Deficiency of nicotinamide adenine dinucleotide phosphate, reduced form oxidase enhances hepatocellular injury but attenuates fibrosis after chronic carbon tetrachloride administration

Abstract

Reactive oxygen species (ROS) activate hepatic stellate cells and enhance fibrogenesis. This study determined the role of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase deficiency in the development of hepatocellular necrosis, inflammation, and apoptosis in relation to fibrosis produced by chronic carbon tetrachloride (CCl(4)) administration. Wild-type (WT) mice or mice with deficiency of the gp91(phox) subunit of NADPH complex (gp91(phox(-/-) )) were subjected to biweekly CCl(4) injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aspartate aminotransferase (AST) was higher after CCl(4) administration in gp91(phox(-/-) ) than in WT mice, correlating with increased necrosis on liver histology. By contrast, more hepatocyte apoptosis was found after CCl(4) in the WT than in the gp91(phox(-/-) ) mice, which was associated with changes in components of the mitochondrial pathway of apoptosis, namely, an increase in the pro-apoptotic BAX protein in the WT, but not in the gp91(phox(-/-) ) mice and also a lower cytosolic cytochrome c in the gp91(phox(-/-) ) mice. There were fewer stellate cells and less fibrosis after CCl(4) in the gp91(phox(-/-) ) as compared with the WT mice. The increase in alpha(1)(I) collagen messenger RNA (mRNA), however, was greater after CCl(4) in the gp91(phox(-/-) ) mice. Matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA increased more in the gp91(phox(-/-) ) than in WT mice after CCl(4.) Tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 increased after CCl(4) only in the gp91(phox(-/-) ) mice.

Conclusion: Decreased hepatic fibrosis after chronic CCl(4) administration in mice with NADPH oxidase deficiency occurs in the setting of greater necrosis and inflammation but decreased apoptosis.

Fig. 1

Necroinflammatory changes and serum aminotransferases after 8 weeks of chronic carbon tetrachloride (CCl₄) administration in wild type (WT) and gp91^phox−/− mice. Liver cell necrosis (N) was evaluated on a scale of 1–4 as follows: 1, occasional; 2, mild scattered; 3, confluent zonal and 4, extensive. Grades for hepatic inflammation (I) were: 1, scattered; 2, mild; 3, moderate and 4, marked. The histological changes were assessed in x 20 magnification fields of slides stained with hematoxylin-eosin (H&E). The values are expressed as means ± SE of 8 determinations per group. *p<0.05 vs. WT for necroinflammatory changes. *p<0.05 vs control for serum aminotransferases.

Fig.2

Histology with sirius red collagen staining of liver sections from (A) WT and (B) gp91^phox−/− mice after 8 weeks of chronic CCl₄ administration. (original magnification × 100). C. Relative densitometry of fibrosis in WT and gp91^phox−/− mice after chronic CCl₄ administration. The degree of fibrosis was measured by sirius red staining and densitometry of the various groups. The density of fibrosis was determined as intensity of sirius red staining divided by the area of the captured field. A total of 24 fields were captured from livers of each group of mice. The values are expressed as means ± SE. * p< 0.01 vs. respective control. ⁺ p<0.01 vs WT + CCl₄. D. Immunofluorescent detection of activated stellate cells in (A) WT and (B) gp91^phox−/− mice after chronic CCl₄ administration. The liver slices were incubated with Cy3 monoclonal α-smooth muscle actin antibody (original magnification × 100).

Fig.3

Liver α₁(I) collagen mRNA and transforming growth factor β (TGFβ) mRNA in WT and gp91^phox−/− mice after chronic CCl₄ administration. The mRNAs were determined by real time quantitative PCR. The relative amounts of mRNA were normalized against β-actin in the same samples. The values are expressed as means ± SE of 8 determinations per group. * p<0.05 vs. control. **p<0.01 vs. control. + p<0.05 vs. WT + CCl₄.

Fig.4

Apoptosis determined by TUNEL staining of liver sections from WT and gp91^phox−/− mice after chronic CCl₄ administration. (A) WT-control; (B) WT + CCl₄; (C) gp91^phox−/− control; (D) gp91^phox−/− + CCl₄. Original magnification × 100.

Fig. 5

Effects of chronic CCl₄ on in WT and gp91^phox−/− mice on A. FAS and B. Activities of caspase 3 and 8. FAS was determined by western blot and quantitated by densitometry. White bars represent 52 kDA and shaded bars 48 kDa Fas. All values are expressed as means ± SE of 8 measurements per group. * p< 0.05 vs. respective control. **p<0.01 vs.respective control; ⁺p<0.05 vs. WT + CCl₄.

Fig. 6

Effects of chronic CCl₄ administration on Bax, Bcl Xs/l Bcl-2 and cytosolic cytochome c in WT and gp91^phox−/− mice. The determinations were done by western blot. The values are expressed as means ± SE of relative densitometry of 8 measurements per group. * p< 0.05 vs. control. **P<0.01 vs. control.

Fig. 7

Effects of chronic CCl₄ administration on matrix metalloproteinase 2 (MMP-2), 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 in WT and gp91^phox−/− mice determined by western blot. *p< 0.05 vs. respective control.

Fig. 8

Necroinflammatory changes and serum aminotransferases after 1 week of CCl₄ administration in wild type (WT) and gp91^phox−/− mice. Liver cell necrosis (N) was evaluated on a scale of 1–4 as follows: 1, occasional; 2, mild scattered; 3, confluent zonal and 4, extensive. Grades for hepatic inflammation (I) were: 1, scattered; 2, mild; 3, moderate and 4, marked. The histological changes were assessed in x 20 magnification fields of slides stained with hematoxylin-eosin (H&E). The values are expressed as means ± SE of 8 determinations per group. **p<0.01 vs. WT for necroinflammatory changes. *p<0.05 and **p<0.01 vs. control for serum aminotransferases.

Fig. 9

Effects of one week of chronic CCl₄ administration on A. Activities of caspase 3 and 8 and (B) on cytosolic and mitochondrial cytochrome c in WT and gp91^phox−/− mice. Cytochrome c was determined by western blot and quantitated by densitometry. The values are expressed as means ± SE of of 8 measurements per group. * p< 0.05 vs. respective control.

Fig. 10

Liver α₁(I) collagen mRNA and transforming growth factor β (TGFβ) mRNA in WT and gp91^phox−/− mice after one week of CCl₄ administration. The mRNAs were determined by real time quantitative PCR. The relative amounts of mRNA were normalized against β-actin in the same samples. The values are expressed as means ± SE. * p< 0.05 vs. respective control. ⁺p<0.05 vs. WT + CCl₄.

Fig. 11

Effects of one week of CCl₄ administration on matrix metalloproteinase 2 (MMP-2), 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 in WT and gp91^phox−/− mice determined by western blot. * p< 0.05 vs. respective control.