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*

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.
Animals ; Cartilage/cytology* ; Chondrocytes/drug effects* ; Chondrogenesis/drug effects* ; Extracellular Matrix/metabolism* ; Quercetin/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Tissue Scaffolds

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

To observe the influence of matrix metalloproteinases-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), CD47, L-selectin and advanced oxidation proteinproducts (AOPP) in osteoarthritis and the intervention of curcumin.A total of 20 male C57BL/6 mice (10.05-15.00 g) were randomly divided into control group, OA group, Cur25 group and Cur50 group (intraperitoneal injected 25 μmol/L or 50 μmol/L of curcumin everyday after modeling). After 4 weeks treatment, we observed the morphological changes of the gross specimen by immunohistochemical method, and observed the ultrastructure of cartilage tissue under electron microscope. The expression of MMP-2, MCP-1 and CD47 were detected by western blotting, and L-selectin and AOPP were detected by ELISA and spectrophotometer, respectively.In the cartilage tissue morphology, the chondrocytes of OA group showed obvious change, while Cur25 and Cur50 groups maintained the good cartilage cell membrane intact. Compared with control group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in OA group, Cur25 group and Cur50 group were increased (all<0.05), while CD47 levels were decreased (all<0.05). Compared with OA group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in Cur25 group and Cur50 group were decreased (all<0.05), while CD47 levels were increased (all<0.05), and such changes were more significant in Cur50 group (all<0.05).The MMP-2, MCP-1, CD47, L-selectin and AOPP are closely associated with the pathology course of OA. Curcumin has protection effect on cartilage, which can relieve joint cartilage degeneration, reduce cartilage inflammation and increase the metabolic activity of chondrocytes.
Advanced Oxidation Protein Products ; metabolism ; Animals ; Biomarkers ; CD47 Antigen ; metabolism ; Cartilage ; chemistry ; drug effects ; pathology ; Chemokine CCL2 ; metabolism ; Chondrocytes ; drug effects ; pathology ; Curcumin ; administration & dosage ; pharmacology ; Cytokines ; L-Selectin ; metabolism ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Mice, Inbred C57BL ; Osteoarthritis ; genetics ; pathology ; physiopathology ; Oxidative Stress

To investigate the effect of diosgenin (Dgn) on chondrocytes and its relation to JAK2/STAT3 signaling pathway in mice with osteoarthritis (OA).Fifteen male C57BL/6 mice were randomly divided into three groups:control group, OA group and OA+Dgn group. After 4 weeks of treatment, the histopathological changes of cartilage tissue were observed by toluidine blue staining under light microscopy and the ultrastructure of chondrocytes was observed under electron microscopy. The primarily cultured chondrocytes of OA mice were randomly divided into 4 groups:(1) OA group, (2) Dgn group, (3) Dgn+AG490 group, (4) AG490 group. The expression of p-JAK2, p-STAT3, Bax, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) were detected by Western blotting, and superoxide dismutase (SOD) was detected using colorimetric method.The morphological observation showed that the chondrocytes of OA group presented considerable pathological changes, while the chondrocytes in OA+Dgn group maintained intact membrane. Electron microscopy observation found obvious injury in cartilage tissues of OA group, while that in OA+Dgn group remained smooth. Compared with OA group, the expressions of p-JAK2 and p-STAT3 in chondrocytes of Dgn group were increased (all<0.05), and the expressions of Bax protein, SDH, COX and SOD were decreased (all<0.05). While compared with Dgn group, the expressions of p-JAK2, p-STAT3, SDH, COX and SOD in chondrocytes of Dgn+AG490 group were decreased (all<0.05), and the expression of Bax protein was increased (<0.05).Diosgenin can inhibit apoptosis and increase mitochondrial oxidative stress capacity of chondrocytes in mice with osteoarthritis, which is closely related to the activation of JAK2/STAT3 signaling pathway.
Animals ; Apoptosis ; drug effects ; Cartilage ; drug effects ; pathology ; Chondrocytes ; chemistry ; drug effects ; pathology ; Diosgenin ; pharmacology ; Electron Transport Complex IV ; metabolism ; Janus Kinase 2 ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria ; drug effects ; genetics ; Osteoarthritis ; genetics ; physiopathology ; Oxidative Stress ; drug effects ; STAT3 Transcription Factor ; drug effects ; Signal Transduction ; Succinate Dehydrogenase ; metabolism ; Superoxide Dismutase ; metabolism ; Tyrphostins ; pharmacology ; bcl-2-Associated X Protein ; metabolism

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

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

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

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