Aims. Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism. Methods. In vitro, interleukin-1 beta (IL-1β) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage. Results. DHCA prevented iNOS and IL-6 from being upregulated by IL-1β. Moreover, the IL-1β-induced upregulation of MMPs could be inhibited by DHCA. Additionally, the administration of DHCA counteracted IL-1β-induced downregulation of aggrecan, collagen II, and SOX9. DHCA protected articular cartilage by blocking the NF-κB and
Aims. The role of N,N-dimethylformamide (DMF) in diabetes-induced osteoporosis (DM-OS) progression remains unclear. Here, we aimed to explore the effect of DMF on DM-OS development. Methods. Diabetic models of mice, RAW 264.7 cells, and bone marrow macrophages (BMMs) were established by streptozotocin stimulation, high glucose treatment, and receptor activator of nuclear factor-κB ligand (RANKL) treatment, respectively. The effects of DMF on DM-OS development in these models were examined by micro-CT analysis, haematoxylin and eosin (H&E) staining, osteoclast differentiation of RAW 264.7 cells and BMMs, H&E and tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA) of TRAP5b and c-terminal telopeptides of type 1 (CTX1) analyses, reactive oxygen species (ROS) analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Cell Counting Kit-8 (CCK-8) assay, and Western blot. Results. The established diabetic mice were more sensitive to ovariectomy (OVX)-induced osteoporosis, and DMF treatment inhibited the sensitivity. OVX-treated diabetic mice exhibited higher TRAP5b and c-terminal telopeptides of type 1 (CTX1) levels, and DMF treatment inhibited the enhancement. DMF reduced RAW 264.7 cell viability. Glucose treatment enhanced the levels of TRAP5b, cathepsin K, Atp6v0d2, and H. +. -ATPase, ROS, while DMF reversed this phenotype. The glucose-increased protein levels were inhibited by DMF in cells treated with RANKL. The expression levels of antioxidant enzymes Gclc, Gclm, Ho-1, and Nqo1 were upregulated by DMF. DMF attenuated high glucose-caused osteoclast differentiation by targeting mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signalling in BMMs. Conclusion. DMF inhibits high glucose-induced osteoporosis by targeting
Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology.Aims
Methods
Bisphosphonates play an important role in the treatment of catabolic bone diseases such as osteoporosis. In addition to their anti-resorptive activity exerted by their proapoptotic effect on osteoclasts, recent data suggest that nitrogen-containing bisphosphonates (N-BP) may also promote osteogenic differentiation by an unknown mechanism. Similar bone-anabolic effects have been attributed to cholesterol-lowering statins, which represent another class of mevalonate pathway inhibitors besides N-BP, suggesting a common mode of action. In vascular endothelial cells statins were recently shown to activate the Mek5/Erk5 mitogen-activated protein kinase cascade, which plays an important role in cellular differentiation, apoptosis or inflammatory processes. Here we evaluated whether N-BPs may also target the Mek5/Erk5 pathway and analysed the consequence of Erk5 activation on bone-relevant gene expression, calcification and osteoblast differentiation. We show that N-BP dose-dependently activate Erk5 in primary human endothelial cells and osteoblasts. The mechanism likely involves farnesyldiphosphate synthase (FDPS) inhibition and subsequent inactivation of the small GTPase Cdc42 since siRNA-mediated knockdown of both genes could reproduce N-BP-induced ERK5 activation. ERK5 activation resulted in regulation of several bone-relevant genes and was required for calcification and osteoblastic differentiation of mesenchymal stems cells as evident by the lack of alkaline phosphatase induction and alizarin-red staining observed upon Erk5 knockdown or upon differentiation initiation in presence of a pharmacological Erk5 inhibitor.Background/Aims
Methods and Results
Aims. Tert-butylhydroquinone (tBHQ) has been identified as an inhibitor of oxidative stress-induced injury and apoptosis in human neural stem cells. However, the role of tBHQ in osteoarthritis (OA) is unclear. This study was carried out to investigate the role of tBHQ in OA. Methods. OA animal model was induced by destabilization of the medial meniscus (DMM). Different concentrations of tBHQ (25 and 50 mg/kg) were intraperitoneally injected in ten-week-old female mice. Chondrocytes were isolated from articular cartilage of mice and treated with 5 ng/ml lipopolysaccharide (LPS) or 10 ng/ml interleukin 1 beta (IL-1β) for 24 hours, and then treated with different concentrations of tBHQ (10, 20, and 40 μM) for 12 hours. The expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured. The expression levels of interleukin 6 (IL-6), IL-1β, and tumour necrosis factor alpha (TNF-α) leptin in plasma were measured using enzyme-linked immunoabsorbent assay (ELISA) kits. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathway proteins, and macrophage repolarization-related markers, were detected by western blot. Results. Tert-butylhydroquinone significantly attenuated cartilage destruction in DMM-induced mice in vivo. It demonstrated clear evidence of inhibiting IL-1β-induced chondrocyte apoptosis, inflammation, and differentiation defect in vitro. Meanwhile, tBHQ inhibited LPS-induced activation of NF-κB and
The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67,
Osteoarthritis, the most common degenerative joint disease, significantly impairs life quality and labor capability of patients. Synovial inflammation, initiated by HMGB1 (High mobility group box 1)-induced activation of macrophage, precedes other pathological changes. As an upstream regulator of NF-κB (nuclear factor-kappa B) and
Complement C5a receptor 1 (C5aR1) has crucial functions in host defense against danger molecules, as does toll-like receptor 2 (TLR2). Both innate immunity receptors interact in immune cells in the context of infectious inflammatory diseases often associated with bone loss, such as periodontitis. C5aR1 plays an important role in bone, as it is expressed on bone cells and strongly upregulated due to bone injury. Importantly, C5aR1-ko mice are protected against arthritis and C5aR1 contributes to bone loss in periodontitis. In contrast, less data exist on the role of TLR2 on osteoblasts, however, it is known that TLR2 is expressed on osteoblasts and contributes to bacterial-induced bone resorption. The aim of this study was to evaluate the interaction of C5aR1 and TLR2 in osteoblasts, including intracellular signaling pathways and gene expression patterns. Primary osteoblasts were isolated from 8–12 week-old WT mice and differentiated for 14 days. Osteoblasts were assessed for expression of C5aR1 and TLR2. Phosphorylation of mitogen-activated protein kinases (MAPK) in response to C5a and Pam3CSK4 (TLR2 agonist) was analyzed by immunoblotting. Gene expression profiling after 30 min and 4 h stimulation of C5a was performed by microarray and candidate genes were validated by quantitative Real-Time PCR (qRT-PCR). Immunoprecipitation was performed using a C5aR1-antibody and C5aR1 and TLR2 were subsequently detected by immunoblotting. Statistics: One way ANOVA p<0.05, n=4–6. We showed that C5aR1 and TLR2 are expressed on osteoblasts and strongly upregulated during differentiation. Via immunoprecipitation, we could show that C5aR1 and TLR2 do physically interact in osteoblasts. We then examined if C5aR1 and TLR2, besides their physical interaction, also act via the same intracellular signaling pathways. Gene expression profiling upon C5a stimulation revealed that the top regulated pathways are related to
Summary Statement. Combination of sorafenib with irradiation achieved synergistic effect with dose reduction in both 143B and HOS cell lines. This demonstrated the potential application of sorafenib in the treatment of osteosarcoma metastasis and radiation resistance. Introduction. More than 20% of patients with osteosarcoma die of the disease within 5 years due to tumour relapse and metastasis. Identifying new treatment that works singly or in combination with conventional therapies is urgently required. We previously found that the Ras/Raf/
Objectives. The aim of this study was to investigate the role of miR-126 in the development of osteoarthritis, as well as the potential molecular mechanisms involved, in order to provide a theoretical basis for osteoarthritis treatment and a novel perspective for clinical therapy. Methods. Human chondrocyte cell line CHON-001 was administrated by different doses of interleukin (IL)-1β to simulate inflammation. Cell viability, migration, apoptosis, IL-6, IL-8, and tumour necrosis factor (TNF)-α expression, as well as expression of apoptosis-related factors, were measured to assess inflammation. miR-126 expression was measured by quantitative polymerase chain reaction (qPCR). Cells were then transfected with miR-126 inhibitor to assess the effect of miR-126 on IL-1β-injured CHON-001 