat, so as to exert the anti-inflammatory and analgesic effects.

Animals ; Arthritis, Experimental ; metabolism ; therapy ; Electroacupuncture ; Glial Cell Line-Derived Neurotrophic Factor ; analysis ; Immunohistochemistry ; Rats ; Rats, Sprague-Dawley

BACKGROUND: Immune regulatory dendritic cells (DCs) play an important role in maintaining self-tolerance. Recent evidences demonstrate that DCs expressing indoleamine 2,3-dioxygenase (IDO), which is involved in tryptophan catabolism, play an important role in immunoregulation and tolerance and induce T cell apoptosis. This study was devised to examine the role of IDO in the oral tolerance induction in collagen-induced arthritis (CIA) mouse model. METHODS: Beginning 2 weeks before immunization, CII was fed six times to DBA/1 mice and the effect on arthritis was assessed. In tolerized mice, CD11c+ DCs were isolated and stimulated with CII, IFN-gamma, and LPS with or without IDO inhibitor, 1-methyl-DL-tryptophan (1-MT) and IDO expression by CD11c+ DCs was analyzed using FACS and RT-PCR. The expression of IDO, MHC II, CD80, and CD86 by CD11c+ DCs were examined using confocal microscopy. Regulatory effect of CD11c+ DCs on Ag-specific T cell proliferative response to CII was examined by mixed lymphocyte reaction (MLR) with or without 1-MT. RESULTS: The proportion of IDO-expressing CD11c+ DCs was slightly higher in tolerized mice than in CIA mice and significantly increased after stimulation with CII, IFN-gamma, and LPS in an IDO- dependent manner. On confocal microscopic examination, the expression of IDO was higher and those of MHC II and CD86 were lower in CD11c+ DCs from tolerized mice compared to those from CIA mice. On MLR, CD11c+ DCs from tolerized mice inhibited T cell proliferative response to CII in an IDO-dependent manner. CONCLUSION: Enhanced IDO expression by CD11c+ DCs from tolerized mice may contribute to the regulation of proliferative response of CII-reactive T cells and could be involved in the induction of oral tolerance to CII.
Animals* ; Apoptosis ; Arthritis ; Arthritis, Experimental ; Dendritic Cells* ; Immunization ; Indoleamine-Pyrrole 2,3,-Dioxygenase* ; Lymphocyte Culture Test, Mixed ; Metabolism ; Mice ; Microscopy, Confocal ; Models, Animal* ; T-Lymphocytes ; Tryptophan

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. It is known that aucubin (AU) exerts anti-inflammatory activity, but its effects and mechanisms in RA are unclear. This study investigated the anti-inflammatory effects and mechanisms of AU in vivo and in vitro. Human fibroblast-like synoviocyte cells from patients with RA (HFLS-RA), RAW264.7 cells, and MC3T3-E1 cells were used to evaluate the effects of AU on migration, invasion, apoptosis, osteoclast differentiation and production. Immunofluorescence was used to observe nuclear translocation of nuclear factor (NF)-κB, the double luciferase reporter gene method was used to observe NF-κB-p65 activity in AU-treated MC3T3-E1 cells. RT-qPCR was used to measure expression of bone metabolism and inflammation-related genes, and western blot was used to measure bone metabolism and NF-κB protein expression levels. Collagen-induced arthritis (CIA) rat model was used for pharmacodynamics study. Arthritis indexes were measured in the ankle and knee, histological staining and Micro-computed tomography were performed on the ankle joints. Also, inflammatory factor gene expression and the levels of NF-κB-related proteins were detected as in vitro. AU effectively inhibited HFLS-RA cell migration and invasion, promoted apoptosis, and inhibited RAW264.7 cell differentiation into osteoclasts, as well as inhibited NF-κB-p65 activity in MC3T3-E1 cells. Notably, AU significantly reduced the gene expression levels of three cell-related inflammatory factors and bone metabolism factors, effectively inhibited the expression of p-Iκκα β, p-IκBα, and p-p65 proteins. In vivo, AU relieved joint inflammation, reduced related inflammatory factors, and inhibited NF-κB signaling. It could be used to treat RA-related synovial inflammation and bone destruction through the NF-κB pathway.
Animals ; Anti-Inflammatory Agents/therapeutic use* ; Arthritis, Experimental ; Arthritis, Rheumatoid/drug therapy* ; Cells, Cultured ; Humans ; Inflammation/pathology* ; Iridoid Glucosides ; NF-kappa B/metabolism* ; Rats ; X-Ray Microtomography

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms. METHODS: Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of miR-124a, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA) and its downstream genes Bcl-2 and Bax. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group. RESULTS: Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all P<0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (P<0.05 or P<0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (P<0.01 or P<0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (P<0.01 or P<0.001), while the expressions of PIK3CA and Bcl-2 were decreased (P<0.05 or P<0.01 or P<0.001), and the ratio of Bcl-2 to Bax was significantly decreased (P<0.01 or P<0.001). CONCLUSIONS Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.
Animals ; Arthritis, Experimental/metabolism* ; Arthritis, Rheumatoid/genetics* ; Cattle ; Cell Proliferation ; Class I Phosphatidylinositol 3-Kinases/metabolism* ; Mice ; Mice, Inbred DBA ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Synovial Membrane ; bcl-2-Associated X Protein/metabolism*

Animals ; Arthritis, Experimental ; metabolism ; pathology ; Breast Neoplasms ; pathology ; Cadherins ; metabolism ; physiology ; Cell Movement ; Female ; Fibroblasts ; cytology ; pathology ; Humans ; Macrophages ; cytology ; pathology ; Neoplasm Invasiveness ; Synovial Membrane ; cytology ; metabolism ; pathology

OBJECTIVETo observe the effects of Xinfeng Capsule (XC) on the cardiac function and the myocardial ultrastructure in rats with adjuvant arthritis (AA).

