BACKGROUND & AIMS: OBJECTIVE:To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS:Expression of iNOS was quantified by quantitative reverse transcriptase-polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS:The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at -289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at -5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. CONCLUSION:These findings demonstrate the association between demethylation of specific NF-κB-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.

背景与目标：

BACKGROUND & AIMS: BACKGROUND:Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation--such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias)--generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. METHODS:Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. RESULTS:We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor). CONCLUSION:Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors.

背景与目标：

BACKGROUND & AIMS: :Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-1beta (0, 1, 10 ng/ml) for 48 h. IL-1beta enhanced expression of IGF-IR levels, as determined by both [125I]-IGF-I binding studies and Western blotting, while reducing the concentration of endogenous IGF-I detected in conditioned media by radioimmunoassay. Western ligand blotting revealed that chondrocytes primarily secreted IGF binding proteins (IGFBPs) with molecular weights of 28-30 and 32-34 kDa, which were identified as IGFBPs 5 and 2, respectively, and that IL-1beta treatment diminished IGFBP-2, the prominent homolog in conditioned media. Northern blot analysis suggested IL-1beta regulation of IGF-I and, to some extent, IGF-IR was mediated by transcription; however, the cytokine did not affect IGFBP-2 expression. To test for evidence of proteolysis by matrix metalloproteinases (MMPs), additional cultures were co-incubated with inhibitors for MMPs 2/9, 3, and 8. IGFBP-2 suppression was partially reversed by gelatinase (MMP-2/9) inhibition. In summary, these findings further delineate the role of IL-1 as a key regulator of the IGF-I system within articular cartilage, demonstrating that regulation occurs through both direct (transcriptional) and indirect (proteolytic) mechanisms.

背景与目标： : Interleukin-1 (IL-1) 和胰岛素样生长因子-I (igf-i) 对关节软骨内基质代谢具有相反作用，被认为在骨关节炎的发病机理中起着重要作用。为了更好地了解这些合成代谢 (igf-i) 和分解代谢 (IL-1) 刺激之间的联系，我们检查了关节软骨细胞 (ACs) 的igf-i信号系统的外源性IL-1调节。来自非关节炎窒息关节的马ACs在单层培养中扩增，在藻酸盐珠中封装10天，并用重组马IL-1beta (0,1，10 ng/ml) 刺激为高密度单层48小时。通过 [125I]-igf-i结合研究和Western印迹测定，IL-1beta增强了igf-ir水平的表达，同时降低了通过放射免疫测定法在条件培养基中检测到的内源性igf-i的浓度。Western配体印迹显示，软骨细胞主要分泌分子量为28-30和32-34 kDa的IGF结合蛋白 (IGFBPs)，分别被鉴定为IGFBPs 5和2，并且IL-1beta治疗减少了IGFBP-2，这是条件培养基中的主要同源物。Northern印迹分析表明igf-i的IL-1beta调节，并且在某种程度上，igf-ir是通过转录介导的; 然而，细胞因子不影响IGFBP-2的表达。为了测试基质金属蛋白酶 (mmp) 蛋白水解的证据，将另外的培养物与mmp 2/9、3和8的抑制剂共同孵育。IGFBP-2抑制被明胶酶 (MMP-2/9) 抑制部分逆转。总之，这些发现进一步描述了IL-1作为关节软骨内igf-i系统的关键调节因子的作用，表明调节通过直接 (转录) 和间接 (蛋白水解) 机制发生。

