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A high-cholesterol diet promotes steatohepatitis and liver tumorigenesis in HCV core gene transgenic mice

  • Xiaojing Wang
  • Naoki TanakaEmail author
  • Xiao Hu
  • Takefumi Kimura
  • Yu Lu
  • Fangping Jia
  • Yoshiko Sato
  • Jun Nakayama
  • Kyoji Moriya
  • Kazuhiko Koike
  • Toshifumi Aoyama
Genotoxicity and Carcinogenicity

Abstract

Previous epidemiological studies have suggested a link between high-cholesterol intake and liver disease progression, including hepatocellular carcinoma (HCC). However, the precise mechanism of hepatotoxicity and hepatocarcinogenesis caused by excessive cholesterol consumption remains unclear. We aimed to investigate the impact of dietary cholesterol using hepatitis C virus core gene transgenic (HCVcpTg) mice, which spontaneously developed HCC with age. Male HCVcpTg mice were treated for 15 months with either a control diet or an isocaloric diet containing 1.5% cholesterol, and liver phenotypes and tumor-associated signaling pathways were evaluated. The high-cholesterol diet-fed HCVcpTg mice exhibited a significantly higher incidence of liver tumors compared with the control diet mice (100% vs. 41%, P < 0.001). The diet induced steatohepatitis with pericellular fibrosis and evoked higher mRNA expression of pro-inflammatory and pro-fibrotic mediators along with enhanced hepatocyte proliferation and greater oxidative and endoplasmic reticulum stress in the liver. Moreover, long-term consumption of cholesterol-rich diet activated nuclear factor-kappa B (NF-κB) and p62/sequestosome 1 (Sqstm1)-nuclear factor erythroid 2 (NRF2) axis, enhanced fibrogenesis, and consequently accelerated hepatic tumorigenesis. In conclusion, these results demonstrate that a high-cholesterol diet facilitates liver tumorigenesis by inducing steatohepatitis, promoting hepatocyte division, and up-regulating cellular stress and pro-inflammatory NF-κB and detoxifying p62/Sqstm1-NRF2 signals. Therefore, high dietary cholesterol should be avoided in HCV-infected patients to prevent development of steatohepatitis, liver fibrosis, and HCC.

Keywords

Steatohepatitis NF-κB p62/Sqstm1-NRF2 axis Fibrogenesis Oxidative stress 

Abbreviations

4-HNE

4-Hydroxy-nonenal

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

ALP

Alkaline phosphatase

αSMA

α-Smooth muscle actin

BAX

B-cell leukemia/lymphoma 2-associated X protein

CHOP

CCAAT/enhancer-binding protein homologous protein

DR5

Tumor necrosis factor receptor superfamily member 10b

ER

Endoplasmic reticulum

FA

Fatty acid

HCC

Hepatocellular carcinoma

F-Chol

Free cholesterol

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HCV

Hepatitis C virus

HCVcpTg

HCV core gene transgenic

HMG-CoA

Hydroxymethylglutaryl-CoA

IL

Interleukin

LXR

Liver X receptor

NASH

Non-alcoholic steatohepatitis

NEFA

Non-esterified fatty acid

NF-κB

Nuclear factor-kappa B

NQO1

NAD(P)H quinone dehydrogenase 1

NRF2

Nuclear factor erythroid 2

PC

phospholipid

PCNA

Proliferating cell nuclear antigen

PPAR

Peroxisome proliferator-activated receptor

qPCR

Quantitative polymerase chain reaction

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEM

Standard error of the mean

Sqstm1

Sequestosome 1

SREBP

Sterol regulatory element-binding protein

T-BA

Total bile acid

T-Chol

Total cholesterol

TG

Triglyceride

TGF-β1

Transforming growth factor-β1

TLR

Toll-like receptor

TNFα

Tumor necrosis factor-α

Notes

Acknowledgements

We appreciate Mr. Trevor Ralph for his English editorial assistance and Dr. Takero Nakajima and Dr. Ruan Guo (Shinshu University School of Medicine) for their invaluable help, advice, instruction, and encouragement. We thank Research Center for Supports to Advanced Science, Shinshu University, for technical supports and animal care. This study was partially supported by JSPS Grants-in-Aid for Scientific Research (KAKENHI Grant number 16K08734).

Author contribution

Design and writing the paper: NT and TA. Performing experiments: XW, NT, XH, TK, YL, FJ, and YS. Analyzing data: XW, NT, XH, TK, YL, and YS. Supervision: JN, KM, and KK. All authors read and approved the final manuscript.

Funding

The authors have declared that no financial support exists.

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflict of interest exists.

Supplementary material

204_2019_2440_MOESM1_ESM.docx (468 kb)
Supplementary material 1 (DOCX 468 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Metabolic RegulationShinshu University School of MedicineMatsumotoJapan
  2. 2.Department of GastroenterologyLishui Hospital, Zhejiang University School of MedicineLishuiPeople’s Republic of China
  3. 3.Research Center for Social Systems, Shinshu UniversityMatsumotoJapan
  4. 4.Department of PathophysiologyHebei Medical UniversityShijiazhuangPeople’s Republic of China
  5. 5.Department of GastroenterologyShinshu University School of MedicineMatsumotoJapan
  6. 6.Department of Molecular PathologyShinshu University School of MedicineMatsumotoJapan
  7. 7.Department of Infection Control and PreventionThe University of TokyoTokyoJapan
  8. 8.Department of GastroenterologyThe University of TokyoTokyoJapan

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