Fig 2: NAG-1/GDF15 inhibits AIM2 inflammasome activation in the FFA-induced hepatocellular steatosis model. (A–B) The expression of AIM2 inflammasome (AIM2, ASC, Caspase-1, IL-1β, and IL-18) at the mRNA (A) and protein level (B) in HepG2 and Huh-7 cells upon treatment with FFA for 24 h as determined using qRT-PCR or Western blotting. (C–D) The mRNA (C) and protein (D) expression of AIM2 inflammasome components in HepG2 and Huh-7 cells as transfected with pcDNA3.1-NAG-1 plasmid with or without FFA treatment. (E, F) The expression of AIM2 inflammasome components at the mRNA (E) and protein level (F) in HepG2 and Huh-7 cells as transfected with NAG-1 siRNA with or without FFA treatment. Data are shown as means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's post hoc test (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs empty vector or negative control of siRNA, without FFA treatment, #P < 0.05, ##P < 0.01 vs empty vector or negative control of siRNA, with FFA treatment.

Fig 3: OZ treatment inhibits SAH-induced NF-κB p65 activation. a, b Western blot analysis of p-IκBa, IκBa in treated mice and primary neurons. OZ treatment significantly restrained the phosphorylation of IκBa and the degradation of IκBa induced by SAH. c, d Nuclear fractions of brain tissue and neurons were extracted. Protein levels of nuclear p-NF-κB p65 and NF-κB p65 in treated mice and primary neurons. OZ administration reduced SAH-induced enhancement of nuclear p-NF-κB p65. e, f mRNA levels of NLRP3, IL-β, and IL-18 in treated mice and primary neurons. Data are expressed as mean ± SD, n = 5 per group. ***P < 0.001 vs Sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs SAH+Vehicle group. †††P < 0.001 vs Control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 vs OxyHb group

Fig 4: Juglanin improves pro-inflammatory cytokine releases caused by lipopolysaccharide (LPS). Serum pro-inflammatory cytokines were measured, including (A) TNF-α, (B) interleukin-1β (IL-1β), (C) IL-18, (D) IL-6, (E) IL-4 and (F) IL-17. Pro-inflammatory cytokines of (G) TNF-α, (H) IL-1β, (I) IL-18, (J) IL-6, (K) IL-4 and (L) IL-17 in lung tissue samples induced by LPS were measured after juglanin administration. The data are shown as mean ± SEM (n=10). *p<0.05 and **p<0.001 vs. the control (Con); +p<0.05, ++p<0.01 and +++p<0.001 vs. LPS-induced mice (LPS).

Fig 5: Cit protects against LPS-induced pulmonary pyroptosis and apoptosis. (A) IL-1β and (B) IL-18 concentration in lung tissue was determined using ELISA kits. (C) Expression of GSDMD-N was normalized to GSDMD-FL. (D) Representative western blot bands of Bax and Bcl-2. (E) Representative TUNEL-positive cells (green) in lung sections. Cell nucleus was stained with DAPI (blue). Scale bar, 100 µm. Data are expressed as the mean ± standard error of the mean (n=6). **P<0.01 vs. con; ##P<0.01 vs. LPS. LPS, lipopolysaccharide; GSDMD, gasdermin D; N, N-terminal; FL, full length; Con, control; Cit, citrulline.