Toll Like Receptor-4 Upregulates Cystathionine β Synthetase Expression Through NF-kB Activation In Rats With Chronic Visceral Hypersensitivity

Background: Abdominal pain or bloating is one of primary symptoms of patents with irritable bowel syndrome(IBS) with mechanisms that as yet are incompletely defined. The aim of this study was to investigate roles of toll-like receptor 4(TLR4) and NF-kB on upregulation of endogenous hydrogen sulfide producing enzyme cystathionine b-synthetase(CBS) and visceral hypersensitivity in a rat model of IBS.Methods:1. Visceral hypersensitivity was induced by colonic injection of 0.5% acetic acid(AA, 0.2ml) in 10-day-old rats. Behavioral response to colorectal distension(CRD) was measured at age of 6 weeks by visual observation of abdominal withdrawal reflex(AWR) by a blinded observer.2. Western blot analysis was employed to measure expressions of TLR4, NF-kB and CBS in colon DRGs from control and “IBS-like” rats induced by neonatal colonic inflammation(NCI).3. Colon-specific DRG neurons were labeled with Dil and acutely dissociated for measuring excitability with patch-clamp techniques.4. Immunofluorescence was employed to determine co-expression of TLR4, NF-kB and CBS.5. Chromatin immunoprecipitation(Ch IP) analysis was employed to determine co-expression and interaction of NF-kB with CBS.Results:1. NCI significantly upregulated TLR4 expression in colon DRGs.2. Intrathecal administration of TLR4 inhibitor, CLI-095, enhanced colorectal distention threshold.3. CLI-095 treatment also reversed hyperexcitability of colon-specific DRG neurons and reduced expression of CBS and p65.4. Immunofluorescence showed TLR4 was coexpressed with CBS in colon-related DRG neurons. CLI-095 treatment also reduced expression of CBS.5. Immunofluorescence also showed TLR4 was coexpressed with NF-kB. Western blotting showed NCI significantly upregulated nucleus p65 expression in colon-related DRGs. And CLI-095 reversed the upregulation of nucleus p65 expression.6. Furthermore, inhibition of p65 by pyrrolidine dithiocarbamate(PDTC) reduced the upregulation of CBS and attenuated visceral hypersensitivity.7. Ch IP experiments showed that NCI significantly increased the ability of CBS binding with p65 DNA consensus site.8. Incubation of cultured DRG neurons with LPS significantly enhanced p65 and CBS expression while inhibition of p65 by pre-incubation of PDTC reversed the upregulation of CBS expression.Conclusion: Our results suggest that activation of TLR4 by NCI upregulated CBS expression, which is mediated by NF-kB signaling pathway, thus contributing to visceral hypersensitivity. The present and future research would identify a specific neurobiological target for the treatment of chronic visceral hyperagesia in functional gastrointestinal disorders.