Fig 2: Caspase 6 deficiency activates TAK1 and inhibits RIPK1 and pyroptosis in fatty livers. Four-week-old Caspase 6 KO mice were fed with HFD for 24 weeks. (A) Caspase 6 expression was detected by western blot assay in fatty livers. (B) Photomicrographs of H&E staining, Oil Red O staining, and Masson staining in liver sections (scale bars, 200, 100, and 100 μm). (C) Liver weight was evaluated. (D) Liver function was evaluated by serum ALT and AST levels (U/L). (E) Liver TG. (F) qRT-PCR analysis of Fas, Srebp-1c, Col1a-1, Col3a-1, and Pdgfa in fatty livers. (G) Immunohistochemistry staining of Ly6G+ neutrophils and immunofluorescence staining of CD11b in liver tissues (scale bar, 50 μm). (H) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in fatty livers. (I) Western blot analysis and relative density ratio of TAK1, RIPK1, p-RIPK1, GSDMD-N, cCaspase 1, and NLRP3 in liver tissues. (J) Immunofluorescence staining of p-RIPK1 in fatty livers (scale bars, 50 μm). ANOVA, Bonferroni multiple comparison test, n = 4–6 per group. All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, hematoxylin and eosin; HFD, high-fat diet; KO, knockout; qRT-PCR, quantitative real time-PCR; TG, triglyceride.

Fig 3: The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and CRISPR-Caspase 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, p-RIPK1, GSDMD-N, cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.

Fig 4: NUAK1 inhibition suppressed Caspase 6 activity and induced TAK1 activation and cell death in mouse MASH models. Four-week-old mice were fed with HFD for 24 weeks, accompanied by the treatment of NUAK1 inhibitor HTH-01-015. (A) Western blot analysis and relative density ratio of active Caspase 6, TAK1, RIPK1, p-RIPK1, GSDMD-N, cCaspase 1, and NLRP3. (B) Immunohistochemistry staining of p-RIPK1 in fatty livers (scale bars, 50 μm). (C) Caspase 6 activity in the liver lysate. (D) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in fatty livers. (E) ELISA analysis of serum IL-1β levels. (F) ELISA analysis of serum IL-18 levels. ANOVA, Bonferroni multiple comparison test, n = 4–6 per group. All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: HFD, high-fat diet; qRT-PCR, quantitative real time-PCR.

Fig 5: RIPK1 is essential for regulating pyroptosis activation and inflammation in response to MASH. Mouse hepatocytes were transfected with RIPK1 KO, GSDMD KO, RIPK1 Activation, and GSDMD Activation vectors, then treated with HTH-01-015, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bars, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Western blot analysis and relative density ratio of RIPK1, GSDMD-N, cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (F) Immunofluorescence staining of NLRP3 (red) and ASC (green) in hepatocytes (scale bars, 20 and 10 μm). (G) qRT-PCR analysis of IL-1β, IL-6, and TNF-α in hepatocytes. (H) Oil Red O staining of PA-stimulated hepatocytes (scale bars, 20 and 10 μm). (I) LDH releasing from hepatocytes. (J) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (K) Western blot analysis and relative density ratio of RIPK1, GSDMD-N, cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (L) Immunofluorescence staining of NLRP3 (red) and ASC (green) in hepatocytes (scale bars, 20 and 10 μm). (M) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. ANOVA, Bonferroni multiple comparison test, n = 4–6 per group. All data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: KO, knockout, LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.