An Experimental Study Of The Pathogenesis Of Colonic Hypersensitivity Of Irritable Bowel Syndrome Evoked By Sodium Butyrate

Irritable bowel syndrome (IBS) is nowadays a health care burden, because it represents one of the most common disorders encountered in gastrointestinal practice and highly affects quality of life. The major features for diagnosing IBS have been defined as (1) abdominal pain or discomfort, (2) altered bowel habits, and (3) the absence of any detectable structural or biochemical abnormality. Chronic or recurrent abdominal pain represents a symptom common to all patients with IBS, and rectal or colonic hypersensitivity is frequently encountered in IBS. Visceral hypersensitivity has been recognized as a characteristic feature of IBS and is gaining more and more interest and has even been regarded once as a biological mark of IBS. Visceral hypersensitivity is defined as lower pain and discomfort threshold and/or greater response to a stimulus. It may be involved in the pathogenesis of abdominal pain or discomfort and seems to result from the sensitization of nerve afferent pathways originating from the DRG. But the exact sensitization mechanism remains unclear. So, it's necessary to study the pathological mechanisms of colorectal hypersensitivity of IBS, and then explore promising targets for treatment of IBS.The disorder responsible for hypersensitivity in IBS may occur at the level of extrinsic peripheral afferent pathways. Comparison of patients with IBS to controls and patients with traumatic injury of the spinal cord suggests that sensitisation in patients with IBS occurs in splanchnic lumbar afferents. Somatostatin and its analogue octreotide have been shown to increase sensory thresholds to rectal and colonic distension in patients with IBS. Conversely, somatostatin also decreases the perception of electrical stimulation of the rectum and the amplitude of evoked potentials recorded at both the cortical and spinal levels. These findings imply that hypersensitivity might also result from sensitisation of peripheral afferent nerves. The plasticity of visceral afferents—that is, alterations in their ability to encode and transmit sensory information—is discussed in the paper by McMahon. In the case of sensory neurones, an increase in their excitability can have major consequences in terms of perceived pain or reflex control. Modulation of their excitability may be particularly relevant in diseases such as IBS.The vanilloid receptor type 1 (VR1 or TRPV1)1 is a vanilloid gated, nonselective cation channel that belongs to the transient receptor potential (TRP) channel superfamily. VR1 is expressed on small diameter neurons within sensory ganglia and accounts for the highly selective action of vanilloids as excitatory agents for nociceptors. In addition to vanilloids, heat and protons also influence vanilloid receptors and nociceptive pathways, and VR1 thus can be viewed as a molecular integrator of chemical and physical stimuli that elicit pain. The modulation of VR1 activity contributes to the sensitization of nociceptors associated with the development of allodynia and hyperalgesia. Beyond the transmission of peripheral nerve influx to the spinal cord, capsaicin-sensitive peptidergic C-fibers are of major importance in the generation of neurogenic inflammation and peripheral nerve sensitization due to retrograde release of neuropeptides (substance P, SP; calcitonin gene-related peptide, CGRP).The treatment of IBS is empirical and often poorly efficient. Research lacks suitable models for studying the pathophysiologic mechanisms of the colonic hypersensitivity and new pharmacologic targets.Recently, Sophie Bourdu has found that enemas with butyrate, a short-chain fatty acid produced by the colonic degradation of alimentary fibers could elicit a colorectal hypersensitivity. This is in line with the observation that colonic butyrate levels were increased in patients with IBS, which makes this model particularly relevant.Therefore, this project was carried out to study the effect of enemas with butyrate on the colorectal hypersensitivity in IBS rat modles. The expression of VR1,SP and CGRP were also evaluated in the rat models so as to explore the neurological mechanisms of colorectal hypersensitivity in IBS. Moreover, the model of cultures of primary dissociate DRG neurons of embryos of rat were established to explore the relationship between butyrate and release of SP and CGRP in DRG neurons and its related mechanism involved in VR1 .These results may have important meaning for further study the mechanisms of the colorectal hypersensitivity in IBS or to develop new targets for treatment of IBS. The abstract of the full text is as follows:Part I Butyrate sensitizes the release of SP and CGRP evoked by capsaicin from primary cultured rat dorsal root ganglion neuronsDRG was dissected out from embryonic 15-day-old Wistar rat and cultured as dissociate cells for 24 h then exposed to butyrate (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L, 10 mmol/L, respectively) for another 48 h. The neurons cultured continuously in media served as normal control. All above cultured samples were processed for detecting expression of mRNA for SP, CGRP and vanilloid receptor 1 (VR1) of DRG neurons by RT-PCR, and VR1 protein expression by Western blot. SP and CGRP basal release levels were measured by radioimmunoassay (RIA). After that, the DRG cells for RIA were stimulated by capsaicin (300 nmol/L) for 5 min and the culture media were harvested for detecting SP and CGRP release levels by RIA. The neurons exposed to vehicle solution served as vehicle controls. The effects of sodium butyrate on DRG neurons were analyzed according to the above data.The results are as follows:(1) The effects of butyrate on SP and CGRP mRNA expression in primary cultured DRG neurons were investigated by RT-PCR. Butyrate at higher dosages promoted SP and CGRP mRNA expression in primary cultured DRG neurons (P<0.001).