cells. Expression of B-cell lymphoma 2 (Bcl-2) and the activity of mitogen-activated protein kinase (MAPK) / Jun N-terminal kinase (JNK) signaling pathway were measured by Western blot to explore the underlying mechanism through which miR-126 affects IL-1β-induced inflammation. Results. After IL-1β administration, cell viability and migration were suppressed while apoptosis was enhanced. Expression of IL-6, IL-8, and TNF-α were all increased, and miR-126 was upregulated. In IL-1β-administrated CHON-001 cells, miR-126 inhibitor suppressed the effect of IL-1β on cell viability, migration, apoptosis, and inflammatory response. Bcl-2 expression was negatively regulated with miR-126 in IL-1β-administrated cells, and thus affected expressions of phosphorylated
Background. Signalling by growth differentiation factor 6 (GDF6/BMP13) has been implicated in the development and maintenance of healthy NP cell phenotypes and GDF6 mutations are associated with defective vertebral segmentation in Klippel-Feil syndrome. GDF6 may thus represent a promising biologic for treatment of IVD degeneration. This study aimed to investigate the effect of GDF6 in human NP cells and critical signal transduction pathways involved. Methods. BMP receptor expression profile of non-degenerate and degenerate human NP cells was determined through western blot, immunofluorescence and qPCR. Phosphorylation statuses of Smad1/5/9 and non-canonical p38
Summary. Anabolic and catabolic signalling processes within IVDs display overlapping pathways, however some pathways were identified as selective to catabolic signalling and inhibition of one of these pathways inhibited some of the catabolic factors induced by IL-1 although NFkB inhibition also affected anabolic expression. Degeneration of intervertebral discs (IVDs) is implicated in 40% of low back pain cases. In the normal disc the balance between anabolic and catabolic processes are carefully balanced. During degeneration this balance is lost in favour of catabolic processes which lead to degradation of the IVD, infiltration of blood vessels and nerves and release of cytokines which sensitise nerves to pain. Interleukin 1 (IL-1) is known to be important in the pathogenesis of IVD degeneration, here we investigated the intracellular signalling pathways activated by IL-1 and those activated by an anabolic factor (CDMP-1) to investigate differential pathways. Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 or CDMP-1 for 30 minutes. Site-specific phosphorylation of 46 signalling molecules were identified using R&D proteome array. The activation of ERK1/2, p38, c-jun, and IkB were confirmed using cell based ELISAs, in addition pNFκB localisation in stimulated cells was determined using immunohistochemisty. Pre-treatment with inhibitors to p38, and NFkB for 30 minutes, followed by stimulation with IL-1 (10ng/mL) or CDMP-1 (10ng/mL) for 24 hours was investigated to determine effects on anabolic and catabolic factors. In addition localisation of phosphorylated c-jun, p38 and NFkB were investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo. Twenty intracellular signalling pathways were activated following CDMP-1 treatment and 8 signalling pathways activated by IL-1. Of note key classical IL-1 signalling pathways p38
Clinical use of glucocorticoids engenders deleterious changes in bone fragility and initiates apoptosis in osteoblasts and osteocytes. The pathways leading to corticosteroid-induced death in bone remain unclear. Similarly little is known about the effects of ‘bone sparing’ bisphosphonates on osteocytes in vivo. We investigated the effects of bisphosphonates (BPs) on dexamethasone (Dex)-induced apoptosis in the murine osteocyte cell line, MLO-Y4 and studied the putative pathways involved by intervention with inhibitors of signalling molecules, such as p42/44
In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK)
Objectives. In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. Methods. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion. Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as
Wear debris from worn cobalt chrome joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the relationship between the amount of DNA damage and the changes in gene expression was investigated in human fibroblasts after exposure to artificial cobalt chrome particles. The comparison was made with different doses of particles, at different time intervals and in fibroblasts of different ages, those that had completed 10 population doublings (10 PD fibroblasts) and those that had completed 35 population doublings (35 PD fibroblasts). The genes (TGF-©¬2, p38
