Gallic Acid Alleviates Gouty Arthritis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis Through Enhancing Nrf2 Signaling

Abstract

Gallic acid is an active phenolic acid widely distributed in plants, and there is compelling evidence to prove its anti-inflammatory effects. NLRP3 inflammasome dysregulation is closely linked to many inflammatory diseases. However, how gallic acid affects the NLRP3 inflammasome remains unclear. Therefore, in the present study, we investigated the mechanisms underlying the effects of gallic acid on the NLRP3 inflammasome and pyroptosis, as well as its effect on gouty arthritis in mice. The results showed that gallic acid inhibited lactate dehydrogenase (LDH) release and pyroptosis in lipopolysaccharide (LPS)-primed and ATP-, nigericin-, or monosodium urate (MSU) crystal-stimulated macrophages. Additionally, gallic acid blocked NLRP3 inflammasome activation and inhibited the subsequent activation of caspase-1 and secretion of IL-1β. Gallic acid exerted its inhibitory effect by blocking NLRP3-NEK7 interaction and ASC oligomerization, thereby limiting inflammasome assembly. Moreover, gallic acid promoted the expression of nuclear factor E2-related factor 2 (Nrf2) and reduced the production of mitochondrial ROS (mtROS). Importantly, the inhibitory effect of gallic acid could be reversed by treatment with the Nrf2 inhibitor ML385. NRF2 siRNA also abolished the inhibitory effect of gallic acid on IL-1β secretion. The results further showed that gallic acid could mitigate MSU-induced joint swelling and inhibit IL-1β and caspase 1 (p20) production in mice. Moreover, gallic acid could moderate MSU-induced macrophages and neutrophils migration into joint synovitis. In summary, we found that gallic acid suppresses ROS generation, thereby limiting NLRP3 inflammasome activation and pyroptosis dependent on Nrf2 signaling, suggesting that gallic acid possesses therapeutic potential for the treatment of gouty arthritis.

Keywords: NLRP3 inflammasome; Nrf2; gallic acid; gouty arthritis; pyroptosis.

Figure 1

Gallic acid prevents cell death in lipopolysaccharide (LPS)-primed and NLRP3 stimuli-treated macrophages. (A) J774A.1 cell were treated with gallic acid for 24 or 48 h, and then cytotoxicity was detected by CCK-8 assay. (B–D) J774A.1 cell were primed with LPS (500 ng/ml) for 4 h and then stimulated with ATP (3 mM) for 1 h with or without gallic acid. (E–I) LPS-primed bone marrow-derived macrophages (BMDMs) were incubated with gallic acid for 30 min and then stimulated with ATP (3 mM) or nigericin (10 μM) for 1 h. (D, E) The culture supernatant was collected to analyze lactate dehydrogenase (LDH) secretion. (B, F, H) Representative immunofluorescence images of cell death as detected by propidium iodide (PI) and Hoechst 33342 staining. (C, G, I) The percentage of PI-positive cells relative to all cells was calculated; 10 randomly chosen fields were quantified. GA, gallic acid. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Gallic acid inhibits NLRP3 inflammasome activation and pyroptosis in murine macrophages. (A, B) Lipopolysaccharide (LPS)-primed J774A.1 cell were stimulated with ATP for 1 h in the presence or absence of gallic acid. (C–F) LPS-primed BMDMs were incubated with gallic acid for 30 min and then stimulated with ATP (C, D) or nigericin (E, F) for 1 h. Supernatants (Sup.) and cell extracts (Lys.) were analyzed by immunoblotting (A, C, E), and IL-1β release in supernatants was also analyzed by ELISA (B, D, F). GA, gallic acid. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Gallic acid blocks ASC oligomerization and speck formation. (A–E) LPS-primed bone marrow-derived macrophages (BMDMs) were incubated with gallic acid for 0.5 h before incubated with ATP or nigericin for 1 h, or monosodium urate (MSU) for 6 h. (C) ASC oligomerization in cross-linked cytosolic pellets from ATP-treated BMDMs was analyzed by immunoblotting. (A, D) Representative immunofluorescence images of ASC speck formation in LPS-primed BMDMs stimulated with ATP or nigericin or MSU in the presence or absence of gallic acid (80 µM). White arrows indicate ASC specks (green). Scale bars, 20 µm. Quantification of macrophages containing ASC speck formation in five random images is shown in (B, E). GA, gallic acid. **P < 0.01, ***P < 0.001.

