cyclin-d1 and Osteoarthritis

Subchondral bone sclerosis is a major feature of osteoarthritis (OA), and bone marrow mesenchymal stem cells (BMSCs) are presumed to play an important role in subchondral bone sclerosis. Accumulating evidence has shown that stromal cell-derived factor-1α (SDF-1α) plays a key role in bone metabolism-related diseases, but its role in OA pathogenesis remains largely unknown. The purpose of this study was to explore the role of SDF-1α expressed on BMSCs in subchondral bone sclerosis in an OA model.. In the present study, C57BL/6J mice were divided into the following three groups: the sham control, destabilization of the medial meniscus (DMM), and AMD3100-treated DMM (DMM + AMD3100) groups. The mice were sacrificed after 2 or 8 weeks, and samples were collected for histological and immunohistochemical analyses. OA severity was assessed by performing hematoxylin and eosin (HE) and safranin O-fast green staining. SDF-1α expression in the OA model was measured using an enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (q-PCR), and immunohistochemistry. Micro-CT was used to observe changes in subchondral bone in the OA model. CD44, CD90, RUNX2, and OCN expression in subchondral bone were measured using q-PCR and immunohistochemistry. In vitro, BMSCs were transfected with a recombinant lentivirus expressing SDF-1α, an empty vector (EV), or siRNA-SDF-1α. Western blot analysis, q-PCR, and immunofluorescence staining were used to confirm the successful transfection of BMSCs. The effect of SDF-1α on BMSC proliferation was evaluated by performing a CCK-8 assay and cell cycle analysis. The effect of SDF-1α on the osteogenic differentiation of BMSCs was assessed by performing alkaline phosphatase (ALP) and alizarin red S (ARS) staining. Cyclin D1, RUNX2 and OCN expression were measured using Western blot analysis, q-PCR, and immunofluorescence staining.. SDF-1α expression in the DMM-induced OA model increased. In the DMM + AMD3100 group, subchondral bone sclerosis was alleviated, OA was effectively relieved, and CD44, CD90, RUNX2, and OCN expression in subchondral bone was decreased. In vitro, high levels of SDF-1α promoted BMSC proliferation and increased osteogenic differentiation. Cyclin D1, RUNX2, and OCN expression increased.. The results of this study reveal a new molecular mechanism underlying the pathogenesis of OA. The targeted regulation of SDF-1α may be clinically effective in suppressing OA progression.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chemokine CXCL12; Core Binding Factor Alpha 1 Subunit; Cyclin D1; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Osteoarthritis; Osteogenesis; Sclerosis

Osteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported.. This paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA.. Under similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1β concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results.. RNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05).. Based on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.

Topics: Animals; Cell Proliferation; Chondrocytes; Cyclin D1; Disease Models, Animal; Gene Expression; Immunoblotting; Immunohistochemistry; Inflammation; Interleukin-1beta; Mice; Osteoarthritis; Proliferating Cell Nuclear Antigen; Rats; RNA-Seq

Osteoarthritis (OA) is an orthopedic inflammatory disease which can cause functional disability and chronic pain. MiRNAs are known to play important roles in OA. To identify the targets for the treatment of OA, bioinformatics analysis was performed to explore differentially expressed miRNAs between OA and normal samples.. Bioinformatics analysis was conducted to identify differentially expressed miRNAs. To mimic OA in vitro, primary chondrocytes were stimulated with IL-1β. Meanwhile, flow cytometry was performed to detect the cell apoptosis and cycle distribution. In addition, protein and mRNA expressions were detected by Western blot and RT-qPCR, respectively. Finally, in vivo model of OA was constructed to investigate the function of miR-892b in OA.. The data indicated that miR-892b was identified to be upregulated in OA samples. Additionally, miR-892b antagomir markedly reversed IL-1β-induced growth decline of chondrocytes via inhibiting the apoptosis. IL-1β notably elevated the expressions of MMP1 and MMP13 and downregulated the level of Aggrecan in chondrocytes, while miR-892b antagomir reversed these phenomena. Meanwhile, cyclin D1 and cyclin D2 were the direct targets of miR-892b. In addition, IL-1β-induced G1 phase arrest in chondrocytes was partially abolished by of miR-892b antagomir. In vivo study indicated miR-892b antagomir could significantly alleviate the symptom of OA in a rat model.. MiR-892b antagomir inhibits the progression of OA via targeting Cyclin D1 and Cyclin D2. Thus, our finding might supply a novel target for OA treatment.

