Abstract
BackgroundGout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown. The aim of this study was to evaluate the oxidative stress in human fibroblast-like synoviocytes (FLS) exposed to monosodium urate (MSU) crystals, which trigger an inflammatory process.MethodsHuman FLS isolated from synovial tissue explants were stimulated with MSU crystals (75 μg/mL) for 24 h. Cellular viability was evaluated by crystal violet staining, apoptosis was assessed using Annexin V, and the cellular content of reactive oxygen species (ROS) and nitrogen species (RNS) (O2-, H2O2, NO) was assessed with image-based cytometry and fluorometric methods. In order to determine protein oxidation levels, protein carbonyls were detected through oxyblot analysis, and cell ultrastructural changes were assessed by transmission electron microscopy.ResultsThe viability of FLS exposed to MSU crystals decreased by 30 % (P < 0.05), while apoptosis increased by 42 % (P = 0.01). FLS stimulated with MSU crystals exhibited a 2.1-fold increase in H2O2 content and a 1.5-fold increase in O2- and NO levels. Oxyblots revealed that the spots obtained from FLS protein lysates exposed to MSU crystals exhibited protein carbonyl immunoreactivity, which reflects the presence of oxidatively modified proteins. Concomitantly, MSU crystals triggered the induction of changes in the morphostructure of FLS, such as the thickening and discontinuity of the endoplasmic reticulum, and the formation of vacuoles and misfolded glycoproteins.ConclusionsOur results prove that MSU crystals induce the release of ROS and RNS in FLS, subsequently oxidizing proteins and altering the cellular oxidative state of the endoplasmic reticulum, which results in FLS apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1012-3) contains supplementary material, which is available to authorized users.
Highlights
Gout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown
Two blinded experts reported similar morphological characteristics in synthetic monosodium urate (MSU) crystals and those obtained from the synovial fluid of patients during an acute gouty attack, and they were unable to differentiate between the two types of crystals
Assessment of cell viability and apoptosis Cell cultures exposed to MSU crystals at a concentration of 75 μg/mL maintained a viability of 77 % ± 0.50, while a concentration of 100 μg/mL resulted in a viability of 54.82 % ± 0.46 compared to unstimulated fibroblast-like synoviocytes (FLS) (P ≤ 0.05) (Fig. 1a)
Summary
Gout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown. Gout is a uric acid (UA) metabolic disorder that promotes the formation and deposition of monosodium urate (MSU) crystals inside joints and periarticular soft tissues as a result of hyperuricemia It is the most common inflammatory arthropathy in young men, and its prevalence is underestimated due to the long asymptomatic phase of the disease [1]. The activation of NADPH oxidase, xanthine oxidase, and nitric oxide synthase enzymes generates hydrogen peroxide (H2O2), superoxide anion (O2-) and nitric oxide (NO), respectively The interaction of these last two molecules promotes the Zamudio-Cuevas et al Arthritis Research & Therapy (2016) 18:117 generation of peroxynitrite (ONOO-), which in turn increases apoptosis, the degradation of connective tissues, and joint damage [7, 8]. Due to the complex interactions that take place within joints among various cell types, including neutrophils, macrophages, mast cells, endothelial cells and synovial fibroblasts, it is possible for synovial fibroblasts to play a role in modulating the inflammatory response to MSU crystals in patients with gout [9, 10]
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