Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

OBJECTIVETo investigate the effect of Flavonoids extracted from Echinps latifolius Tausch(FELT) on rheumatoid arthritis (RA) in rat model.

METHODFifty SD rats were randomly divided into model group, control group, and low, medium, and high-dose FELT groups (n=10 in each group). Complete Freund's adjuvant (0.1 mL) was used to induce RA in rats. FELT in doses of 50 mg/kg, 100 mg/kg, 150 mg/kg was given to rats in low, medium and high-dose FELT groups by gavage, and same volume of PBS was given to rats in control group. The arthritis score and the paw swelling score were measured to evaluate the therapeutic effect of FELT. Real time qPCR was used to detect the mRNA expression of fibronectin and MMP3 in synovial tissue and the mRNA expression of caspase 3, Bcl-2 and Bcl-2 associated X protein (Bax) in fibroblast-like synoviocytes (FLS).

RESULTSThe arthritis score and the paw swelling score were significantly decreased in three FELT groups compared to RA model rats (P <0.05). The relative expression levels of FN and MMP3 mRNA in synovium of three FELT-treatment groups were significantly lower than those in model group (1.80, 1.76 and 1.67 vs 2.53; 1.69, 1.46 and 1.45 vs 2.67, respectively, all P <0.05). The relative expression levels of Bax and caspase 3 mRNA in FLSs of three FELT groups were higher than those in model group (0.56, 0.58 and 0.60 vs 0.30; 0.54, 0.56 and 0.59 vs 0.29, respectively, all P <0.05); while the relative expression levels of Bcl-2 mRNA in FELT groups were lower than that in model group (2.20, 2.08 and 2.08 vs 4.04, respectively, P <0.05).

CONCLUSIONFELT may inhibit the synovium proliferation in RA model rats through promoting the FLS apoptosis.

Animals ; Apoptosis ; Arthritis, Rheumatoid ; drug therapy ; Caspase 3 ; metabolism ; Disease Models, Animal ; Echinops Plant ; chemistry ; Fibroblasts ; metabolism ; Flavonoids ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; bcl-2-Associated X Protein ; metabolism

Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.
Animals ; Arthritis ; Arthritis, Rheumatoid ; metabolism ; prevention & control ; Benzylisoquinolines ; pharmacology ; therapeutic use ; Cell Movement ; drug effects ; Cell Proliferation ; Cells, Cultured ; Disease Models, Animal ; Down-Regulation ; Fibroblasts ; drug effects ; metabolism ; Humans ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plant Roots ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; Stephania ; chemistry ; Synovial Membrane ; cytology ; drug effects ; metabolism ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVE: To explore the eff ect of pulchinenoside (PULC) on fi broblast-like synoviocytes (FLS) apoptosis in adjuvant arthritis (AA) rats. METHODS: A total of 60 SD rats were randomly divided into 8 groups: A normal control group, an AA group, a low PULC group (50 mg/kg), a middle PULC group (100 mg/kg) or a high PULC group (150 mg/kg) and an ibuprofen (8 mg/kg) group (n=10 per group). FLS from the AA rats was cultured. The expression of Bcl-2, Bax, caspase-3 and the FLS proliferation were detected by the real time qPCR and MTT, respectively. The expression of IL-6 and IL-8 in culture medium was detected by ELISA. RESULTS: Compared with the AA group, the Bcl-2 expression was down-regulated (all P<0.05), the Bax and caspase-3 expression was up-regulated (all P<0.05), and the FLS proliferation was inhibited (all P<0.05). The IL-6 and IL-8 expression was suppressed in the FLS in the PULC groups at different dosages (all P<0.05) as well as in the ibuprofen group (P<0.05). CONCLUSION PULC may inhibit the FLS proliferation in AA rats by increase in FLS apoptosis.
Animals ; Apoptosis ; drug effects ; Arthritis, Experimental ; Caspase 3 ; metabolism ; Fibroblasts ; cytology ; drug effects ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pulsatilla ; chemistry ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the role of natural killer-22 (NK-22) cells in the synovial fluid in the proliferation of fibroblast-like synoviocytes (FLS) in patients with rheumatoid arthritis (RA) and explore the possible signal pathway involved.

METHODSNK-22 cells in the SF of RA patients were sorted by flow cytometry. NK-22 cells were cultured for two weeks and the purity was detected by flow cytometry before stimulation with 20 ng/ml phorbol 12-myristate 13-acetate and 0.5 µmol/L ionomycin for 4 h. The level of interleukin-22 (IL-22) in the culture medium supernatant was then measured with ELISA. The proliferation of FLS in the presence of the culture supernatant of NK-22 cells was assessed with MTT assay at 24, 48 and 72 h, and the effect of IL-22 antibody on FLS proliferation was also observed. Real-time PCR and Western blotting were used to detect Stat3 mRNA and p-Stat3 protein levels, respectively, in the FLS exposed to rhIL-22 and AG490.

RESULTSNK-22 cells were successfully sorted by flow cytometry with a purity exceeding 90%. The levels of IL-22 in the supernatant of NKp44(+)NK cell culture averaged 1273.42∓254.48 pg/ml. The FLS proliferated rapidly 24, 48, and 72 h after the addition of culture supernatant of NK-22 cells (P<0.05). IL-22 antibody obviously inhibited the proliferation of FLS induced by NK-22 cell culture supernatant (P<0.05). Exposure of the FLS to rhIL-22 obviously increased cellular Stat3 expression levels, which were significantly lowered by the addition of AG490 (P<0.05).