BACKGROUND & AIMS: :Alterations in cell morphology involve changes in the actin cytoskeleton and play crucial roles in determining chondrocyte phenotypes. Although the effects of simvastatin (SV) have been demonstrated in various cell types, the mechanisms and effects of SV on chondrocyte differentiation and actin cytoskeletal rearrangement are still unclear. Here, we investigated the roles of actin filament rearrangement on SV-induced differentiation of rabbit articular chondrocytes. Treatment with SV caused actin remodeling in comparison with that in untreated chondrocytes, as determined by immunofluorescence staining. Moreover, treatment with cytochalasin D (CD) and jasplakinolide (JAS), which modulate actin filament formation, resulted in reorganization of the actin cytoskeleton compared with that induced by SV in chondrocytes. In addition, CD synergistically enhanced the SV-induced increase in type II collagen expression, whereas JAS dramatically inhibited SV-induced differentiation. We also found that differentiation via SV-dependent actin cytoskeleton changes was regulated by the extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase pathways. These results demonstrated that actin cytoskeletal rearrangement by SV regulated type II collagen expression and suggested that ERK-1/2 and p38 kinase pathways may play important roles in SV-induced type II collagen expression by altering actin cytoskeletal reorganization in rabbit articular chondrocytes.

背景与目标： : 细胞形态的改变涉及肌动蛋白细胞骨架的改变，并在确定软骨细胞表型中起关键作用。尽管辛伐他汀 (SV) 的作用已在各种细胞类型中得到证实，但SV对软骨细胞分化和肌动蛋白细胞骨架重排的机制和作用仍不清楚。在这里，我们研究了肌动蛋白丝重排在SV诱导的兔关节软骨细胞分化中的作用。通过免疫荧光染色确定，与未处理的软骨细胞相比，SV治疗引起肌动蛋白重塑。此外，与SV诱导的软骨细胞相比，调节肌动蛋白丝形成的细胞松弛素D (CD) 和jasplakinolide (JAS) 治疗可导致肌动蛋白细胞骨架的重组。此外，CD协同增强SV诱导的II型胶原表达的增加，而JAS显着抑制SV诱导的分化。我们还发现，通过SV依赖性肌动蛋白细胞骨架变化的分化受细胞外信号调节激酶 (ERK)-1/2和p38激酶途径的调节。这些结果表明，SV引起的肌动蛋白细胞骨架重排调节了II型胶原的表达，并表明ERK-1/2和p38激酶途径可能通过改变兔关节软骨细胞的肌动蛋白细胞骨架重组在SV诱导的II型胶原表达中起重要作用。

BACKGROUND & AIMS: :Osteoarthritis (OA) is a chronic and prevalent degenerative musculoskeletal disorder, which is characterized by articular cartilage degradation and joint inflammation. MicroRNA-203a (miR-203a) has been shown to be involved in multiple pathological processes during OA, but little is known about its function in chondrocyte extracellular matrix (ECM) degradation. In the present study, we aimed to elucidate the effects of miR-203a on articular cartilage degradation and joint inflammation. We observed that the miR-203a level was significantly up-regulated in OA tissues and in an in vitro model of OA, respectively. Inhibition of miR-203a significantly alleviated the interleukin (IL)-1β-induced inflammatory response and ECM degradation in chondrocytes. Moreover, mothers against decapentaplegic homolog 3 (Smad3), a key factor in maintaining chondrocyte homeostasis, was identified as a putative target of miR-203a in chondrocytes. More importantly, inhibition of Smad3 impaired the inhibitory effects of the miR-203a on IL-1β-induced inflammatory response and ECM degradation. Collectively, these results demonstrated that miR-203a may contribute to articular cartilage degradation of OA by targeting Smad3, suggesting a novel therapeutic target for the treatment of OA.

背景与目标： : 骨关节炎 (OA) 是一种慢性且普遍的退行性肌肉骨骼疾病，其特征是关节软骨退化和关节炎症。MicroRNA-203a (miR-203a) 已被证明参与OA期间的多个病理过程，但对其在软骨细胞细胞外基质 (ECM) 降解中的功能知之甚少。在本研究中，我们旨在阐明miR-203a对关节软骨退化和关节炎症的影响。我们观察到，在OA组织和OA的体外模型中，miR-203a水平分别显着上调。抑制miR-203a可显著减轻白细胞介素 (IL)-1β 诱导的软骨细胞炎症反应和ECM降解。此外，抗去胸截瘫同源物3 (Smad3) 是维持软骨细胞稳态的关键因素，被确定为软骨细胞miR-203a的推定靶标。更重要的是，Smad3的抑制损害了miR-203a对il-1β 诱导的炎症反应和ECM降解的抑制作用。总的来说，这些结果表明，miR-203a可能通过靶向Smad3来促进OA的关节软骨降解，从而为OA的治疗提供了新的治疗靶标。

BACKGROUND & AIMS: The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC alpha was essentially the same as that of type II collagen during de- and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC alpha, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC alpha in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC alpha did not affect type II collagen levels, suggesting that PKC alpha expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by down-regulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of N-cadherin, alpha-catenin, and beta-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.

背景与目标： 软骨细胞的分化表型在体外培养过程中通过称为 “去分化” 的过程迅速丧失。在这项研究中，我们研究了蛋白激酶C (PKC) 和细胞外信号调节蛋白激酶 (ERK) 在维持分化的软骨细胞表型中的作用。从兔关节软骨分离的软骨细胞在连续单层培养后进行去分化，停止II型胶原表达和蛋白聚糖合成，这通过在藻酸盐凝胶中培养去分化细胞而被逆转。PKC α 在去分化和再分化过程中的表达模式与II型胶原的表达模式基本相同，因为在去分化过程中表达降低，而在再分化过程中表达增加。与PKC α 相反，去分化过程中ERK活性增加了15倍。这种增强的活性在再分化过程中终止。0代软骨细胞中PKC α 的下调导致去分化。然而，PKC α 的过表达并未影响II型胶原水平，这表明PKC α 的表达不足以维持分化的表型。然而，PD98059对ERK的抑制增强了第0代细胞中II型胶原的表达和蛋白聚糖的合成，延缓了单层培养过程中的去分化，并逆转了PKC下调引起的去分化。与PKC依赖性的ERK调节软骨形成不同，PKC和ERK独立调节软骨细胞去分化，这通过观察证实，PKC下调和ERK抑制分别不会改变ERK磷酸化和PKC表达。此外，在表型改变过程中与II型胶原相反调节的N-钙粘蛋白，α-连环蛋白和 β-连环蛋白的表达在软骨形成过程中受到PKC和ERK的调节，但没有去分化，支持这两种蛋白激酶调节软骨细胞分化和维持分化表型的独特机制。

BACKGROUND & AIMS: :Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2alpha; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10(-8) to 10(-4)M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G(0)/G(1) phase. Upon a 24h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10(-4)M). Replenishments of PGE1, PGE2 or PGF2alpha could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG-independent mechanisms. COX-2 selective NSAIDs showed less deleterious effects on chondrocytic proliferation and death.

背景与目标： : 以前的报道表明非甾体抗炎药 (NSAIDs) 抑制骨修复。我们先前的研究进一步发现，酮洛尔可延迟软骨内骨的形成，并且关键的有效时机是在修复的早期。此外，我们发现NSAIDs抑制了培养的成骨细胞的增殖并诱导了细胞死亡。在这项研究中，我们假设NSAIDs可能会影响软骨细胞的增殖和死亡，这在软骨内骨形成的早期阶段起着重要作用。非选择性NSAIDs，吲哚美辛，酮咯酸，双氯芬酸和吡罗昔康; cyclooxygenase-2 (COX-2) 选择性NSAIDs，塞来昔布和DFU (罗非考昔的类似物); 前列腺素 (PGs)，PGE1，PGE2和pgf2α; 以及每个NSAID加上每个PG。研究了NSAIDs对胎鼠epi关节软骨细胞增殖，细胞周期动力学，细胞毒性和细胞死亡的影响。结果表明，除DFU外，所有测试的NSAIDs均在覆盖理论治疗浓度的浓度范围 (10(-8) 至10(-4)M) 下抑制胸苷掺入软骨细胞。NSAIDs在G(0)/G(1) 期阻止了细胞周期。处理24小时后，软骨细胞培养物中的四种非选择性NSAIDs显着诱导了LDH泄漏和细胞死亡 (凋亡和坏死)。然而，除了较高浓度的塞来昔布 (10(-4)M) 外，COX-2抑制剂对软骨细胞的细胞毒性无显着影响。补充PGE1，PGE2或PGF2alpha不能逆转NSAIDs对软骨细胞增殖和细胞毒性的影响。在这项研究中，我们发现治疗浓度的非选择性NSAIDs导致软骨细胞增殖抑制和细胞死亡，这可能是先前研究发现的NSAIDs延迟骨修复过程中软骨内骨化的原因之一。此外，NSAIDs的这些作用可能通过PG独立机制起作用。COX-2选择性NSAIDs对软骨细胞增殖和死亡的有害作用较小。

