The dynamics of cell death patterns and regeneration during acute liver injury in mice

Abstract

Acute liver injury is a serious clinical syndrome with multiple causes and unclear pathological process. Here, CCl₄ - and D-galactosamine/lipopolysaccharide (D-gal/LPS)-induced acute liver injury was established to explore the cell death patterns and determine whether or not liver regeneration occurred. In CCl₄ -induced hepatic injury, three phases, including the early, progressive, and recovery phase, were considered based on alterations of serum transaminases and liver morphology. Moreover, in this model, cytokines exhibited double-peak fluctuations; apoptosis and pyroptosis persisted throughout all phases; autophagy occurred in the early and the progressive phases; and sufficient and timely hepatocyte regeneration was observed only during the recovery phase. All of these phenomena contribute to mild liver injury and subsequent regeneration. Strikingly, only the early and progressive phases were observed in the D-gal/LPS model. Slight pyroptosis occurred in the early phase but diminished in the progressive phase, while apoptosis, reduced autophagy, and slight but subsequently diminished regeneration occurred only during the progressive phase, accompanied by a strong cytokine storm, resulting in severe liver injury with high mortality. Taken together, our work reveals variable modes and dynamics of cell death and regeneration, which lead to different consequences for mild and severe acute liver injury, providing a helpful reference for clinical therapy and prognosis.

Keywords: acute liver injury; cell death; cytokines; hepatocyte; prognosis; regeneration.

Fig. 1

CCl₄‐ and D‐gal/LPS‐induced acute liver injury models. Mice were injected with either CCl₄ (olive oil as control) or D‐gal/LPS (saline as control) to induce acute liver injury and were sacrificed at different time points. Macroscopic appearance and H&E staining of the liver tissues for the CCl₄ (A)‐ and D‐gal/LPS (E)‐treated mice; serum ALT (B, F), AST (C, G), and LDH (D, H) were measured. Scale bar, 400 μm for 10× and 100 μm for 40×. The data were expressed as the mean ± SEM for at least triplicate experiments. P values were analyzed by Student’s t‐test. *P < 0.05 vs control.

Fig. 2

Cytokine expression in CCl₄‐induced acute liver injury model mice. The expression of inflammation‐related genes TNF‐α (A), IL‐1β (B), Mcp‐1 (C), IL‐10 (D), and IL‐6 (E) was evaluated by qRT‐PCR. The data were expressed as the mean ± SEM for at least triplicate. P values were analyzed by Student’s t‐test. *P < 0.05 vs control.

Fig. 3

Expression of apoptosis‐, pyroptosis‐, and autophagy‐related genes in liver tissues of CCl₄‐induced acute liver injury model mice. (A) The protein level of apoptosis genes in liver tissues was determined by western blot; (B) TUNEL detection of apoptotic gene expression in liver tissue; the protein level of pyroptosis genes in liver tissue (C) and KCs (D) determined by western blot; and (E) the protein level of autophagy‐related gene in liver tissue determined by western blot. GAPDH was used as an internal control. Scale bar, 400 μm for 10× and 100 μm for 40×. The data were expressed as the mean ± SEM for at least triplicate experiments. P values were analyzed by Student’s t‐test. *P < 0.05 vs control.

Fig. 4

Expression of proliferation‐associated genes in CCl₄‐induced acute liver injury model mice. RNA levels of Pcna detected using qRT‐PCR in liver tissue (A) and hepatocytes (E); protein level of PCNA and cyclin D1 detected using western blot in liver tissue (B‐C) and hepatocytes (F, G), GAPDH was used as an internal control; (D) expression of PCNA detected in liver tissue using IHC. Scale bar, 400 μm for 10× and 100 μm for 40×. The data were expressed as the mean ± SEM for at least triplicate experiments. P values were analyzed by Student’s t‐test. *P < 0.05 vs control.

Fig. 5

Cytokine expression in D‐gal/LPS‐induced acute liver injury model mice. The expression of inflammation‐related genes TNF‐α (A), IL‐1β (B), Mcp‐1 (C), IL‐10 (D), and IL‐6 (E) was evaluated by qRT‐PCR. The data were expressed as the mean ± SEM for at least triplicate experiments. P values were analyzed by Student’s t‐test. *P < 0.05 vs control.

Fig. 6

Expression of apoptosis, pyroptosis, autophagy, and proliferation in liver tissues of mice in D‐gal/LPS‐induced acute liver injury model mice. (A) The protein level of apoptosis genes in liver tissues was determined by western blot; (B) TUNEL detection of apoptotic gene expression in liver tissue; (C) the protein level of pyroptosis genes in liver tissue determined by western blot; (D) the protein level of autophagy‐related gene in liver tissue determined by western blot; (E) the protein level of regeneration‐related genes in liver tissue determined by western blot. GAPDH was used as an internal control. Scale bar, 400 μm for 10× and 100 μm for 40×. The data were expressed as the mean ± SEM for at least triplicate experiments. P values were analyzed by Student’s t‐test. *P < 0.05 vs control.

Fig. 7

Schematic diagram illustrating the progress of CCl₄‐ and D‐gal/LPS‐induced acute liver injury model mice.