RESUMEN
BACKGROUND: Matrix metalloproteinases are catabolic enzymes that play a key role in the articular cartilage degeneration evident in degenerative and inflammatory conditions of articular cartilage. The aim of this study is to assess the ability of pravastatin to modify matrix metalloproteinase (MMP) messenger RNA (mRNA) expression and enzyme activity in a culture of normal human chondrocytes stimulated by interleukin-1ß. MATERIALS AND METHODS: Normal human chondrocytes were stimulated with interleukin (IL)-1ß for 6 h to induce MMP expression, simulating a catabolic state, and then treated with pravastatin (1, 5 and 10 µM) for a further 18 h before cell lysates and supernatants were harvested. Cells stimulated with IL-1ß but not treated with pravastatin served as controls. Real-time polymerase chain reaction (PCR) was used to assess expression of MMP-3 and MMP-9 mRNA. MMP enzyme activity was assessed using a fluorescent MMP-specific substrate. Statistical analysis was performed using analysis of variance (ANOVA). RESULTS: MMP-3 and MMP-9 mRNA expression was reduced at all concentrations tested with statistically significant trends in reduction (p = 0.002 and <0.001, respectively). Analysis of culture supernatants revealed that pravastatin treatment led to a reduction in total MMP activity but not to a statistically significant degree (p = 0.07). CONCLUSIONS: Treatment with pravastatin of stimulated human chondrocytes leads to significant down-regulation of selected MMP genes and a non-significant reduction in MMP enzyme activity. Our results provide further evidence that statins may have a role to play in future treatment of disease affecting articular chondrocytes.
Asunto(s)
Cartílago Articular/enzimología , Condrocitos/enzimología , Regulación hacia Abajo/efectos de los fármacos , Interleucina-1beta/farmacología , Metaloproteinasas de la Matriz/genética , Pravastatina/farmacología , ARN Mensajero/genética , Western Blotting , Cartílago Articular/citología , Células Cultivadas , Condrocitos/citología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Metaloproteinasas de la Matriz/biosíntesis , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto™ (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast viability, function and gene expression compared to enoxaparin, a commonly used LMWH. METHODS: Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 µg/ml) or enoxaparin (1, 10 and 100 µg/ml). The effect of each drug on osteoblast function was evaluated by measuring alkaline phosphatase activity. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). RESULTS: Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast viability. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. CONCLUSIONS: These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function.
Asunto(s)
Anticoagulantes/farmacología , Enoxaparina/farmacología , Inhibidores del Factor Xa , Morfolinas/farmacología , Osteoblastos/efectos de los fármacos , Tiofenos/farmacología , Anciano , Fosfatasa Alcalina/metabolismo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Osteoblastos/enzimología , Osteocalcina/genética , Osteocalcina/metabolismo , ARN Mensajero/metabolismo , RivaroxabánRESUMEN
PURPOSE: We aimed to assess the effect on chondrocyte viability of adding magnesium to a variety of commonly available local anesthetic agents. METHODS: Human chondrocytes were grown under standard culture conditions. Cells were exposed to a local anesthetic agent with the addition of magnesium (10%, 20%, or 50%). Cells were also exposed to the varying concentrations of magnesium and 0.9% saline solution. Untreated cells served as controls. The CellTiter 96 AQueous One Solution Cell Proliferation Assay was used to assess for cell viability 24 hours after exposure. One-way analysis of variance was used to test for statistical significance. RESULTS: Magnesium sulfate alone was no more toxic than normal saline solution (P > .3) compared with untreated cells. The addition of magnesium to the local anesthetic agents resulted in greater cell viability than when cells were treated with a local anesthetic alone (lidocaine [P = .033], levobupivacaine [P = .007], bupivacaine [P < .001], and ropivacaine [P < .001]). CONCLUSIONS: Our findings support the use of magnesium either alone or in combination with a local anesthetic rather than a local anesthetic alone, and this represents a potential strategy for the reduction of chondrocyte toxicity associated with intra-articular local anesthetic administration after arthroscopy. CLINICAL RELEVANCE: The addition of magnesium to a local anesthetic results in a reduced toxic effect to the articular chondrocyte. This may represent a potential approach to intra-articular analgesia.
Asunto(s)
Anestésicos Locales/farmacología , Condrocitos/efectos de los fármacos , Sulfato de Magnesio/farmacología , Amidas/farmacología , Amidas/toxicidad , Anestésicos Locales/toxicidad , Bupivacaína/análogos & derivados , Bupivacaína/farmacología , Bupivacaína/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/citología , Humanos , Levobupivacaína , RopivacaínaRESUMEN
BACKGROUND: Ischaemic preconditioning (IPC) has emerged as a method of reducing ischaemia-reperfusion injury. However, the complex mechanism through which IPC elicits this protection is not fully understood. The aim of this study was to investigate the genomic response induced by IPC in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the IPC mechanism. METHODS: Twenty patients, undergoing primary total knee arthroplasty, were randomly assigned to IPC (n = 10) and control (n = 10) groups. Patients in the IPC group received ischaemic preconditioning immediately prior to surgery. IPC was induced by three five-minute cycles of tourniquet insufflation interrupted by five-minute cycles of reperfusion. A muscle biopsy was taken from the operative knee of control and IPC-treated patients at the onset of surgery and, again, at one hour into surgery. The gene expression profile of muscle biopsies was determined using the Affymetrix Human U113 2.0 microarray system and validated using real-time polymerase chain reaction (RT-PCR). Measurements of C-reactive protein (CRP), erythrocyte sedimentation (ESR), white cell count (WCC), cytokines and haemoglobin were also made pre- and post-operatively. RESULTS: Microarray analysis revealed a significant increase in the expression of important oxidative stress defence genes, immediate early response genes and mitochondrial genes. Upregulation of pro-survival genes was also observed and correlated with a downregulation of pro-apoptotic gene expression. CRP, ESR, WCC, cytokine and haemoglobin levels were not significantly different between control and IPC patients. CONCLUSIONS: The findings of this study suggest that IPC of the lower limb in total knee arthroplasty patients induces a protective genomic response, which results in increased expression of immediate early response genes, oxidative stress defence genes and pro-survival genes. These findings indicate that ischaemic preconditioning may be of potential benefit in knee arthroplasty and other musculoskeletal conditions.