BACKGROUND & AIMS: :Estrogen hormones are important for cartilage homeostasis, but nothing is known regarding the expression and role of the membrane G protein-coupled estrogen receptor (GPER), G protein-coupled receptor 30 (GPR30), in adult articular chondrocytes. Using immunohistochemistry of cartilage sections, quantitative real-time polymerase chain reaction and Western blot of chondrocyte extracts, we found that these cells express GPR30. Nonetheless, the pattern of bands detected by two distinct antibodies does not overlap, suggesting that the proteins detected represent partially degraded forms of the receptor. Treatment with GPR30 agonists did not induce Akt or ERK1/2 phosphorylation, two known GPR30-activated signaling pathways, suggesting that GPR30 is not functional in human chondrocytes. Therefore, the protective anti-osteoarthritic role of estrogen hormones in cartilage homeostasis is likely independent of GPR30. This study was performed using human cartilage collected from the distal femoral condyles of multiorgan donors at the Bone and Tissue Bank of the University and Hospital Center of Coimbra.

背景与目标： : 雌激素激素对软骨稳态很重要，但关于成年关节软骨细胞中膜g蛋白偶联雌激素受体 (GPER)，g蛋白偶联受体30 (GPR30) 的表达和作用尚不清楚。使用软骨切片的免疫组织化学，实时定量聚合酶链反应和软骨细胞提取物的蛋白质印迹，我们发现这些细胞表达gpr30。尽管如此，两种不同抗体检测到的条带模式不会重叠，这表明检测到的蛋白质代表了受体的部分降解形式。用GPR30激动剂治疗不会诱导Akt或ERK1/2磷酸化，这是两种已知的GPR30-activated信号通路，表明GPR30在人软骨细胞中不起作用。因此，雌激素在软骨稳态中的保护性抗骨关节炎作用可能与gpr30无关。这项研究是使用从科英布拉大学和医院中心的骨骼和组织库的多器官供体的股骨远端con收集的人类软骨进行的。

BACKGROUND & AIMS: OBJECTIVE:To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. METHODS:Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. RESULTS:Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. CONCLUSION:Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.

背景与目标：

BACKGROUND & AIMS: BACKGROUND:This study aimed to investigate the role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) and related molecular mechanisms, in osteoarthritis (OA). METHODS:Cartilage tissues of OA patients and healthy volunteers were isolated and cultured. After transfection with the appropriate constructs, chondrocytes were classified into Blank, pcDNA3.1-NC, pcDNA3.1-MEG3, si-NC, si-MEG3, pcDNA3.1-NC + mimics NC, pcDNA3.1-MEG3 + mimics NC, pcDNA3.1-NC + miR-361-5p mimics and pcDNA3.1-MEG3 + miR-361-5p mimics groups. qRT-PCR was used to detect the expression of MEG3, miR-361-5p and FOXO1. Western blot, luciferase reporter assay, RIP, CCK-8, and flow cytometry analysis were performed to reveal the morphology, proliferation, and apoptotic status of cartilage cells. Histological analysis and immunostaining were conducted in the OA rat model. RESULTS:Expression of MEG3 and FOXO1 was significantly decreased in OA compared with the normal group, while the expression of miR-361-5p was increased. MEG3 might serve as a ceRNA of miR-361-5p in OA chondrocytes. Moreover, using western blot analyses and the CCK-8 assay, MEG3 was shown to target miR-361-5p/FOXO1, elevate cell proliferation, and impair cell apoptosis. Functional analysis in vivo showed that MEG3 suppressed degradation of the cartilage matrix. CONCLUSION:MEG3 can contribute to cell proliferation and inhibit cell apoptosis and degradation of extracellular matrix (ECM) via the miR-361-5p/FOXO1 axis in OA chondrocytes.

