Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 21, 2020; 26(47): 7513-7527
Published online Dec 21, 2020. doi: 10.3748/wjg.v26.i47.7513
Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway
Zhi Wang, Zhen-Xing Cheng, Simon T Abrams, Zi-Qi Lin, Edwin Yates, Qian Yu, Wei-Ping Yu, Ping-Sheng Chen, Cheng-Hock Toh, Guo-Zheng Wang
Zhi Wang, Zhen-Xing Cheng, Wei-Ping Yu, Ping-Sheng Chen, Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing 210009, Jiangsu Province, China
Zhi Wang, Qian Yu, Department of Gastroenterology, Zhongda Hospital, Nanjing 210009, Jiangsu Province, China
Zhen-Xing Cheng, Simon T Abrams, Cheng-Hock Toh, Guo-Zheng Wang, Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool L69 7BE, United Kingdom
Zhen-Xing Cheng, Department of Gastroenterology, The First Affiliated Hospital, Anhui Medical University, Hefei 230032, Anhui Province, China
Zi-Qi Lin, Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Edwin Yates, Department of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom
Cheng-Hock Toh, Roald Dahl Haemostasis & Thrombosis Ctr, Royal Liverpool University Hospital, Liverpool L69 7BE, United Kingdom
Author contributions: Wang Z conceived the study; Cheng ZX assisted animal experiments and hydroxyproline measurements; Lin ZQ and Abrams ST assisted in performing in vitro experiments; Yates E synthesized and characterised non-anticoagulant heparin; Abrams ST helped edit figures; Yu Q, Yu WP, Chen PS, Toh CH and Wang GZ supervised the work and were involved in data analysis and manuscript writing; and all authors have read and agreed to the published version of the manuscript.
Supported by Key R & D Program of Jiangsu Province, No. BE2019712; British Heart Foundation, No. PG/14/19/30751 and No. PG/16/65/32313.
Institutional review board statement: The study was reviewed and approved by Medical School of Southeast University.
Institutional animal care and use committee statement: All procedures were performed according to State laws and monitored by local inspectors, and approved by the Animal Research Ethics Committee at the Medical School of the Southeast University.
Conflict-of-interest statement: No conflict interested has been claimed by any author.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (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:
Corresponding author: Ping-Sheng Chen, MBChB, MD, PhD, Professor, Research Dean, Department of Pathology and Pathophysiology, Medical School, Southeast University, No. 87 Dingjiaqiao, Nanjing 210009, Jiangsu Province, China.
Received: August 27, 2020
Peer-review started: August 27, 2020
First decision: October 17, 2020
Revised: November 8, 2020
Accepted: December 6, 2020
Article in press: December 6, 2020
Published online: December 21, 2020

Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated.


To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis.


In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated.


Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice.


Extracellular histones potentially enhance fibrogenesis via the TLR4–MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

Keywords: Liver fibrosis, Extracellular histones, Non-anticoagulant heparin, TLR4, MyD88, CCl4

Core Tip: This study fills the gap between recurrent liver injury and liver fibrosis. When liver cells die, histones are released. High levels of extracellular histones not only cause secondary liver injury, but also activate the TLR4-MyD88 signaling pathway to enhance collagen I production and liver fibrosis. Binding of non-anticoagulant heparin (NAHP) to extracellular histones reduces histone toxicity, alleviates liver injury, and prevents histones from activating the TLR4-MyD88 signaling pathway. These results may explain why NAHP reduces liver fibrosis in this animal model, although further investigations are required.