Topics: Chondrocytes; Cyclin D1; Cyclin D2; Down-Regulation; Humans; MicroRNAs; Osteoarthritis; Transcriptional Activation; Up-Regulation

This study aims to investigate whether SNHG16 (small nucleolar RNA host gene 16) can promote the progression of osteoarthritis (OA) by regulating the microRNA-93-5p/Cyclin D1 (CCND1) axis, thereby finding new therapeutic targets for the treatment of OA.. A total of 23 OA patients and 23 patients undergoing lower extremity amputation were enrolled in this study. We collected their cartilage tissues from knee joint for isolating chondrocytes. The relative levels of SNHG16, CCND1 and microRNA-93-5p in cartilage tissues of OA patients and controls were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The regulatory effect of SNHG16 on proliferative potential of chondrocytes was evaluated by Cell Counting Kit-8 (CCK-8) and colony formation assay, respectively. Cell cycle progression was examined using flow cytometry. Dual-Luciferase reporter gene assay was conducted to verify the binding between SNHG16 with microRNA-93-5p and microRNA-93-5p with CCND1. Rescue experiments were performed to elucidate whether SNHG16 regulated CCND1 expression by targeting microRNA-93-5p.. The expressions of SNHG16 and CCND1 upregulated, while microRNA-93-5p downregulated in cartilage tissues of OA patients relative to controls. Correlation regression analyses showed a negative expression correlation between SNHG16 and microRNA-93-5p, as well as CCND1 and microRNA-93-5p in OA patients. On the contrary, SNHG16 expression was positively correlated to CCND1 expression in OA. The knockdown of SNHG16 suppressed viability, cloning ability and cell cycle progression, but induced apoptosis in chondrocytes. Dual-Luciferase reporter gene assay showed that SNHG16 could bind to microRNA-93-5p. SNHG16 knockdown markedly upregulated the expression of microRNA-93-5p. Moreover, the knockdown of microRNA-93-5p reversed the inhibited viability due to SNHG16 knockdown. Transfection of microRNA-93-5p mimics markedly inhibited CCND1 expression. Importantly, CCND1 overexpression reversed the inhibitory effect of SNHG16 knockdown on chondrocyte viability.. SNHG16 promotes the development of OA by regulating microRNA-93-5p/CCND1 axis.

Topics: Apoptosis; Case-Control Studies; Cell Cycle; Cell Proliferation; Cells, Cultured; Chondrocytes; Cyclin D1; Down-Regulation; Gene Knockdown Techniques; Humans; MicroRNAs; Molecular Mimicry; Osteoarthritis; RNA-Binding Motifs; RNA, Small Nucleolar; Transfection; Up-Regulation

This study investigated the effects of cyclin D1 gene silencing on cell proliferation and apoptosis of interleukin-1β (IL-1β)-induced osteoarthritis (OA) chondrocytes. Chondrocytes from healthy sprague-dawley rats were divided into blank, OA model (chondrocytes underwent IL-1β inducement), OA trial (chondrocytes underwent IL-1β inducement with cyclin D1-shRNA treatment), and negative control (NC; chondrocytes underwent IL-1β inducement and control-shRNA treatment) groups. Cell proliferation was assessed by CCK-8 assay, and cell cycle and apoptosis by flow cytometry. qRT-PCR and Western blotting were performed to detect cyclin D1 and apoptosis-related factors expression levels. Chondrocyte proliferation increased after 72-96 h after incubation. The OA trial group exhibited reduced cell proliferation at 48, 72, and 96 h after treatment. The OA model, OA trial, and NC groups all contained more cells arrested in G1 phase and had higher apoptosis rates than the blank group. Additionally, the OA trial group contained more cells arrested in G1 phase, with increased apoptosis rates compared to the OA model and NC groups. The OA model group had lowest expression of cyclin D1 whereas the blank group contained the highest among the four groups. qRT-PCR also showed that the OA model, OA trial, and NC groups all had increased expression levels of Bax and reduced expression levels of Bcl-2 and P53 compared to the blank group, whereby by the OA group had the most significant change. The combined evidence in our study shows that cyclin D1 gene silencing suppresses proliferation and induces apoptosis of rat chondrocytes in IL-1β-induced OA. J. Cell. Biochem. 119: 290-299, 2018. © 2017 Wiley Periodicals, Inc.

