Grape seed-derived procyanidins alleviate gout pain via NLRP3 inflammasome suppression

Abstract

Background: Gout is one of the common inflammatory arthritis which affects many people for inflicting unbearable pain. Macrophage-mediated inflammation plays an important role in gout. The uptake of monosodium urate (MSU) crystals by macrophages can lead to activation of NOD-like receptors containing a PYD 3 (NLRP3) inflammasome, thus accelerating interleukin (IL)-1β production. Reactive oxygen species (ROS) promoted development of the inflammatory process through NLRP3 inflammasome. Our study aimed to find a food-derived compound to attenuate gout pain via the specific inhibition of the NLRP3 inflammasome in macrophages.

Methods: CD-1 mice were used to evaluate the degree of pain and the swelling dimension of joints after an intra-articular (IA) MSU injection in the ankle. The murine macrophage cell line Raw 264.7 was used to investigate the effects of procyanidins and the mechanism underlying such effects. Histological analysis was used to measure the infiltration of inflammatory cells. ROS produced from Raw 264.7 cells were evaluated by flow cytometry. Cell signaling was measured by Western blot assay and immunofluorescence.

Results: Procyanidins significantly attenuated gout pain and suppressed ankle swelling. Procyanidins also inhibited MSU-induced activation of the NLRP3 inflammasome and increase of IL-1β. Furthermore, procyanidins decreased ROS levels in Raw 264.7 cells.

Conclusions: Suppression of the NLRP3 inflammasome in macrophages contributes to the amelioration of gout pain by procyanidins.

Keywords: Gout pain; Macrophages; NLRP3 inflammasome; Procyanidins.

Fig. 1

Procyanidins suppressed MSU-induced NLRP3 inflammasome activation in RAW macrophages. a The cells were stimulated by LPS (1 μg/ml) for 6 h and then stimulated with MSU crystals for another 6 h. Procyanidins were added 20 min before LPS administration. Western blot samples were prepared from the supernatant (n = 4). b The cell extracts were collected at 12 h following LPS treatment from Raw 264.7 cells; procyanidins were added 20 min before LPS (n = 4). c The cells were stimulated by LPS (1 μg/ml) for 6 h and then stimulated with MSU crystals for another 6 h. Procyanidins (10 μM) were added 20 min before LPS. The supernatant (SN) and cell lysis fractions (Input) of Raw 264.7 cells were collected respectively (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 vs. naive; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the MSU-treated group

Fig. 2

Procyanidins suppressed MSU-induced ROS production in macrophages. a The levels of ROS were assessed by calculating the ratio of positive-staining cells among 10,000 cells using flow cytometry. Raw 264.7 cells were primed with procyanidins (10 μM) for 20 min and then stimulated by MSU crystals for 3 h. b MTT assay: Raw 264.7 cells were treated with various doses of procyanidins (1, 5, and 10 μM) for 24 h

Fig. 3

Procyanidins suppressed MSU-induced gout pain in mice. Mice were treated with various doses of procyanidins (PO) 20 min before the injection of MSU crystals (0.5 mg/10 μL). a Mechanical allodynia was performed to evaluate the effect of procyanidins (n = 8). b Time course of changes in MSU-induced ankle swelling (n = 8). c Representative photographs of mouse ankles at 24 h following MSU injection. Bar: 5 mm (d) hematoxylin- and eosin-stained sections of the ankle joints obtained 24 h after MSU injection. Bar: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the MSU-treated group

Fig. 4

Procyanidins suppressed NLRP3 inflammasome activation in ankle periarticular tissue. Mice were treated with various doses of procyanidins (15, 30, and 60 mg/kg, PO) 20 min before the injection of MSU crystals (0.5 mg/10 μL). Western blot samples were collected 24 h after MSU injection (n = 4). Expression of pro- and cleaved IL-1β (a), pro- and cleaved caspase-1 (b), and NLRP3 (c) in ankle periarticular tissue are shown. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the MSU-treated group

Fig. 5

Procyanidins inhibited central sensitization. a Phosphorylation of NMDA receptors (n = 4). b Phosphorylation of p38, ERK, and JNK (n = 4). c Immunofluorescence analysis of c-fos in the dorsal horn of the spinal cord (n = 5). The quantification of c-fos immunofluorescence is represented as the number of c-fos-positive cells in the superficial dorsal horns. Bar: 80 μm *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the MSU-treated group