Easl immuno poster
1
A HIGH FAT DIET AND PARENTERAL IRON CAUSES INCREASED INFLAMMATION AND OXIDATIVE STRESS BUT LESS STEATOSIS IN AN OBESE, DIABETIC MOUSE MODEL OF NAFLD James E. Nelson 1 , Bryan Maliken 1 , Barjinder Dhillon 1 , Heather Klintworth 1 , Matthew M. Yeh 2 , Kris V. Kowdley 1,2 1 Center for Liver Disease, Digestive Disease Institute, and Benaroya Research Institute, Virginia Mason Medical Center Seattle WA. 2 Dept of Pathology, University of Washington Medical Center, Seattle WA METHODS Lepr db/db mice were fed either a 71% high-fat diet (61% unsaturated fat) or normal chow for 8 weeks. A subset of mice on both diets were administered a single dose of 1.25 mg/g wt Fe-dextran by IP injection at the start of the study. Histological features of NASH and iron deposition were scored using NASH CRN scoring criteria. Malondialdehyde (MDA), iron and hydroxyproline were assessed in liver tissue. Liver and adipose tissue were collected for expression of proinflammatory, lipid and oxidative stress genes, determined by RT- PCR, normalized to GAPDH mRNA 0 1 2 3 Steatosis (0-3) Mean Score # 0 1 2 3 4 Lobular Inflammation (0-3) Mean Score †# †# 0 0.4 Hepatocellular Ballooning (0-2) Mean Score 0 2 4 NAFLD Activity Score (0-8) †# † p<0.05 vs Normal Chow; # p<0.05 vs HFD High Fat Diet (20x) High Fat Diet + Iron (20x) 0 1 2 3 4 Hepatic IL6 expression Relative IL6/GAPDH mRNA †# 0 1 2 3 4 Hepatic TNFα expression Relative TNFα/GAPDH mRNA †# † p<0.05 vs Normal Chow; # p<0.05 vs HFD 0 1.5 3 Hepatic Srebp1c Relative Srebp1c/GAPDH mRNA †# # 0 1000 2000 3000 4000 5000 6000 7000 8000 Hepatic Iron Concentration HIC µg iron/g liver tissue †# †# Normal Chow HFD Parenteral Iron HFD +Parenteral Iron 0 3 6 9 Hepatic HAMP expression Relative HAMP/GAPDH mRNA †# † BACKGROUND Iron is thought to contribute to NAFLD progression by increasing oxidative stress as a catalyst for the production of reactive oxygen species. Iron may also potentiate progression of NAFLD by altering insulin signaling and lipid metabolism. The aim of this study was to examine the effects of parenteral iron loading in an obese, diabetic murine model of NAFLD. FIGURE 1: Histology Scores IGURE 2: Liver Histology and Iron Staining H&E stain Iron stain IGURE 3: Relationship of Iron and Hepcidin Expression 0 1 2 3 4 Hepatic Heme Oxygenase-1 Expression Relative HO-1/GAPDH mRNA †# †# 0 10 20 30 40 50 Hepatic Malondialdehyde MDA (umoles/g tissue) †# †# 0 2 4 6 8 10 Hepatic Hydroxyproline Assay ug HP/gram tissue †# † † p<0.05 vs Normal Chow; # p<0.05 vs HFD FIGURE 5: Relationship of Iron, Oxidative Stress and Collagen Production FIGURE 4: Relationship of Iron and Inflammatory and Lip Gene Expression B CONCLUSIONS PI administration causes a strong inflammatory response PI administration leads to KC activation and phagocytosis of iron PI caused decreased steatosis PI induces oxidative stress and initiates fibrogenesis Combination of HFD and PI led to more inflammation, oxidative stress and collagen production than either iron or HFD alone
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Transcript of Easl immuno poster
