Ammonium glycyrrhizin counteracts liver injury caused by lipopolysaccharide/amoxicillin-clavulanate potassium

Abstract

We treated isolated chicken primary hepatocytes with lipopolysaccharide/amoxicillin clavulanate potassium (LPS/AC) to model liver injury and investigate its underlying mechanisms. We also used this model to assess the cytoprotective effects of compound ammonium glycyrrhizin (CAG) in vitro. LPS/AC-induced injury decreased cell viability and increased the activity of serum aspartate transaminase and alanine transaminase. Levels of superoxide dismutase, glutathione, and glutathione peroxidase were lower than control, while levels of the oxidative product malondialdehyde and reactive oxygen species were higher. Treatment with CAG for 24 h ameliorated these changes. Caspase-3 activity assays and flow cytometry revealed increased apoptosis in the model group. However, apoptosis decreased after CAG treatment, as confirmed by Hoechst 33342 staining. We also observed changes in mitochondrial ultrastructure. Real-time PCR and western blot analyses showed that CAG treatment downregulated LPS/AC-induced RNA expression of caspase-3, caspase-9, bax, cytochrome c, and fas, and upregulated the expression of bcl-2. Mitochondrial cytochrome c was released into the cytosol and the inner mitochondrial membrane potential (ΔΨm) was decreased. Our results highlight CAG as a potential therapeutic agent to counteract LPS/AC-induced liver injury.

Keywords: amoxicillin clavulanate potassium; antioxidant; chicken primary hepatocytes; compound ammonium glycyrrhizin; lipopolysaccharide.

Figure 1. Cell viability assay for cultured chicken hepatocytes

(A) Graph indicating that cell viability was ∼90%. (B) Image showing that most hepatocytes cultured for 6 h adhered to the bottom of the culture plate. (C–D) Hepatocytes cultured at 24 h and 48 h, exhibiting differentiation into irregular polygonal shapes.

Figure 2. Effects of LPS/AC treatment on hepatocytes

(A) Variations in hepatocyte viability after LPS/AC treatment for 24 h. MTT assay showing that LPS/AC treatment induces a decrease in cell viability in a dose-dependent manner. Values are expressed as the mean ± SD (n = 3) and the IC50 was 30 μg/mL of LPS + 100 μg/mL of AC. (B) Effect of different concentrations of LPS/AC treatment (24 h) on hepatocyte cytomorphology as visualized with an inverted phase-contrast microscope (20×). a: Normal cells; b: 30 μg/mL of LPS + 60 μg/mL of AC; c: 30 μg/mL of LPS + 80 μg/mL of AC; d: 30 μg/mL of LPS + 100 μg/mL of AC; e: 30 μg/mL of LPS + 120 μg/mL of AC; f: 30 μg/mL of LPS + 140 μg/mL of AC.

Figure 3

The effects of CAG on (A) cell viability, (B) alanine transaminase (ALT) activity, and (C) aspartate transaminase (AST) activity. The hepatocytes were treated with 30 μg/mL of LPS + 100 μg/mL of AC after exposure to CAG for 24 h. For the LPS/AC-treated groups, “−” and “+” represent the cells in culture medium and those treated with 30 μg/mL of LPS + 100 μg/mL of AC, respectively. For the CAG-treated groups, “−” represent cells treated without CAG. Values are expressed as the mean ± SD. (n = 3). ^# < 0.05, ^## < 0.01 compared to the control; * < 0.05, ** < 0.01 compared to the model.

Figure 4. Effects of the CAG treatment on the levels of oxidative stress, cellular antioxidant enzymes, and caspase-3 activity

(A) SOD, (B) GSH, (C) MDA, (D) caspase-3, and (E) GSH-Px activity, and (F) reactive oxygen species (ROS) levels in the supernatant. Values are expressed as the mean ± SD. (n = 3). ^# < 0.05, ^## < 0.01 compared to the control; * < 0.05, ** < 0.01 compared to the model.

Figure 5. Changes in apoptosis rate in chicken primary hepatocytes as determined by FCM

Change in the percentage of apoptotic cells for (A) control group, (B) model group, (C) 1 μg/mL CAG treatment group, (D) 10 μg/mL CAG treatment group, and (E, F) 100 μg/mL CAG treatment group. ^# < 0.05, ^## <0.01 compared to the control; * < 0.05, ** < 0.01 compared to the model group.

Figure 6. Hoechst 33342 staining

Images of cultured hepatocytes stained with Hoechst 33342. (A) Control, (B) model, and (C) 100 μg/mL CAG treatment groups.

Figure 7. Effect of CAG on LPS/AC-induced changes in the expression of apoptosis-related genes in hepatocytes

β-actin was used as a reference. mRNA expression of (A) caspase-3, (B) bax, (C) bcl-2, (D) fas, (E) caspase-9, and (F) cytochrome c. The 2^–ΔΔCt method was used to quantify the expression levels of each gene. Values are expressed as the mean ± SD (n = 3). ^# < 0.05, ^## < 0.01 compared to the control; * < 0.05, ** < 0.01 compared to the model.

Figure 8. Effect of CAG on LPS/AC-induced changes in the expression of apoptosis-related proteins in hepatocytes

(A) western blot bands, (B) caspase-3, (C) caspase-9, (D) bcl-2, (E) bax, and (F) cytochrome c. β-actin was used as reference. Bands analyzed by ImageJ. The data are expressed as the mean ± SD (n = 3). ^# < 0.05, ^## < 0.01 compared to the control; * < 0.05, ** < 0.01 compared to the model group.

Figure 9. Ultrastructural features of chicken primary hepatocytes

(A) Control and (B) model groups. Arrows point to mitochondria.

Figure 10. Effect of CAG on LPS/AC-induced changes in the expression of cytoplasmic and mitochondrial cyt c in hepatocytes

(A) western blot bands, (B) cytoplasm cyt, and (C) mitochondria cyt c. β-actin was used as a reference. Bands analyzed by ImageJ. The data are expressed as the mean ± SD (n = 3). ^## < 0.01 compared to control group; ** < 0.01 compared to the model group.

Figure 11. Effect of CAG on LPS/AC-induced changes in the mitochondrial membrane potential of chicken liver hepatocytes

ΔΨm was measured in the (A) control group, (B) model group, (C) 100 μg/mL CAG treatment group. (D) Percentage of green fluorescence. The data are expressed as the mean ± SD (n = 3). ^## < 0.01 compared to the control group; * < 0.05 compared to the model group.

Figure 12. Effects of CAG treatment on p38 MAPK phosphorylation

Three groups of cells were tested; namely, the control, the model and the 100 μg/mL CAG treatment groups. ^# < 0.05, ^## < 0.01 compared to the control group; * < 0.05, ** < 0.01 compared to the model group.