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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

Arthritis Research & Therapy 1/2018

Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

Zeitschrift:
Arthritis Research & Therapy > Ausgabe 1/2018
Autoren:
Charles McWherter, Yun-Jung Choi, Ramon L. Serrano, Sushil K. Mahata, Robert Terkeltaub, Ru Liu-Bryan
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s13075-018-1699-4) contains supplementary material, which is available to authorized users.

Abstract

Background

Arhalofenate acid, the active acid form of arhalofenate, is a non-agonist peroxisome proliferator-activated receptor γ (PPARγ) ligand, with uricosuric activity via URAT1 inhibition. Phase II studies revealed decreased acute arthritis flares in arhalofenate-treated gout compared with allopurinol alone. Hence, we investigated the anti-inflammatory effects and mechanisms of arhalofenate and its active acid form for responses to monosodium urate (MSU) crystals.

Methods

We assessed in-vivo responses to MSU crystals in murine subcutaneous air pouches and in-vitro responses in murine bone marrow-derived macrophages (BMDMs) by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE/Western blot, immunostaining, and transmission electron microscopy analyses.

Results

Oral administration of arhalofenate (250 mg/kg) blunted total leukocyte ingress, neutrophil influx, and air pouch fluid interleukin (IL)-1β, IL-6, and CXCL1 in response to MSU crystal injection (p < 0.05 for each). Arhalofenate acid (100 μM) attenuated MSU crystal-induced IL-1β production in BMDMs via inhibition of NLRP3 inflammasome activation. In addition, arhalofenate acid dose-dependently increased activation (as assessed by phosphorylation) of AMP-activated protein kinase (AMPK). Studying AMPKα1 knockout mice, we elucidated that AMPK mediated the anti-inflammatory effects of arhalofenate acid. Moreover, arhalofenate acid attenuated the capacity of MSU crystals to suppress AMPK activity, regulated expression of multiple downstream AMPK targets that modulate mitochondrial function and oxidative stress, preserved intact mitochondrial cristae and volume density, and promoted anti-inflammatory autophagy flux in BMDMs.

Conclusions

Arhalofenate acid is anti-inflammatory and acts via AMPK activation and its downstream signaling in macrophages. These effects likely contribute to a reduction of gout flares.
Zusatzmaterial
Additional file 1: A-769662 activated AMPK downstream targets involved in regulation of mitochondrial function. BMDMs were treated with direct AMPK activator A-769662 (100 μM) for 1 h before being stimulated with MSU crystals (0.2 mg/mL) for 6 or 18 h in RPMI containing 1% FBS. Western blot analysis was carried out to examine phosphorylation and expression of AMPKα, expression of SIRT1, PGC-1α, and TFAM, and expression of TXN1, TXN2, and TXNIP from 18-h treatment cells (A), and expression of LC3 and p62 from 6-h treatment cells (B). Data shown in A and B are representative of three individual experiments. (PDF 753 kb)
13075_2018_1699_MOESM1_ESM.pdf
Additional file 2: Arhalofenate acid promoted autophagy flux. BMDMs were treated with arhalofenate acid (100 μM) for 1 h before being stimulated with MSU crystals (0.2 mg/mL) for 6 h in RPMI containing 1% FBS. Immunofluorescence microscopy was carried out to visually identify p62 puncta (green) and lysosomes (LAMP1, red), and determine co-localization (yellow) of p62 and LAMP1 (A, 63×). The numbers of yellow punctae per cell were counted and presented in a graph (B). Data in A are representative of three individual experiments. Data in B are the mean ± SD of 200 cells examined for each condition. The p values represent comparisons between none and MSU crystals alone, or between MSU crystals alone and MSU crystals plus arhalofenate acid. (PDF 38585 kb)
13075_2018_1699_MOESM2_ESM.pdf
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