ABSTRACT
OBJECTIVE: Experimental and clinical data suggest that statins may protect bone by inhibiting bone resorption and/or stimulating bone formation. Interleukin-6 (IL-6) is produced by osteoblasts, and potently stimulates osteoclast activation playing a key role in normal bone resorption as well as in post-menopausal and inflammation-driven osteoporosis. Although statins inhibit IL-6 production from different cell types, currently no data exist on osteoblasts. The aim of the study was to evaluate the effect of rosuvastatin on IL-6 production by human osteoblasts. METHODS: Osteoblasts from osteoarthritic patients were incubated with rosuvastatin (0.1-10 µmol/L)±IL-1ß, and IL-6 production was evaluated as cytokine concentration in the culture medium (ELISA), as well as mRNA expression in the cells (qPCR). Putative intracellular mechanisms of the drug, such as blocking HMG-CoA-reductase, and interference in the prenylation process were investigated by the addition of mevalonate and isoprenoids. The effect of rosuvastatin±IL-1ß on the anti-resorptive molecule osteoprotegerin (OPG) was also assessed (ELISA). RESULTS: Rosuvastatin significantly reduced IL-6 levels in the osteoblast culture medium, both in unstimulated and IL-1ß-stimulated cells. This effect was reversed by mevalonate or geranylgeraniol, but not farnesol. Moreover, the drug decreased both spontaneous and IL-1ß-induced IL-6 mRNA expression in osteoblasts. Conversely, rosuvastatin did not affect OPG levels in the culture medium. CONCLUSION: Our results show that rosuvastatin decreases IL-6 production by osteoblasts, thereby suggesting a possible inhibiting activity on osteoclast function in an indirect way. These data may provide further rationale for employing rosuvastatin to beneficially affect bone metabolism in post-menopausal women and possibly in inflammation-driven osteoporosis.
Subject(s)
Fluorobenzenes/pharmacology , Interleukin-1beta/pharmacology , Interleukin-6/genetics , Osteoblasts/drug effects , Osteoblasts/physiology , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Aged , Bone Resorption/drug therapy , Bone Resorption/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Culture Media/pharmacology , Diterpenes/pharmacology , Farnesol/pharmacology , Femur Head/cytology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Inflammation/drug therapy , Inflammation/metabolism , Interleukin-6/metabolism , Mevalonic Acid/pharmacology , Middle Aged , Osteoblasts/cytology , Osteoporosis/drug therapy , Osteoporosis/metabolism , Osteoprotegerin/metabolism , RNA, Messenger/metabolism , Rosuvastatin Calcium , Terpenes/pharmacologyABSTRACT
The aim of this in vitro study was to examine the possible effect of [2-methyl-5-(4-methylsulphonyl)phenyl-1-phenyl-3-(2-n-propyloxyethyl)]-1H-pyrrole (VA441), a new selective cyclooxygenase (COX)-2 inhibitor, on human osteoarthritic (OA) chondrocyte cultivated in the presence or absence of interleukin-1ß (IL-1ß). In particular, we assessed the effects of 1 and 10 µM of VA441, celecoxib, and indomethacin on cell viability, COX-2 and inducible nitric oxide synthase (iNOS) gene expression, prostaglandin E(2) (PGE(2)) production, and nitric oxide (NO) and metalloproteinase-3 (MMP-3) release. Furthermore, we carried out morphological assessment by transmission electron microscopy (TEM). The presence of IL-1ß led to a significant increase in PGE(2), MMP-3, and NO production, as well as a significant increase in gene expression of COX-2 and iNOS. All the drugs tested had a statistically significant inhibitory effect on PGE(2) production and gene expression of COX-2 stimulated by IL-1ß. VA441 and celecoxib significantly suppressed IL-1ß-stimulated MMP-3 and NO and iNOS gene expression in a dose-dependent manner, while indomethacin did not show any significant effect on MMP-3 and NO production or on iNOS gene expression. TEM demonstrated that IL-1ß severely alters the structure of chondrocytes; after co-incubation with VA441 or celecoxib, the cells recovered their ultrastructure. Our data suggest that VA441 and celecoxib may have a beneficial effect on chondrocyte metabolism.
Subject(s)
Chondrocytes/drug effects , Cyclooxygenase 2 Inhibitors/pharmacology , Pyrroles/pharmacology , Sulfones/pharmacology , Aged , Cell Survival/drug effects , Cells, Cultured , Chondrocytes/metabolism , Cyclooxygenase 2/genetics , Dinoprostone/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Interleukin-1beta/pharmacology , Matrix Metalloproteinase 3/metabolism , Middle Aged , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , OsteoarthritisSubject(s)
Arthritis, Rheumatoid/genetics , MicroRNAs/genetics , T-Lymphocytes/metabolism , Adult , Aged , Aged, 80 and over , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cell Separation , Female , Gene Expression Regulation , Humans , Male , MicroRNAs/metabolism , Middle AgedABSTRACT
It is well known that estrogens are implicated in the pathogenesis of osteoarthritis. Raloxifene is a selective estrogen receptor modulator used in the treatment of osteoporosis, though little is known about the possible effects of raloxifene on cartilage metabolism. The aim of our study was to evaluate the possible in vitro effects of raloxifene in human osteoarthritis chondrocytes cultivated in the presence or absence of Interleukin-1 beta (IL-1ß) (5 ng/ml). The effects of 0.1 µM and 1 µM of raloxifene in the culture medium were assessed using an immuno-enzymatic method for proteoglycans and metalloproteinase-3 (MMP-3), and the Griess method for nitrite. Gene expression of inducible Nitric Oxide Synthase (iNOS) was detected by real-time PCR. A morphological analysis was performed by transmission electron microscopy (TEM). Cell viability was significantly (P<0.01) reduced by the IL-1ß, and restored to basal levels by raloxifene at both of the concentrations used. The presence of IL-1 ß led to a significant decrease (P<0.01) in proteoglycan levels as well as a significant increase of MMP-3 and NO (P<0.01). When the cells were co-incubated with IL-1ß and raloxifene, a significant and dose-dependent increase in proteoglycans and a reduction of MMP-3 and nitric oxide (NO) were detected. iNOS was noticeably expressed in IL-1ß stimulated chondrocytes, while raloxifene decreased in a very significant manner the gene expression of iNOS at both of the concentrations used. The results of the biochemical evaluation were confirmed by TEM. Our data suggest that raloxifene may have a potential chondroprotective role in osteoarthritis.
Subject(s)
Chondrocytes/metabolism , Chondrocytes/pathology , Cytoprotection/drug effects , Interleukin-1beta/pharmacology , Raloxifene Hydrochloride/pharmacology , Selective Estrogen Receptor Modulators/pharmacology , Aged , Cell Proliferation/drug effects , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/ultrastructure , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Enzymologic/drug effects , Humans , Matrix Metalloproteinase 3/metabolism , Microscopy, Electron, Transmission , Middle Aged , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/genetics , Osteoarthritis/pathology , Proteoglycans/metabolismABSTRACT
The aim of the present study was to demonstrate the mitogenic and differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes in mono- and three-dimensional cultures. In order to assess if PRPr supplementation could maintain the chondrocyte phenotype or at least inhibit the cell de-differentiation even after several days in culture, we performed a proteomic study on several cell cultures independently grown, for different periods of time, in culture medium with FCS, human serum (HS), and releasates obtained from PRP and platelet-poor plasma (PPP). We found that PRP treatment actually induced in chondrocytes the expression of proteins (some of which novel) involved in differentiation.
