BACKGROUNDThere are few reports of a biological role for glycosyltransferases in the infiltration of osteoarthritic synovitis. The aim of this research was to investigate the expression and cellular location of β-1,4-galactosyltransferase I (β-1,4-GalT-I) in a surgically-induced rat model of knee osteoarthritis (OA), and explore the role of β-1,4-GalT-I in the pathogenesis of OA.

METHODSMale Sprague-Dawley rats were randomly divided into three groups: OA group, sham group and normal group. The model of OA was established in the right knees of rats by anterior cruciate ligament transaction (ACLT) with partial medial meniscectomy. Fibroblast-like synoviocytes (FLSs) obtained from normal rat synovial tissue were cultured. The expression of β-1,4-GalT-I mRNA in the synovial tissue, articular cartilage and FLSs treated with tumor necrosis factor-α (TNF-α) were assayed by real-time PCR. Western-blotting and immunohistochemisty were used to observe the expression of β-1,4-GalT-I at the protein level. Double immunofluorescent staining was used to define the location of the β-1,4-GalT-I with macrophage-like synoviocytes, FLSs, neutrophils, and TNF-α in the OA synovium. The alteration of TNF-α in FLSs which were treated with lipopolysaccharide (LPS) and β-1,4-GalT-I-Ab were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe mRNA and protein expression of β-1,4-GalT-I increased in synovial tissue of the OA group compared with the normal and sham groups at two and four weeks after the surgery, however, no significant difference appeared in the articular cartilage. Immunohistochemistry also indicated that the β-1,4-GalT-I expression in OA synovium at four weeks after surgery increased sharply compared with the control group. β-1,4-GalT-I co-localized with macrophage-like synoviocytes, FLSs, neutrophils and TNF-α in rat OA synovitis. Moreover, in vitro β-1,4-GalT-I mRNA in FLSs was affected in a dose- and time-dependent manner in response to TNF-α stimulation. ELISA revealed that the expression of TNF-α was attenuated in FLSs in vitro when treated with anti β-1,4-GalT-I antibody.

CONCLUSIONβ-1,4-GalT-I may play an important role in the inflammation process of rat OA synovial tissue which would provide the foundation for further researching into the concrete mechanism of β-1,4-GalT-I in OA synovitis.

Animals ; Blotting, Western ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Galactosyltransferases ; genetics ; metabolism ; Immunohistochemistry ; Knee Joint ; enzymology ; pathology ; surgery ; Male ; Osteoarthritis, Knee ; enzymology ; genetics ; pathology ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; enzymology ; Synovitis ; enzymology ; etiology

The aim of this study was to investigate the expression and localization of cyclooxygenase-1 and -2 (COX-1 and COX-2) in synovial tissues from patients with rheumatoid arthritis (RA). Synovial tissues from 9 patients with RA and 5 patients with osteoarthritis (OA) were examined for COX-1 and COX-2 expressions by immunohistochemical staining using 2 polydonal COX-1 and COX-2 antibodies. In RA synovia, synovial lining cells showed intense immunostaining for COX-1, whereas slight to moderate staining was observed in inflammatory cells, stromal fibroblast-like cells and vascular endothelial cells. There was no significant difference in COX-1 expression between RA and OA synovia. The localization of COX-2 expression dearly differed from that of COX-1 expression, being most intense in inflammatory cells. However, there was no difference in COX-1 and COX-2 expressions between RA and OA synovial tissues. Our observations support that inflammatory mechanisms modulated by COX-1 and COX-2 in chronic RA synovium might be similar to those in chronic OA synovium.
Adult ; Aged ; Arthritis, Rheumatoid/pathology ; Arthritis, Rheumatoid/enzymology* ; Cell Division ; Female ; Fibrin/metabolism ; Human ; Isoenzymes/metabolism ; Isoenzymes/biosynthesis* ; Male ; Middle Age ; Neutrophil Infiltration ; Osteoarthritis/enzymology ; Prostaglandin-Endoperoxide Synthase/metabolism ; Prostaglandin-Endoperoxide Synthase/biosynthesis* ; Stromal Cells/pathology ; Stromal Cells/enzymology ; Synovial Membrane/pathology ; Synovial Membrane/enzymology*

Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular-superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC-SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1beta, TNFalpha, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.
Animals ; Arthritis, Experimental/blood/*enzymology/metabolism ; *Arthritis, Rheumatoid/enzymology/pathology ; Fibroblasts/metabolism ; Gene Expression Regulation ; Inflammation/pathology ; Interleukin-1beta/blood/metabolism ; Joints/enzymology/pathology ; Matrix Metalloproteinases/blood/metabolism ; Mice ; Mice, Transgenic ; Reactive Oxygen Species/metabolism ; *Superoxide Dismutase/genetics/metabolism ; Synovial Fluid/*enzymology ; Synovial Membrane/pathology

OBJECTIVETo observe the influence of intra-articular injection of sodium hyaluronate (SH) on the expression of inducible nitric oxide synthase (iNOS) in the synovium and nitric oxide (NO) content in synovial fluid of rabbits with traumatic osteoarthritis (OA).

METHODSSixteen white rabbits underwent unilateral anterior cruciate ligament transection and were randomly divided into 2 groups 5 weeks after the operation. Rabbits in the experimental group received intra-articular injection of 0.3 ml of 1% SH, once a week for 5 weeks. Animals in the control group were treated under the same conditions using physiological saline. All the animals were sacrificed at the 10th week after surgery. The mRNA expression of iNOS in the synovium was analyzed using reverse transcription-polymerase chain reaction. The content of NO in the synovial fluid was assayed.

RESULTSThe level of iNOS expression of the synovium in the experimental group was lower than that in control group (0.47+/-0.09 vs. 0.65+/-0.12, t equal to 3.45, P less than 0.01). Compared with control group, the content of NO decreased significantly in synovial fluid of SH injection group (134.11 micromolar/L +/- 12.47 micromolar/L vs. 152.17 micromolar/L +/- 15.69 micromolar/L, t equal to 2.55, P less than 0.05).

CONCLUSIONSSH significantly decreases the content of NO in the synovial fluid of rabbits with traumatic OA. SH may exert the effect on synovial fluid NO level as a result of the suppression of iNOS expression in the synovium. It may be one of the mechanisms of the therapeutic effect of SH on early traumatic OA.

Animals ; Anterior Cruciate Ligament Injuries ; Hyaluronic Acid ; pharmacology ; therapeutic use ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type II ; analysis ; Osteoarthritis ; drug therapy ; etiology ; metabolism ; Rabbits ; Random Allocation ; Synovial Fluid ; chemistry ; Synovial Membrane ; enzymology

OBJECTIVETo 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.

METHODSThirty-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.

RESULTSArthritis 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.

CONCLUSIONSPADI4 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.

Animals ; Arthritis, Experimental ; enzymology ; metabolism ; Blotting, Western ; Collagen ; administration & dosage ; Female ; Hydrolases ; metabolism ; Immunohistochemistry ; Protein Tyrosine Phosphatase, Non-Receptor Type 22 ; metabolism ; Protein-Arginine Deiminases ; Rats ; Rats, Wistar ; Synovial Membrane ; enzymology ; metabolism

This paper is aimed to investigate the effect of titanium (Ti) particles and tumor necrosis factor alpha (TNF-alpha) on the expressions of MMP-1, 2, 3 in human synovial cells, so as to explore the possible mechanism of osteolysis post-operation of metal-on-metal total joint arthroplasty in human synovial cells induced by Ti particles. In vitro cell cultures, human synovial cells were treated by Ti particles and/or TNF-alpha. The total RNA was isolated at 2 hours after the treatment. The gene expression of MMP-1, 2, 3 was analyzed by Semi-quantitative Reverse-transcriptional PCR and quantitative real-time PCR. Cell supernatant was collected at 12, 24, 48 hours after the treatment and Gelatin zymography was performed to detect the activity of MMP-2. Compared to those in the control group (untreated), Ti particles and TNF-alpha increased the gene expression of MMP-1, 2, 3 respectively (P < 0.05), and the effect of combination of the two was even more significant (P < 0.01). The trend of activities of MMP-2 is similar with gene expression. Ti particles and TNF-alpha increased MMP-2 activities by 1.3 times and 1.5 times respectively (P < 0.05), and the combination of the two increased by 1.7 times (P < 0.01). Ti particles and TNF-alpha-induced the stimulation of MMP-1, 2, 3 expressions and MMP-2 activities in human knee joint synovial cells may be involved in aseptic loosening after metal-on-metal arthroplasty through increasing the degradation of bone matrix and declining of osseous support structure mechanics.
Cells, Cultured ; Humans ; Joint Prosthesis ; Knee Joint ; cytology ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 3 ; genetics ; metabolism ; Matrix Metalloproteinases ; genetics ; metabolism ; Particle Size ; Prosthesis Failure ; adverse effects ; RNA ; genetics ; metabolism ; Synovial Membrane ; cytology ; enzymology ; Titanium ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo observe the metabolic, regulatory and anti-oxidative effects of modified Bushen Huoxue Decoction (BSHXD), a Chinese herbal medicine for kidney (Shen)-reinforcement and blood-activation, on an osteoarthritis (OA) rabbit model.

