Ganoderic acid A ameliorates non-alcoholic streatohepatitis (NASH) induced by high-fat high-cholesterol diet in mice

Abstract

Non-alcoholic steatohepatitis (NASH) is becoming a huge global health problem. Previous studies have revealed that ganoderic acids have hepatoprotective and hypocholesterolemic effects. In the present study, to evaluate the anti-NASH activity of ganoderic acid A (GAA), male 6-week-old C57BL/6J mice were divided into the following four groups, which were administered different diets: Normal diet (ND group), high-fat high-cholesterol diet (HFHC group), HFHC diet supplemented with 25 mg/kg/day (GAAL group) or 50 mg/kg/day of GAA (GAAH group). After 12 weeks of GAA treatment, histopathological results revealed that compared with that of the HFHC group, GAA significantly inhibited fat accumulation, steatosis, inflammation and fibrosis in the liver. GAA effectively reduced serum aspartate transaminase and alanine transaminase levels compared with the HFHC model. Furthermore, the endoplasmic reticulum (ER) stress-responsive proteins, including glucose-regulated protein 78, phosphorylated (p)-eukaryotic initiation factor-2α and p-JNK, were significantly suppressed by GAA, while ERp57, p-MAPK and p-AKT were significantly increased after GAA treatment. Taken together, it was concluded that GAA could resist HFHC diet-induced NASH. In terms of its underlying mechanism, GAA could improve liver inflammation and fibrosis by inhibiting hepatic oxidative stress and the ER stress response induced by HFHC.

Keywords: endoplasmic reticulum stress; ganoderic acid A; high-fat high-cholesterol diet; inflammation; non-alcoholic streatohepatitis.

Figure 1

GAA improves the metabolic profiles of HFHC-fed mice. (A) Body weight, (B) food intake, (C) TG, (D) TC, (E) LDL-c, (F) HDL-c, (G) AST and (H) ALT levels were determined in murine blood. Mice were fed an ND or HFHC diet with or without indicated doses of GAA treatment (n=6-8). (I) The chemical structure and molecular weight of GAA. Assays were repeated three times. Data are expressed as the mean ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the HFHC group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GAA, ganoderic acid A; GAAH, GAA 50 mg/kg/day; GAAL, GAA 25 mg/kg/day; HDL-c, high density lipoprotein-cholesterol; HFHC, high-fat high-cholesterol; LDL-c, low density lipoprotein-cholesterol; MW, molecular weight; ns, not significant; ND, normal diet; TC, total cholesterol; TG, triglyceride.

Figure 2

GAA alleviates hepatic steatosis in HFHC-fed mice. (A) Livers were stained with Oil red O. (B) The area of Oil red O was determined using ImageJ 1.8.0 software. (C) The ratio of HW/BW was determined. (D and E) Hepatic TC and TG levels were evaluated. Mice were fed an ND or HFHC diet with or without indicated doses of GAA treatment (n=6-8). Assays were repeated three times. Data are expressed as the mean ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the HFHC group. GAA, ganoderic acid A; GAAH, GAA 50 mg/kg/day; GAAL, GAA 25 mg/kg/day; HFHC, high-fat high-cholesterol; HW/BW, liver weight to body weight; ND, normal diet; ns, not significant; TC, total cholesterol; TG, triglyceride.

Figure 3

GAA reduces the hepatic inflammation response in HFHC-fed mice. (A) H&E staining of liver tissue. (B and C) CD68 and F4/80 levels in liver were detected by immunohistochemistry. (D and E) Relative levels of CD68 and F4/80 were measured. (F) IL-1β, (G) TNFα and (H) IL-6 levels in murine blood. mRNA levels of (I) IL-1β, (J) TNFα and (K) IL-6 levels in liver tissue. Mice were fed an ND or HFHC diet with or without indicated doses of GAA treatment (n=6-8). Assays were repeated three times. Data are expressed as the mean ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the HFHC group. GAA, ganoderic acid A; GAAH, GAA 50 mg/kg/day; GAAL, GAA 25 mg/kg/day; H&E, hematoxylin and eosin; HFHC, high-fat high-cholesterol; ND, normal diet; ns, not significant.

Figure 4

GAA decreases the hepatic fibrosis in HFHC-fed mice (A) SR staining of liver tissue. (B) The relative intensity of SR staining. mRNA levels of (C) α-SMA, (D) TGF-β and (E) MMP-13 levels in murine liver tissue. Mice were fed an ND or HFHC diet with or without indicated doses of GAA treatment (n=6-8). Assays were repeated three times. Data are expressed as the mean ± SEM. ^**P<0.01 and ^***P<0.001 vs. the HFHC group. GAA, ganoderic acid A; GAAH, GAA 50 mg/kg/day; GAAL, GAA 25 mg/kg/day; HFHC, high-fat high-cholesterol; ND, normal diet; α-SMA, α-smooth muscle actin; SR, Sirius Red.

Figure 5

GAA hepatoprotection is associated with hepatic oxidative stress and the ER stress response. (A) MDA and (B) SOD levels were detected in murine livers. (C and G) Western blotting was performed to determine the relative protein levels of (D) GRp78, (E) p-eIF-2α, (F) p-JNK, (H) ERp57, (I) p-MAPK and (J) p-AKT. Mice were fed an ND or HFHC diet with or without indicated doses of GAA treatment (n=6-8). Assays were repeated three times. Data are expressed as the mean ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the HFHC group. eIF-2α, eukaryotic initiation factor-2α GAA, ganoderic acid A; GAAH, GAA 50 mg/kg/day; GAAL, GAA 25 mg/kg/day; GRp78, glucose-regulated protein 78; HFHC, high-fat high-cholesterol; MDA, malondialdehyde; ND, normal diet; ns, not significant; p, phosphorylated; SOD, superoxide dismutase.