Objectives Obesity is a major risk factor for the development of

Objectives Obesity is a major risk factor for the development of osteoarthritis (OA) that is associated with a state of low-grade inflammation and increased circulating adipokines and free fatty acids (FFA). and human articular cartilage explants cultured with FFA with or without IL-1β. Results Palmitate but not oleate induced caspase activation and MPC-3100 cell death in IL-1β-stimulated normal chondrocytes and upregulated and expression in chondrocytes and fibroblast-like synoviocytes through toll-like receptor-4 signaling. In cartilage explants palmitate induced chondrocyte death IL-6 release and extracellular matrix degradation. Palmitate synergized with IL-1β in stimulating proapoptotic and proinflammatory cellular responses. MPC-3100 Pharmacological inhibition of caspases or TLR-4 signaling reduced palmitate and IL-1β-induced cartilage damage. Conclusions Palmitate acts as a pro-inflammatory and catabolic factor that in synergy with IL-1β induces chondrocyte apoptosis and articular cartilage breakdown. Collectively our data suggest that elevated levels of ActRIB saturated FFA often found in obesity may contribute to OA pathogenesis. and was assessed in normal and OA human being chondrocytes treated with oleate or palmitate with or without IL-1β. In regular chondrocytes palmitate however not oleate improved and mRNA (Numbers 2A-C) aswell as IL-6 secretion (suggest ± SD; BSA: 1.3 ± 1.1 ng/ml; palmitate: 2.5 ± 0.4 ng/ml; IL-1β: 32.4 ± 1.8 ng/ml; palmitate+IL-1β: 43.4 ± 4.1 ng/ml; BSA vs palmitate p=0.049; IL-1β MPC-3100 palmitate+IL-1β p=0.0036). When cells had been incubated with both palmitate and IL-1β the manifestation of and was synergistically improved (p<0.05) (Figures 2A B). This synergy had not been noticed for since co-treatment with palmitate avoided IL-1β-induced upregulation (Shape 2C). OA chondrocytes exhibited identical and gene manifestation patterns as regular chondrocytes (Numbers 2D-F). Incubation with FFAs didn't alter MMP13 ADAMTS4 and collagen type 2 gene manifestation whatever the existence of IL-1β in regular or OA articular chondrocytes (Numbers 3A-C G-E). Nevertheless whereas levels had been unchanged upon FFA treatment in regular chondrocytes palmitate considerably (p<0.05) downregulated expression in OA chondrocytes treated with IL-1β (Numbers 3D H). Shape 2 Free of charge fatty proinflammatory and acids mediators manifestation in human being articular MPC-3100 chondrocytes and fibroblast-like synoviocytes. and mRNA amounts evaluated by qPCR in regular human being articular chondrocytes (A-C) osteoarthritic articular chondrocytes ... Shape 3 Manifestation of extracellular matrix (ECM) proteins and proteases in human being articular chondrocytes treated with palmitate oleate and IL-1β. and mRNA amounts evaluated by qPCR in regular human being articular chondrocytes (A-D) ... In addition we analyzed and expression in human fibroblast-like synoviocytes treated with palmitate or oleate alone or in combination with IL-1β. Palmitate but not oleate significantly (p<0.01) increased and expression and co-treatment with IL-1β further enhanced this effect (Figures 2G-H). FFA alone did not modify expression and significantly (p<0.01) decreased IL-1β-induced upregulation (Figure 2I). To determine whether palmitate effects are receptor-mediated we tested CLI-095 a pharmacological inhibitor of TLR-4 signaling (30) which completely blocked and expression induced by palmitate but not by IL-1β in normal articular chondrocytes and fibroblast-like synoviocytes (Figures 4A-D). Figure 4 Effects of TLR-4 signaling inhibition in human articular chondrocytes and fibroblast-like synoviocytes treated with palmitate and IL-1β. (A) and (B) gene expression assessed by qPCR in normal human chondrocytes treated with palmitate ... Palmitate induces chondrocyte death and extracellular matrix damage in bovine cartilage MPC-3100 explants To evaluate long-term palmitate effects on articular cartilage integrity bovine cartilage explants were treated with palmitate or oleate alone or combined with IL- 1β. Palmitate but not oleate significantly (p<0.05) increased cell death in cartilage explants as evidenced by a decrease in cell viability and an increase in cleaved-PARP staining particularly in the cartilage surface (Figures 5A-D). This effect was synergistically enhanced by IL-1β. ECM breakdown was assessed by Safranin-O staining and analysis of GAGs levels in the media. Palmitate but not oleate significantly (p<0.05) decreased Safranin-O staining in cartilage explants and increased GAGs release into the medium (Figures 5E F). This catabolic effect of palmitate was significantly (p<0.05) enhanced in explants stimulated with IL-1β. Figure 5 Free fatty acids effects in bovine and human cartilage explants..