Fig 2: In BMDMs treated with C16:0 + MSU, Sirt3 deficiency accelerated NLRP3 inflammasome activation. a The protein levels of NLRP3, Caspase-1, and IL-1β in the cell lysates were evaluated by Western blot. Mature IL-1β (p17) and p20 fragments of Caspase-1 were detected in cell culture supernatants. b Total cell lysates were obtained in Triton X-100–containing buffer. Insoluble (pellet) fractions were cross-linked with DSS to capture ASC oligomers. The soluble and insoluble fractions were analyzed by Western blot with ASC antibody. c Representative LCM images of BMDMs stained with ASC antibody. DAPI staining nuclei. Scale bar, 40 μm. *P < 0.05. NS shows no statistical difference

Fig 3: Sirt3 deficiency exacerbated MSU crystal-induced inflammation in vivo. a Representative plots of migrated leukocytes (CD45+) and neutrophils (CD11b+Gr-1.+) in the peritoneal fluid were detected by FCM. The number of migrated leukocytes and neutrophils were quantified and compared among the groups on the right. b The levels of IL-1β and CCL2 in the peritoneal fluid were detected by ELISA. c Paw swelling index. d Representative microscopic images of mouse paw sections stained with HE (magnification 20 × 10). n = 5 mice for each group. *P < 0.05

Fig 4: Sirt3 deficiency increased mtROS production and NF-κB activation in BMDMs treated with C16:0 + MSU. a Sirt3 protein levels of BMDMs from Sirt3+/+ and Sirt3−/− mice. b The statistical analysis graph of mitochondrial ROS generation. c Mitochondrial ROS levels were measured by LCM image using a Mito-SOX probe. Red fluorescence represented mitochondrial ROS level. Blue shows nuclei staining with DAPI. Scale bar: 40 μm. d Mitochondrial respiration (oxygen consumption rate, OCR) was measured using Seahorse XFe24 Analyzer. e Quantification of basal respiration, maximal respiration, and OCR-coupled ATP production were analyzed (n = 5 biological replicates). f ATP Lite Luminescence Assay kit was used to detect global ATP production (n = 4 biological replicates). g Cytosolic and nuclear protein extracted from BMDMs were respectively reacted with anti-phosphorylated NF-κB P65 (p-P65) and anti-NF-κB P65 by western blotting. *P < 0.05. NS means no statistical difference

Fig 5: MSU crystals stimulation reduced the expression and deacetylation activity of Sirt3. a Sirt3 mRNA and protein levels were detected in PBMCs of health controls (HC), patients with acute gout (AG), and intermittent gout (IG). Sirt3 mRNA detection (HC, n = 24), (AG, n = 24), (IG, n = 24); GAPDH was used as an internal reference. Sirt3 protein detection (HC, n = 6), (AG, n = 6), (IG, n = 6), TUBULIN as an internal reference. b BMDMs were respectively stimulated with C16:0, MSU, or C16:0 + MSU for 12 h and Western blot was used to detect Sirt3 protein expression. c The protein levels of Sirt3 in the mouse paw treated with MSU crystals or vehicle. Data presented as mean ± SD (n = 5 mice per group). *P < 0.05. NS means no statistical difference. d Sirt3 agonists (Viniferin) inhibited the acetylation level of mitochondrial proteins in BMDMs treated with Viniferin (1 μM) and C16:0 + MSU crystals for 12 h. Acetyl-conjugated proteins in mitochondrial proteins were blotted with acetylated-lysine antibody. e Viniferin treatment decreased K68 lysine acetylation of mitochondrial SOD2 protein and mitochondrial proteins were analyzed by Western blot in BMDMs. f The activity of SOD in the mitochondrial proteins of BMDMs was detected with WST-8 method. Data are mean ± SD. Three independent repetitive experiments were conducted for each result. *P < 0.05. NS represents P > 0.05