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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 NCI upregulates CBS expression,which is mediated by the NF-kB signaling pathway,thus contributing to visceral hypersensitivity.
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 Sprague Dawley 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 di stention 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 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, incubated with 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; <0.05), while the inhibition of p65 via pre-incubation with PDTC 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-kB signaling pathway, contributed to visceral hypersensitivity.