Figure 4

Gallic acid suppresses nigericin-induced NLRP3 inflammasome activation and pyroptosis dependent on Nrf2. (A–F) Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) were incubated with Nrf2 inhibitor ML385 (2 μM) for 30 min, treated with gallic acid (80 μM) for 30 min, and then stimulated with nigericin (10 μM). (A) Representative immunofluorescence images of MitoSOX and MitoTracker-stained BMDMs. Scale bars, 10 μm. (B) The relative fluorescence intensity of MitoSOX and MitoTracker was compared with that of control BMDMs. (E) The percentage of PI-positive cells relative to total cells was calculated; 10 randomly chosen fields were quantified. Data are shown as means ± sem (n = 10). (C) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by western blotting. (D) ELISA of IL-1β levels in supernatants. (F) Co-Immunoprecipitation was applied to analyze the interaction between NEK7 and NLRP3. GA, gallic acid. **P < 0.01, ***P < 0.001.

Figure 5

Gallic acid decreases monosodium urate (MSU)-induced NLRP3 inflammasome activation partly through Nrf2. (A–E) LPS-primed BMDMs were treated with ML385 for 30 min, incubated with gallic acid for 30 min, and then stimulated with MSU for 6 h. (A) Representative IF images of cell death indicated by PI and Hoechst 33342 staining. The percentage of PI-positive cells relative to all cells was calculated; 10 randomly chosen fields were quantified. (B) Representative IF images of MitoTracker-stained BMDMs. Scale bars, 10 μm. The relative fluorescence intensity of MitoTracker was compared with that of control BMDMs. (C) The culture supernatant was obtained to analyze LDH release. (D) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by immunoblotting. (E, F) ELISA kit was used to detect IL-1β levels in culture supernatants. GA, gallic acid. **P < 0.01, ***P < 0.001.

Figure 6

Gallic acid alleviates monosodium urate (MSU)-induced NLRP3 inflammasome activation in vivo. (A–D) C57BL/6J mice were treated with an intra-articular injection of MSU crystals (1 mg/mouse) in the presence of gallic acid (100 mg/kg) or colchicine (1 mg/kg) for 24 h. Representative ankle photographs were shown in (A), Scale bars, 3.5 mm. (B) Joint swelling was measured at different time points. (C) Joint culture supernatant medium was measured by the IL-1β ELISA kit. Data are shown as means ± sem (n = 6 mice). (D) Hematoxylin and eosin (H&E)-stained infiltrated leukocytes (black arrow) in joint tissues. (E, F) Immunohistochemical of IL-1β, caspase 1 p20, and Nrf2 were acquired in the indicated groups. Scale bars, 100 μm. GA, gallic acid. ***P < 0.001.

Figure 7

Gallic acid reduces monosodium urate (MSU)-induced inflammatory cell infiltration in vivo. (A–D) C57BL/6J mice were treated with an intra-articular injection of MSU crystals (1 mg/mouse) with gallic acid (100 mg/kg) colchicine (1 mg/kg) for 24 h. (A) Representative immunohistochemistry images of F4/80 and/or Ly6G stained knee joint sections. Scale bars, 100 μm. (B) Representative immunofluorescence images of F4/80 and/or Ly6G stained knee joint sections. Scale bars, 50 μm. (C) The percentage of F4/80- or Ly6G -positive cells relative to total cells was calculated. Data are shown as means ± sem (n = 6 mice). (D) The separate cells from the knee joint were stained with CD11b-FITC, F4/80-PE, and Ly6G-Percp-Cy5.5 to analyze macrophages and neutrophils subset by flow cytometry. (E) The percentage of CD11b+F4/80+ and CD11b+Ly6G+ cells relative to total cells was calculated. Data are shown as means ± sem (n = 6 mice). GA, gallic acid. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.