METHODSA rabbit model for knee joint OA was established by the classic Hulth's method. The OA model rabbits were randomized into 5 groups: the model control group, the positive control group treated with glucosamine sulfate, and the three BSHXD treated groups treated respectively with low, moderate, and high doses of BSHXD. In addition, a normal control group and a sham-operated group were set up. Experimental animals were sacrificed after a 7-week treatment, and pathological changes in cartilaginous tissue were estimated using the Mankin criteria. Hydroxyproline (Hyp) and malonaldehyde (MDA) contents in blood serum and urine, as well as superoxide dismutase (SOD) activity and nitric oxide (NO) content in blood serum and knee joint synovial homogenates were detected.

RESULTSMankin scoring showed insignificant statistical differences between the various treatment groups (P >0.05), but all were better than the model control group (P <0.05). Serum and urinary contents of Hyp and MDA as well as serum and synovial levels of NO were significantly lower, but the SOD activity in blood serum and synovial tissue was higher in the BSHXD treated groups than in the model group P <0.01); the effect of BSHXD was dose-dependent to some extent.

CONCLUSIONThe modified BSHXD shows an effect of improving cartilage metabolism in experimental rabbits with OA, and possesses osteo-chondric protective effects in antagonizing peroxidation injury.

Animals ; Antioxidants ; pharmacology ; therapeutic use ; Cartilage, Articular ; drug effects ; pathology ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hydroxyproline ; blood ; urine ; Male ; Malondialdehyde ; metabolism ; Nitric Oxide ; blood ; Osteoarthritis ; blood ; drug therapy ; metabolism ; pathology ; Rabbits ; Superoxide Dismutase ; blood ; Synovial Membrane ; drug effects ; enzymology ; pathology

AIMTo study the effects of lipopolysaccharide (LPS), the supernatant of U937 cells stimulated with LPS and dexamethasone on matrix metalloproteinase-9 (MMP-9) expression in the synoviocyte from patients with rheumatoid arthritis(RA).

METHODSFibroblast-like cells (FLS) from the joint tissue of patients with rheumatoid arthritis were cultured and incubated for 24 h with LPS (1 mg.L-1) or the supernatant of U937 cells stimulated with LPS (1 mg.L-1) for 24 h. Dexamethasone was added to the supernatant of U937 cells and FLS was incubated for 24 h. The activity of MMP-9 was analyzed by gelatin zymography. Protein expression of MMP-9 was detected by Western blot using special polyclonal antibodies. The mRNA expression of MMP-9 was detected by RT-PCR.

RESULTSThe expression of MMP-9 was not markedly changed in FLS treated with LPS. The MMP-9 activity, MMP-9 secretion and MMP-9 mRNA expression were significantly increased in FLS cultured with the supernatant from U937 cell treated with LPS. Dexamethasone markedly inhibited the activity, protein secretion and mRNA expression of MMP-9 in FLS cultured with the supernatant from U937 cell stimulated with LPS, and the inhibitory effects were increased as the concentration of dexamethasone increased.

CONCLUSIONLPS did not directly affect the expression of MMP-9 in FLS, but it was found to indirectly cause the increase of MMP-9 expression in FLS by stimulating U937 cell. Dexamethasone was found to inhibit this increase of MMP-9 expression.

Anti-Inflammatory Agents ; pharmacology ; Arthritis, Rheumatoid ; pathology ; Cell Division ; drug effects ; Cells, Cultured ; Dexamethasone ; pharmacology ; Fibroblasts ; pathology ; Gene Expression ; drug effects ; Humans ; Lipopolysaccharides ; pharmacology ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; metabolism ; RNA, Messenger ; drug effects ; genetics ; Synovial Membrane ; drug effects ; enzymology ; pathology ; U937 Cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.
Anti-Inflammatory Agents/immunology/pharmacology ; Cell Line, Tumor ; Cyclic GMP/analogs & derivatives/immunology/metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors/metabolism ; Humans ; Interleukin-1beta/*metabolism ; Male ; Nitric Oxide/*biosynthesis/genetics/immunology ; Nitric Oxide Synthase Type II/*biosynthesis/genetics/immunology ; Phosphodiesterase Inhibitors/immunology/*pharmacology ; Piperazines/immunology/*pharmacology ; Purines/immunology/pharmacology ; Signal Transduction/drug effects/genetics/immunology ; Sulfones/immunology/*pharmacology ; Synovial Membrane/enzymology/immunology

OBJECTIVETo investigate the effect of Ermiao Recipe (, EMR) with medicinal guide Angelicae Pubescentis Radix (APR) on the homing of bone marrow stem cells (BMSCs) to focal zone in osteoarthritis (OA) rats.

METHODSForty-eight Sprague-Dawley rats were randomly assigned to the sham-operated, model, EMR, and EMR plus APR groups (12 rats in each group). The OA rat model was induced by anterior cruciate ligament transection and medial meniscus resection. All rats were injected with recombinant human granulocyte colonystimulating factor [rhG-CSF, 30 μg/(kg·d) for continuous 7 days], and rats in the EMR and EMR plus APR groups were treated with EMR or EMR plus APR at 1.6 or 1.9 g/(kg·d) for 3 or 6 weeks, respectively. Cartilage histopathologic changes were observed by hematoxylin and eosin staining. Chondrocytes apoptosis and cartilage matrix components were tested by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay and special staining. Interleukin-1β (IL-1 β), tumor necrosis factor α (TNF-α), bone morphogenetic protein 2 (BMP-2), and transforming growth factor beta-1 (TGF-β1) in serum were detected by enzyme-linked immunosorbent assay or radioimmunoassay assay. Matrix metalloproteinase (MMP)-13, tissue inhibitors of metalloproteinase (TIMP)-1, bromodeoxyuridine (BrdU), cluster of differentiation 34 (CD34), and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemistry assay.

RESULTSEMR and EMR plus APR significantly inhibited articular cartilage damage and synovium inflammation in OA rats at 3 or 6 weeks of treatment, the most obvious changes in these parameters were found in the EMR plus APR group. At 6 weeks, compared with EMR treatment, EMR plus APR remarkably inhibited chondrocytes apoptosis and the release of IL-1β and TNF-α, obviously decreased MMP-13 expression, and significantly increased expressions of proteoglycan, collagen, type II collagen and TIMP-1, serum levels of BMP-2 and TGF-β1 as well as expressions of BrdU, CD34 and SDF-1 in cartilage articular (P<0.01 or P<0.05).

CONCLUSIONThe medicinal guide APR improved the therapeutic effects of EMR on OA rats by promoting directional homing of BMSCs to focal zone.

Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; Bone Morphogenetic Protein 2 ; blood ; Bromodeoxyuridine ; metabolism ; Cartilage, Articular ; drug effects ; enzymology ; pathology ; Chemokine CXCL12 ; metabolism ; Chondrocytes ; drug effects ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Humans ; Interleukin-1beta ; blood ; Knee Joint ; drug effects ; pathology ; Male ; Matrix Metalloproteinase 13 ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; blood ; Osteoarthritis ; blood ; drug therapy ; pathology ; Rats, Sprague-Dawley ; Synovial Membrane ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transforming Growth Factor beta1 ; blood ; Tumor Necrosis Factor-alpha ; blood