背景与目标：

BACKGROUND & AIMS: OBJECTIVE:Metabolic disorders and inflammation of chondrocytes are major pathological changes in aging cells and osteoarthritis (OA). Recent studies demonstrated age-related mitochondrial dysfunction may be a key contributing factor in the development of OA. Mitofusin 2 (MFN2) is a key regulator of mitochondrial fusion, cell metabolism, autophagy and apoptosis. This study was performed to ascertain whether MFN2 was involved in the aging of chondrocytes and OA. METHODS:Metabolic measurements were taken in rat chondrocytes between different ages (3-week, 5-month, 12-month). MFN2 activity was detected in both human and rat chondrocytes during aging and OA. Then, knockdown of MFN2 with small interfering RNA (siRNA) was performed to confirm whether MFN2 contributes to metabolic changes. Lentiviruses were used to establish MFN2-overexpression/knockdown OA models both in vivo and in vitro to confirm whether MFN2 contributes to OA progress. Further, regulatory mechanism of MFN2 was assessed and interaction between MFN2 and PARKIN was performed. RESULTS:A metabolic shift to mitochondrial respiration was confirmed in rat chondrocytes during aging. MFN2 expression was elevated in both human and rat chondrocytes during aging and OA. Knockdown of MFN2 with siRNA reversed the age-related metabolic changes in rat chondrocytes. Overexpression of MFN2 exacerbated inflammation and OA progress, while knockdown of MFN2 ameliorated inflammation and OA progress. Further, MFN2 could be ubiquitinated by PARKIN, declined PARKIN expression during aging and OA might result in elevated MFN2 expression. CONCLUSIONS:Elevated MFN2 contributes to metabolic changes and inflammation during aging of rat chondrocytes and osteoarthritis.

背景与目标：

BACKGROUND & AIMS: PURPOSE:Accumulating evidence suggests that microRNA-145 (miR-145) plays an important role in osteoarthritis (OA), which is a chronic progressive joint disease. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes metastasis in cancers and functions as a sponge for miR-145. However, the role of MALAT1/miR-145 in OA pathogenesis has not yet been elucidated. MATERIALS AND METHODS:The expression of MALAT1 and miR-145 was examined by quantitative real-time PCR; the interaction between miR-145, MALAT1 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was verified by luciferase reporter assay. Correlations among MALAT1, miR-145, and ADAMTS5 were analyzed by Spearman rank analysis. Chondrocytes viability and cartilage extracellular matrix (ECM) degradation were investigated with cell viability assay and Western blotting analyzing expression of ADAMTS5, collagen type 2 alpha 1 (COL2A1), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). RESULTS:MALAT1 was upregulated, and miR-145 was downregulated in OA samples and IL-1β-induced chondrocytes. Mechanically, miR-145 could directly bind to MALAT1 and ADAMTS5. Moreover, miR-145 expression was negatively correlated with MALAT1 and ADAMTS5 expression in OA patients, whereas MALAT1 and ADAMTS5 expression was positively correlated. Functionally, overexpression of MALAT1 inhibited chondrocyte viability and promoted cartilage ECM degradation in IL-1β-induced chondrocytes. In support thereof, MALAT1 silencing and miR-145 upregulation exerted the opposite effect in IL-1β-induced chondrocytes. Moreover, the effect of MALAT1 was counteracted by miR-145 upregulation, and ADAMTS5 restoration could abate miR-145 effects. CONCLUSION:An MALAT1/miR-145 axis contributes to ECM degradation in IL-1β-induced chondrocytes through targeting ADAMTS5, suggesting that MALAT1/miR-145/ADAMTS5 signaling may underlie human OA pathogenesis.