Purpose: Chondrosarcoma is the second most common malignancy in bone and results from unregulated growth of mesenchymal cells.. Chondrosarcoma does not appear to respond either to chemotherapy or radiation. Currently chondrosarcoma has no effective treatment and a new approach to adjuvant therapy for this tumor is urgently needed. We have previously reported that Matrix metalloproteinase -1 (MMP-1)gene expression is an independent predictor of survival in chondrosarcoma.(Scully, 2000). We have reported also that the expression of the large TNC splice variant may correlate with malignancy and poor clinical outcome in human chondrosarcoma and be implicated in the process of basement membrane invasion. The aim of the current study was to confirm that Tenascin-C large splice variant(TNC320) stimulates matrix metalloproteinase-1(MMP-1)expression and invasive potential and to elucidate molecular mechanisms underlying this activation. Method: The chondrosarcoma cell line was recultured in alginate beads. (Guo et al, 1989)The beads contained exogenous proteins according to design. Alginate beads were dissociated later by chelation and the cells were pelleted by centrifugation. Gel electrophoresis and Western blotting were performed to demonstrate the expression of MMP-1 protein in the cells cultured with treatments. MMP-1 protein was detected by mouse antiMMP-1 monoclonal antibody from Chemicon. QPCR and RT-PCR were used to detect MMP-1mRNA expression with different treatments Transactivation of MMP-1 luc construct was detected in transiently transfected JJ012 human chondrosarcoma cultured cells. Reporter gene, Collagenase, Cell invasion assays and MMP-ELISA were performed for this study. Results: The analysis of gene expression in cultured cells grown under different condition indicated significant increases of MMP-1mRNA steady-state levels in the cells with TNC 320 treatment. Gel electrophoresis demonstrated augmented MMP-1 protein in cells cultured with TNC 320. The result was confirmed by examining MMP-1 promoter transactivation of 30fold in comparison with control and other treatments. Both invasion and collagenase assays demonstrated 3 fold difference in the cells treated with TNC 320. Experiments with constitutively active expression kinases indicated that MMP-1 expression induced by TN320 was associated with
Irisin is a hormone-like myokine released from skeletal muscle during exercise. It has also been reported that irisin levels in serum and synovial fluid of knee osteoarthritis (OA) patients were negatively correlated with OA severity. We hypothesized that irisin might play a role in the cartilage homeostasis mediated by physical activity. Therefore, this study aims to explore the cross talk between skeletal muscle and cartilage tissues in human with OA mediated by the myokine irisin. Human articular OA chondrocytes were isolated, expanded and cultured in micro-mass 3-D culture system. Pellets were cultured with or without r-Irisin, and then activated by protein inhibitors of p38-MAPK signalling pathway. After one week the amount of GAG content was evaluated. Quantitative gene expression of Coll-X and Coll-II was performed. WB was utilized to detect expressions of p38-MAPK signalling pathway and Coll-X and Coll-II. In the current study, chondrocytes cultured in r-Irisin showed a significant higher GAG/DNA content compared to control (p<0.05). Moreover, r-Irisin promoted a significant increase of the expression collagen type II and decrease of collagen type X in (p<0.05). This OA chondrocytes recovery was abrogated by the p38
Purpose: Mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients are not well characterized and little is known of how they are regulated. Recent evidence indicates that a major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human MSCs from OA patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). However, the intracellular pathways for transducing signals that regulate hypertrophy in MSCs remain unclear. In chondrocytes, this pathway is mediated by mitogen activated protein kinase (MAPK) p38. The aim of this study was to determine the phosphorylation levels of ERK/p38
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders. We examine the inhibitory effect of a high affinity, glycosyl-phosphatidylinositol-anchored TIMP variant named ‘T1PrαTACE’ on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation.Aims
Methods