Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 28, 2018; 24(48): 5477-5490
Published online Dec 28, 2018. doi: 10.3748/wjg.v24.i48.5477
Mismatched effects of receptor interacting protein kinase-3 on hepatic steatosis and inflammation in non-alcoholic fatty liver disease
Waqar Khalid Saeed, Dae Won Jun, Kiseok Jang, Sang Bong Ahn, Ju Hee Oh, Yeon Ji Chae, Jai Sun Lee, Hyeon Tae Kang
Waqar Khalid Saeed, Dae Won Jun, Department of Gastroenterology, Hanyang University College of Medicine, Seoul 04763, South Korea
Kiseok Jang, Department of Pathology, Hanyang University College of Medicine, Seoul 04763, South Korea
Sang Bong Ahn, Department of Internal Medicine, Eulji University, Daejeon 34824, South Korea
Ju Hee Oh, Yeon Ji Chae, Jai Sun Lee, Hyeon Tae Kang, Department of Translational Medicine, Hanyang University College of Medicine, Seoul 04763, South Korea
Author contributions: Saeed WK drafted the manuscript; Jun DW designed the research and supervised the project; Oh JH, Chae YJ, Lee JS, and Kang HT collected the data; Lee JS analyzed the data; Jang K analyzed the histology data; Ahn SB supervised and revised the manuscript.
Supported by National Research Foundation of Korea (NRF), funded by the South Korean Government, No. NRF-2017M3A9C8028794.
Institutional animal care and use committee statement: All the experimental procedures were approved by the Hanyang University Institutional Animal Care and Use Committee of (HY-IACUC-16-0075).
Conflict-of-interest statement: The authors have declared no conflicts of interest.
Data sharing statement: No additional data is available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Dae Won Jun, MD, PhD, Professor, Department of Internal Medicine, Hanyang University College of Medicine, 222-1 Wangsimni-ro, Seongdong-gu, Seoul 04763, South Korea. noshin@hanyang.ac.kr
Telephone: +82-2-22908338 Fax: +82-2-9720068
Received: August 27, 2018
Peer-review started: August 27, 2018
First decision: October 24, 2018
Revised: November 8, 2018
Accepted: November 13, 2018
Article in press: November 13, 2018
Published online: December 28, 2018
Abstract
AIM

To validate the effects of receptor interacting protein kinase-3 (RIP3) deletion in non-alcoholic fatty liver disease (NAFLD) and to clarify the mechanism of action.

METHODS

Wild-type (WT) and RIP3 knockout (KO) mice were fed normal chow and high fat (HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha (TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine (C-X-C motif) ligand (CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated.

RESULTS

RIP3KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3KO mice compared to HF diet fed WT mice. RIP3KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers (microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3KO mice. The overall NAFLD Activity Score was the same between WT and RIP3KO mice; however, RIP3KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals (CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pan-caspase inhibitor (necroptotic condition) treatment in monocytes. Neutrophil chemokines (CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes.

CONCLUSION

RIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.

Keywords: Necroptosis, Receptor interacting protein kinase-3, Mixed lineage kinase domain-like protein, Non-alcoholic fatty liver disease, Steatosis

Core tip: Receptor interacting protein kinase-3 (RIP3) deletion was associated with increased fatty change, hepatic tissue triglycerides, body weight, and serum aspartate aminotransferase and alanine aminotransferase levels. Very-low-density lipoproteins secretion markers, including apolipoprotein-B, microsomal triglyceride transfer protein, and protein disulfide isomerase, were suppressed with RIP3 deletion. High fat diet fed RIP3KO mice had reduced expressions of tumor necrosis factor alpha and neutrophil chemokines [Chemokine (C-X-C motif) ligands: CXCL1, and CXCL2] compared to high fat diet fed wild-type mice. In vitro analysis suggests that necroptotic stimulation [lipopolysaccharide + N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone] increased CXCL1/2 expression in monocytes. Treatment with RIP3 inhibitor (GSK’843) decreased the expression of CXCL1/2 as well as interleukin-6.