背景与目标：

BACKGROUND & AIMS: OBJECTIVE:This study aimed to explore potential microRNAs (miRNAs), which participate in the pathological process of condylar hyperplasia (CH) through targeting specific proliferation- and apoptosis- related genes of chondrocytes. METHODS:Insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R) and B-cell CLL/lymphoma 2 (BCL2) in CH cartilage were detected by real-time polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence. MiRanda and TargetScanS algorithms were used to predict certain miRNAs in CH chondrocytes concurrently modulating the above three genes. MiR-15b was screened and identified using real-time PCR. After transfection of miR-15b mimics or inhibitor into CH chondrocytes, expression of the above three genes was detected by real-time PCR and western blot, meanwhile, cell proliferation and apoptosis was examined by CCK8, cell cycle assays, flow cytometry and Hoechst staining. Dual luciferase activity was performed to identify the direct regulation of miR-15b on IGF1, IGF1R and BCL2. RESULTS:Expression of IGF1, IGF1R and BCL2 increased in CH cartilage. Seven microRNAs concurrently correlated with IGF1, IGF1R and BCL2. Among them, only miR-15b significantly changed in CH chondrocytes. Overexpression of miR-15b in CH chondrocytes suppressed the expression of IGF1, IGF1R and BCL2, while it increased when miR-15b was knockdown. Furthermore, miR-15b suppressed their expression by directly binding to its 3'-UTR in these cells. Besides, miR-15b hampered chondrocytes proliferation through targeting IGF1 and IGF1R and accelerated chondrocytes apoptosis through targeting BCL2. CONCLUSION:Suppressed miR-15b contributed to enhanced proliferation capacity and weakened apoptosis of chondrocytes through augmentation of IGF1, IGF1R and BCL2, thereby resulting in development of CH.

背景与目标：

BACKGROUND & AIMS: :Proinflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) enhance degradation of cartilage-specific, type II collagen by matrix metalloproteinase-13 (MMP-13). We investigated the previously unknown role of H-Ras and reactive oxygen species (ROS) in the cytokine induction of MMP-13 gene expression in human articular chondrocytes by using pharmacological inhibitors, RNA interference (RNAi) and antioxidants. Manumycin A, an inhibitor of H-Ras farnesylation by farnesyltransferase, suppressed IL-1β- and TNF-α-induced MMP-13 mRNA and protein expression. Small interfering RNA (siRNA)-mediated H-Ras silencing down-regulated MMP-13 mRNA and protein induction by IL-1β and TNF-α. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase/NOX) inhibitor, diphenyleneiodonium (DPI) suppressed cytokine-induced MMP-13 expression and superoxide production. Apocynin, another NOX inhibitor, also diminished MMP-13 induction. Deoxyglucose an antimetabolite of glucose metabolism reduced MMP-13 increase. Role of NOX-mediated ROS production was reaffirmed by the observation that the antioxidants, trolox, nordihydroguaiaretic acid (NDGA), quercetin and resveratrol downregulated cytokine-induced MMP-13 mRNA and protein expression. These results provide strong pharmacological and genetic evidence for the implication of H-Ras and NADPH oxidase-generated superoxide production in MMP-13 gene regulation by IL-1β and TNF-α. These proteins could be potentially targeted for therapeutic inhibition of MMP-13-driven cartilage erosion by using H-Ras and NOX inhibitors and antioxidants.

