Promotion of mitochondrial energy metabolism during hepatocyte apoptosis in a rat model of acute liver failure

Abstract

Hepatocyte apoptosis and energy metabolism in mitochondria have an important role in the mechanism of acute liver failure (ALF). However, data on the association between apoptosis and the energy metabolism of hepatocytes are lacking. The current study assessed the activity of several key enzymes in mitochondria during ALF, including citrate synthase (CS), carnitine palmitoyltransferase‑1 (CPT‑1) and cytochrome c oxidase (COX), which are involved in hepatocyte energy metabolism. A total of 40 male Sprague‑Dawley rats were divided into five groups and administered D‑galactosamine and lipopolysaccharide to induce ALF. Hepatic pathology and terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling examinations indicated that hepatocyte apoptosis was observed at 4 h and increased 8 h after ALF. Hepatocyte necrosis appeared at 12 h and was significantly higher at 24 h with inflammatory cell invasion. The results measured by electron microscopy indicated that ultrastructural changes in mitochondria began at 4 h and the mitochondrial outer membrane was completely disrupted at 24 h resulting in mitochondrial collapse. The expression of CS, CPT‑1 and COX was measured and analyzed using assay kits. The activity and protein expression of CS, CPT‑1 and COX began to increase at 4 h, reached a peak at 8 h and decreased at 12 h during ALF. The activities of CS, CPT‑1 and COX were enhanced during hepatocyte apoptosis suggesting that these enzymes are involved in the initiation and development of ALF. Therefore, these results demonstrated that energy metabolism is important in hepatocyte apoptosis during ALF and hepatocyte apoptosis is an active and energy‑consuming procedure. The current study on how hepatocyte energy metabolism affects the transmission of death signals may provide a basis for the early diagnosis and development of an improved therapeutic strategy for ALF.

Figure 1

Histological examination of acute liver damage induced by D-galactosamine/lipopolysaccharide treatment in rats. Liver sections were stained with hematoxylin and eosin. ALF 4 h, hepatic tissue underwent ballooning degeneration without inflammatory invasion in the sinusoids; ALF 8 h, hepatocyte apoptosis and partial hyperemia was visible in the sinusoids and piecemeal necrosis and inflammatory invasion was observed; ALF 12 h, a large quantity of hepatocytes underwent piecemeal necrosis and there was rupture of hepatic cords; ALF 24 h, marked necrosis of hepatocytes and severe hyperemia was observed in the sinusoids, the boundary of hepatocytes became obscure, the fiber mesh scaffold collapsed and there was clear inflammatory invasion. ALF, acute liver failure.

Figure 2

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling detection of apoptosis in rat livers during the early stages of ALF. (A–C) Apoptotic cells were visible 4 and 8 h after D-galactosamine/lipopolysaccharide treatment in rats and were located predominantly around the central vein, as indicated by arrows; (D) 10 fields of view were randomly selected from sections of every sample and the apoptotic cells were counted under a lens (magnification, ×200) to calculate the number of apoptotic cells in every 100 cells. ALF, acute liver failure.

Figure 3

Morphological alterations in liver mitochondria during ALF. Control group, the mitochondria were round or oval with complete membrane structures and concentric circles or longitudinally arranged ridges that were tidy and compact; ALF 4 h, the mitochondria were reduced in number, fairly structured, relatively complete and had a complete structure of ridges that had a relatively tidy arrangement but with concentrated stroma; ALF 8 h, the mitochondria were swollen and clearly damaged but had somewhat complete outer membranes and disorganized ridge structures; ALF 12 h, the mitochondria were clearly swollen and their inner structures were not easily visible; ALF 24 h, the outer membrane was disrupted and the mitochondria had collapsed. ALF, acute liver failure.

Figure 4

Activities of CS, CPT-1 and COX in liver mitochondria and their protein expression level in liver tissue. (A–C) The activity of CS, CPT-1 and COX in liver mitochondria began to increase at 4 h, peaked at 8 h and began to decrease at 12 h. (D) Western blotting demonstrated that the protein expression levels of CS, CPT-1 and COX were upregulated at 4 h and then decreased after 12 h in liver tissue. ALF, acute liver failure; CS, citrate synthase; CPT-1, carnitine palmitoyltransferase-1; COX, cytochrome c oxidase; D-GalN, D-galactosamine; LPS, lipopolysaccharide.