A HIGH FAT DIET AND PARENTERAL IRON CAUSES INCREASED INFLAMMATION AND OXIDATIVE STRESS BUT LESS STEATOSIS IN AN OBESE, DIABETIC MOUSE MODEL OF NAFLD
James E. Nelson1, Bryan Maliken1, Barjinder Dhillon1, Heather Klintworth1, Matthew M. Yeh2, Kris V. Kowdley 1,2
1Center for Liver Disease, Digestive Disease Institute, and Benaroya Research Institute, Virginia Mason Medical Center Seattle WA. 2Dept of Pathology, University of Washington Medical Center, Seattle WA
METHODSLeprdb/db mice were fed either a 71% high-fat diet (61% unsaturated fat) or normal chow
for 8 weeks.A subset of mice on both diets were administered a single dose of 1.25 mg/g wt Fe-
dextran by IP injection at the start of the study. Histological features of NASH and iron deposition were scored using NASH CRN
scoring criteria. Malondialdehyde (MDA), iron and hydroxyproline were assessed in liver tissue. Liver and adipose tissue were collected for expression of proinflammatory, lipid and
oxidative stress genes, determined by RT-PCR, normalized to GAPDH mRNA
00.5
11.5
22.5
3Steatosis (0-3)
Mea
n Sc
ore
#
00.51
1.52
2.53
3.5Lobular Inflammation (0-3)
Mea
n Sc
ore †#†#
00.20.4
Hepatocellular Ballooning (0-2)
Mea
n Sc
ore
024
NAFLD Activity Score (0-8)
†#
† p<0.05 vs Normal Chow; # p<0.05 vs HFD
High Fat Diet (20x) High Fat Diet + Iron (20x)
00.5
11.5
22.5
33.5
Hepatic IL6 expression
Rel
ativ
e IL
6/G
APD
H
mR
NA
†#
00.5
11.5
22.5
33.5 Hepatic TNFα expression
Rel
ativ
e TN
Fα/G
APD
H
mR
NA
†#
† p<0.05 vs Normal Chow; # p<0.05 vs HFD
0123
Hepatic Srebp1c
Rel
ativ
e Sr
ebp1
c/G
APD
H
mR
NA
†##
010002000300040005000600070008000
Hepatic Iron Concentration
HIC
µg
iron/
g liv
er ti
ssue †#
†#
Normal
Chow H
FD
Parenter
al Iro
n
HFD +Pare
nteral
Iron
0
2
4
6
8
Hepatic HAMP expression
Rel
ativ
e H
AM
P/G
APD
H m
RN
A †#†
BACKGROUNDIron is thought to contribute to NAFLD progression by increasing oxidative stress as a catalyst for the production of reactive oxygen species. Iron may also potentiate progression of NAFLD by altering insulin signaling and lipid metabolism. The aim of this study was to examine the effects of parenteral iron loading in an obese, diabetic murine model of NAFLD.
FIGURE 1: Histology Scores
FIGURE 2: Liver Histology and Iron Staining
H&E stain
Iron stain
FIGURE 3: Relationship of Iron and Hepcidin Expression
0
1
2
3
4
Hepatic Heme Oxygenase-1 Ex-pression
Rel
ativ
e H
O-1
/GA
PDH
mR
NA
†#†#
05
101520253035404550
Hepatic Malondialdehyde
MD
A (u
mol
es/g
tiss
ue) †#
†#
0123456789
10 Hepatic Hydroxyproline Assay
ug H
P/gr
am ti
ssue
†#
†
† p<0.05 vs Normal Chow; # p<0.05 vs HFD
FIGURE 5: Relationship of Iron, Oxidative Stress and Collagen Production
FIGURE 4: Relationship of Iron and Inflammatory and Lipid Gene Expression
B
CONCLUSIONSPI administration causes a strong inflammatory responsePI administration leads to KC activation and phagocytosis of ironPI caused decreased steatosisPI induces oxidative stress and initiates fibrogenesisCombination of HFD and PI led to more inflammation, oxidative stress and
collagen production than either iron or HFD alone
