2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.
Animals ; Cartilage, Articular/*cytology ; Cell Dedifferentiation/*drug effects ; Cell Nucleus/drug effects/metabolism ; Chondrocytes/*cytology/drug effects/enzymology/*metabolism ; Deoxyglucose/*pharmacology ; Endoplasmic Reticulum/drug effects/pathology ; Glycogen Synthase Kinase 3/metabolism ; Mutant Proteins/metabolism ; Protein Transport/drug effects ; Proteoglycans/metabolism ; Rabbits ; Signal Transduction/*drug effects ; beta Catenin/*metabolism

OBJECTIVETo study the mechanism of action of Tougu Xiaotong Capsule (透骨消痛胶囊, TGXTC) ex vivo in suppressing chondrocyte (CD) apoptosis induced by sodium nitroprussiate (SNP).

METHODSThirty New Zealand rabbits, 2 months old, were randomized by lottery into five groups, six in each: the blank group treated with saline, the positive control group treated with Zhuanggu Guanjie Pill (壮骨关节丸, 70 mg/kg), and the three experimental groups, EGA, EGB, and EGC, treated with low dose (35 mg/kg), moderate dose (70 mg/kg), and high dose (140 mg/kg) of TGXTC, respectively. All treatments were administered via gastrogavage twice a day for 3 days. Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared. CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining. SNP of various final concentrations (0, 0.5, 1.0, and 2.0 mmol/L) was used to induce CD apoptosis, and the dosage-effect relationship of SNP in inducing CD apoptosis was determined. Serum samples from the blank, control, and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP. Cell apoptosis was determined by Hoechst 33342 staining, viability of CDs was quantified by MTT, CD apoptosis rate was determined by annexin V-FITC/PI staining, levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR, and contents of caspase-3 and caspase-9 proteins were determined by colorimetry.

RESULTSCD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner. The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study. Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis, decrease in p53 mRNA expression, inhibition of catalytic activities of caspase-3 and caspase-9, and increase in Bcl-2 mRNA expression when compared with the serum from the blank group (P<0.05).

CONCLUSIONTGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression, and inhibition of caspase-3 and caspase-9 catalytic activities.

Animals ; Apoptosis ; drug effects ; Biocatalysis ; drug effects ; Capsules ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Male ; Models, Biological ; Nitroprusside ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Rabbits ; Reproducibility of Results ; Serum ; chemistry ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 β (IL-1 β)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis.

METHODSSerum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 β stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 β-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 β, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction.

RESULTSIL-1 β stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 β, TNF-α, MMP-3 and MMP-13. However, the IL-1 β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs.

CONCLUSIONZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

Animals ; Base Sequence ; Blotting, Western ; Caveolins ; metabolism ; Chondrocytes ; drug effects ; enzymology ; metabolism ; DNA Primers ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Profiling ; Interleukin-1beta ; physiology ; MAP Kinase Signaling System ; Male ; RNA, Messenger ; genetics ; Rabbits ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo investigate the in vitro effect of caffeic acid phenethyl ester (CAPE), a NF-κB inhibitor, on the apoptosis of osteoarthritic (OA) chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9).

METHODSAnnexin V-FITC/propidium iodide (PI) labeling and western blotting were used to observe and determine the apoptosis in TNFα-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants.

RESULTSIt was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFα for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated (from proMMP-9 to active MMP-9), and the physiologic calcium concentration (2.5 mmol/L) could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFα for 24 h. The stimulatory effect of TNFα just on proMMP-9 was counteracted significantly by CAPE.

CONCLUSIONNF-κB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFα (a pro-apoptotic factor). Therefore, therapeutic NF-κB inhibitor was a 'double-edged swords' to the apoptosis of chondrocytes and the secretion of MMP-9.

Aged ; Apoptosis ; drug effects ; Caffeic Acids ; pharmacology ; therapeutic use ; Calcium ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; secretion ; Drug Evaluation, Preclinical ; Female ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; NF-kappa B ; antagonists & inhibitors ; Osteoarthritis ; drug therapy ; enzymology ; Phenylethyl Alcohol ; analogs & derivatives ; pharmacology ; therapeutic use ; Tumor Necrosis Factor-alpha ; pharmacology

IL-1beta is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1beta-induced matrix metalloproteinase-2 (MMP- 2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.
Chondrocytes/drug effects/*enzymology/metabolism ; Dinoprostone/analysis/*metabolism ; Gelatinase A/analysis/*biosynthesis ; Humans ; Indoles/pharmacology ; Interleukin-1/*pharmacology ; Isoenzymes/antagonists & inhibitors/metabolism ; Nitrobenzenes/pharmacology ; Phosphorylation/drug effects ; Prostaglandin-Endoperoxide Synthase/metabolism ; Protein Kinase C/antagonists & inhibitors/metabolism ; Research Support, Non-U.S. Gov't ; Sulfonamides/pharmacology ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism

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