Fig 2: MiR-326 inhibited chondrocyte pyroptosis by targeting HDAC3 and activating the STAT1/NF-?B p65 signaling pathway. (a) Overexpression of HDAC3 could counteract the accelerated proliferation of OA chondrocytes induced by miR-326 mimics; (b) overexpression of HDAC3 could counteract the enhanced migration of OA chondrocytes induced by miR-326 mimics; (c) overexpression of HDAC3 could counteract the increased mRNA levels of chondrogenic genes (COL2A1, SOX9, Agg, and Prg4) induced by miR-326 mimics in OA chondrocytes; (d) the dual luciferase gene report confirmed the targeting relationship between miR-326 and HDAC3; (e) overexpression of HDAC3 could reverse the reduction of proinflammatory factors (IL-1ß, IL-18, IL-6, and TNF-a) in OA chondrocyte culture medium induced by miR-326 mimics; (f) Overexpression of HDAC3 could reverse the decrease of DNA-binding activity of NF-?B p65 in OA chondrocytes induced by miR-326 mimics; (g) overexpression of HDAC3 could reverse the decrease of Caspase-1 activity in OA chondrocytes induced by miR-326 mimics; (h) immunofluorescence results showed that overexpression of HDAC3 could reverse the decrease level of GSDMD and cleaved Caspase-1 in OA chondrocytes induced by miR-326 mimics; (i) Western blot results showed that overexpression of HDAC3 could counteract the activation of STAT1/NF-?B p65 signaling pathway in OA chondrocytes induced by miR-326 mimics; (j) Western blot results showed that overexpression of HDAC3 could reverse the decrease level of pyroptosis-related proteins (NLRP3, ASC, GSDMD, Caspase-1, IL-1ß, and IL-18) in OA chondrocytes induced by miR-326 mimics; *P < 0.01, **P < 0.01.

Fig 3: Canagliflozin inhibits NLRP3 inflammasome activation through up-regulating Bif-1. (A–C) Effects of CAN on Bif-1, p62 and the ratio of LC3-II/LC3-I. J774A.1 cells were transfected with Bif-1 siRNA or negative control siRNA using Lipofectamine 3000. Twenty-four hours after transfection, cells were primed with LPS (500 ng/ml) for 6 h in the presence or absence of CAN (10 µM), then stimulated with 5 mM ATP for 30 min. Whole cell lysis and supernatant were collected. Western blotting analysis of (D,E) Bif-1, (F–H) pro-IL-1ß and pro-IL-18 were presented. (I) LDH release in supernatant was assessed. (J,K) ELISA of IL-1ß and IL-18 in supernatant. Nor, normal control group; LPS/ATP (L/A), untreated LPS/ATP control group; CAN, CAN-treated LPS/ATP group. NC, negative control group; si-Bif-1, group treated with siRNA of Bif-1. Data were expressed as mean ± SD (n = 3). Differences with a probability value of <0.05 were considered significant. (B,C,G,H) *p < 0.05, **p < 0.01, ***p < 0.001 vs Nor; #p < 0.05, ##p < 0.01, ###p < 0.001 vs L/A. (E) and (I–K) *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Inhibition of HDAC3 or STAT1/NF-?B p65 signaling pathway counteracts pyroptosis induced by silence of miR-326 in chondrocytes. (a) Immunofluorescence results showed that inhibition of HDAC3 or STAT1/NF-?B p65 could counteract the increase of GSDMD and cleaved Caspase-1 induced by miR-326 inhibitor in chondrocytes; (b) inhibition of HDAC3 or STAT1/NF-?B p65 could counteract the increase of Caspase-1 activity induced by miR-326 inhibitor in chondrocytes; (c) inhibition of HDAC3 or STAT1/NF-?B p65 could counteract the increase of proinflammatory factors (IL-1ß, IL-18, IL-6, and TNF-a) induced by miR-326 inhibitor in chondrocytes; (d) Western blot results showed that inhibition of HDAC3 or STAT1/NF-?B p65 could counteract the increase level of pyroptosis-related proteins (NLRP3, ASC, GSDMD, Caspase-1, IL-1ß, and IL-18) induced by miR-326 inhibitor in chondrocytes; *P < 0.01, **P < 0.01.

Fig 5: Card9 deficiency results in excessive activation of NLRP3 inflammasome in vitro.A, B BMDMs were obtained from WT mice and Card9-/- mice. Cells were unstimulated (medium) or pretreatment with LPS (100 ng/ml) for 3 h, and then respectively treated with ATP (5 mM, 1 h), MSU (500 µg/ml, 2 h) or Nigericin (10 µM, 2 h). A ELISA analysis of IL-1ß and IL-18 secretion in the culture supernatant. B Western blot analysis of cell lysates. C–E BMDMs were obtained from WT mice and Card9-/- mice, cells were unstimulated (medium) or were primed with 100 ng/ml LPS for 3 h, and then treated with ATP (5 mM, 1 h). C Lysates of BMDMs immunoprecipitated with anti-ASC and then conducted immunoblot analysis. D, E Representative immunofluorescence images of ASC speck formation in BMDMs and quantification. Data are shown as mean ± SEM. Scale bars, 0.2 µm. Each group was representative of at least three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.