Galangin Improved Non-Alcoholic Fatty Liver Disease in Mice by Promoting Autophagy

Abstract

Background: Previous studies have shown that curcumin derivatives can improve the fatty degeneration of liver tissue that occurs in nonalcoholic fatty liver disease (NAFLD). However, the specific mechanism for that improvement remains unclear. We examined whether the curcumin derivative galangin could reduce the fatty degeneration of liver tissue in mice with NAFLD by inducing autophagy, from the perspective of both prevention and treatment.

Methods: C57BL/6J mice were randomly assigned to a prevention group (given galangin and a HFD simultaneously) or a treatment group (given galangin after being fed an HFD). The prevention group was treated with galangin (100 mg/kg/d) or an equal volume of normal saline (NS) while being fed an HFD. Some mice were treated with an autophagy inhibitor (3-methyladenine, 3-MA; 30 mg/kg/biwk, i.p.) while being fed an HFD and galangin. HepG2 cells were cultured in DMEM medium containing both free fatty acids and galangin.

Results: Galangin was found to reduce the fatty degeneration of liver tissue induced by eating an HFD at both the prevention and treatment levels, and that effect might be related to an enhancement of hepatocyte autophagy. Inhibition of autophagy by 3-MA blocked the protective effect of galangin on hepatic steatosis. At the cellular level, galangin reduced lipid accumulation and enhanced the level of hepatocyte autophagy.

Conclusion: In vitro and in vivo studies showed that galangin cannot only improve pre-existing hepatic steatosis but also prevent the development of stenosis by promoting hepatocyte autophagy.

Keywords: 3-methyladenine; autophagy; free fatty acid; galangin; nonalcoholic fatty liver disease.

Figure 1

Preventive administration of galangin reduced hepatic steatosis. Mice were fed a HFD or a normal control diet (NC) for 8 weeks. Galangin (100 mg/kg/d) was given orally for 8 weeks. (A) ALT and (B) AST levels. (C) Weights of the mice in each group. (D) Wet weight of the liver. (E) TG content in liver tissue. (F) CHOL content in liver tissue. (G) Liver sections stained with HE or ORO (x400). The displayed value represents the mean value ± SEM (n = 4). Asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) indicate statistically significant differences.

Figure 2

Galangin administration reduced hepatic steatosis in NAFLD mice. After the mice were fed HFD or control normal diet for 8 weeks, galangin (100 mg/kg/d) was given orally for the next 4 weeks. (A) ALT and (B) AST levels. (C) Weights of the mice in each group. (D) Wet weight of the liver. (E) TG content in liver tissue. (F) CHOL content in liver tissue. (G) Liver sections stained with HE or ORO (x400). The displayed value represents the mean value ± SEM (n = 4). Asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) indicate statistically significant differences.

Figure 3

Effect of galangin at different concentrations on NAFLD cell viability. Asterisks (*P < 0.05) indicate statistically significant differences.

Figure 4

Galangin reduced FFA-induced lipid accumulation in HepG2 cells. (A) TG content in HepG2 cells in the prevention group. (B) TG content in HepG2 cells in the treatment group. (C) ORO staining (x400) was used to observe lipid deposits in each group of HepG2 cells. The displayed value represents the mean value ± SEM (n = 3). Asterisks (**P < 0.01, ***P < 0.001) indicate statistically significant differences.

Figure 5

Galangin induced hepatocyte autophagy in vivo and in vitro. Western blotting for mTOR, LC3-I/II, Beclin1, Atg3, AMPKα1, and pAMPKα1 expression was performed in the following groups: (A) C57BL/6 mice were fed a normal diet (NC) or HFD for 8 weeks, and galangin (100 mg/kg/d) H+G) or normal saline (H+NS) was given by stomach irrigation. Some of the mice that were fed galangin and a HFD diet were also injected with 3-MA (30 mg/kg, three times per week, IP) (H+G+3-MA) before their liver tissues were harvested. (B) C57BL/6 mice were fed a normal diet (NC) or HFD for 8 weeks and then given galangin (100 mg/kg/d) (H+G) or normal saline (H+NS). Some of the mice fed galangin and a HFD were also injected with 3-MA (30 mg/kg three times per week, IP) (H+G+3-MA). Some of the mice were fed only a HFD for another 4 weeks (HFD) before their liver tissues were harvested. (C) HepG2 cells were induced by free fatty acids (FFAs) for 24 h and then treated with galangin (100 μM) (FFA+G) and vehicle (DMSO) (FFA+vehicle). In order to study the effect of galangin on autophagy, a 3-MA (5 mM) co-treatment group (FFA+G+3-MA) was added for comparison. (D) HepG2 cells were treated with galangin (100 μM) for 12 h (FFA+G) or with vehicle (DMSO) plus FFAs (FFAs+vehicle) after 24 hours of FFA induction. In order to study the effect of galangin on autophagy, a 3-MA (5 mM) co-treatment group (FFA+G+3-MA) was partially added to the FFA+G treatment group for comparison.

Figure 6

Effect of galangin on liver inflammation and lipid metabolism-related gene expression in mice in the prevention group. Mice were fed a normal diet (NC) or HFD for 8 weeks, and galangin (100 mg/kg, stomach irrigation) (H+G) was administered each day at the same time. The HFD-fed mice were co-treated with galangin and 3-methyladenine (3-MA, 30 mg/kg, IP, three times per week) (H+G+3-MA) prior to being sacrificed. Liver inflammation- and lipid metabolism-related gene expression in mouse liver tissue was detected by real-time PCR. (A) TNF-α mRNA expression. (B) Fatty acid translocase (CD36) mRNA expression. (C and D) SREBP1c (C) and ChREBP (D) mRNA expression. (E and F) PPARα (E) mRNA expression and expression of its target gene, CPT1a (F). Each value represents a mean value ± SEM (n = 3). The asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) indicate a statistically significant difference.

Figure 7

Effects of galangin on liver inflammation and lipid metabolism-related gene expression in mice in the treatment group. Mice were fed a normal diet (NC) or high-fat diet (HFD) for 8 weeks. Galangin (100 mg/kg, stomach irrigation) (H+G) was administered each day for another 4 weeks. The HFD-fed mice were co-treated with galangin and 3-methyladenine (3-MA, 30 mg/kg, IP, three times per week) (H+G+3-MA) for another 4 weeks prior to being sacrificed. Liver inflammation- and lipid metabolism-related gene expression in samples of mouse liver tissue was detected by real-time PCR. (A) TNF-α mRNA expression. (B) Fatty acid translocase (CD36) mRNA expression. (C and D) SREBP1c (C) and ChREBP (D) mRNA expression. (E and F) PPARα (E) mRNA expression and CPT1a mRNA expression (F). Values represent the mean value ± SEM (n = 3). The asterisks (**P < 0.01, ***P < 0.001) indicate a statistically significant difference.