ANGPT1–GABARAP axis modulates NLRP3 inflammasome–mediated pyroptosis in Crohn’s disease

Figures

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  Figure 1. GABARAP is associated with reduced Crohn’s disease risk. (A) Two-sample MR analysis of pyroptosis-related proteins in relation to Crohn’s disease risk. Points denote odds ratios (ORs) and 95% confidence intervals (CIs); horizontal error bars indicate 95% CIs, and the dashed line indicates OR = 1. (B) Forest plot of SNP-level effect estimates for GABARAP on CD risk, with pooled inverse-variance-weighted (IVW) and MR-Egger estimates shown on the log odds ratio scale. (C) Scatter plot of SNP-specific effects on GABARAP (exposure) versus CD (outcome), with fitted lines showing MR estimates from different MR methods. (D) Funnel plot of SNP-level estimates against their standard errors; symmetry indicates no marked evidence of directional horizontal pleiotropy. (E) Leave-one-out analysis of the MR estimate; each point reflects exclusion of a single SNP.

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  Figure 2. ANGPT1–GABARAP axis is associated with Crohn’s disease risk. Two-step MR analysis suggested a putative genetic relationship between ANGPT1 and GABARAP. ANGPT1 showed a positive association with GABARAP, whereas GABARAP was linked to reduced CD risk. Mediation analysis suggested a partial statistical indirect association involving GABARAP, representing 22.97% of the total effect. These estimates should be interpreted as genetically predicted associations rather than evidence of a definitive causal hierarchy.

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  Figure 3. ANGPT1–GABARAP axis is associated with pyroptosis activation in DSS-induced colitis. (A) Body weight change (% of baseline). (B) Disease activity index (DAI). (C) Representative colon morphology. (D) Colon length. (E) Representative H&E-stained colon sections. Scale bars as indicated. (F) Histological scores. (G) LDH activity in colon tissue homogenates normalized to total protein content. (H) Relative mRNA expression of ANGPT1, GABARAP, IL18 and IL1B. (I) Western Blotting analysis of NLRP3, ANGPT1, GSDMD-FL, GSDMD-N, pro-Caspase-1, Caspase-1 (p10), GABARAP and β-actin. (J–N) Quantification of NLRP3 (J), ANGPT1 (K), GSDMD cleavage (GSDMD-N/GSDMD-FL) (L), Caspase-1 activation (Caspase-1 (p10)/pro-Caspase-1) (M), and GABARAP (N), normalized to β-actin. (O) ELISA quantification of ANGPT1, IL-18, and IL-1β in colon tissue. Mice received 2% DSS in drinking water for 7 days followed by regular water for 3 days; controls received regular water throughout. Western Blotting quantification was normalized to the corresponding β-actin loading control. Data are shown as mean ± SD (n ≥ 5). Statistical significance was assessed using an unpaired two-tailed Student’s t-test.

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  Figure 4. GABARAP knockdown enhances NLRP3 inflammasome-associated pyroptotic signaling in cell models. (A, F) Representative western blots of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase-1, Caspase-1 (p10), GABARAP, and β-actin in THP-1-derived macrophages and NCM460 cells. (B–E, G–J) Quantification of NLRP3 (B, G), GSDMD cleavage [GSDMD-N/GSDMD-FL; (C, H)], Caspase-1 activation [Caspase-1 (p10)/pro-Caspase-1; (D, I)], and GABARAP (E, J), normalized to β-actin. (K, L) ELISA quantification of GABARAP, IL-18, and IL-1β in culture supernatants. After 48 h of siRNA transfection, cells were stimulated with LPS (1 μg/mL, 4 h) followed by nigericin (10 μM, 2 h); unstimulated cells served as controls. Representative western blots are shown, and densitometric quantification was based on three independent transfection experiments. NLRP3, GSDMD, and Caspase-1/GABARAP were detected on separate membranes with corresponding β-actin controls. Data are shown as mean ± SD (n = 3) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

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  Figure 5. ANGPT1 attenuates pyroptotic signaling accompanied by GABARAP restoration. (A, G) Representative western blots of NLRP3, ANGPT1, GSDMD-FL, GSDMD-N, pro-Caspase-1, Caspase-1 (p10), GABARAP, and β-actin in THP-1-derived macrophages and NCM460 cells. (B–F, H–L) Quantification of NLRP3 (B, H), ANGPT1 (C, I), GSDMD cleavage [GSDMD-N/GSDMD-FL; (D, J)], Caspase-1 activation [Caspase-1 (p10)/pro-Caspase-1; (E, K)], and GABARAP (F, L), normalized to β-actin. (M, N) ELISA quantification of ANGPT1, GABARAP, IL-18, and IL-1β in culture supernatants. After 48 h of siRNA transfection, cells were treated with recombinant human ANGPT1 (rhANGPT1; 200 ng/mL, 30 min), followed by LPS (1 μg/mL, 4 h) and nigericin (10 μM, 2 h), as indicated; unstimulated cells served as controls. Representative western blots are shown, and densitometric quantification was based on three independent transfection experiments. NLRP3, ANGPT1/GSDMD, and Caspase-1/GABARAP were detected on separate membranes with corresponding β-actin controls. Data are shown as mean ± SD (n = 3) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.