(2) The effects of butyrate on VR1 mRNA and VR1 protein expression in primary cultured DRG neurons were investigated by RT-PCR and Western blot, respectively. An increase expression of VR1 mRNA and VR1 protein in primary cultured DRG neurons were induced by butyrate in a dose-dependent manner.(3) To test whether butyrate can increase sensitivity of capsaicin on the release of SP or CGRP from DRG neurons in vitro, DRG neurons were incubated with butyrate at different concentrations for 48 h and then stimulated by 300 nmol/L capsaicin for 5 min. Butyrate at different concentrations could not alter the basal neuropeptide release. Butyrate at higher concentrations (1 mmol/L, 10 mmol/L) significantly enhanced neuropeptide release evoked by capsaicin as compared with that at lower concentrations (0.01 mmol/L, 0.1 mmol/L) of butyrate and without butyrate (P<0.05). Vehicle solution could not increase neuropeptide release as compared with basal release.The results indicate that butyrate may promote the expression of mRNA for SP, CGRP and increase sensitivity of capsaicin on SP and CGRP release from primary cultured rat dorsal root ganglion neurons. Butyrate did not alter the basal release of SP, CGRP evoked by capsaicin.. The promotion of VR1 mRNA and VR1 protein expression by butyrate implicated that VR1 may be involved in the mechanisms of sensory neuropeptide release evoked by capsaicin. So, fibers, commonly believed to be beneficial in IBS, should induce or increase symptoms in patients with IBS and must therefore be used with care. Part II An experimental study of the effects of VR1 and neuropeptide on colorectal hypersensitivity in IBS rats enemas with butyrateMale Wister rats weighing 200-220 g were used in this study. Rats were maintained in laboratory conditions for 1 week before each experiment. The animals were housed 4 per cage with food and water available ad libitum. Great care was taken, particularly with regard to housing conditions, to avoid or minimize discomfort to the animals.For each enema, a catheter (2-mm Fogarty catheter) was placed into the colon at 7 cm from the anus, and the animals received 1 ml of sodium butyrate solution (200 mmol/L) at neutral pH (pH 6.9) twice daily for 3 days. Pressure-induced behavioral response to CRD were successively assessed on days 3, 6, 9, 12, 15 and 18 after the first enema for the same animals.To explore mechanisms of enemas with butyrate on the colorectal hypersensitivity in IBS rat modles, another group of animal was used. The blood samples and L₆-S₂ DRG were taken from the rats on day 9 after the first enema. The levels of CGRP and SP in plasma were determined by methods of radioimmunoassay. CGRP and SP mRNA, CGRP and SP protein in DRG were determined by methods of semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western Blot (WB), respectively. In the present study, SP(4 mg/kg),CGRP (500 ng/kg), L733060 (3 mg/kg), CGRP8-37 (20μg/kg) or capsaicin (50 mg/kg) was used to evaluate the role of SP, CGRP on the colorectal hypersensitivity in IBS rat modles and the role of VRl pathway in regulation of the synthesis and release of SP , CGRP. These results may have important meaning for further study the mechanisms of the colorectal hypersensitivity in IBS or to develop new targets for treatment of IBS.The series of experiments was divided into 10 groups: (1) the sham group, (2) the NaB group, (3) the L733060+NaB group, (4) the CGRP8-37+NaB group, (5) the capsaicin + NaB group. (6) the SP group. (7) the CGRP group, (8) the NaB + L733060 group. (9) the NaB +CGRP8-37 group, (10) the NaB + capsaicin group.The results are as follows:(1) Butyrate enemas induced a sustained colorectal hypersensitivity and, but no macroscopic and histologic modifications of the colonic mucosa, as observed in patients with IBS. The effect was completely abolished by pretreatment with L733060,CGRP8-37 or capsaicin.(2) Enemas with NaB induced the expression of SP, CGRP, VR1 mRNA and protein in DRG The level of SP, CGRP in plasma was significantly elevated on day 9 after the first enema. Apparent changes of SP, CGRP, VR1 mRNA and protein level at above mentioned time points were observed.(3) CGRP or SP intravenous injection induced gastric hypersensitivity.(4) L733060 (3 mg/kg, injected intravenously), CGRP8-37(20μg/kg, injected intravenously) and capsaicin (50 mg/kg, injected subcutaneously) significantly increased the CRD thresholds and brought them back to those of healthy animals.The results indicate that the release of SP, CGRP plays an important role in the colorectal hypersensitivity induced by enemas with NaB. The cause of increasing release of SP, CGRP was that the synthesis of SP, CGRP was increased; on the other hand, the increased expression of VR1 elevated the DRG neurons' sensitivity. SP receptor antagonist may prevent the occurrence of colorectal hypersensitivity, but has no effect on existed colorectal hypersensitivity. CGRP8-37 and capsaicin have prevention and therapeutical effect with colorectal hypersensitivity, may provide new promising targets for treatment of IBS.