Fig 2: NUAK1 is upregulated in the livers of patients with MASH or mouse MASH models. (A) NUAK1 mRNA in the liver tissues of human MASLD (NAS <5) and MASH (NAS ≥5), compared with normal individuals, as determined by qRT-PCR (n = 18 per group). (B) NUAK1 protein levels in the liver tissues of human normal individuals and MASH, as determined by western blot (n = 5 per group). (C) NUAK1 immunohistochemistry staining in liver specimens of patients with MASH and normal individuals (scale bar: 50 µm). (D) Correlation between NUAK1 mRNA and liver TGF-β expression in human MASH livers. (E–G) mRNA and protein levels of NUAK1 were detected in the livers of mice fed with HFD or MCD compared with NCD. (H) Double immunofluorescence staining of NUAK1 (green) and HNF-4α (red) in liver samples of mice fed on NCD or HFD (scale bar, 50 μm). DAPI was used to visualize nuclei (blue). 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; MCD; methionine-choline–deficient diet; NAS, nonalcoholic fatty liver disease activity score; NCD, normal chow diet; qRT-PCR, quantitative real time-PCR.

Fig 3: NUAK1 inhibition alleviates liver steatosis and fibrosis on HFD-induced MASH in mice. Four-week-old mice were fed with HFD for 24 weeks, accompanied by treatment of NUAK1 inhibitor HTH-01-015 (20 mg/kg). (A) Schematic protocol of the experiment. (B, C) Liver function was evaluated by serum ALT and AST levels (U/L). (D) Liver weight was evaluated. (E) Representative histological staining (H&E) of liver tissue (scale bar, 200 μm). (F) Oil Red O staining of liver tissues (scale bar, 100 μm). (G) Masson staining of liver tissues and fibrosis score (scale bar, 100 μm). (H) Immunohistochemistry staining of α-SMA of liver tissues (scale bar, 50 μm). (I) Immunofluorescence staining of CD11b of liver tissues (scale bar, 50 μm). (J) Liver TC and TG. (K) qRT-PCR analysis of Fas, Srebp-1c, Col1a1, Col3a1, and Pdgfa in fatty livers. 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; qRT-PCR, quantitative real time-PCR; TC, total cholesterol; TG, triglyceride.

Fig 4: 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.