The Effects of Berberine on Concanavalin A-Induced Autoimmune Hepatitis (AIH) in Mice and the Adenosine 5'-Monophosphate (AMP)-Activated Protein Kinase (AMPK) Pathway

Abstract

BACKGROUND Berberine, a herbal extract, has been reported to protect against inflammatory disorders. The adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway can be activated by berberine and inhibited by the synthetic, reversible AMP-competitive inhibitor, Compound C. The aim of this study was to investigate the effects of berberine on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) in mice via the AMPK pathway. MATERIAL AND METHODS BALB/c mice were treated with berberine, with or without Compound C, followed by treatment with Con A. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Liver tissue histology was performed to evaluate hepatic injury and AIH. Cytokine levels in serum and hepatic tissue were measured by enzyme-linked immunoassay (ELISA) and used quantitative polymerase chain reaction (qPCR). Levels of phosphorylated acetyl coenzyme-A carboxylase (ACC), representing AMPK activation, were detected by Western blotting. RESULTS Serum ALT and AST levels were significantly reduced by berberine (100 and 200 mg/kg/day) in mice with Con A-induced hepatitis. Berberine also reduced Con A-induced hepatocyte swelling, cell death, and infiltration of leukocytes. Serum levels of tumor necrosis factor (TNF)-alpha, interferon (IF)-gamma, interleukin (IL)-2, and IL-1beta were reduced by berberine pre-treatment; levels of serum IL-10, an anti-inflammatory cytokine, was elevated. These protective effects of berberine on Con-A-induced AIH were reversed by treatment with Compound C. CONCLUSIONS In a murine model of Con A-induced AIH, berberine treatment reduced hepatic injury via activation of the AMPK pathway. Further studies are recommended to determine the potential therapeutic role for berberine in AIH.

Figure 1

Berberine reduced concanavalin A (Con A)-induced autoimmune hepatitis (AIH)-induced and immunological hepatic injury in mice. After the mice were gavaged (fed through the mouth via a tube) with solvent or berberine (50, 100, 200 mg/kg) for three days, they were intravenously injected with 20 mg/kg concanavalin A (Con A). Eight hours following injection, the serum and liver samples were collected. (A, B) Illustrate the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, respectively. C is a photomicrograph of the mouse liver tissue section stained with hematoxylin and eosin (H&E) (magnification, ×200). Data are expressed as mean ±SD (n=6, ** P<0.01 vs. control group, ^## P<0.01 vs. Con A group).

Figure 2

Effects of berberine on serum cytokines in concanavalin A (Con A)-stimulated mice. After the mice were gavaged (fed through the mouth via a tube) with solvent or berberine (50, 100, 200 mg/kg) for three days, they were injected intravenously with 20 mg/kg of Con A. Eight hours following injection, serum samples were obtained and used to detect TNF-α (A), IFN-γ (B), IL-1β (C), IL-2 (D) and IL-10 (E) with enzyme-linked immunosorbent assay (ELISA) (n=8). ** P<0.01 vs. the control group, ^## P<0.01 vs. the Con A group.

Figure 3

Effects of berberine on the expression of cytokines in the liver tissue of concanavalin A (Con A)-stimulated mice. After the mice were gavaged (fed through the mouth via a tube) with solvent or 50, 100, and 200 mg/kg berberine for three days, they were injected intravenously with 20 mg/kg of concanavalin A (Con A). At 8 hours after Con A injection, the TNF-α (A), IFN-γ (B), IL-1β (C), IL-2 (D) and IL-10 (E) mRNA levels in liver tissue were detected by quantitative polymerase chain reaction (qPCR) (n=6). ** P<0.01 vs. the control group, ^## P<0.01 vs. the Con A group.

Figure 4

Berberine activated adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) in mouse liver tissue. (A, C) Mice were treated with 50, 100, and 200 mg/kg of berberine for three days. After treatment with berberine for 8 hours, Western blot analysis was used to detect the phosphorylation of acetyl coenzyme-A carboxylase (ACC). (B, D) Con A-injected mice were pretreated with 100 mg/kg berberine, with or without the adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (20 mg/kg) for three days. At 8 hours after Con A injection, ACC phosphorylation levels were detected by Western blot analysis. The western blot bands were semi-quantified with NIH Image J software and normalized with internal control, β-actin (C, D) (n=6). ** P<0.01 vs. the control group, ^## P<0.01 vs. the Con A group, ^§§ P<0.01 vs. the Con A + berberine group.

Figure 5

Effects of Compound C on serum cytokines in berberine-treated mice. Concanavalin A (Con A)-injected mice were pretreated with 100 mg/kg berberine with or without 20 mg/kg Compound C for three days. At 8 hours after Con A injection, the serum and liver samples were collected. Serum alanine aminotransferase (ALT) (A) and aspartate aminotransferase (AST) (B) levels were detected with an enzyme-linked immunoassay (ELISA). C is a photomicrograph of the mouse liver tissue section stained with hematoxylin and eosin (H&E) (magnification, ×200), data are expressed as the mean ±SD (n=6). ** P<0.01 vs. the control group, ^## P<0.01 vs. the Con A group, ^§§ P<0.01 vs. the Con A plus berberine group.

Figure 6

Effects of Compound C on serum cytokines in berberine-treated mice. Concanavalin A (Con A)-injected mice were pretreated with 100 mg/kg berberine with or without 20 mg/kg of Compound C for three days. At 8 hours after Con A injection, the serum TNF-α (A), IFN-γ (B), IL-1β (C), IL-2 (D), and IL-10 (E) levels were detected with an enzyme-linked immunoassay (ELISA) (n=6). ** P<0.01 vs. the control group, ^## P<0.01 vs. the Con A group, ^§§ P<0.01 vs. the Con A plus berberine group.

Figure 7

Effect of Compound C on liver cytokine expression in berberine-treated mice. Mice injected with concanavalin A (Con A)- were pretreated with 100 mg/kg berberine with or without 20 mg/kg Compound C for three days. At 8 hours after Con A injection, the TNF-α (A), IFN-γ (B), IL-1β (C), IL-2 (D), and IL-10 (E) mRNA levels in liver tissue were detected by quantitative polymerase chain reaction (qPCR) (n = 6). ** P<0.01 vs. the control group, ^## P<0.01 vs. the Con A group, ^§§ P<0.01 vs. the Con A plus berberine group.