Blockade of 11β-hydroxysteroid dehydrogenase type 1 ameliorates metabolic dysfunction-associated steatotic liver disease and fibrosis

Abstract

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme involved in the conversion of cortisone to active cortisol in the liver. Elevated cortisol levels can trigger oxidative stress, inflammation, and hepatocyte damage, highlighting the importance of 11β-HSD1 inhibition as a potential therapeutic approach. This study aimed to explore the effects of INU-101, an inhibitor of 11β-HSD1, on the development of metabolic dysfunction-associated steatotic liver disease (MASLD) and fibrosis. Our findings demonstrated that INU-101 effectively mitigated cortisol-induced lipid accumulation, reactive oxygen species generation, and hepatocyte apoptosis. Furthermore, 11β-HSD1 inhibition suppressed hepatic stellate cell activation by modulating β-catenin and phosphorylated SMAD2/3. INU-101 administration significantly reduced hepatic lipid accumulation and liver fibrosis in mice fed fast-food diet. This study suggests that INU-101 holds promise as a clinical candidate for treating MASLD and fibrosis, offering potential therapeutic benefits by targeting the intricate processes involving 11β-HSD1 and cortisol regulation in the liver.

Keywords: 11β-HSD1; Cortisol; Cortisone; Lipid accumulation; Liver fibrosis.

Fig. 1

Cortisol induces hepatic lipid accumulation and subsequent cell death (A) 11βHSD1 mRNA expression in primary hepatocytes, KCs, and HSCs. (B–C) ELISA for detecting conversion of cortisone to cortisol in Hep3B cells and primary hepatocyte. (D–E) BODIPY staining and quantification of cortisol in HepG2 cells. (F–G) mRNA expression of SREBP1 and ChREBP in Hep3B cells treated with cortisol. (H) Quantification of AMPK and p-AMPK protein levels in Hep3B cells following cortisol treatment. The original image was provided in Figure S3 (I-J) Flow cytometry analysis of mitochondrial ROS production in Hep3B cells after cortisol treatment. (K–L) Flow cytometry analysis of apoptosis in Hep3B cells after cortisol treatment. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 2

Blockade of 11β-HSD1 prevents cortisone-mediated lipid accumulation and subsequent cell death (A) ELISA showing cortisone conversion to cortisol following co-treatment with INU-101 in Hep3B cells. (B–C) BODIPY staining and quantification of cortisone following co-treatment with INU-101 in HepG2 cells. (D–E) SREBP1 and ChREBP mRNA expression following co-treatment of cortisone with INU-101 in Hep3B cells. (F) Quantification of AMPK and p-AMPK protein levels in Hep3B cells following cortisone and INU-101 treatment. The original image was provided in Figure S4 (G-H) Flow cytometry analysis of mitochondrial ROS production in Hep3B cells after cortisone and INU-101 treatment. (I–J) Flow cytometry analysis of apoptosis detection in Hep3B cells after cortisone and INU-101 treatment. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 3

Cortisol signaling links hepatocyte-derived DAMPs to HSC activation (A) Schematic co-culture system. (B) Protein levels of β-catenin, p-SMAD2/3, and SMAD2/3 in LX2 cells after cell supernatant treatment. The original image was provided in Figure S5 (C-D) TQuantitative analysis of β-catenin/actin and p-SMAD2/3/SMAD2/3. (E) mRNA expression of pro-fibrosis- and proliferation-related genes (including ACTA2, COL4A1, FN1, TIMP1, C-MYC and CCND1) in LX2 cells after cell supernatant treatment. (F) Protein levels of β-catenin, p-SMAD2/3, and SMAD2/3 in LX2 cells after co-treatment with supernatant and INU-101. The original image was provided in Fig. S6. (H) Quantification of β-catenin, p-SMAD2/3, and SMAD2/3. The original image was provided in Fig. S6. (I) mRNA expression of pro-fibrosis- and proliferation-related genes (including ACTA2, COL4A1, FN1, TIMP1, C-MYC and CCND1) in LX2 cells after co-treatment with supernatant and INU-101. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 4

11β-HSD1 mediates cortisol-induced HSC activation by regulating β-catenin signaling (A) Protein level of β-catenin in cortisol-treated LX2 cells. The original image was provided in Figure S7 (B-E) mRNA expression of pro-fibrosis- and proliferation-related genes (including ACTA2, COL4A1, FN1, C-MYC and CCND1) in LX2 cells. (F) Cell proliferation assay of LX2 after treatment with different concentrations of cortisol. (G) Protein levels of β-catenin. The original image was provided in Figure S8 (H-L) mRNA expression of pro-fibrosis- and proliferation-related genes in LX2 cells after treatment with cortisol or cortisol combined with β-catenin knockdown. (M) Cell proliferation assay of LX2 cells after treatment with cortisol or cortisol combined with β-catenin knockdown. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 5

Pharmacological inhibition of 11β-HSD1 ameliorates FFD-induced MASLD and fibrosis (A) Liver weight of mice fed for 24 -weeks with normal chow diet (NCD), FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (B) Liver-to-body weight ratio of 2mice fed for 4-weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (C) Serum ALT levels of 24 -weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (D) Serum cholesterol levels of 24 -weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (E) Serum triglyceride levels of mice fed for 24-weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (F) Macroscopic observation, H&E staining and Sirius red staining of liver tissues from mice fed for 24-weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (G) NAS scoring of liver tissues from mice fed for 24 -weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101. (H) Quantification of Sirius red staining of liver tissue from mice fed for 24 -weeks with NCD, FFD, FFD + UDCA, FFD + OCA, and FFD + INU-101 liver tissue. (I–L) mRNA expression of pro-fibrosis-related genes, such as COL1A1, LOX, TIMP1 and COL4A1, in liver tissue. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.