Rabbit Articular Chondrocytes Research Articles

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro.

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Resveratrol protects rabbit articular chondrocyte against sodium nitroprusside-induced apoptosis via scavenging ROS.

This study aims to investigate the mechanism by which resveratrol (RV) prevents sodium nitroprusside (SNP)-induced chondrocyte apoptosis, which is a characteristic feature of osteoarthritis(OA). Rabbit articular chondrocytes were pre-incubated with 100μM RV for 18h before 1.5mM SNP co-treatment for 6h. Cell viability was evaluated by CCK-8. Annexin V/PI double staining and Hoechst 33258 staining were used to determine the fashion of SNP-induced chondrocytes death. Mitochondrial membrane potential (ΔΨm) was measured by using flow cytometry (FCM) with TMRM and Rhodamine 123 staining. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were confirmed by FCM analysis with DCFH-DA and DAF-FM DA staining. Cytoskeleton proteins of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red were validated by confocal microscopy. SNP induced time- and dose-dependent chondrocytes apoptosis with decline of ΔΨm, activation of caspases as well as cytoskeletal remodeling. SNP induced a significant induction of both ROS and NO. RV remarkably prevented SNP-induced ROS production and apoptosis as well as cytoskeletal remodeling, but did not prevent SNP-induced NO production. Pretreatment with NO scavengers did not significantly prevent SNP-induced apoptosis and cytoskeletal remodeling. SNP induces NO-independent ROS production which dominates rabbit articular chondrocyte apoptosis, and RV protects chondrocytes against SNP-induced apoptosis via scavenging ROS instead of NO.

Luteolin Inhibits the Activity, Secretion and Gene Expression of MMP-3 in Cultured Articular Chondrocytes and Production of MMP-3 in the Rat Knee.

We investigated whether luteolin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as production of MMP-3 in the rat knee to evaluate the potential chondro-protective effects of luteolin. Rabbit articular chondrocytes were cultured in a monolayer and IL-1β-induced gene expression levels of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen were measured by reverse transcription - polymerase chain reaction (RT-PCR). Effects of luteolin on interleukin-1β (IL-1β)-induced secretion and enzyme activity of MMP-3 in rabbit articular chondrocytes were investigated by western blot analysis and casein zymography, respectively. The effect of luteolin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) luteolin inhibited the gene expression levels of MMP-3, MMP-1, MMP-13, ADAMTS-4 and ADAMTS-5. However, it increased the gene expression level of collagen in rabbit articular chondrocytes; (2) luteolin inhibited the secretion and activity of MMP-3; (3) luteolin inhibited in vivo production of MMP-3 protein. These results suggest that luteolin can regulate the gene expression, secretion and activity of MMP-3, by directly acting on articular chondrocytes.

In vitro effect of a synthesized sulfonamido-based gallate on articular chondrocyte metabolism

Autologous chondrocyte implantation (ACI) is a promising strategy for cartilage repair and reconstitution. However, limited cell numbers and the dedifferentiation of chondrocytes present major difficulties to the success of ACI therapy. Therefore, it is important to find effective pro-chondrogenic agents that restore these defects to ensure a successful therapy. In this study, we synthesized a sulfonamido-based gallate, namely N-[4-(4,6-dimethyl-pyrimidin-2-ylsulfamoyl)-phenyl]-3,4,5-trihydroxy-benzamide (EJTC), and investigated its effects on rabbit articular chondrocytes through an examination of its specific effects on cell proliferation, morphology, viability, GAG synthesis, and cartilage-specific gene expression. The results show that EJTC can effectively promote chondrocyte growth and enhance the secretion and synthesis of cartilage ECM by upregulating the expression levels of the aggrecan, collagen II, and Sox9 genes. The expression of the collagen I gene was effectively downregulated, which indicates that EJTC inhibits chondrocytes dedifferentiation. Chondrocyte hypertrophy, which may lead to chondrocyte ossification, was also undetectable in the EJTC-treated groups. The recommended dose of EJTC ranges from 3.125μg/mL to 7.8125μg/mL, and the most profound response was observed with 7.8125μg/mL. This study may provide a basis for the development of a novel agent for the treatment of articular cartilage defects.

Resveratrol regulates type II collagen and COX-2 expression via the ERK, p38 and Akt signaling pathways in rabbit articular chondrocytes

Resveratrol, a naturally occurring polyphenolic phytoalexin antioxidant compound present in grapes and red wine, has been reported to induce various biochemical responses. It has been shown to possess anti-aging, anti-inflammatory and anti-proliferative activities in several cell types. However, the effects of resveratrol in normal cells, including chondrocytes, have not yet been clearly elucidated. The aim of the present study was to evaluate the effects of resveratrol on differentiation and inflammation in rabbit articular chondrocytes and to investigate the underlying mechanism of action. Rabbit articular chondrocytes were treated with 20 μM resveratrol for different time periods or with various concentrations of resveratrol for 24 h. It was observed that the expression levels of type II collagen and sulfated proteoglycan, as determined by western blot analysis and Alcian blue staining, respectively, increased following treatment with resveratrol in a concentration-dependent manner at concentrations up to 20 μM and then decreased at higher concentrations. The expression levels of cyclooxygenase (COX-2) and prostaglandin E2 (PGE2) began to increase at 10 min after the addition of resveratrol, reached peak levels at 3 h and decreased from the peak level thereafter, as determined by western blot analysis and PGE2 assay, respectively. It was also demonstrated that resveratrol caused phosphorylation of mitogen-activated protein kinase proteins [extracellular signal-regulated kinases (ERK), p38 and c-Jun N-terminal kinases (JNK)] and Akt in rabbit articular chondrocytes. The inhibition of ERK, p38 kinase, phosphoinositide 3-kinase (PI3K) and Akt with PD98059, SB203580, LY294002 and triciribine, respectively, suppressed resveratrol-induced type II collagen and COX-2 expression. However, inhibition of JNK with SP600125 produced no clear changes in the expression levels of type II collagen and COX-2. The results suggest that resveratrol in articular chondrocytes stimulates differentiation and inflammation via the ERK, p38 and Akt signaling pathways.

Overexpression of MicroRNA-25 by Withaferin A Induces Cyclooxygenase-2 Expression in Rabbit Articular Chondrocytes

Increasing evidence supports the role of microRNAs (miRNA) in the regulation of inflammation in various human disorders. Several recent studies have demonstrated that microRNA-25 (miR-25) has multiple functions, and it affects the expression of inflammatory mediators. Withaferin A (WFA), a natural compound derived from the medicinal plant Withania somnifera, has shown the potential to be an effective drug for arthritis treatment in several preclinical and clinical studies. We investigated the role of miR-25 in the WFA-mediated up-regulation of cyclooxygenase-2 (COX-2) expression in rabbit articular chondrocytes. WFA induced COX-2 expression in a dose-dependent manner as analyzed by western blot analysis and immunofluorescence staining in rabbit articular chondrocytes. WFA up-regulated miR-25 expression as determined by real-time PCR. Overexpression of miR-25 in the presence of WFA increased the expression of COX-2 compared to that observed with just WFA treatment alone, as indicated by western blot analysis and Real-time PCR. Moreover, silencing of miR-25 by anti-miR25 inhibited COX-2 expression in a dose-dependent manner. Since miR-25 up-regulation by WFA treatment induced the expression of COX-2 in rabbit articular chondrocytes, these findings collectively suggest that miR-25 mediates the WFA-induced inflammatory responses in chondrocytes.

The biological effects of sex hormones on rabbit articular chondrocytes from different genders.

The aim of this study was to investigate the biological effects of sex hormones (17β-estradiol and testosterone) on rabbit articular chondrocytes from different genders. We cultured primary rabbit articular chondrocytes from both genders with varying concentration of sex hormones. We evaluate cell proliferation and biochemical functions by MTT and GAG assay. The chondrocyte function and phenotypes were analyzed by mRNA level using RT-PCR. Immunocytochemical staining was also used to evaluate the generation of collagen-II. This study demonstrated that 17β-estradiol had greater positive regulation on the biological function and gene expressions of articular chondrocytes than testosterone, with the optimal concentrations of 10−6 and 10−7 M, particularly for female chondrocytes.

