1. Volume 129, Issue 5, Pages 1663-1674 (November 2005)
NF-κB Activation, Rather Than TNF, Mediates Hepatic Inflammation in a Murine Dietary Model of Steatohepatitis 
Aileen dela Peña, Isabelle Leclercq, Jacqueline Field, Jacob George, Brett Jones, Geoffrey Farrell 
Gastroenterology 
Volume 129, Issue 5, Pages (November 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Evolution of steatohepatitis in MCD diet-fed wt, TNF−/−, and TNFR-1−/− mice. Liver sections from CTL diet-fed wt (A), TNF−/− (B), and TNFR-1−/− (C) mice show no abnormalities. In contrast, liver sections from 10-day MCD diet-fed wt (D), TNF−/− (E), and TNFR-1−/− (F) mice and 5-week MCD diet-fed wt (G) and TNF−/− (H) mice show increasing severity of steatosis and inflammatory recruitment with duration of dietary feeding. There are no apparent differences between the genotypes. Arrow, inflammatory infiltrate; asterisk, fat-laden hepatocyte; P, portal tract, and V, central vein. H&E staining, original magnification ×20. Sections are representative of at least 4 replicate experiments in each group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Effects of MCD dietary feeding on hepatic triglyceride and lipoperoxide levels, steatosis and necroinflammatory scores, polymorph count, and serum ALT in wt, TNF−/−, and TNFR-1−/− mice. Female wt, TNF−/−, and TNFR-1−/− mice were fed either the MCD or CTL diet for predetermined experimental periods. Hepatic triglyceride levels (A) and steatosis score (B) exhibit a similar temporal pattern between all genotypes, whereas necroinflammatory score (C) and polymorph count (D) only show significant increases after 5 weeks of MCD dietary feeding. Biochemical analysis of serum ALT levels (E) and hepatic TBARS (F) indicate that MCD dietary feeding for 10 days induces significant hepatocellular injury and lipid peroxidation, respectively. No apparent differences between the genotypes are significant. Data (mean ± SD) from groups of n = 4–7 mice. *P < .05, **P < .001, ***P < for MCD vs CTL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Feeding the MCD diet activates NF-κB in wt, TNF−/−, and TNFR-1−/− mice. (A) Representative EMSA of hepatic nuclear extracts from individual wt, TNF−/−, and TNFR-1−/− animals at indicated times of the dietary regime. (B) Supershift analysis with p65 and p50 antibodies show the presence of active p65:p50 heterodimer complexes and inhibitory p50:p50 homodimers in a representative 10-day MCD/wt sample. (C) EMSA of nuclear protein from cell fractions (hepatocytes or nonparenchymal cells, NPC) from livers of wt mice fed the MCD or CTL diet for 10 days. Densitometric analysis of p65:p50 (D) and p50:p50 (E) bands reveals that the p65:p50 “active” heterodimer binds with greater intensity than the p50:p50 “repressive” homodimer. Data (mean ± SD) from groups of n = 4–7 mice. *P < .05, ***P < for MCD vs CTL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Feeding the MCD diet induces hepatic ICAM-1 expression in wt, TNF−/−, and TNFR-1−/− mice. Hepatic ICAM-1 mRNA (A) and protein (B) levels are induced by MCD dietary feeding, indicating that “activated” NF-κB is present in both wt and TNFR-1−/− mice. In TNF−/− mice, ICAM-1 expression is more variable but does not appear to be significantly different to the other genotypes. Data (mean ± SD) for groups of n = 4–7 mice. *P < .05 for MCD vs CTL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Expression of the mIκB superrepressor in vivo and suppression of NF-κB activity. Representative immunohistochemistry for hemagglutinin (HA) on liver sections from mice infected with Ad5LacZ (A) or Ad5mIκB (B) for 12 days shows expression of the HA-tagged mIκB, with strongly positive hepatocytes (black arrows) and nonparenchymal cells (red arrows). Original magnification ×20. (C) Representative Western blot for total (mutant plus native) IκBα and densitometric analysis confirms the trend (not significant) for increased hepatic IκB protein expression in MCD diet-fed mice infected with Ad5mIκB. (D) Representative EMSA showing the mIκB “superrepressor” prevents MCD diet-induced NF-κB activation. Data (mean ± SD) for groups of n = 4 or 5 mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Hepatic mIκB expression prevents up-regulation of proinflammatory mediators during intake of the MCD diet for 10 days. In mice infected with the control vector (Ad5LacZ), MCD dietary feeding caused the expected induction of hepatic ICAM-1 mRNA (A), ICAM-1 protein (B), and TNF mRNA (C). Values for all these indices of NF-κB transcriptional activation were normalized by infection with the mIκB superrepressor adenovirus. Data (mean ± SD) for groups of n = 4 or 5 mice. *P < .05, ***P < for MCD vs CTL, #P < .05, ##P < .001, ###P < for Ad5mIκB vs Ad5LacZ.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
The mIκB superrepressor prevents inflammatory infiltration in mice fed the MCD diet for 10 days. (A) Liver sections from mice infected with Ad5mIκB that received the CTL diet for 10 days showed no abnormalities. (B) In contrast, liver sections from mice infected with Ad5LacZ and fed the MCD diet for 10 days showed the usual lipid accumulation and inflammatory infiltration (cf, Figure 7B with Figures 1D–F). However, mice infected with Ad5mIκB “superrepressor” for 12 days (C) appeared to have less focal inflammation despite a similar extent of steatosis (see Table 3 for group data on steatosis, necroinflammatory scores, and polymorph count). Arrow, inflammatory infiltrate; asterisk, steatosis; P, portal tract; V, central vein. Representative histology from groups of n = 4 or 5 mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions