• Applied Microscopy
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• 제24권1호
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• pp.59-76
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• 1994

Fine structure of the distal femoral epiphysis of growing mouse was studied by electron microscopy. The first morphological evidence of developing secondary center of ossification in the distal femoral epiphysis was found at newborn mouse. Ossification center was in the form of multiple foci of calcification and its cells were represented by remnant of degenerated cells within large lacunae that were separated by mineralized cartilaginous septa. Endochondral ossification beneath the articular cartilage proceeded in a less orderly manner than metaphyseal endochondral ossification. Columns of hypertrophied chondrocytes were not distinctly parallel to intercellular mineralized septa in all direction. Hypertrophied chondrocytes in the inner zone of the epiphseal center of ossification showed disintegrated. Resorption of mineralized cartilaginous septa was undertaken by perivascular cells and multinucleated chondroclasts. Resorption of the calcified cartilage was restricted to the region of ruffled border of the chondroclast. Growth along the metaphyseal side of the epiphyseal center of ossification was different from that along the articular surface. As the secondary center expanded toward the metaphyseal side, many vascular buds penetrated unmineralized cartilaginous septa and invaded viable chondrocytes. Many hypertrophied chondrocytes bodering the metaphyseal side of bone center remained viable after they became embedded in mineralized cartilaginous septa. This result suggested that the hypertrophied.

• 생명과학회지
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• 제21권4호
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• pp.486-493
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• 2011

Nitric oxide (NO) donors는 heme oxygenase-1 (HO-1)의 강력한 유도제이다. 그러나 NO donors에 의한 HO-1의 발현이 NO donor에 의해 방출되는 NO에 의한 직접적인 영향인지는 불분명하다. 본 연구에서 흰쥐의 무릎으로부터 분리 배양한 관절연골세포에서 HO-1의 발현에 NO donors의 영향을 조사하였다. NO donors (SIN-1, SNAP 그리고 SNP)는 HO-1의 mRNA와 단백질의 합성을 크게 증가시켰다. 그리고 NO의 표적 분자인 guanylate cyclase와 protein kinase G의 관련성을 살펴본 결과, NO donors에 의한 Nrf2와 HO-1의 발현증가와는 무관한 것으로 보였다. 흥미롭게도, NO scavenger인 carboxy-PTIO와 SOD mimetic TEMPOL은 NO donors에 의한 HO-1의 발현을 억제하였다. 게다가, peroxynitrite scavenger인 MnTBAP에 의해서도 Nrf2와 HO-1의 발현이 완전히 억제되었다. Peroxynitrite는 NO와 superoxide의 반응에 의해 세포 내에서 자연적으로 형성되는 물질이므로 peroxynitrite가 관절연골세포에서 HO-1의 발현에 직접적인 영향을 주는지를 관찰하였다. 관절연골세포에 peroxynitrite를 처리한 결과, 시간과 농도 의존적으로 Nrf2와 HO-1의 발현을 크게 증가시켰다. 본 실험 자료 는 NO donors에 의한 HO-1의 발현증가는 방출되는 NO의 직접적인 영향이라기 보다는 NO와 superoxide의 반응으로 형성되는 peroxynitrite에 의해 유도된다는 것을 시사한다.

• BMB Reports
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• 제51권10호
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• pp.508-513
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• 2018

Fibronectin fragments found in the synovial fluid of patients with osteoarthritis (OA) induce the catabolic responses in cartilage. Nuclear high-mobility group protein Box 1 (HMGB1), a damage-associated molecular pattern, is responsible for the regulation of signaling pathways related to cell death and survival in response to various stimuli. In this study, we investigated whether changes induced by 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) in HMGB1 expression influences the pathogenesis of OA via an HMGB1-modulated autophagy signaling pathway. Human articular chondrocytes were enzymatically isolated from articular cartilage. The level of mRNA was measured by quantitative real-time PCR. The expression of proteins was examined by western blot analysis, immnunofluorescence assay, and enzyme-linked immunosorbent assay. Interaction of proteins was evaluated by immunoprecipitation. The HMGB1 level was significantly lower in human OA cartilage than in normal cartilage. Although 29-kDa FN-f significantly reduced the HMGB1 expression at the mRNA and protein levels 6 h after treatment, the cytoplasmic level of HMGB1 was increased in chondrocytes treated with 29-kDa FN-f, which significantly inhibited the interaction of HMGB1 with Beclin-1, increased the interaction of Bcl-2 with Beclin-1, and decreased the levels of Beclin-1 and phosphorylated Bcl-2. In addition, the level of microtubule-associated protein 1 light chain 3-II, an autophagy marker, was down-regulated in chondrocytes treated with 29-kDa FN-f, whereas the effect was antagonized by mTOR knockdown. Furthermore, prolonged treatment with 29-kDa FN-f significantly increased the release of HMGB1 into the culture medium. These results demonstrated that 29-kDa FN-f inhibits chondrocyte autophagy by modulating the HMGB1 signaling pathway.

