Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice

Figure 1 Dose- and time-dependent liver damage, apoptosis and reactive oxygen species generation. A: Carbon tetrachloride (CCl₄)-induced hepatic injury as measured by alanine aminotransferase (ALT) levels. Values are mean ± SE, n = 5, ^aP < 0.05 vs control, ^cP < 0.05 vs 12 h at the same dose; B: Hepatic apoptosis was examined by terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining. For each sample, five randomly selected fields at 200× magnification were evaluated. Values are mean ± SE, n = 5; ^eP < 0.05 vs 100 μL/kg body weight group at the same time point; C: Generation of reactive oxygen species as measured by malondialdehyde (MDA) in both caspase-12^+/+ and -12^-/- mouse livers 12, 24 and 48 h following CCl₄ treatment (300 μL/kg body weight). Values are mean ± SE, n = 8; ^gP < 0.05 vs controls.

Figure 2 Carbon tetrachloride induced endoplasmic reticulum stress in the liver. The representative Western blots show that there was an increase in endoplasmic reticulum stress response in CCl₄-treated mice. Glucose-regulated protein 78 (GRP78), CCAAT/-enhancer-binding protein homologous protein (CHOP) and serine/threonine-protein kinase/endoribonuclease (IRE-1) were upregulated in the livers of both caspase-12^+/+ and -12^-/- mice treated with CCl₄.

Figure 3 Localization of procaspase-12 in the subcellular components of liver cells and the activation of caspase-12 by carbon tetrachloride. A: Procaspase-12 was detected only in the microsome fraction but not in the nuclear, mitochondria and soluble fractions; B: Activation of caspase-12 was detected in the liver of caspase-12^+/+ mice at 12 and 24 h after CCl₄ treatment. Western blot was performed using an antibody recognizing both pro- and active forms of caspase-12. Procaspase-12 (55 kDa) was cleaved to the active form (36 kDa). The representative blot was from the results of three experiments.

Figure 4 Genotyping and confirmation of the lack of caspase-12 in the caspase-12^-/- mice. A: Genotyping result indicates the absence of wild-type allele and presence of mutant allele in the caspase-12^-/- mice; B: Reverse transcription polymerase chain reaction confirmed the absence of caspase-12 mRNA in caspase-12^-/-; C: Western blot showing the absence of caspase-12 protein in caspase-12^-/- mice.

Figure 5 Attenuated carbon tetrachloride-induced hepatic apoptosis and liver injury in caspase-12^-/- mice. A: Terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining to evaluate carbon tetrachloride (CCl₄)-induced hepatocyte apoptosis in caspase-12^-/- mice. Five randomly selected fields at 200× magnification were evaluated for each tissue section. The numbers of apoptotic cells were counted as TUNEL-positive cells/mm². Values are mean ± SE, n = 8; ^aP < 0.05 vs controls, ^cP < 0.05 vs caspase-12^+/+; B: CCl₄-induced liver injury as measured by serum alanine aminotransferase (ALT) levels. Values are mean ± SE, n = 8; ^eP < 0.05 vs controls, ^gP < 0.05 vs caspase-12^+/+ mice; C: Representative sections of TUNEL staining under fluorescence microscope (200 ×). TUNEL-positive cells were identified by the nuclear fluorescence staining.

Figure 6 Activation of caspases and cytochrome C release in mice treated with carbon tetrachloride. Western blot analysis of A: Caspase-12; B: Caspase-3; C: Cytochrome C release from mitochondria; D: Casapse-9; E: Caspase-8; F: Actin as control in CCl₄-treated and control caspase-12^+/+ and caspase-12^-/- mice; G: Densitometric analyses of cytosolic cytochrome C and cleaved caspase-3 and -9 were performed from four independent Western blot experiments. The values were normalized to actin for each individual sample. Values are mean ± SE; ^aP < 0.05 vs CCl₄-treated caspase-12^+/+.