Daphnetin alleviates inflammation and promotes autophagy via the AMPK/mTOR pathway in gouty arthritis

Abstract

Gouty arthritis (GA) is an inflammatory disease resulting from monosodium urate (MSU) crystal deposition in joints and surrounding tissues. Daphnetin (DAP) is a coumarin derivative with potent anti-inflammatory activity. Nonetheless, whether DAP can protect against MSU-induced acute GA remains unclarified. In this study, C57BL/6 mice were injected intra-articularly with MSU crystal suspension to induce acute GA. THP-1 cells were stimulated with MSU to mimic the microenvironment of GA in vitro. Hematoxylin-eosin staining was conducted to observe the pathological changes in mouse synovial tissues. ELISA and RT-qPCR were employed for inflammatory cytokine level determination. Immunofluorescence staining was performed to estimate LC3 expression in THP-1 cells. Western blotting was used for protein expression analysis. The results showed that DAP pretreatment mitigated MSU-elicited ankle joint swelling and synovial damage in mice. Moreover, DAP hindered proinflammatory factor expression and promoted autophagy in MSU-stimulated GA mice and THP-1 cells. Mechanistically, DAP induced AMPK activation and mTOR inactivation. Blocking AMPK signaling counteracted DAP-mediated effects on inflammation and autophagy in MSU-stimulated THP-1 cells. In conclusion, DAP prevents MSU-elicited GA by alleviating inflammation and enhancing autophagy via AMPK/mTOR signaling transduction.

Keywords: autophagy; daphnetin; gouty arthritis; monosodium urate crystal.

FIGURE 1

Daphnetin attenuates MSU‐induced articular swelling in gouty arthritis mice. (A) The scheme for the experimental model. (B) Representative images of ankles 24 h after MSU injection. (C) Measurement of increased ankle circumference at different time points after MSU injection. (D) Representative images of H&E staining showing histopathological changes in the synovial tissues of the ankle joint. n = 6/group. MSU, monosodium urate. ***p < 0.001 versus control; ##p < 0.01 versus MSU.

FIGURE 2

Daphnetin alleviates the inflammatory response in MSU‐induced mice. (A–C) ELISA for determining serum levels of IL‐6, IL‐1β, and TNF‐α in each group. (D–F) ELISA for evaluating the concentrations of IL‐6, IL‐1β, and TNF‐α in the synovial tissues of the ankle joint. n = 6/group. MSU, monosodium urate. ***p < 0.001 versus control; #p < 0.05, ##p < 0.01, ###p < 0.001 versus MSU.

FIGURE 3

Daphnetin activates autophagy in MSU‐induced mice. (A) Western blotting for estimating autophagy‐related protein levels in mouse synovial tissues. (B–D) Quantitative results of western blotting. n = 6/group. MSU, monosodium urate. *p < 0.05, **p < 0.01 versus control; ##p < 0.01, ###p < 0.001 versus MSU.

FIGURE 4

Daphnetin reduces proinflammatory factor levels in MSU‐stimulated THP‐1 cells. (A) CCK‐8 assay for evaluating THP‐1 cell viability under treatment with various concentrations of DAP for 24 h. (B) CCK‐8 assay for assessing THP‐1 cell viability under treatment with vehicle, MSU, or MSU plus DAP (10, 20, or 40 μM). (C) Evaluation of lactate dehydrogenase release in the culture medium of each group. (D–F) ELISA for determining proinflammatory cytokine concentrations in indicated THP‐1 cells. (G–I) RT‐qPCR analysis of cytokine mRNA levels in THP‐1 cells of each group. DAP, daphnetin; MSU, monosodium urate. *p < 0.05 versus DAP (0 μM); ***p < 0.001 versus vehicle; #p < 0.05, ##p < 0.01, ###p < 0.001 versus model + DAP (0 μM).

FIGURE 5

DAP enhances autophagy in monosodium urate‐treated THP‐1 cells. (A) Western blotting showing protein levels of autophagy‐associated markers in indicated THP‐1 cells. (B–D) Quantification of relative protein levels. (E) Representative images of IF staining for detecting LC3 expression in THP‐1 cells. DAP, daphnetin. *p < 0.05 versus vehicle; #p < 0.05, ##p < 0.01 versus model + DAP (0 μM).

FIGURE 6

DAP regulates AMPK/mTOR signaling transduction. (A–C) Western blotting showing (p)‐AMPK and (p)‐mTOR protein expression in the mouse synovial tissues of each group. (D–F) Western blotting depicting (p)‐AMPK and (p)‐mTOR protein expression in THP‐1 cells. DAP, daphnetin. *p < 0.05, **p < 0.01 versus vehicle; ##p < 0.01 versus model + DAP (0 μM).

FIGURE 7

Inhibition of AMPK signaling reverses DAP‐mediated effects on autophagy and inflammation in THP‐1 cells. (A–C) Western blotting showing (p)‐AMPK and (p)‐mTOR protein expression in monosodium urate‐stimulated THP‐1 cells pretreated with DAP or DAP + CC (an AMPK inhibitor). (D–G) Western blotting for estimating autophagy‐related protein levels in indicated THP‐1 macrophages. (H) RT‐qPCR analysis of cytokine mRNA expression in THP‐1 cells. DAP, daphnetin. ***p < 0.001 versus vehicle; ##p < 0.01, ###p < 0.001 versus DAP.