Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 28, 2015; 21(28): 8615-8628
Published online Jul 28, 2015. doi: 10.3748/wjg.v21.i28.8615
TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity
Bo Yuan, Wei-Hong Tang, Li-Juan Lu, Yuan Zhou, Hong-Yan Zhu, You-Lang Zhou, Hong-Hong Zhang, Chuang-Ying Hu, Guang-Yin Xu
Bo Yuan, Wei-Hong Tang, Yuan Zhou, Hong-Yan Zhu, You-Lang Zhou, Hong-Hong Zhang, Chuang-Ying Hu, Guang-Yin Xu, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, Institute of Neuroscience, The Second Affiliated Hospital, Soochow University, Suzhou 215123, Jiangsu Province, China
Li-Juan Lu, Department of Pain Management, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China
Author contributions: Yuan B performed experiments, analyzed data, and prepared figures and the manuscript; Tang WH performed experiments, analyzed data and prepared figures; Lu LJ analyzed data, prepared figures and the manuscript; Zhou Y performed experiments and analyzed data; Zhu HY performed experiments and analyzed data; Zhou YL performed experiments; Zhang HH performed experiments and analyzed data; Hu CY analyzed data and prepared the manuscript; Xu GY designed experiments, prepared figures and edited the manuscript; Yuan B, Tang WH and Lu LJ contributed equally to this work.
Supported by National Natural Science Foundation of China, No. 81230024, No. 81471137 (to GYX) and No. 31400947 (to HHZ); and Priority Academic Program Development of Jiangsu Higher Education Institutions.
Institutional review board statement: The study was reviewed and approved by Soochow University Institutional Review Board.
Institutional animal care and use committee statement: Animal care and handling of the animals were approved by the Institutional Animal Care and Use Committee at Soochow University and were in strict accordance with the guidelines of the International Association for the Study of Pain.
Conflict-of-interest statement: All authors have no conflict of interest related to the manuscript.
Data sharing statement: Technical appendix, statistical code, and dataset were available from the corresponding author. Participants gave informed consent for data sharing. No additional data are 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/
Correspondence to: Guang-Yin Xu, MD, PhD, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, Institute of Neuroscience, The Second Affiliated Hospital, Soochow University, 199 Ren-Ai Road, Suzhou 215123, Jiangsu Province, China. guangyinxu@suda.edu.cn
Telephone: +86-512-65882817 Fax: +86-512-65883602
Received: February 9, 2015
Peer-review started: February 10, 2015
First decision: March 10, 2015
Revised: April 3, 2015
Accepted: May 20, 2015
Article in press: May 21, 2015
Published online: July 28, 2015
Processing time: 170 Days and 17.7 Hours
Abstract

AIM: To investigate the roles of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB on cystathionine β synthetase (CBS) expression and visceral hypersensitivity in rats.

METHODS: This study used 1-7-wk-old male Sprague-Dawley rats. Western blot analysis was employed to measure the expression of TLR4, NF-κB and the endogenous hydrogen sulfide-producing enzyme CBS in colon dorsal root ganglia (DRG) from control and “irritable bowel syndrome” rats induced by neonatal colonic inflammation (NCI). Colon-specific DRG neurons were labeled with Dil and acutely dissociated to measure excitability with patch-clamp techniques. Immunofluorescence was employed to determine the co-expression of TLR4, NF-κB and CBS in DiI-labeled DRG neurons.

RESULTS: NCI significantly upregulated the expression of TLR4 in colon-related DRGs (0.34 ± 0.12 vs 0.72 ± 0.02 for the control and NCI groups, respectively, P < 0.05). Intrathecal administration of the TLR4-selective inhibitor CLI-095 significantly enhanced the colorectal distention threshold of NCI rats. CLI-095 treatment also markedly reversed the hyperexcitability of colon-specific DRG neurons and reduced the expression of CBS (1.7 ± 0.1 vs 1.1 ± 0.04, P < 0.05) and of the NF-κB subunit p65 (0.8 ± 0.1 vs 0.5 ± 0.1, P < 0.05). Furthermore, the NF-κB-selective inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the upregulation of CBS (1.0 ± 0.1 vs 0.6 ± 0.1, P < 0.05) and attenuated visceral hypersensitivity in the NCI rats. In vitro, incubation of cultured DRG neurons with the TLR4 agonist lipopolysaccharide significantly enhanced the expression of p65 (control vs 8 h: 0.9 ± 0.1 vs 1.3 ± 0.1; control vs 12 h: 0.9 ± 0.1 vs 1.3 ± 0.1, P < 0.05; control vs 24 h: 0.9 ± 0.1 vs 1.6 ± 0.1, P < 0.01) and CBS (control vs 12 h: 1.0 ± 0.1 vs 2.2 ± 0.4; control vs 24 h: 1.0 ± 0.1 vs 2.6 ± 0.1, P < 0.05), whereas the inhibition of p65 via pre-incubation with PDTC significantly reversed the upregulation of CBS expression (1.2 ± 0.1 vs 0.6 ± 0.0, P < 0.01).

CONCLUSION: Our results suggest that the activation of TLR4 by NCI upregulates CBS expression, which is mediated by the NF-κB signaling pathway, thus contributing to visceral hypersensitivity.

Keywords: Irritable bowel syndrome; Dorsal root ganglion; Visceral hypersensitivity; Hydrogen sulfide; Toll-like receptors; Nuclear factor-kappa B

Core tip: This study investigated the roles of toll-like receptor 4 (TLR4) and its signaling pathway in the pathogenesis of visceral hypersensitivity in a rat model of irritable bowel syndrome (IBS). We demonstrated that activation of TLR4 upregulates the expression of the endogenous hydrogen sulfide-synthesizing enzyme cystathionine β synthetase. This upregulation was mediated by enhanced translocation of nuclear factor-κB in dorsal root ganglion cells, through which it contributed to the visceral hypersensitivity in IBS-like rats. Our results suggest TLR4 plays a major role in the development of functional visceral pain and hence suggest a novel mechanism and potential therapeutic target for the treatment of chronic visceral pain in patients with IBS.