Topics: Animals; Apoptosis; Chondrocytes; Cyclin D1; Disease Models, Animal; Gene Silencing; Interleukin-1beta; Osteoarthritis; Rats; Rats, Sprague-Dawley

Articular chondrocytes derived from osteoarthritic tissues (OA HAC) show a severely reduced chondrogenic commitment. This impairment undermines their use for tissue-engineered cartilage repair, which relies on cell proliferation and growth to meet therapeutic needs, but also on efficient cell plasticity to recover the chondrogenic phenotype. Reversine (Rev), a 2,6-disubstituted purine inhibitor of spindle-assembly checkpoints, was described to convert differentiated mesenchymal cells to their undifferentiated precursors. We hypothesized that Rev exposure could divert OA HAC to more plastic cells, re-boosting their subsequent commitment. HAC were enzymatically released from OA cartilage specimens, expanded for 2 weeks and treated with 5 μm Rev in dimethylsulphoxide (DMSO) or with DMSO alone for 6 days. Cell growth was assessed using the AlamarBlue

Topics: Aged; Aged, 80 and over; Cartilage, Articular; Cell Cycle Checkpoints; Cell Proliferation; Cell Shape; Cells, Cultured; Chondrocytes; Cyclin D1; Dimethyl Sulfoxide; Female; Gene Expression Regulation; Humans; Ki-67 Antigen; Male; Morpholines; Osteoarthritis; Phenotype; Purines; RNA, Messenger; Smad3 Protein; SOX9 Transcription Factor

The study was aimed to investigate the impact of CX3CR1 expression on the proliferation and apoptosis of osteoarthrosis (OA) chondrocytes through Wnt/ β-catenin pathway.. The expression levels of CX3CR1 and Wnt/β-catenin pathway-related genes mRNA and protein in OA chondrocytes were examined by qRT-PCR and western blot. MTT and flow cytometry (FCM) assays were employed to assess cell proliferation, cell cycle and apoptosis. XAV-939, a Wnt/β-catenin pathway inhibitor, was used to inhibit the pathway.. CX3CR1 was significantly overexpressed in OA cartilages than that in normal articular cartilages (P < 0.05). After siCX3CR1 transfection, the expression level of Wnt 3, nuclear β-catenin, Cyclin D1, MMP-13 and phosphorylated GSK-3β significantly increased, while cytoplasm β-catenin, GSK-3β and phosphorylated β-catenin expression was inhibited (P<0.05). XAV-939abolished the effects of siCX3CR1 on proliferation, apoptosis and cell cycle progression of OA chondrocytes (P<0.05).. CX3CR1 regulated chondrocyte proliferation and apoptosis through Wnt/β-catenin signaling pathway. The overexpression of CX3CR1 in chondrocytes of OA may be closely related to the pathogenesis and progression of OA.

Topics: Aged; Apoptosis; beta Catenin; Cartilage, Articular; Cell Cycle; Cell Line; Cell Proliferation; Chondrocytes; CX3C Chemokine Receptor 1; Cyclin D1; Female; Humans; Male; Middle Aged; Osteoarthritis; RNA, Messenger; Wnt Signaling Pathway

Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d-glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucose's effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS-Glc), and the study aimed to investigate whether CTS-Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS-Glc-regulated proliferation of human chondrocytes. We found that CTS-Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose- or chitosan-only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS-Glc-increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP-1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin-dependent kinase-4 and -6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS-Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS-Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients.

