OBJECTIVES: To explore the mechanism by which Tougu Xiaotong Capsule (TXC) promotes chondrogenic differentiation and cartilage repair in mice with osteoarthritis (OA). METHODS: Fifty 8-week-old male C57BL mice were randomly divided into normal control group, cartilage damage (induced by subchondral ring-shaped drilling) model group and TXC treatment groups at low, moderate and high doses (184, 368 and 736 mg/kg, respectively). Saline (in normal control and model groups) and TXC were administered after modeling by daily gavage for 6 consecutive weeks. The changes of cartilage damage in the mice were assessed by measuring thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) and using micro-CT, modified safranine O and fast green staining, HE staining, and qPCR. Primary cultures of mouse synovial mesenchymal stem cells (SMSCs) with lentivirus vector transfection for interfering CXCL12, TXC treatment, or both for 24 h were examined for chondrogenic differentiation using immunofluorescence staining, scratch assay, immunocytochemistry, and Western blotting. RESULTS: In mouse models with cartilage damage, TXC treatment at the moderate dose significantly alleviated joint pain, promoted cartilage repair, and upregulated the mRNA expression levels of CXCL12, GDF5, collagen II, aggrecan, Comp and Sox9 in the cartilage tissue. In primary mouse SMSCs, CXCL12 knockdown resulted in significant reduction of GDF5 protein expression, migration ability and Sox9 protein expression, and these changes were obviously reversed by TXC treatment. CONCLUSIONS TXC promotes chondrogenic differentiation of mouse SMSCs to promote repair of cartilage damage in mice by activating the CXCL12/GDF5 pathway.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Osteoarthritis/metabolism* ; Male ; Growth Differentiation Factor 5/metabolism* ; Mice, Inbred C57BL ; Mice ; Chemokine CXCL12/metabolism* ; Signal Transduction/drug effects* ; Cell Differentiation/drug effects* ; Cartilage, Articular/drug effects* ; Mesenchymal Stem Cells/cytology*

OBJECTIVETo investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease (KBD) and in an established T-2 toxin- and selenium (Se) deficiency-induced rat model.

METHODSCartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and mRNA levels were determined by real-time reverse transcription polymerase chain reaction.

RESULTSIncreased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and mRNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet.

CONCLUSIONT-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.

Adolescent ; Animals ; Apoptosis ; drug effects ; Biomarkers ; Cartilage, Articular ; physiopathology ; Child ; Chondrocytes ; physiology ; Female ; Humans ; Kashin-Beck Disease ; etiology ; physiopathology ; Male ; Matrilin Proteins ; genetics ; metabolism ; Models, Animal ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Selenium ; deficiency ; T-2 Toxin ; pharmacology

SRT1720, a new discovered drug, was reported to activate silent information regulator 1 (SIRT1) and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside (SNP) (2.5 mmol/L) and divided into five groups: 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group (0 μmol/L SRT1720). MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) were detected by Western blotting and the expression levels of SIRT1, type II collagen, and aggrecan mRNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the mRNA expression levels of type II collagen and aggrecan increased ({ptP}<0.05), and the expression levels of p53, NF-κB and bax decreased (P<0.05). It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.
Aggrecans ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Cartilage, Articular ; cytology ; drug effects ; metabolism ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; metabolism ; Chromatin ; chemistry ; drug effects ; metabolism ; Collagen Type II ; genetics ; metabolism ; Gene Expression Regulation ; Heterocyclic Compounds, 4 or More Rings ; pharmacology ; Nitroprusside ; toxicity ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; genetics ; metabolism ; Primary Cell Culture ; Rabbits ; Signal Transduction ; drug effects ; genetics ; Sirtuin 1 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVEAlthough chondroprotective activities have been documented for polysaccharides, the potential target of different polysaccharide may differ. The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo, especially on the expression of type II collagen.

METHODSChondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type II collagen. Chondrocyte viability was assessed after being treated with HBP-A in different concentrations. Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope (SEM). The constructs were treated with HBP-A and then injected to nude mice subcutaneously. Six weeks after transplantation, the specimens were observed through transmission electron microscopy (TEM). The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTs-5), aggrecan and type II collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction (PCR). The expression of type II collagen and matrix metalloproteinases-3 (MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay (ELISA), respectively.

RESULTSMMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration. In morphological study, there were significant appearance of collagen in those constructs treated by HBP-A. Accordingly, in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs, the expression of type II collagen was increased significantly in HBP-A group when compared with control group (P<0.001).

CONCLUSIONSThe study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3, ADAMTs-5, and increasing of type II collagen expression.

ADAM Proteins ; genetics ; metabolism ; Aggrecans ; genetics ; metabolism ; Alginates ; pharmacology ; Animals ; Cartilage, Articular ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; metabolism ; ultrastructure ; Collagen Type II ; genetics ; metabolism ; Female ; Glucans ; pharmacology ; Glucuronic Acid ; pharmacology ; Hexuronic Acids ; pharmacology ; Hydrogel, Polyethylene Glycol Dimethacrylate ; pharmacology ; Immunohistochemistry ; Matrix Metalloproteinase 3 ; metabolism ; Mice, Nude ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Tissue Engineering ; methods

Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.
Animals ; Anti-Inflammatory Agents/administration & dosage ; Apoptosis/drug effects/physiology ; Cartilage, Articular/cytology/drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/*metabolism ; Dose-Response Relationship, Drug ; MAP Kinase Kinase 4/*metabolism ; Phosphatidylinositol 3-Kinases/*metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rabbits ; Reactive Oxygen Species/*metabolism ; Withanolides/*administration & dosage

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.
ADAM Proteins/antagonists & inhibitors ; Cartilage, Articular/*drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/metabolism ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Glycosaminoglycans/*metabolism ; Humans ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase Inhibitors/pharmacology ; Oncostatin M/metabolism ; Osteoarthritis, Knee/drug therapy/genetics/metabolism ; Procollagen N-Endopeptidase/antagonists & inhibitors

This study is to investigate the effect of Vam3, a dimeric derivative of resveratrol, on SNP-induced apoptosis and its potential mechanism in rat articular chondrocytes. Isolated rat articular chondrocytes were treated with sodium nitroprusside (SNP), a NO donor, to induce apoptosis. Apoptosis percentage was evaluated by Annexin V-PI and nucleus fracture was examined by DAPI staining. Level of intracellular reactive oxygen species (ROS) was detected using 2, 7'-dichlorofluorescin diacetate (DCFH-DA) as a fluorescence probe by fluorescence microplate reader. The change in mitochondrial membrane potential was detected by TMRE staining. Expressions of SIRT1, acetylated p53 (ac-p53), cleaved caspase 9 and cleaved caspase 3 were determined by Western blotting. It showed that Vam3 up to 10 micromol x L(-1) could significantly reduce SNP-induced rat articular chondrocytes apoptosis (P < 0.01) and nucleus fracture, inhibit the increase of intracellular ROS level (P < 0.01) and reverse the decrease in mitochondrial membrane potential (P < 0.01). Simultaneously, Vam3 could upregulate the expression of SIRT1, deacetylate p53, and inhibit the cleavage of caspase 9 and caspase 3 (P < 0.01) of rat articular chondrocytes exposed to SNP. This study indicates Vam3 could protect rat articular chondrocytes against SNP-induced apoptosis, perhaps through the upregulation of SIRT1 and deacetylation of p53.
Animals ; Apoptosis ; drug effects ; Arabidopsis Proteins ; pharmacology ; Cartilage, Articular ; cytology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Nitric Oxide Donors ; antagonists & inhibitors ; pharmacology ; Nitroprusside ; pharmacology ; Qa-SNARE Proteins ; pharmacology ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Sirtuin 1 ; metabolism ; Tumor Suppressor Protein p53 ; 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

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