Withaferin A-caused production of intracellular reactive oxygen species modulates apoptosis via PI3K/Akt and JNKinase in rabbit articular chondrocytes.

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.Graphical

3D dynamic culture of rabbit articular chondrocytes encapsulated in alginate gel beads using spinner flasks for cartilage tissue regeneration.

Cell-based therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. In the present study, the effects of an integrated three-dimensional and dynamic culture on rabbit articular chondrocytes were investigated. Cells (passages 1 and 4) were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Subcutaneous implantation of the cell-laden beads was performed to evaluate the ectopic chondrogenesis. It was found that cells remained viable after 35 days in the three-dimensional dynamic culture. Passage 1 cells demonstrated a proliferative growth in both media. Passage 4 cells showed a gradual reduction in DNA content in growth medium, which was attenuated in chondrogenic medium. Deposition of glycosaminoglycans (GAG) was found in all cultures. While passage 1 cells generally produced higher amounts of GAG than passage 4 cells, GAG/DNA became similar on day 35 for both cells in growth media. Interestingly, GAG/DNA in growth medium was greater than that in chondrogenic medium for both cells. Based on GAG quantification and gene expression analysis, encapsulated passage 1 cells cultured in growth medium displayed the best ectopic chondrogenesis. Taken together, the three-dimensional and dynamic culture for chondrocytes holds great potential in cartilage regeneration.

Resveratrol Inhibits Nitric Oxide-Induced Apoptosis via the NF-Kappa B Pathway in Rabbit Articular Chondrocytes

Resveratrol (trans-3,4'-trihydroxystillbene), a naturally occurring polyphenolic antioxidant found in grapes and red wine, elicits diverse biochemical responses and demonstrates anti-aging, anti-inflammatory, and anti-proliferative effects in several cell types. Previously, resveratrol was shown to regulate differentiation and inflammation in rabbit articular chondrocytes, while the direct production of nitric oxide (NO) in these cells by treatment with the NO donor sodium nitroprusside (SNP) led to apoptosis. In this study, the effect of resveratrol on NO-induced apoptosis in rabbit articular chondrocytes was investigated. Resveratrol dramatically reduced NO-induced apoptosis in chondrocytes, as determined by phase-contrast microscopy, the MTT assay, FACS analysis, and DAPI staining. Treatment with resveratrol inhibited the SNP-induced expression of p53 and p21 and reduced the expression of procaspase-3 in chondrocytes, as detected by western blot analysis. SNP-induced degradation of I-kappa B alpha (IκB-α) was rescued by resveratrol treatment, and the SN50 peptide-mediated inhibition of NF-kappa B (NF-κB) activity potently blocked SNP-induced caspase-3 activation and apoptosis. Our results suggest that resveratrol inhibits NO-induced apoptosis through the NF-κB pathway in articular chondrocytes.

The COX-2 Selective Blocker Etodolac Inhibits TNFα-Induced Apoptosis in Isolated Rabbit Articular Chondrocytes

Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl− current (ICl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl− conductance. The TNFα-evoked Cl− current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners.Treatment with N-acetyl-l-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes.

Improvement of PHBV Scaffolds with Bioglass for Cartilage Tissue Engineering

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) have proven to be possible matrices for the three-dimensional growth of chondrocyte cultures. However, the engineered cartilage grown on these PHBV scaffolds is currently unsatisfactory for clinical applications due to PHBV’s poor hydrophilicity, resulting in inadequate thickness and poor biomechanical properties of the engineered cartilage. It has been reported that the incorporation of Bioglass (BG) into PHBV can improve the hydrophilicity of the composites. In this study, we compared the effects of PHBV scaffolds and PHBV/BG composite scaffolds on the properties of engineered cartilage in vivo. Rabbit articular chondrocytes were seeded into PHBV scaffolds and PHBV/BG scaffolds. Short-term in vitro culture followed by long-term in vivo transplantation was performed to evaluate the difference in cartilage regeneration between the cartilage layers grown on PHBV and PHBV/BG scaffolds. The results show that the incorporation of BG into PHBV efficiently improved both the hydrophilicity of the composites and the percentage of adhered cells and promoted cell migration into the inner part the constructs. With prolonged incubation time in vivo, the chondrocyte-scaffold constructs in the PHBV/BG group formed thicker cartilage-like tissue with better biomechanical properties and a higher cartilage matrix content than the constructs in the PHBV/BG group. These results indicate that PHBV/BG scaffolds can be used to prepare better engineered cartilage than pure PHBV.

Characterization of chondrocyte scaffold carriers for cell‐based gene therapy in articular cartilage repair

Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in three-dimensional scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and open-cell polylactic acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At 4 weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs.

Mesenchymal Stem Cells Downregulate Articular Chondrocyte Differentiation in Noncontact Coculture Systems: Implications in Cartilage Tissue Regeneration

While chondrogenesis of mesenchymal stem cells (MSCs) in vitro has been extensively studied, their participation in cartilage tissue repair remains unresolved. This study was designed to elucidate if MSCs affect the phenotype of articular chondrocytes (ACs). A combination of noncontact coculture modes was developed. Human or rabbit MSCs and rabbit ACs (rACs) were encapsulated in alginate hydrogel beads [three-dimensional (3D)] or cultured in a monolayer [two-dimensional (2D)] and subsequently cocultured in the Transwell(®) system. After coculture, cell morphology, growth, deposition of the cartilaginous extracellular matrix (ECM), and gene expression of rACs were investigated. It was found that upon coculture without a cell-cell contact, both 2D and 3D cultured MSCs dramatically induced the morphological transformation of 2D cultured rACs from round to a spindle-like shape, and however inhibited the generation of cellular aggregates of 3D cultured rACs. Most strikingly, a coculture resulted in a significantly less deposition of the cartilaginous ECM, including glycosaminoglycans and collagen type II by both 2D and 3D cultured rACs. Importantly, similar observations were achieved for rACs cultured in an MSC-conditioned medium, confirming the definite paracrine interactions between MSCs and rACs. Based on the analysis of gene expression, this phenotypic change of rACs was not identical as the dedifferentiation. To the best of our knowledge, this is a first study demonstrating that MSCs could downregulate chondrocytic differentiation of ACs and warrants considerations in cartilage tissue repair.

Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte

The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real-time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage.

Sulforaphane (SFN) regulates dedifferentiation and cyclooxygenase-2 (COX-2) expression via MAPkinase pathway in rabbit articular chondrocytes

Sulforaphane (SFN), a member of the isothiocyanate family, is a biologically active compound extracted from cruciferous vegetables such as broccoli, cauliflower, radishes and cabbage. Although it possesses potent anti-inflammatory and anticancer properties, the specific effects and detailed mechanisms of SFN remain poorly understood. To investigate whether SFN regulates dedifferentiation and inflammation, chondrocytes were treated with SFN, which induced dedifferentiation and reduced inflammation, as indicated by the reduction of type II collagen and cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner, as determined by Western blot analysis and RT-PCR. Synthesis of sulfated proteoglycan and production of prostaglandin E2 (PGE2) were decreased by treatment of SFN, as was detected by Alcian blue staining and immunohistochemical staining, and immunofluorescence staining and PGE2 assay, respectively. Additionally, treatment of SFN stimulated activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated-kinase (ERK-1/2) and p38 kinase. Inhibition of ERK-1/2 with PD98059 rescued SFN-induced a loss of type II collagen expression, whereas inhibition of p38 kinase with SB203580 recovered SFN-reduced COX-2 expression, as determined by the Western blot analysis, immunohistochemical staining and immunofluorescence staining. Taken together, these results indicate that SFN regulates type II collagen via ERK-1/2 and COX-2 expression via p38kinase in rabbit articular chondrocytes.