METHODSSixty rats were randomly divided into the normal control group, the model group, the methotrexate (MTX) treated group, the Tripterygium Glycosides Tablet (TGT) treated group, and the XC treated group, 12 in each group. Except those in the normal control group, rats in the rest groups were induced to establish the AA model by intradermally injecting Freund's complete adjuvant into their right paws. The medication was started from the 19th day. Normal saline was administered to rats in the normal control group and the model group. MTX, TGT, and XC was respectively administered to rats in the MTX, TGT, and XC groups. The medication lasted for 30 days. The swelling degree of voix pedis, arthritis index (AI), the heart function, serum levels of cytokines, and the myocardial ultrastructure were observed.

RESULTS(1) Compared with the normal control group, the swelling degree of voix pedis and AI significantly increased (P < 0.01), the body weight significantly decreased (P < 0.05) in the model group and the other 3 treated groups. (2) Compared with the model group, the heart index (HI), left ventricular systolic pressure (LVSP), and left ventricular end diastolic pressure (LVEDP) significantly decreased (P < 0.05, P < 0.01), the maximum rate of left ventricular pressure of development or decline (+/- dp/dt(max)) significantly increased (P < 0.05, P < 0.01). Compared with the MTX treated group, the LVSP and LVEDP significantly decreased (P < 0.05), and +/- dp/dtmax significantly increased (P < 0.05). (3) Compared with the model group, TNF-alpha, brain natriuretic peptide (BNP), and IL-17 significantly decreased (P < 0.05, P < 0.01); IL-10, CD4+, CD4+ CDA25+ significantly increased (P < 0.05, P < 0.01). Compared with the MTX treated group, IL-17 significantly decreased (P < 0.05), CD4+ CD25+ expression significantly increased in the XC group (P < 0.05). (4) Transmission electron microscopy showed that myocardial ultrastructure was basically contact in the XC treated group, approaching to that of the normal control group.

CONCLUSIONSDecreased cardiac function and damaged myocardial ultrastructure existed in AA rats. XFC could ameliorate the swelling degree of voix pedis and AI, as well as improve the heart function. Its mechanisms might be correlated with down-regulating serum levels of inflammatory factors, up-regulating the expressions of anti-inflammation factors, thus improving the myocardial ultrastructure and protecting injured myocardial cells.

Animals ; Arthritis, Experimental ; drug therapy ; metabolism ; physiopathology ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Myocardium ; metabolism ; ultrastructure ; Phytotherapy ; Rats ; Rats, Wistar ; Tripterygium

OBJECTIVETo investigate the effects of intrathecal injection of ginsenoside Rg1 at different doses on the changes of the behavior and the expressions of excitatory amino-acid transporter 1 (EAAT1), i. e., glutamate-aspartate transporter (GLAST) in the spinal dorsal horn of the arthritis rats with chronic morphine tolerance, and further to explore its mechanisms for morphine tolerance.

METHODSAfter successful intrathecal injection, an adjuvant arthritis model was established in 36 healthy male SD rats. They were randomly divided into 6 groups, 6 in each group. They were intrathecally injected with 10 microL normal saline (Group NS), 10 microg morphine (Group M), 10 microg morphine + 50 microg ginsenoside Rg1 (Group MG50), 10 microg morphine +100 microg ginsenoside Rg1 (Group MG100), 10 microg morphine + 200 microg ginsenoside Rg1 (Group MG200), and 100 microg ginsenoside Rg1 (Group G100), respectively. The normal saline and morphine were intrathecally injected twice daily, while ginsenoside Rg1 at different doses was intrathecally injected once daily, for 7 successive days. Fifty percent mechanical paw withdrawal threshold (PWT) was dynamically detected to evaluate their behaviors. The rats were sacrificed on day 7 after medication. The L3-L5 segment of the spinal cord was isolated for determining the expression of GLAST in the spinal dorsal horn using immunofluorescence staining.

RESULTSThe PWT of Group M was significantly higher than that of Group NS on the 1st and 3rd day after medication (P < 0.05). But it was gradually shortened along with the increasing days of medication. There was no statistical difference between Group M and Group NS on the 7th day (P > 0.05), indicating the formation of morphine tolerance. The PWT of Group MG100 also showed a decreasing tendency, but obviously slower than that of Group M (P < 0.05). The PWT of Group G100 was higher than that of Group NS (P < 0.05). Compared with Group NS, the expression of GLAST in the spinal dorsal horn of rats in Group M was down-regulated (P < 0.01). Compared with Group M, the expression of GLAST in the spinal dorsal horn of rats in Group MG100 and Group G100 was up-regulated (P < 0.05).

CONCLUSIONSSingle application of ginsenoside Rg1 showed mild antinociceptive effect in adjuvant-induced arthritis rats. Intrathecal injection of 100 microg ginsenoside Rg1 could attenuate the formation of morphine tolerance. Its mechanisms might be correlated with up-regulating of the expression of GLAST.

Amino Acid Transport System X-AG ; metabolism ; Animals ; Arthritis, Experimental ; metabolism ; Drug Tolerance ; Ginsenosides ; administration & dosage ; pharmacology ; Injections, Spinal ; Male ; Morphine ; pharmacology ; Pain Measurement ; Rats ; Rats, Sprague-Dawley