Palmatine Protects Against MSU-Induced Gouty Arthritis via Regulating the NF-κB/NLRP3 and Nrf2 Pathways

Abstract

Purpose: Gouty arthritis could be triggered by the deposition of monosodium uric acid (MSU) crystals. Palmatine (PAL), a protoberberine alkaloid, has been proven to possess compelling health-beneficial activities. In this study, we aimed to explore the effect of PAL on LPS plus MSU crystal-stimulated gouty arthritis in vitro and in vivo.

Methods: PMA-differentiated THP-1 macrophages were primed with LPS and then stimulated with MSU crystal in the presence or absence of PAL. The expression of pro-inflammatory cytokines and oxidative stress-related biomarkers and signal pathway key targets were determined by ELISA kit, Western blot, immunohistochemistry and qRT-PCR, respectively. In addition, the anti-inflammatory and antioxidant activities of PAL on MSU-induced arthritis mice were also evaluated.

Results: The results indicated that PAL (20, 40 and 80 μM) dose-dependently decreased the mRNA expression and levels of pro-inflammatory cytokines (interleukin-1beta (IL-1β), IL-6, IL-18 and tumor necrosis factor alpha (TNF-α)). The levels of superoxide dismutase (SOD) and glutathione (GSH) were remarkably enhanced, while the level of malondialdehyde (MDA) was reduced. Western blot analysis revealed that PAL appreciably inhibited NF-κB/NLRP3 signaling pathways through inhibiting the phosphorylation of p-65 and IκBα, blocking the expression of NLRP3, ASC, IL-1β and Caspase-1, as well as enhancing the antioxidant protein expression of Nrf2 and HO-1. In vivo, PAL attenuated MSU-induced inflammation in gouty arthritis, as evidenced by mitigating the joint swelling, and decreasing the productions of IL-1β, IL-6, IL-18, TNF-α and MDA, while enhancing the levels of SOD and GSH. Moreover, PAL further attenuated the infiltration of neutrophils into joint synovitis.

Conclusion: PAL protected against MSU-induced inflammation and oxidative stress via regulating the NF-κB/NLRP3 and Nrf2 pathways. PAL may represent a potential candidate for the treatment of gouty arthritis.

Keywords: NF-κB/Nrf2 signal pathways; NLRP3 inflammasome; gouty arthritis; palmatine.

Figure 1

(A) Chemical structure of PAL. (B) Effect of PAL on cell viability of THP-1 cells. Data are shown as mean ± SD (n = 3).

Figure 2

Effects of PAL and Col (1 µM) on the levels of IL-1β (A), TNF-α (B), IL- 6 (C) and IL-18 (D) in LPS plus MSU-activated THP-1 cells. Data are shown as mean ± SD (n = 3); ^##P < 0.01 vs Control group; **P < 0.01 vs Model group.

Figure 3

Effects of PAL and Col (1 µM) on the mRNA expression of IL-1β (A), TNF-α (B) IL-6 (C) and IL-18 (D) in LPS plus MSU-activated THP-1 cells. Data are shown as mean ± SD (n = 3); ^##P < 0.01 vs Control group, **P < 0.01 vs Model group.

Figure 4

Effects of PAL and Col (1 µM) on the levels of SOD (A), MDA (B) and GSH (C) in LPS plus MSU-activated THP-1 cells. Data are shown as mean ± SD (n = 3); ^##P < 0.01 vs Control group; *P < 0.05, **P < 0.01 vs Model group.

Figure 5

Effects of PAL and Col (1 µM) on NF-κB/NLRP3 and Nrf2 signaling pathways in LPS plus MSU-activated THP-1 cells. (A) Representative Western blotting images of p-p65, p65, p-IKBα, IKBα and β-actin. (B) Representative Western blotting images of NLRP3, ASC, IL-1β, Caspase-1 and β-actin. (C) Representative Western blotting images of Nrf2, HO-1 and β-actin. Changes in the relative protein expression levels of p-p65/p65 (D), p-IKBα/IKBα (E), NLRP3 (F), ASC (G), IL-1β (H), Caspase-1 (I), Nrf2 (J) and HO-1 (K). Data are shown as mean ± SD (n = 3); ^##P < 0.01 vs Control group; *P < 0.05, **P < 0.01 vs Model group.

Figure 6

PAL suppressed the MSU-induced gouty arthritis. Either 25 mg/kg (L), 50 mg/kg (M) and 100 mg/kg (H) of PAL, PBS (Con) or 1 mg/kg of Col (positive control group) was orally administered to mice. After 1 h, either monosodium urate (MSU) crystal in 0.1 mg/10 µL of PBS or PBS alone was injected into the left knee joint of each mouse. PAL decreased the MSU crystal-induced acute gout inflammation in mice: (A) Schematic diagram of in vivo experimental protocol. (B) Joint swelling gain at different time points. (C) Hematoxylin and eosin staining of leukocytes in joint tissues (black arrow). (D) Histologic score. Data are expressed as mean ± SD (n = 6), ^##P < 0.01 vs Control group; *P < 0.05, **P < 0.01 vs Model group.

Figure 7

Levels of IL-1β (A), TNF-α (B), IL-6 (C) and IL-18 (D) in joint tissues, as measured by ELISA. Data are expressed as mean ± SD (n = 6), ^##P < 0.01 vs Control group; *P < 0.05, **P < 0.01 vs Model group.

Figure 8

Effects of PAL and Col (1 mg/kg) on the levels of SOD (A), MDA (B) and GSH (C) in joint tissue. Data are shown as mean ± SD (n = 6); ^##P < 0.01 vs Control group; **P < 0.01 vs Model group.

Figure 9

Effects of PAL and Col (1 mg/kg) on the inflammatory cell infiltration in vivo. (A) Representative immunohistochemistry images of knee joint sections stained with Ly6G or F4/80 (red arrow). (B) The percentage of Ly6G or F4/80 positive cells relative to total cells was calculated. Data are shown as mean ± SD (n = 3). ^##P < 0.01 vs Control group; **P < 0.01 vs Model group.

Figure 10

Summary scheme of the mechanisms underlying the beneficial effect of PAL in ameliorating MSU-induced inflammation and oxidative stress. On the one hand, PAL inhibited the activation of NF-κB/NLRP3 signaling to ameliorate inflammation response. On the other hand, PAL activated Nrf2 and promoted its nuclear translocation, thereby alleviating oxidative stress.