Topics: Adaptor Proteins, Signal Transducing; Adsorption; Aged; Antirheumatic Agents; Cell Culture Techniques; Cell Line; Cell Proliferation; Chitosan; Chondrocytes; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Fatty Acid Synthase, Type I; Female; Glucose; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Membranes, Artificial; Middle Aged; mTOR Associated Protein, LST8 Homolog; Multiprotein Complexes; Osteoarthritis; Protein Kinase Inhibitors; Regulatory-Associated Protein of mTOR; RNA Interference; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Time Factors; TOR Serine-Threonine Kinases; Transfection

Berberine chloride (BBR) is an isoquinoline derivative alkaloid isolated from medicinal herbs, including Coptis chinensis and Berberis aristate. This compound plays significant roles in the treatment of osteoarthritis (OA). The purpose of this study was to investigate the effects of BBR on the proliferation of sodium nitroprusside (SNP)-stimulated chondrocytes in vitro, the articular cartilage in a rat OA model, as well as to discuss the molecular mechanisms underlying these effects. In vitro, we demonstrated that BBR led to cell proliferation, increased the cell population in S-phase and decreased that in G0/G1-phase; moreover, the F-actin remodeling in SNP-stimulated chondrocytes were prevented. In addition, BBR markedly up-regulated β-catenin, c-Myc, and cyclin D1 expression of genes and proteins, and down-regulated glycogen synthase kinase-3β (GSK-3β) and matrix metalloproteinase-7 (MMP-7) expression. Notably, inhibition of the Wnt/β-catenin pathway by XAV939 partially blocked these effects. The in vivo results suggested that BBR promoted β-catenin protein level and enhanced proliferating cell nuclear antigen (PCNA) expression in osteoarthritic rat cartilage. In conclusion, these findings indicate that BBR promotes SNP-stimulated chondrocyte proliferation by promoting G1/S phase transition and synthesis of PCNA in cartilage through activation of Wnt/β-catenin signaling pathway.

Topics: Animals; Berberine; Cartilage, Articular; Cell Cycle; Cell Proliferation; Chondrocytes; Cyclin D1; Cytoskeleton; DNA-Binding Proteins; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3 beta; Lymphoid Enhancer-Binding Factor 1; Nitroprusside; Osteoarthritis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Transcription Factor 4; Transcription Factors; Wnt Signaling Pathway

Duhuo Jisheng Decoction (DHJSD), a well known traditional Chinese folk medicine, is used for eliminating stagnation, removing blood stasis, promoting blood circulation and alleviating pain; it is commonly used for the treatment of various diseases, including osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effects of OA remain unclear. In the present study, the effects of DHJSD on the morphology of articular cartilage and the G1/S cell cycle progression in chondrocytes, as well as the underlying mechanisms, were investigated. A total of 27 two‑month‑old male Sprague Dawley rats were randomly divided into 3 groups: the control group (no papain-induced OA; received an equivalent amount of saline only), the model group (papain-induced OA; received an equivalent amount of saline only) and the DHJSD group [papain-induced OA; received a clinical oral dose of DHJSD (9.3 g/kg/day)]. After 8 consecutive weeks of treatment, the morphological changes in articular cartilage were observed under an optical microscope and by transmission electron microscopy (TEM) and the mRNA and protein expression levels of cyclin D1, CDK4, CDK6, retinoblastoma protein (Rb) and p16 were measured by RT‑PCR and immunohistochemistry, respectively. Treatment with DHJSD significantly improved the arrangement of collagen fibers in the articular cartilage, as well as its structure and reduced cell degeneration compared with the model group. The mRNA and protein expression levels of cyclin D1, CDK4, CDK6 and Rb in the DHJSD‑treated group were significantly increased compared with those in the model group, whereas p16 expression was significantly downregulated. Taken together, these results indicate that DHJSD treatment promotes chondrocyte proliferation by promoting the G1/S checkpoint transition in the cell cycle and by upregulating the expression of cyclin D1, CDK4, CDK6 and Rb and downregulating the expression of p16 and this may, in part, explain its clinical efficacy in the treatment of osteoarthritis.

Topics: Animals; Cartilage, Articular; Chondrocytes; Cyclin D1; Cyclin-Dependent Kinase 6; G1 Phase; Immunohistochemistry; Male; Microscopy, Electron, Transmission; Osteoarthritis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction