ML335 inhibits TWIK2 channel-mediated potassium efflux and attenuates mitochondrial damage in MSU crystal-induced inflammation.

BACKGROUND: Activation of the NLRP3 inflammasome is critical in the inflammatory response to gout. Potassium ion (K+) efflux mediated by the TWIK2 channel is an important upstream mechanism for NLRP3 inflammasome activation. Therefore, the TWIK2 channel may be a promising therapeutic target for MSU crystal-induced inflammation. In the present study, we investigated the effect of ML335, a known K2P channel modulator, on MSU crystal-induced inflammatory responses and its underlying molecular mechanisms. METHODS: By molecular docking, we calculated the binding energies and inhibition constants of five K2P channel modulators (Hydroxychloroquine, Fluoxetine, DCPIB, ML365 and ML335) with TWIK2. Intracellular potassium ion concentration and mitochondrial function were assessed by flow cytometry. The interaction between MARCH5 and SIRT3 was demonstrated by immunoprecipitation and Western blotting assay. MSU suspensions were injected into mouse paw and peritoneal cavity to induce acute gout model. RESULTS: ML335 has the highest binding energy and the lowest inhibition constant with TWIK2 in the five calculated K2P channel modulators. In comparison, among these five compounds, ML335 efficiently inhibited the release of IL-1ß from MSU crystal-treated BMDMs. ML335 decreased MSU crystal-induced K+ efflux mainly dependent on TWIK2 channel. More importantly, ML335 can effectively inhibit the expression of the mitochondrial E3 ubiquitin ligase MARCH5 induced by MSU crystals, and MARCH5 can interact with the SIRT3 protein. ML335 blocked MSU crystal-induced ubiquitination of SIRT3 protein by MARCH5. In addition, ML335 improved mitochondrial dynamics homeostasis and mitochondrial function by inhibiting MARCH5 protein expression. ML335 attenuated the inflammatory response induced by MSU crystals in vivo and in vitro. CONCLUSION: Inhibition of TWIK2-mediated K+ efflux by ML335 alleviated mitochondrial injury via suppressing March5 expression, suggesting that ML335 may be an effective candidate for the future treatment of gout.

Maresin1 ameliorates MSU crystal-induced inflammation by upregulating Prdx5 expression.

BACKGROUND: Maresin1 (MaR1) is a potent lipid mediator that exhibits significant anti-inflammatory activity in the context of several inflammatory diseases. A previous study reported that MaR1 could suppress MSU crystal-induced peritonitis in mice. To date, the molecular mechanism by which MaR1 inhibits MSU crystal-induced inflammation remains poorly understood. METHODS: Mousebone marrow-derived macrophages (BMDMs) were pretreated with MaR1 and then stimulated with FAs (palmitic, C16:0 and stearic, C18:0) plus MSU crystals (FAs + MSUc). In vivo, the effects of MaR1 treatment or Prdx5 deficiency on MSUc induced peritonitis and arthritis mouse models were evaluated. RESULTS: The current study indicated that MaR1 effectively suppressed MSUc induced inflammation in vitro and in vivo. MaR1 reversed the decrease in Prdx5 mRNA and protein levels induced by FAs + MSUc. Further assays demonstrated that MaR1 acceleratedPrdx5 expression by regulating the Keap1-Nrf2 signaling axis. Activation of AMPK by Prdx5 improved homeostasis of the TXNIP and TRX proteins and alleviated mitochondrial fragmentation. In addition, Prdx5 overexpression inhibited the expression of CPT1A, a key enzyme for fatty acid oxidation (FAO). Prdx5 protected against defects in FA + MSUc induced FAO and the urea cycle. CONCLUSION: MaR1 treatment effectively attenuated MSUc induced inflammation by upregulating Prdx5 expression. Our study provides a new strategy by which Prdx5 may help prevent acute gout attacks.

Sirt3 improves monosodium urate crystal-induced inflammation by suppressing Acod1 expression.

BACKGROUND: Previous studies have revealed that Sirt3 deficiency is associated with several inflammatory responses. The purpose of this study is to investigate the role and potential molecular mechanisms of Sirt3 in the inflammation induced by monosodium urate (MSU) crystals. METHODS: The Sirt3 expression level in the peripheral blood mononuclear cells (PBMCs) of patients with gout was measured. Function and molecular mechanism of Sirt3 in MSU crystal-induced inflammation were investigated in bone marrow-derived macrophages (BMDMs), C57BL/6 mouse, and Sirt3-/- mouse. RESULTS: Sirt3 expression was decreased in the PBMCs of patients with gout. Sirt3 agonist (Viniferin) inhibited the acetylation levels of mitochondrial proteins including the SOD2 protein. RNA sequencing, bio-informatics analysis, RT-PCR, and Western blot demonstrated that Sirt3 could suppress the expression of Acod1 (Irg1), which plays an important role in gout. In BMDMs treated with palmitic acid (C16:0) plus MSU crystals, Acod1 knockdown repressed mitochondrial reactive oxygen species (mtROS) over-production, macrophage migration, and mitochondrial fragmentation, and Acod1 improved AMPK activity. The over-expression of Acod1 did not significantly affect the level of itaconic acid, but greatly decreased the levels of some important intermediate metabolites of the tricarboxylic acid (TCA) cycle. These data indicate that Acod1 exerts a pro-inflammatory role in MSU crystal-induced inflammation and is independent of the metabolic level of itaconic acid. Sirt3 deficiency exacerbates inflammatory response induced by MSU crystals in vitro and in vivo. CONCLUSION: The current study has shown that Sirt3 can alleviate the MSU crystal-induced inflammation by inhibiting the expression of Acod1.

Dynamin-Related Protein 1 Is Involved in Mitochondrial Damage, Defective Mitophagy, and NLRP3 Inflammasome Activation Induced by MSU Crystals.

Excessive generation of reactive oxygen species (ROS) has great impacts on MSU crystal-induced inflammation. Drp1-dependent mitochondrial fission is closely associated with mitochondrial ROS levels. However, whether Drp1 signaling contributes to MSU crystal-induced inflammation remains unclear. Mice bone marrow-derived macrophages (BMDMs) were primed with LPS and then stimulated with MSU suspensions for 12 h. The protein levels associated with mitochondrial dynamics, oxidative stress, and mitophagy were detected by Western blot. BMDMs were loaded with MitoTracker Green probe to detect mitochondrial morphology. To measure mitochondrial reactive oxygen species (ROS) and total ROS levels, cells were loaded, respectively, with MitoSOX and DHE probes. The effects of Mito-TEMPO, an antioxidant that targets the mitochondria or DRP1 inhibitor (Mdivi-1) on MSU crystal-induced peritonitis and arthritis mouse models, were evaluated. Our study revealed that MSU crystal stimulation resulted in elevation of mitochondrial fragmentation of BMDMs. Treatment with Mito-TEMPO or Drp1 knockdown significantly ameliorated the mitochondrial damage induced by MSU crystals. BMDMs exposure to MSU crystals increased the expression of auto/mitophagy marker proteins and promoted the fusion of mitophagosomes with lysosomes, leading to accumulation of mitolysosomes. Drp1 knockdown alleviated defective mitophagy and activation of the NLRP3 inflammasome in MSU crystal-treated BMDMs. This study indicates that there is crosstalk between mitochondrial ROS and Drp1 signaling in MSU crystal-induced inflammation. Drp1 signaling is involved in MSU crystal-induced mitochondrial damage, impaired mitophagy and NLRP3 inflammasome activation.