Original Research
Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2)

https://doi.org/10.1016/j.jcmgh.2017.11.016Get rights and content
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Background & Aims

Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis.

Methods

Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals.

Results

TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice.

Conclusions

Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

Keywords

NASH
Nrf2
TBE-31

Abbreviations used in this paper

ACACA
acetyl-CoA carboxylase alpha
ACLY
ATP citrate lyase
ACOT7
acetyl-CoA thioesterase 7
ACOX2
acetyl-CoA oxidase 2
ADRP
adipose differentiation-related protein
AP-1
activator protein 1
ApoB
apolipoprotein B
ATF4
activating transcription factor-4
ATF6
activating transcription factor-6
BCL-2
B-cell lymphoma
BIP
binding immunoglobulin protein
CAT
catalase
CD36
cluster of differentiation 36
CDDO
2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
C/EBP
CCAAT/enhancer-binding protein
CES1G
carboxylesterase 1g
CHOP
C/EBP homologous protein
ChREBP
carbohydrate-responsive element-binding protein
COL1A1
collagen, type I, alpha-1
COX2
cyclooxygenase-2
CPT1A
carnitine palmitoyltransferase 1a
DGAT2
diacylglycerol acyltransferase-2
DMSO
dimethyl sulfoxide
eIf2α
eukaryotic translation initiation factor 2A
ER
endoplasmic reticulum
FASN
fatty acid synthase
FXR
farnesoid X receptor
GCLC
glutamate-cysteine ligase catalytic
GCLM
glutamate-cysteine ligase modifier
GPX2
glutathione peroxidase-2
GSH
reduced glutathione
GSSG
oxidized glutathione
GSTA4
glutathione S-transferase Alpha-4
GSTM1
glutathione S-transferase Mu-1
GTT
glucose tolerance test
H&E
hematoxylin and eosin
HF
high-fat
HFFr
high-fat diet with fructose in drinking water
HF30Fr
high-fat diet with 30% fructose in drinking water
HF55Fr
high-fat diet with 55% fructose in drinking water
HMOX1
heme oxygenase-1
IRE1α
inositol requiring kinase-1α
IκB
inhibitor of NF-κB
IKK
IκB kinase
ITT
insulin tolerance test
JNK1
c-Jun N-terminal kinase 1
Keap1
Kelch-like ECH-associated protein-1
LXRα
liver X receptor α
MCD
methionine- and choline-deficient
MCP-1
monocyte chemotactic protein-1
MGPAT
mitochondrial glycerol-3-phosphate acetyltransferase
MPO
myeloperoxidase
MTTP
microsomal triglyceride transfer protein
NAFLD
non-alcoholic fatty liver disease
NAS
NAFLD activity score
NASH
nonalcoholic steatohepatitis
NF-κB
nuclear factor-κB
NOS2
nitric oxide synthase-2
NQO1
NAD(P)H:quinone oxidoreductase 1
Nrf2
NF-E2 p45-related factor 2
p58IPK
p58 inhibitor of the PKR kinase
PARP
poly ADP ribose polymerase
PCR
polymerase chain reaction
PDI
protein disulfide isomerase
PERK
PRK-like endoplasmic reticulum kinase
PPARα
peroxisome proliferator-activated receptor α
PPARγ
peroxisome proliferator-activated receptor γ
PRDX6
peroxiredoxin 6
PTGR1
prostaglandin reductase-1
PTT
pyruvate tolerance test
qRT-PCR
quantitative reverse transcriptase PCR
RC
regular chow
SCAD
short-chain acyl-CoA dehydrogenase
SCD1
stearoyl-CoA desaturase-1
SFN
sulforaphane
SHP
small heterodimer partner
SLC7A11
solute carrier family 7 member 11
α-SMA
alpha smooth muscle actin
SREBP-1c
sterol regulatory element-binding protein-1c
TGFβ
transforming growth factor beta-1
TNF-α
tumor necrosis factor-α
TXN1
thioredoxin-1
TXNRD1
thioredoxin reductase-1
UPR
unfolded protein response
XBP1
X-box binding protein-1

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Author contributions David J. Harrison, acquired, analyzed, and interpreted pathology data; obtained funding; drafting of the manuscript. Dorothy Kisielewski, study design, acquired data. Diane M. Cassidy, acquired data. Alison D. McNeilly, acquired data. Jennifer R. Gallagher, acquired data. Shaun V. Walsh, acquired data. Tadashi Honda, synthesized TBE-31 and obtained funding. Rory J. McCrimmon, obtained funding, supervised staff. Albena T. Dinkova-Kostova, obtained funding, supervised staff, drafting of the manuscript. Michael L. J. Ashford, study concept and design, obtained funding, drafting of manuscript. John F. Dillon, study concept and design, supervised staff, obtained funding, corrected drafts of manuscript. John D. Hayes, study concept and design, interpreted data, supervised staff, obtained funding, wrote all drafts of manuscript and approved final text.

Conflicts of interest The authors disclose no conflicts.

Funding Supported by grants MR/J001465/1 (JDH, MLJA, JFD, and RJMcC) and MR/N009851/1 (JDH, MLJA, ATD-K, and DJH) from the Medical Research Council of the United Kingdom and grants from Stony Brook Foundation and Reata Pharmaceuticals, Inc (TH).