Monosodium urate crystals induce oxidative stress in human synoviocytes

Abstract

Background: Gout 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.

Methods: Human 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.

Results: The 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.

Conclusions: Our 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.

Keywords: Gout; Monosodium urate crystals; Oxidative stress; Synoviocytes.

Fig. 1

Cellular response to the presence of monosodium urate (MSU) crystals. a Cell viability after a 24 h treatment. b Cell morphological changes after MSU crystal exposure. The arrows indicate the intracellular vacuoles of MSU crystals. c Apoptosis is revealed by Annexin V detection (yellow arrows) in synoviocytes exposed to MSU crystals and H₂O₂ (100 μM). Additionally, columns show quantification of the apoptotic cells by flow cytometry. Values are expressed as the mean ± standard deviation *P < 0.05 vs control

Fig. 2

Monosodium urate (MSU) crystals increase reactive oxygen species (ROS) in synoviocytes. Arrows indicate intracellular H₂O₂ formation, which is revealed by DCFH oxidation (green fluorescence) in untreated fibroblast-like synoviocytes (FLS) (a); FLS treated with MSU crystals at 24 h (b), and FLS treated with H₂O₂ at 30 minutes (c). Arrows indicate O₂ ^- intracellular production by oxidation of dihydroethidium (DHE) (red fluorescence) in untreated FLS (d); FLS treated with MSU crystals (e), and FLS treated with H₂O₂ (f). Bars show quantification of DCFH and DHE fluorescence: data are reported as units of arbitrary fluorescence (UAF) (g). Values are expressed as the mean ± standard deviation; *P < 0.05 vs control

Fig. 3

Nitric oxide (NO) production in synoviocytes. a Detection of NO in untreated fibroblast-like synoviocytes (FLS). b FLS treated with monosodium urate (MSU) crystals. c FLS treated with H₂O₂. Arrows indicate fluorescence produced by intracellular NO. d Bars show NO quantification by Tali image-based cytometer. Values are expressed as the mean ± standard deviation; *P < 0.05 vs control

Fig. 4

Oxidized proteins assay. a Representative oxyblot of fibroblast-like synoviocytes (FLS) proteins from control group (line 1); FLS proteins exposed to monosodium urate (MSU) crystals (line 2); and FLS proteins from the positive control sample (line 3). b Oxidation scan with fluorescence detector. Results are representative of independent experiments with cells from different patients

Fig. 5

Ultrastructural changes in synoviocytes. A Ultrastructure of an untreated fibroblast-like synoviocytes (FLS). a Magnified view of the section is indicated by a black box showing the nucleus (N), endoplasmic reticulum (ER) and vacuoles (V) highlighted with arrows. B FLS treated with monosodium urate (MSU) crystals at 75 μg/mL exhibiting N, swollen vesicular structures of different sizes, and MSU crystal cavity. b A high-magnification image showing misfolded proteins (MP) aggregates and ER indicated with arrows. C FLS treated with H₂O₂ at 100 μM showing N. c A magnified view of the section is indicated by a black box showing MP aggregates, ER and N. Results are representative of one of five separate experiments with FLS from different patients