CONCLUSIONNK-22 cells in the SF of RA patients can produce high concentrations of IL-22 to promote the proliferation of FLS through the STAT3 signal pathway.

Arthritis, Rheumatoid ; metabolism ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; Humans ; Interleukins ; metabolism ; Killer Cells, Natural ; cytology ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Synovial Fluid ; cytology ; Synovial Membrane ; cytology

The role of pulchinenoside (PULC) in the regulation of MeCP2 expression was investigated in RA model rats. Adjuvant arthritis rats were used as RA model rats, and fibroblast-like synoviocytes (FLS) from the RA model rats were cultured. The effect of 100 mg x kg(-1) PULC gavage treatment on the MeCP2 expression and the effect of MeCP2 siRNA on the expression of SFRP2 and β-catenin were detected by real time qPCR and Western blotting. The role of PULC in the FLS proliferation was detected by MTT. The results showed that the MeCP2 expression was down-regulated, the SFRP2 expression was up-regulated and the FLS proliferation was inhibited in FLS after therapy. MeCP2 siRNA significantly inhibited the MeCP2 expression, up-regulated the SFRP2 expression and inhibited the β-catenin expression in FLS from RA model rats. PULC may increase the SFRP2 expression, inhibit the Wnt signaling and inhibit the FLS proliferation in FLS from the RA model rats by inhibiting the MeCP2 expression.
Animals ; Arthritis, Rheumatoid ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Male ; Methyl-CpG-Binding Protein 2 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; genetics ; 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

The progress of research on the the anterior cruciate ligament (ACL) wound healing demonstrates that the synovial tissue in the knee joint plays a very important role in the healing process of injured ACL. Therefore, the molecular response mechanisms of lysyl oxidase (LOX) and matrix metalloproteina (MMP) in normal/injured ACL fibroblast cells could be considered to perform the major analysis function of injured ACL healing mechanism. The mRNA expressions of LOXs and MMPs and the activity expressions of MMP-2 in ACL fibroblasts co-cultured with synovial cells were analyzed by quantitative real-time PCR and zymography. The results showed that co-culture could regulate the mRNA expressions of LOXs and MMPs in the ACL fibroblasts cells. These results suggest that the differential expressions of LOXs and MMP-1, 2, 3 in co-cultured ACL indicate that interaction crosstalk do exist between ACL cells and synovial cells and provide a theoretical basis for subsequent exploration of the mechanisms and treatment of ACL injury and repair.
Anterior Cruciate Ligament ; cytology ; Anterior Cruciate Ligament Injuries ; Coculture Techniques ; Fibroblasts ; cytology ; metabolism ; Humans ; Knee Injuries ; physiopathology ; Knee Joint ; cytology ; Matrix Metalloproteinases ; genetics ; metabolism ; Protein-Lysine 6-Oxidase ; genetics ; metabolism ; Synovial Membrane ; cytology ; Wound Healing ; physiology

Arthritis, Rheumatoid ; metabolism ; Cells, Cultured ; HSP72 Heat-Shock Proteins ; pharmacology ; Humans ; Interleukin-10 ; metabolism ; Synovial Membrane ; cytology ; drug effects ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo study the effect of intracellular reactive oxygen species (ROS) levels on T cell activation and apoptosis of synovial cells in collagen induced arthritis (CIA) rats, and to explore the mechanism of Fengshining Capsule (FSN) in the treatment of rheumatoid arthritis (RA).

METHODSSixty rats were randomly divided into the normal control group, the CIA model group, the Tripterygium Poly-glycoside Tablet (TPT) group, the low dose FSN group (at the daily dose of 0.33 g/kg), the middle dose FSN group (at the daily dose of 0.66 g/kg), and the high dose FSN group (at the daily dose of 1.32 g/kg), 10 in each group. T lymphocyte subsets were detected by flow cytometry. The content of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in plasma of rats were detected by ELISA. Its expression of hydroxyl radicals was detected by ultraviolet spectrophotometry. Caspase-3 and Caspase-9 protein expressions were measured by Western blot.

RESULTSCompared with the CIA model group, the levels of ROS were elevated in each dose FSN group (P < 0.01). The level of CD4+ / CD8 was significantly reduced in the middle dose FSN group (P < 0.01). The content of IFN-gamma was obviously lowered in each dose FSN group (P < 0.01), while that of IL-4 was obviously elevated in the high dose FSN group (P < 0.01). Meanwhile, the expression of Caspase-9 and Caspase-3 significantly increased in each dose FSN group (P < 0.05). Besides, the average gray scale of Caspase-9 was significantly higher in the low and middle FSN groups than in the TPT group (P < 0.05, P < 0.01).

CONCLUSIONThe mechanism of FSN for regulating the immune hyperfunction and inhibiting the proliferation of synovial cells in CIA rats might be associated with up-regulating in vivo ROS levels.

Animals ; Apoptosis ; drug effects ; Arthritis, Rheumatoid ; metabolism ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Lymphocyte Activation ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Synovial Membrane ; cytology ; pathology ; T-Lymphocytes ; drug effects ; metabolism