Chrysophanol-8-O-glucoside protects mice against acute liver injury by inhibiting autophagy in hepatic stellate cells and inflammatory response in liver-resident macrophages

Abstract

Acute liver failure (ALF) is an unfavorable condition characterized by the rapid loss of liver function and high mortality. Chrysophanol-8-O-glucoside (CPOG) is an anthraquinone derivative isolated from rhubarb. This study aims to evaluate the protective effect of CPOG on lipopolysaccharide (LPS)/D-GalN-induced ALF and its underlying mechanisms. LPS/D-GalN-induced mice ALF model and LPS treatment model in RAW 264.7 and LX2 cells were established. It was found that CPOG ameliorated LPS/D-GalN-induced liver injury and improved mortality as indicated by Hematoxylin-eosin (H&E) staining. Molecularly, qPCR and ELISA results showed that CPOG alleviated LPS/D-GalN-induced release of alanine aminotransferase and aspartate transaminase and the secretion of TNF-α and IL-1β in vivo. LPS/D-GalN-induced intracellular ROS production was also attenuated by CPOG in liver tissue. Further, CPOG attenuated ROS generation and inhibited the expression of p-IκB and p-p65 as well as the expression of TNF-α and IL-1β stimulated by LPS in RAW 264.7 cells. In addition, CPOG alleviated LPS-induced up-regulation of LC3B, p62, ATG5 and Beclin1 by attenuating ROS production and inhibiting MAPK signaling in LX2 cells. Taken together, our data indicated that the CPOG protected against LPS/D-GalN-induced ALF by inhibiting oxidative stress, inflammation response and autophagy. These findings suggest that CPOG could be potential drug for the treatment of ALF in clinic.

Keywords: acute liver injury; autophagy; chrysophanol-8-O-glucoside; lipopolysaccharide; oxidative stress.

FIGURE 1

CPOG ameliorates LPS/D-GalN-induced ALF. (A) Chemical structure of CPOG. (B) Kaplan-Meier method was used to create the survival curves after LPS/D-GalN injection. (C–G) Liver sections were subjected to H&E staining. Representative photographs were shown from vehicle mice (C), mice treated with CPOG (20 mg/kg) (D), mice treated with CPOG (40 mg/kg) (E), mice treated with NAC (F), mice treated with LPS/D-GalN (G), mice treated with LPS/D-GalN and NAC (H), mice treated with LPS/D-GalN and CPOG (20 mg/kg) (I), mice treated with LPS/D-GalN and CPOG (40 mg/kg) (J). Arrows represent pathological changes in liver tissue. (K) Histological scores of liver sections were determined. Values represent mean ± S.D. one-way ANOVA test was used to determine the significances. *p <0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

CPOG attenuated inflammatory response in LPS/D-GalN-induced ALF model. (A,B) 8 h after LPS/D-GalN injection, the serum levels of ALT and AST were detected. (C,D) Serum levels of TNF-α and IL-1βwere detected by ELISA at 8 h after LPS/D-GalN injection. (E) ROS levels in liver tissue were determined by CM-H2DCFDA at 8 h after LPS/D-GalN injection. Values represent mean ± S.D. Significance was determined by one-way ANOVA test. Data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

CPOG inhibited the expression of LC3B and p62 in LPS/D-GalN-induced ALF model. (A–H) Immunofluorescence imaging was performed with the indicated antibodies. The nucleus was stained with DAPI (blue color). Representative photographs were shown from vehicle mice (A), mice treated with CPOG (20 mg/kg) (B), mice treated with CPOG (40 mg/kg) (C), mice treated with NAC (D), mice treated with LPS/D-GalN (E), mice treated with LPS/D-GalN and NAC (F), mice treated with LPS/D-GalN and CPOG (20 mg/kg) (G), mice treated with LPS/D-GalN and CPOG (40 mg/kg) (H). Scale bar = 20 µm.

FIGURE 4

CPOG inhibited the expression of ATG5 and Beclin1 in LPS/D-GalN-induced ALF model. (A,B) Immunohistochemistry assay was performed with the indicated antibodies. The nucleus was stained with DAPI (blue color). Representative photographs were shown from vehicle mice, mice treated with CPOG (20 mg/kg), mice treated with CPOG (40 mg/kg), mice treated with NAC, mice treated with LPS/D-GalN, mice treated with LPS/D-GalN and NAC, mice treated with LPS/D-GalN and CPOG (20 mg/kg), mice treated with LPS/D-GalN and CPOG (40 mg/kg). Scale bar = 100 µm.

FIGURE 5

CPOG alleviated LPS-induced oxidative stress and inflammation response in RAW264.7 cells. (A) RAW264.7 cells were treated with indicated concentrations of CPOG and intracellular ROS level was detected with CM-H2DCFDA. (B–F) RAW264.7 cells were treated with 1 μg/ml LPS in the presence or absence of 48 μM CPOG for 4 h. The expression of indicated proteins was detected by Western blot (B). p65 cell location was detected by immunofluorescence assay (C). Expression of TNF-α (D) and IL-1β (E) was detected by RT-PCR. Intracellular ROS level was detected with CM-H2DCFDA (F). (G) RAW264.7 cells were incubated with 48 μM CPOG for 4 h before treated with 500 μM H₂O₂ for 30 min. The expression of indicated proteins was detected by Western blot. Values represent mean ± S.D. Significance was determined by one-way ANOVA test. Data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

CPOG alleviated LPS-induced oxidative stress and autophagy in LX2 cells. (A) LX2 cells were treated with indicated concentrations of CPOG and intracellular ROS level was detected with CM-H2DCFDA. (B,C) LX2 cells were treated with 1 μg/ml LPS in the presence or absence of 48 μM CPOG for 4 h. The expression of indicated proteins was detected by Western blot (B). Formation of LC3B puncta was detected by immunofluorescence assay (C). Scale bar = 10 µm. (D,E) LX2 cells were incubated with LY3214 for 12 h before treated with 1 μg/ml LPS for 4 h. The expression of indicated proteins was detected by Western blot (D). Formation of LC3B puncta was detected by immunofluorescence assay (E). Scale bar = 10 µm. (F) LX2 cells were treated with 1 μg/ml LPS in the presence or absence of 48 μM CPOG for 4 h. Intracellular ROS level was detected with CM-H2DCFDA. (G) LX2 cells were incubated with 48 μM CPOG for 4 h before treated with 500 μM H₂O₂ for 30 min. The expression of indicated proteins was detected by Western blot. Values reWresent mean ± S.D. Significance was determined by one-way ANOVA test. Data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.