title: TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Bo
authors.initials: B
authors.lastname: Yuan
authors.affiliation: 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.
authors.forename: Wei-Hong
authors.initials: WH
authors.lastname: Tang
authors.affiliation: 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.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Lu
authors.affiliation: 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.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: 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.
authors.forename: Hong-Yan
authors.initials: HY
authors.lastname: Zhu
authors.affiliation: 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.
authors.forename: You-Lang
authors.initials: YL
authors.lastname: Zhou
authors.affiliation: 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.
authors.forename: Hong-Hong
authors.initials: HH
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Chuang-Ying
authors.initials: CY
authors.lastname: Hu
authors.affiliation: 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.
authors.forename: Guang-Yin
authors.initials: GY
authors.lastname: Xu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003106:Colon / Q000294:innervation*; D003541:Cystathionine beta-Synthase / Q000378:metabolism*; D004195:Disease Models, Animal; D005727:Ganglia, Spinal / Q000187:drug effects / Q000201:enzymology* / Q000503:physiopathology; D006930:Hyperalgesia / Q000201:enzymology* / Q000503:physiopathology / Q000517:prevention & control; D043183:Irritable Bowel Syndrome / Q000188:drug therapy / Q000201:enzymology* / Q000503:physiopathology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009474:Neurons / Q000201:enzymology; D058748:Pain Perception; D017288:Pain Threshold; D011759:Pyrrolidines / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013859:Thiocarbamates / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015854:Up-Regulation; D059265:Visceral Pain / Q000201:enzymology* / Q000503:physiopathology / Q000517:prevention & control
chemical_list: D008070:Lipopolysaccharides; D011759:Pyrrolidines; C498145:Rela protein, rat; D013859:Thiocarbamates; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; C020972:pyrrolidine dithiocarbamic acid; D003541:Cystathionine beta-Synthase
keywords: Dorsal root ganglion; Hydrogen sulfide; Irritable bowel syndrome; Nuclear factor-kappa B; Toll-like receptors; Visceral hypersensitivity