Composite ammonium glycyrrhizin has hepatoprotective effects in chicken hepatocytes with lipopolysaccharide/enrofloxacin-induced injury

Abstract

Composite ammonium glycyrrhizin (CAG) has anti-inflammatory activity. Lipopolysaccharide (LPS) and enrofloxacin (ENR) induce liver damage; however, the mechanism underlying LPS/ENR-induced hepatic injury remains to be elucidated. In the present study, the mechanism of LPS/ENR-induced liver injury was investigated in vitro and the protective effects of CAG were also evaluated. Primary chicken hepatocytes were isolated and a model of LPS/ENR-induced hepatocyte injury was established. mRNA and protein expression levels were evaluated by reverse transcription-quantitative polymerase chain reaction and western blot, respectively. LPS/ENR exposure significantly increased supernatant aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In the LPS/ENR-treated group, glutathione (GSH) and the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were significantly increased. Flow cytometry results revealed that the apoptotic rate significantly increased in the LPS/ENR-treated group compared with the control, while treatment with CAG given 24 h prior to LPS/ENR caused a significant decrease in the apoptotic rate compared with the model group. Furthermore, CAG treatment reversed LPS/ENR-associated alterations in the mRNA and protein expression of Caspase-3, apoptosis regulator Bcl-2 (Bcl-2) and Bcl-2 associated X-protein. The mitochondrial membrane potential significantly decreased and the mitochondrial microstructure was notably altered following exposure to LPS/ENR compared with the control. In conclusion, these results suggested that LPS/ENR-treated hepatocytes were damaged via apoptotic signaling pathways and CAG prevented LPS/ENR-induced hepatocyte injury.

Keywords: composite ammonium glycyrrhizim; drug protection; lipopolysaccharide/enrofloxacin; liver injury.

Figure 1

(A) Effect of different concentrations of LPS/ENR on cytomorphology (magnification, x20) and hepatocyte viability. Images of (a) normal cells; and cells treated with (b) 30/40 µg/ml; (c) 30/60 µg/ml; (d) 30/80 µg/ml; (e) 30/100 µg/ml; and (f) 30/120 µg/ml LPS/ENR. The effect of CAG on (B) cell viability, (C) AST, (D) ALT, (E) GSH, (F) SOD and (G) CAT activity was The effect of CAG on (H) GPx and (I) MDA activity was determined. LPS/enrofloxacin was given at a dose of 30/80 µg/ml. Hepatocytes were collected following CAG treatment for 24 h. Data are expressed as the mean ± standard deviation (n=3). ^##P<0.01 vs. the control group; ^*P<0.05, ^**P<0.01 vs. the model group treated with 30/80 µg/ml LPS/ENR. LPS or L, lipopolysaccharide; CAG, composite ammonium glycyrrhizin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; MDA, malondialdehyde; ENR or E, enrofloxacin.

Figure 2

(A) Apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining via flow cytometry assay in hepatocytes. Apoptosis was measure in (a) control group, (b) model group, (c) CAG group (25 µg/ml), (d) CAG group (50 µg/ml), (e) CAG group (100 µg/ml), (f) CAG group (200 µg/ml) and (g) CAG group (400 µg/ml). (B) Analysis of the total percentage of early apoptotic cells in different groups. Data are expressed as the mean ± standard deviation (n=3). ^##P<0.01 vs. the control group; ^*P<0.05, ^**P<0.01 vs. the model group. LPS, lipopolysaccharide; CAG, composite ammonium glycyrrhizin; model group, cells treated with 30/80 µg/ml LPS/enrofloxacin.

Figure 3

Effects of CAG treatment on the expression of (A) Caspase-3, (B) Bax and (C) Bcl-2. (D) Western blot analysis. Quantitative analysis of protein expression levels of (E) Bcl-2, (F) Caspase-3 and (G) Bax. β-actin was used as the reference gene. (H) Caspase-3 activity. Data are expressed as the mean ± standard deviation (n=3). ^##P<0.01 vs. the control group; ^*P<0.05, ^**P<0.01 vs. the model group treated with 30/80 µg/ml LPS/enrofloxacin. LPS, lipopolysaccharide; CAG, composite ammonium glycyrrhizin; Bcl-2, apoptosis regulator Bcl-2; Bax, Bcl-2 associated X-protein.

Figure 4

Ultrastructural features of chicken primary hepatocytes. (A) Control, (B) model group, (C) CAG group (25 µg/ml), (D) CAG group (50 µg/ml), (E) CAG group (100 µg/ml), (F) CAG group (200 µg/ml) and (G) CAG group (400 µg/ml). The arrows indicate mitochondria. (H) ΔΨm was measured using a fluorescent microscope (magnification, x100). In the model group the green fluorescence was increased compared with the red fluorescence compared with control group, CAG relieved these alterations. CAG, composite ammonium glycyrrhizin.