The effect of palmitate on human articular chondrocytes is partially reversed by oleic acid

Abstract

Purpose: Osteoarthritis is a heterogeneous disease and metabolic factors contribute substantially to its pathogenesis. Conditions such as obesity and metabolic syndrome have been directly related to osteoarthritis regardless of the mechanical damage. In this regard, saturated fatty acids such as palmitate are particularly interesting, since their circulating levels are elevated in these conditions. Palmitate causes apoptosis, inflammation and mitochondrial damage in peripheral tissues. Thus, the aim of this work is to analyze the effects of palmitate and oleate (monoinsaturated fatty acid) on human chondrocytes Methods: The TC28a2 chondrocyte cell line was used to analyze the effect of 0.4mM and 0.7mM palmitate (PA) and palmitate/oleate (PA/O) 1:2 for 12 hours. Isolated RNA was retrotranscribed to cDNA to analyze the gene expression of metalloproteinases (MMP-1, MMP-3, MMP-13), Interleukin 6 (IL6) and Cyclooxygenase 2 (COX2). We also analyzed reactive oxygen species (ROS) production by flow cytometry using MitosoxTM. Total and mitochondrial ATP were measured using ATP Bioluminescence Assay Kit CLS II. Apoptosis was analyzed using the Annexin V FITC/IP Apoptosis detection kit and Caspase 3,7 and 9 activity by means of a luminescense assay (Promega). Data analysis was performed with SPSS software(v19) and qBase plus (Biogazelle). Statistical significance was declared at p<0.05 Results: PA stimulation induced a significant overexpression of MMP-3 (0.4mM/basal=4.70±0.37; 0.7mM/basal=5.62±1.87), MMP-13 (0.4mM/basal=1.2±0.13; 0.7mM/basal=2.04±0.63), IL6 (0.4mM/basal=4.07±0.35; 0.7mM/basal=2.87±0.23) and COX2(0.4mM/basal=1.6±0.65; 0.7mM/basal=2.2±0.85) (p<0.05) Mitochondrial superoxide anion was significantly increased by 6.68-fold (0.4mM PA) and 6.46-fold (0.7mM PA)compared tothe non-stimulated (PA-free) cells (p<0.05). Both total and mitochondrial ATP production were significantly decreased in PA-stimulated cells: PA/basal ratio 0.45 ± 0.04 (0.4mM PA) and 0.67± 0.1 (0.7mM PA)for total ATP and 0.55±0.07 (0.4mM PA) and 0.31±0.29 (0.7mM PA)for mitochondrial ATP (p<0.05) A significant increase of apoptosis was also detected (0.4mM PA/basal ratio=2.32±0.04; 0.7Mm PA/basal ratio=2.7±0.22) (p<0.05); besidescaspases3/7 (0.4mM PA/basal ratio=2.37±0.19; 0.7mM PA/basal ratio=4.32±1.07) and caspase 9 activity (0.4mM PA/basal ratio=2.3±0.24; 0.7mM PA/basal ratio=2.83±0.5) significantly increased after PA stimulation too (p<0.05). After pre-incubation with 5mM NAC (N-Acetylcisteine) the apoptotic effects of PA stimulation were significantly reversed (p<0.05) Co-stimulation with PA/O 1:2 partially reversed the effects of PA; the expression levels of MMP-3, MMP-13 and IL-6 as well as mitochondrial superoxide production andcaspases 3/7 and 9 activity were significantly reduced in relation to PA-stimulated cells (p<0.05); on the contrary, total and mitochondrial ATP showed a non-significant increase in relation to PA-stimulated cells Conclusions: Palmitate overload causes an increased inflammatory response and mitochondrial superoxide anion productionas well as decreased levels of total and mitochondrial ATP. Besides, palmitate-induced apoptosis is mediated by increased mitochondrial oxidative stress.The effects of saturated fatty acids,related to obesity and metabolic syndrome, are partially reversed by monoinsaturated fatty acids such as Oleic acid and could help understand the metabolic phenotype of osteoarthritis