• BMB Reports
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• 제54권10호
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• pp.528-533
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• 2021

Osteoarthritis (OA) is a degenerative disorder that can result in the loss of articular cartilage. No effective treatment against OA is currently available. Thus, interest in natural health products to relieve OA symptoms is increasing. However, their qualities such as efficacy, toxicity, and mechanism are poorly understood. In this study, we determined the efficacy of avenanthramide (Avn)-C extracted from oats as a promising candidate to prevent OA progression and its mechanism of action to prevent the expression of matrix-metalloproteinases (MMPs) in OA pathogenesis. Interleukin-1 beta (IL-1β), a proinflammatory cytokine as a main causing factor of cartilage destruction, was used to induce OA-like condition of chondrocytes in vitro. Avn-C restrained IL-1β-mediated expression and activity of MMPs, such as MMP-3, -12, and -13 in mouse articular chondrocytes. Moreover, Avn-C alleviated cartilage destruction in experimental OA mouse model induced by destabilization of the medial meniscus (DMM) surgery. However, Avn-C did not affect the expression of inflammatory mediators (Ptgs2 and Nos) or anabolic factors (Col2a1, Aggrecan, and Sox9), although expression levels of these genes were upregulated or downregulated by IL-1β, respectively. The inhibition of MMP expression by Avn-C in articular chondrocytes was mediated by p38 kinase and c-Jun N-terminal kinase (JNK) signaling, but not by ERK or NF-κB. Interestingly, Avn-C added with SB203580 and SP600125 as specific inhibitors of p38 kinase and JNK, respectively, enhanced its inhibitory effect on the expression of MMPs in IL-1β treated chondrocytes. Taken together, these results suggest that Avn-C is an effective candidate to prevent OA progression and a natural health product to relieve OA pathogenesis.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1258-1266
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• 2005

The present study was carried out in order to assess the protective effects of calycosin-7-O-$\beta$-D-glucopyranoside, isolated from Astragali radix (AR), on hyaluronidase (HAase) and the recombinant human interleukin-$1\beta$ (IL-$1\beta$)-induced matrix degradation in human articular cartilage and chondrocytes. We isolated the active component from the n-butanol soluble fraction of AR (ARBu) as the HAase inhibitor and structurally identified as calycosin-7-O-$\beta$-D-glucopyranoside by LC-MS, IR, ${1}^H$ NMR, and ${13}^C$ NMR analyses. The $IC_{50}$ of this component on HAase was found to be 3.7 mg/ml by in vitro agarose plate assay. The protective effect of ARBu on the matrix gene expression of immortalized chondrocyte cell line C28/I2 treated with HAase was investigated using a reverse transcription polymerase chain reaction (RT-PCR), and its effect on HAase and IL-$1\beta$-induced matrix degradation in human articular cartilage was determined by a staining method and calculating the amount of degraded glycosaminoglycan (GAG) from the cultured media. Pretreatment with calycosin-7-O-$\beta$-D-glucopyranoside effectively protected human chondrocytes and articular cartilage from matrix degradation. Therefore, calycosin-7-O-$\beta$-D-glucopyranoside from AR appears to be a potential natural ant-inflammatory or antii-osteoarthritis agent and can be effectively used to protect from proteoglycan (PG) degradation.

• Macromolecular Research
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• 제17권9호
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• pp.703-708
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• 2009

Silk fibroin scaffolds were examined as a biomaterial option for tissue-engineered cartilage-like tissue. In tissue engineering for cartilage repair using a scaffold, initial chondrocyte-material interactions are important for the following cell behaviors. In this study, the surface of nanofibrous silk fibroin (NSF) meshes was modified by a microwave-induced argon plasma treatment in order to improve the cytocompatibility of the meshes used as cartilaginous grafts. In addition, the effects of a plasma treatment on the cellular behavior of chondrocytes on NSF were examined. The plasma treatment resulted in an increase in the hydrophilicity of NSF meshes suggesting that the cytocompatibility of the mesh might be improved. Furthermore, the human articular chondrocytes showed higher viability on the surface-modified NSF meshes. These results suggest that the surface modification of NSF meshes by plasma can enhance the cellular behavior of chondrocytes and may be used in tissue engineering.

• Journal of Veterinary Science
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• 제19권6호
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• pp.735-743
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• 2018

We investigated the effect of transforming growth factor beta 1 ($TGF-{\beta}1$) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with $TGF-{\beta}1$ at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of $TGF-{\beta}1$. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by $TGF-{\beta}1$ stimulation was dose and time dependent. $TGF-{\beta}1$ was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in $TGF-{\beta}1$ treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the $TGF-{\beta}1$-treated scaffolds. Together, our results suggest that $TGF-{\beta}1$ has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

• Journal of Ginseng Research
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• 제36권2호
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• pp.161-168
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• 2012

The abnormal maturation and ossification of articular chondrocytes play a central role in the pathogenesis of osteoarthritis (OA). Inhibiting the enzymatic degradation of the extracellular matrix and maintaining the cellular phenotype are two of the major goals of interest in managing OA. Ginseng is frequently taken orally, as a crude substance, as a traditional medicine in Asian countries. Ginsenoside $Rb_1$, a major component of ginseng that contains an aglycone with a dammarane skeleton, has been reported to exhibit various biological activities, including anti-inflammatory and anti-tumor effects. However, a chondroprotective effect of ginsenoside $Rb_1$ related to OA has not yet been reported. The purpose of this study was to demonstrate the chondroprotective effect of ginsenoside $Rb_1$ on the regulation of pro-inflammatory factors and chondrogenic genes. Cultured rat articular chondrocytes were treated with 100 ${\mu}M$ ginsenoside $Rb_1$ and/or 500 ${\mu}M$ hydrogen peroxide ($H_2O_2$) and assessed for viability, reactive oxygen species production, nitric oxide (NO) release, and chondrogenic gene expression. Ginsenoside $Rb_1$ treatment resulted in reductions in the levels of pro-inflammatory cytokine and NO in $H_2O_2$-treated chondrocytes. The expression levels of chondrogenic genes, such as type II collagen and SOX9, were increased in the presence of ginsenoside $Rb_1$, whereas the expression levels of inflammatory genes related to chondrocytes, such as MMP1 and MMP13, were reduced by approximately 50%. These results suggest that ginsenoside $Rb_1$ has potential for use as a therapeutic agent in OA patients.

• 대한의생명과학회지
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• 제15권2호
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• pp.141-146
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• 2009

Paclitaxel, an antimicrotubule agent, binds to beta-tubulin in the microtubule and stabilizes the polymer, thereby repressing dynamic instability. Here, we have demonstrated that microtubule cytoskeletal architecture involved in regulation of the COX-2 expression in chondrocyte treated with paclitaxel. Paclitaxel enhanced COX-2 expression and prostaglandin E2 production, as indicated by the Western blot analysis, reverse transcriptase PCR(RT-PCR) and immunofluorescence staining, and $PGE_2$ assay, respectively. In our previous data have shown that paclitaxel treatment stimulated activation of ERK-1/2 and p38 kinase(Im et al., 2009). SB203580, an inhibitor of p38 kinase, blocked the induction of COX-2 expression by paclitaxel. Also PD98059, an inhibitor of ERK-1/2 kinase was blocked the induced COX-2 expression. These results indicate that activation of ERK-1/2 and p38 kinase is required for COX-2 expression induced by paclitaxel in rabbit articular chondrocytes.

• 대한의생명과학회지
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• 제15권1호
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• pp.67-72
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• 2009

Microtubule-interfering agents (MIAs), including paclitaxel, have been attributed in part to interference with microtubule assembly, impairment of mitosis, and changes in cytoskeleton. But the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. This study investigated the effect of paclitaxel on differentiation such as type II collagen expression and sulfated proteoglycan accumulation in rabbit articular chondrocytes. Paclitaxel caused differentiated chondrocyte phenotype as demonstrated by increment of type II collagen expression and proteoglycan synthesis Paclitaxel treatment stimulated activation of ERK-1/2 and p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced paclitaxel-induced differentiation, whereas inhibition of p38 kinase with SB203580 suppressed paclitaxel-induced differentiation. Our findings suggest that ERK-1/2 and p38 kinase oppositely regulate paclitaxel-induced differentiation in chondrocytes.