OBJECTIVETo study the effect and related mechanism of seaweed polysaccharide (SP) on apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis (RA-FLS).

METHODSRA-FLS were in vitro cultured using modified tissue culture method. Effect of SP (0, 15, 20, and 25 mg/mL, respectively) at different time points (0, 3, 4, and 5 days, respectively) on the proliferation and apoptosis of RA-FLS, and protein expressions of Caspase-3, Bax, and Bcl-2 was detected by cell counting kit-8 (CCK-8) assay, Hoechst 33258 staining assay, TUNEL assay, and Western blot, respectively.

RESULTSCompared with 0 mg/mL SP at the same time point, the proliferation of RA-FLS was inhibited, and the apoptosis was promoted 3, 4, and 5 days after intervened by 15, 20, and 25 mg/mL SP, respectively (P<0.01) in time- and dose-dependent manners. RA-FLS Bax protein expression was up-regulated, Bcl-2 protein expression down-regulated, Caspase-3 activated and split by 15, 20, and 25 mg/mL SP, respectively for 4 days (P<0.05, P<0.01). Besides, the changes were in a dose-dependent manner.

CONCLUSIONSSP could inhibit RA-FLS proliferation and induce its apoptosis in dose- and time-dependent manners. Its apoptosis mechanism might be through up-regulating intracellular Bax protein expression and down-regulating Bcl-2 protein expression, thus influencing the mitochondrion signaling pathway, further promoting Caspase-3 activation and split, resulting in the apoptosis of RA-FLS.

Apoptosis ; drug effects ; Arthritis, Rheumatoid ; pathology ; Caspase 3 ; metabolism ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Humans ; Polysaccharides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Seaweed ; Synovial Membrane ; cytology ; drug effects ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the mechanism of anti-death receptor 5-10 (AD5-10) combined with epirubicin in treating rheumatoid arthritis (RA).

METHODSWe detected the cell viability of the fibroblast-like synoviocytes (FLS) from RA patients with MTT. The expression level of apoptosis signaling pathways protein, p53, and p21 were evaluated with Western blot.

RESULTSWe found that epirubicin, at different doses, could enhance the effect of AD5-10 on FLS, promoting the apoptosis of FLS. The expression levels of caspase-3, -8, -9, c-FLIP, Bcl-2, p53, and p21 in the FLS changed after epirubicin treatment.

CONCLUSIONEpirubicin may coordinate with AD5-10 in inducing FLS apoptosis through affecting the levels of p53, p21, c-FLIP, and Bcl-2.

Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Arthritis, Rheumatoid ; drug therapy ; metabolism ; pathology ; Cells, Cultured ; Epirubicin ; pharmacology ; Humans ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; immunology ; Synovial Membrane ; cytology ; drug effects ; metabolism

Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.
Apoptosis/*drug effects/genetics ; Apoptosis Regulatory Proteins/*genetics/metabolism ; Arthritis, Rheumatoid/genetics/metabolism/*pathology ; Cells, Cultured ; Drug Evaluation, Preclinical ; Fibroblasts/drug effects/metabolism/pathology ; Humans ; Hydrogen Peroxide/*pharmacology ; Proto-Oncogene Proteins/*genetics/metabolism ; RNA, Small Interfering/*pharmacology ; Synovial Membrane/cytology/drug effects/metabolism/*pathology ; Transcription Factors/*antagonists & inhibitors/genetics ; Transcriptional Activation/drug effects ; Transfection

OBJECTIVETo explore the therapeutic effects and mechanisms of Zhubi capsule on adjuvant arthritis (AA) in rats.

METHODThe model of rat was induced by intradermal injection of CFA. The perimeter of ankle joint was measured at different time points. Con A-induced splencyte proliferation was examined by MIT assay and the expression of IL-1, IL-6 in synovium was determined by immunohistochemical method.

RESULTAfter the preventive administration of Zhubi capsule (5.2, 2.6, 1.3 g x kg x d(-1), x 25 d), it was found that Zhubi capsule significantly inhibited the primary and secondary ankle joint swelling. The increased Con A-induced splencyte proliferation reaction and the activated IL1, IL-6 expression of AA rat were suppressed by the treatment with Zhubi capsule.

