TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity  

作　　者：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 

机构地区：[1]Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, Institute of Neuroscience,The Second Affiliated Hospital, Soochow University [2]Department of Pain Management, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School

出　　处：《World Journal of Gastroenterology》2015年第28期8615-8628,共14页世界胃肠病学杂志（英文版）

基　　金：Supported by National Natural Science Foundation of China,No.81230024,No.81471137(to GYX)and No.31400947(to HHZ);Priority Academic Program Development of Jiangsu Higher Education Institutions

摘　　要：AIM:To investigate the roles of toll-like receptor 4(TLR4) and nuclear factor(NF)-κB on cystathionine βsynthetase(CBS) expression and visceral hypersensitivity in rats.METHODS:This study used 1-7-wk-old male SpragueDawley rats.Western blot analysis was employed to measure the expression of TLR4,NF-kB 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-kB and CBS in Dil-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 colonspecific DRG neurons and reduced the expression of CBS(1.7 ± 0.1 vs 1.1 ± 0.04,p < 0.05) and of the NF-kB subunit p65(0.8 ± 0.1 vs 0.5 ± 0.1,P< 0.05).Furthermore,the NF-KB-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 vs1.3 ± 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 NCIAIM:To investigate the roles of toll-like receptor 4(TLR4) and nuclear factor(NF)-κB on cystathionine βsynthetase(CBS) expression and visceral hypersensitivity in rats.METHODS:This study used 1-7-wk-old male SpragueDawley rats.Western blot analysis was employed to measure the expression of TLR4,NF-kB 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-kB and CBS in Dil-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 colonspecific DRG neurons and reduced the expression of CBS(1.7 ± 0.1 vs 1.1 ± 0.04,p < 0.05) and of the NF-kB subunit p65(0.8 ± 0.1 vs 0.5 ± 0.1,P< 0.05).Furthermore,the NF-KB-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 vs1.3 ± 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

关 键 词：IRRITABLE BOWEL syndrome DORSAL rootganglion VISCERAL HYPERSENSITIVITY Hydrogen sulfide TOLL-LIKE receptors Nuclear factor-kappa B 

分 类 号：R574.4[医药卫生—消化系统]