背景与目标： : 促炎性细胞因子，例如interleukin-1 β (IL-1β) 和肿瘤坏死因子 α (TNF-α)，可增强基质metalloproteinase-13 (MMP-13) 对软骨特异性II型胶原蛋白的降解。我们通过使用药理学抑制剂，RNA干扰 (RNAi) 和抗氧化剂研究了H-Ras和活性氧 (ROS) 在细胞因子诱导人关节软骨细胞MMP-13基因表达中的作用。Manumycin A是一种通过法尼基转移酶对H-Ras法尼基化的抑制剂，可抑制IL-1β 和TNF-α 诱导的MMP-13 mRNA和蛋白表达。小干扰RNA (siRNA) 介导的H-Ras沉默通过IL-1β 和TNF-α 下调的MMP-13 mRNA和蛋白质诱导。烟酰胺腺嘌呤二核苷酸磷酸氧化酶 (NADPH氧化酶/NOX) 抑制剂，二苯乙腈 (DPI) 抑制细胞因子诱导的MMP-13表达和超氧化物产生。另一种NOX抑制剂Apocynin也减少了MMP-13诱导。脱氧葡萄糖葡萄糖代谢的抗代谢物MMP-13增加而降低。通过观察抗氧化剂，trolox，去甲二氢愈创木酸 (NDGA)，槲皮素和白藜芦醇下调细胞因子诱导的MMP-13 mRNA和蛋白表达，重申了NOX介导的ROS产生的作用。这些结果为H-Ras和NADPH氧化酶产生的超氧化物产生在IL-1β 和TNF-α MMP-13基因调控中的意义提供了强有力的药理学和遗传学证据。通过使用H-Ras和NOX抑制剂和抗氧化剂，这些蛋白质可以潜在地靶向治疗性抑制MMP-13-driven软骨侵蚀。

BACKGROUND & AIMS: :Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H2 to PGE2 downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription-polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH2 to PGE2 was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-gamma, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1beta or TNF-alpha. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF1alpha and thromboxane B2, the production of PGE2 was greater after chondrocytes were stimulated by IL-1beta or TNF-alpha. Conversion of PGH2 to PGE2 (PGES activity) was significantly increased in the lysate from IL-1beta-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE2 synthesis in patients with OA.

背景与目标： : 前列腺素E合酶 (PGES)，包括膜相关PGES (mPGES)-1，mPGES-2和胞质PGES (cPGES) 的同工酶，是最近发现的花生四烯酸级联反应的末端酶。PGES将前列腺素 (PG)H2转化为环氧合酶 (COX) 下游的PGE2。我们研究了骨关节炎 (OA) 患者关节软骨细胞中PGES同工酶的表达。用各种细胞因子处理软骨细胞，并通过逆转录聚合酶链反应和Northern印迹分析PGES同工酶mRNA的表达,而蛋白质印迹用于蛋白质表达。mPGES-1的亚细胞定位通过免疫荧光显微镜确定。花生四烯酸或PGH2向PGE2的转化通过酶联免疫吸附法测量。最后，免疫组织化学检测mPGES-1蛋白在OA关节软骨中的表达。白介素 (IL)-1β 或肿瘤坏死因子 (TNF)-α 可显着诱导软骨细胞mPGES-1 mRNA的表达，而其他细胞因子，如IL-4，IL-6，IL-8，IL-10和干扰素-γ，没有作用。COX-2在相同条件下也被诱导，尽管其表达模式不同。cPGES的表达，mPGES-2，并且COX-1 mRNA不受IL-1beta或TNF-α 的影响。mPGES-1和COX-2的亚细胞定位在核周区域几乎重叠。与6-keto-pgf1α 和血栓烷B2相比，IL-1beta或TNF-α 刺激软骨细胞后，PGE2的产生更大。IL-1beta-stimulated软骨细胞裂解液中PGH2向PGE2的转化 (PGES活性) 显着增加，并被MK-886抑制。对mPGES-1活性有抑制作用。OA患者关节软骨中的软骨细胞对mPGES-1的免疫染色呈阳性。这些结果表明，mPGES-1可能在OA的发病机理中很重要。它也可能是特异性调节OA患者PGE2合成的治疗策略的潜在新靶标。