Gallotannin causes differentiation and inflammation via ERK-1/-2 and p38 kinase pathways in rabbit articular chondrocytes

Gallotannin (GT) is a type of tannic acid, derived from plant polyphenols, that is an agonist of plant defense mechanisms. Tannins have two types of structure; condensed tannins are a polymer of flavonoid units, while hydrolysable tannins are carbohydrates. GT is used in medical agents for its anti‑viral, anti‑bacterial and anti‑parasitic effects. The present study investigated the effects of GT on differentiation and inflammation in rabbit articular chondrocytes. GT caused differentiation and inflammatory responses in the rabbit articular chondrocytes. GT treatment induced the expression of type Ⅱ collagen and sulfated proteoglycan, as determined by western blot analysis and alcian blue staining, respectively, in a dose‑ and time‑dependent manner. Additionally, treatment with GT increased the expression of cyclooxygenase‑2 (COX‑2) and the production of prostaglandin E2 (PGE2), as determined by western blot analysis and PGE2 assay. GT was confirmed to cause phosphorylation of ERK‑1/‑2 and p38 kinase. Inhibition of pERK with PD98059 promoted GT‑induced type Ⅱ collagen expression. However, the inhibition of p38 with SB203580 suppressed GT‑induced COX‑2 expression and PGE2 production. In summary, the results demonstrated that GT‑induced ERK‑1/‑2 and p38 kinase have opposite effects on differentiation and inflammation in rabbit articular chondrocytes.

Sulforaphane inhibits proliferation by causing cell cycle arrest at the G2/M phase in rabbit articular chondrocytes

Sulforaphane(SFN), a natural compound extracted from cruciferous vegetables, exhibits potent anticancer activity in various types of tumor cells. However, the effect of SFN on the proliferation of chondrocytes is not well understood. In the present study, we addressed the mechanism of action by which SFN suppresses proliferation. We demonstrate that SFN causes an irreversible arrest in cell proliferation, as determined by trypan blue dye exclusion assay and flow cytometric analysis. SFN induced cell cycle arrest at the G2/Mphase by downregulation of cyclinB, cdc2 and cdc25c and upregulation of p21WAF1/CIP1 and p53 in a dose- and time-dependent manner, as determined by western blot analysis. Our data suggest that SFN regulates cell cycle arrest at the G2/M phase in rabbit articular chondrocytes.

PCB126 induces apoptosis of chondrocytes via ROS-dependent pathways

Chondrocyte apoptosis represents an important component in the osteoarthritis (OA) pathogenesis. This study sought to investigate the potential of polychlorinated biphenyl (PCB)126, the most potent and ubiquitous environmental pollutant of PCB congeners, on chondrocyte apoptosis and its mechanism of action. Rabbit articular chondrocytes cultured from tibial and femoral in cartilage were exposed to PCB126. Productions of reactive oxygen species (ROS) and nitric oxide (NO) and nuclear factor-kB (NF-kB) binding activity were measured. After 24h exposure to PCB126, the apoptotic cell death was detected by caspase-3 activity, enzyme-linked immunosorbent assay (ELISA) using antibodies against DNA and histone, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) staining. PCB126 generated ROS, which was blocked by the antioxidants (N-acetylcystein and trolox), or the aryl hydrocarbon receptor (AhR) inhibitor, α-naphthoflavone (α-NF). PCB126 exposure also increased NO production and NF-kB binding activity in the chondrocytes, which were blocked by the iNOS inhibitor, N-monomethyl-l-arginine (l-NMMA). All apoptosis detection techniques used in this study revealed an increase of apoptotic effects by PCB126 exposure, which was blocked by inhibitors of ROS or iNOS. This is the first report to demonstrate the potential of a PCB congener to induce chondrocytes apoptosis, which could be an initial process in cartilage degradation. PCB may be an initiator of chondrocyte apoptosis, which is closely linked to degradation of cartilage in OA pathogenesis. This study may contribute to identifying the possible causes of arthritis in our environment.