Le Carbone prevents liver damage in non-alcoholic steatohepatitis-hepatocellular carcinoma mouse model via AMPKα-SIRT1 signaling pathway activation

Abstract

Le Carbone (LC), a fiber-enriched activated charcoal dietary supplement, claimed to be effective against inflammation associated with colitis, trimethylaminuria, and sclerosis. The study aimed to investigate the underlying mechanisms of LC to protect liver damage and its progression in non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC) mice. To induce this model, C57BL/6J male baby mice were injected with a low-dose of streptozotocin and fed with a high-fat diet (HFD) 32 during 4 weeks-16 weeks of age. The LC suspension was administered orally at a dose of 5 mg/mouse/day started at the age of 6 weeks and continued until 16 weeks of age along with HFD32 feeding. At the end of the experiment, serum and liver tissues were collected for the biochemical, histological, and molecular analysis. We found that LC suspension improved the histopathological changes, serum aminotransferases in NASH mice. The hepatic expression of metabolic proteins, p-AMPKα and sirtuin 1, and proteins responsible for β-oxidation of fatty acids, peroxisome proliferator-activated receptor (PPAR) γ coactivator-α, PPARα were significantly repressed in NASH mice. LC treatment markedly restored these expressions. LC treatment significantly reduced the hepatic proteins expressions of PPARγ, tissue inhibitor of metalloproteinases 4, p47phox, p-JNK, p-ERK1/2, glypican-3, and prothrombin in NASH mice. Our findings demonstrate that LC prevents the liver damage and progression of NASH, possibly by enhancing the AMPK-SIRT1 signaling pathway.

Keywords: AMPKα; Le carbone; Non-alcoholic steatohepatitis; PPARα; SIRT1.

Figure 1

Experimental protocol. Experimental protocol used in the present study. Streptozotocin (STZ) was injected s. c.to the 2 days old pups and then allowed to grow under breast milk. At 4 weeks of age the mice were given either normal diet or high-fat diet (HFD) 32. Le Carbone suspension (LC) treatment was given to the NASH + LC group mice from 6 weeks of age to16 weeks of age. All the mice were sacrificed at 16 weeks of age.

Figure 2

Le Carbone attenuates the clinicopathology in NASH-HCC mice. (A), Representative macroscopic appearance of livers (circles: liver tumors). (B), Histogram for the ratio of liver weight (LW) and body weight (BW) in g/Kg. (C), H&E staining (yellow arrow: macro vesicular steatosis, black arrow: micro vesicular steatosis, red arrow: hypertrophy, circles: inflammatory cells). Scale bars, 50 μm. (D), Histogram of non-alcoholic fatty liver disease (NAFLD) activity score. (E), Fibrosis deposition by Masson trichrome staining (blue area, black arrow). Scale bars, 50 μm. (F), percentage of fibrosis in each group. Data are mean ± SEM, (n = 6/group). Normal, age matched normal mice; NASH, untreated NASH-HCC mice; NASH + LC, NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis was carried out using One-way ANOVA followed by Tukey's test where ∗∗p < 0.01, ∗∗∗p < 0.001vs Normal, and #p < 0.05, ##p < 0.01, ###p < 0.001vs NASH.

Figure 3

Effect of Le Carbone on hepatic AMPKα, SIRT1, GLUT4, PGC1α and PEPCK-C expression in NASH-HCC mice. Representative Western blots show specific bands for hepatic (A), p-AMPKα (for AMPKα); (B), SIRT1 (for β-actin); (C), GLUT4; (D), PGC1α; and (E), PEPCK-C and the representative histograms show the band densities with relative to GAPDH. Each bar represents mean ± SEM. Normal, age matched normal mice; NASH, untreated NASH-HCC mice; NASH + LC, NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis was carried out using One-way ANOVA followed by Tukey's test where, ∗p < 0.05, ∗∗p < 0.01 vs Normal, and #p < 0.05, ##p < 0.01 vs NASH.

Figure 4

Le Carbone attenuates hepatic lipogenesis in NASH-HCC mice. Western blots show specific bands for hepatic (A), PPARα (for β-actin); (B), PPARγ; (C), ADRP; and (D), TIMP4 and the representative histograms show the band densities with relative to GAPDH. Each bar represents mean ± SEM. Normal, age matched normal mice; NASH, untreated NASH-HCC mice; NASH + LC, NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis was carried out using One-way ANOVA followed by Tukey's test where, ∗p < 0.05, ∗∗p < 0.01vs Normal, #p < 0.05, ##p < 0.01vs NASH.

Figure 5

Effect of LC on the expression of CD36 positive cells in NASH-HCC liver. (A–B), Immunohistochemical staining of CD36 positive cells and their quantitative data. Data are mean ± SEM, (n = 6/group). Normal, age matched normal mice; NASH, untreated NASH-HCC mice; NASH + LC, NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day. Scale bars, 50 μm. Statistical analysis was carried out using One-way ANOVA followed by Tukey's test where, ∗∗p < 0.01vs Normal, #p < 0.05 vs NASH.

Figure 6

Effect of Le Carbone on hepatic HO-1, p47phox, Nrf2, p-JNK and p-ERK1/2 expression in NASH-HCC mice. Representative Western blots show specific bands for (A), HO-1; (B), p47phox; (C), Nrf2; (D), p-JNK (for JNK); (E), p-ERK1/2 (ERK1/2) and the representative histograms show the band densities with relative to GAPDH. Each bar represents mean ± SEM. Normal, age matched normal mice; NASH, untreated NASH-HCC mice; NASH + LC, NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis was carried out using One-way ANOVA followed by Tukey's test where, ∗p < 0.05, ∗∗p < 0.01 vs Normal, #p< 0.05, ##p < 0.01 and ###p < 0.001 vs NASH.

Figure 7

Effect of LC on glypican-3 and prothrombin in NASH-HCC liver. Western blots show specific bands for hepatic (A), Glypican-3; and (B), prothrombin (for β-actin), and the representative histograms show the band densities with relative to GAPDH. Each bar represents mean ± SEM. Normal, age matched normal mice; NASH, untreated NASH-HCC mice; NASH + LC, NASH-HCC mice treated with Le Carbone suspension at 5 mg/mouse/day. Statistical analysis was carried out using One-way ANOVA followed by Tukey's test where, ∗p < 0.05, ∗∗p < 0.01 vs Normal, ##p < 0.01vs NASH.