CONCLUSIONZhubi capsule has a therapeutic effect on AA rats, and its mechanism is related to the immunoregulative function.

Animals ; Ankle Joint ; pathology ; Arthritis, Experimental ; metabolism ; pathology ; Capsules ; Cell Proliferation ; drug effects ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Interleukin-1 ; metabolism ; Interleukin-6 ; metabolism ; Male ; Materia Medica ; administration & dosage ; pharmacology ; Membrane Fluidity ; drug effects ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Spleen ; cytology ; Synovial Membrane ; drug effects

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

OBJECTIVETo observe the effects of Dioscornin Tablet (DT) containing serum on nuclear factor of kappa B (NF-kappaB) p65, signal transducer and activator of transcription 3 (STAT3), and vascular endothelial growth factor (VEGF) mRNA expressions in rats' synovial cell strain 364 (RSC-364) induced by interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-alpha), and to investigate the underlying mechanisms for DT to inhibit angiogenesis of rheumatoid arthritis (RA).

METHODSIn this experiment, the vehicle control group, the cell model group, the DT containing serum group, and the positive control group (Tripterygium containing serum) were set up. The DT containing serum and the Tripterygium containing serum were prepared. The RA cell model was established by IL-17 combined TNF-alpha induced injury in RSC-364. The RA cells were intervened by DT containing serum and Tripterygium containing serum respectively. The DNA binding activity of NF-kappaB p65 was detected using TransAM NF-kappaB p65. The expression of STAT3 was observed using Western blot. The VEGF mRNA expressions were detected by real-time quantitative PCR.

RESULTSCompared with the vehicle control group, the NF-kappaB p65 activity, the expressions of STAT3 and VEGF mRNA increased significantly in RSC-364 induced by IL-17 +TNF-alpha (P < 0.01, P < 0.05). Compared with the model group, the NF-kappaB p65 activity, the expressions of STAT3 and VEGF mRNA decreased significantly in the DT containing serum group and the positive control group (P < 0.01, P < 0.05). There was no statistical difference between the two groups (P > 0.05).

CONCLUSIONDT inhibited the VEGF mRNA expression through inhibiting the NF-kappaB p65 activity and the STAT3 protein expression in the Janus kinase (JAK)-signal transducer and activating transcription factor pathway, thus inhibiting the angiogenesis of RA.

Animals ; Arthritis, Rheumatoid ; pathology ; Cells, Cultured ; Diosgenin ; analogs & derivatives ; pharmacology ; Interleukin-17 ; adverse effects ; Male ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; pharmacology ; Rats ; Rats, Wistar ; STAT3 Transcription Factor ; metabolism ; Serum ; Signal Transduction ; Synovial Membrane ; cytology ; drug effects ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; adverse effects ; Vascular Endothelial Growth Factor A ; metabolism

AIMTo study the effect and mechanism of action of total glucosides of paeony (TGP) on synoviocytes from rats with collagen-induced arthritis (CIA).

METHODSChicken type II collagen was used to induce CIA in rats. Synoviocytes were separated by incubation with collagenase and trypsin, and its ultrastructural changes were observed under transmission electron microscope. Synoviocyte proliferation was determined by 3-(4,5-dimethylthiazal-2yl) 2,5- diphenyltetrazoliumbromide (MTT) assay, and IL-1 activity in synoviocytes supernatant was measured by thymocyte proliferation assay. TNFa and PGE, produced by synoviocytes were determined by radioimmunoassay.

RESULTSTGP was shown to protect CIA rats against the ultrastructural damages of synoviocytes. Meanwhile, TGP also suppressed the excessive synoviocyte proliferation and over-production of IL-1, TNFalpha and PGE2.

CONCLUSIONTGP has inhibitory effect on hyperfunctional synoviocytes of CIA rats and its mechanism of action may be related with the inhibition of abnormal proliferation and secretion of synoviocytes.

Animals ; Arthritis, Experimental ; chemically induced ; metabolism ; pathology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type II ; Dinoprostone ; metabolism ; Glucosides ; isolation & purification ; pharmacology ; Interleukin-1 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Paeonia ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; metabolism ; ultrastructure ; Tumor Necrosis Factor-alpha ; metabolism

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