Rat Model Of Irritable Bowel Syndrome Research Articles

Effect of ERK1/2 pathway in the expression of MHC-Ⅱof intestinal dendritic cells in viscerally hypersensitive rats

To study the phenotypic alteration of intestinal dendritic cells (DC) in a rat model of irritable bowel syndrome (IBS) and the change of mitogen-activated protein kinase (MAPK) signaling pathway, in order to explore the potential mechanism of ERK1/2 pathway mediation in abnormal DC immune response. IBS rat model was established by combining neonatal maternal separation and colorectal distension in 10 SD rats, and 10 healthy rats served as controls. Visceral sensitivity was evaluated with abdominal withdrawal reflex (AWR). Mesenteric lymph node DC (MLNDC) was isolated and purified by magnetic label-based technique after modeling. Expression of surface major histocompatibility complex (MHC)-Ⅱin control rats was determined by flow cytometric analyses. Western-blot was used to determine the expression of MHC-Ⅱ, p-p38, p38, phosphorylated extracellular regulated protein kinase (p-ERK1/2), ERK1/2, phosphorylated c-Jun N-terminal kinase (p-JNK), and JNK in MLNDC. Visceral sensitivity was significantly higher in the IBS group than in the control group. The purity of the OX62 positivity MLNDC following magnetic sorting was about 85.57%±7.67%. MLNDC in the control group expressed high level of MHC-Ⅱ. The expression of MHC-Ⅱ and p-ERK1/2 in MLNDC in the IBS group were higher than those in the control group (1.05±0.13 vs 0.67±0.18, t=-2.973, P=0.041; 3.21±0.48 vs 2.34±0.85, t=-3.130, P=0.035); while there was no significant difference in the expressions of p-JNK and p-p38 compared with the control groups (0.95±0.17 vs 0.76±0.36, t=0.808, P=0.464; 1.07±1.13 vs 1.19±0.91, t=0.137, P=0.897). The intestinal DC in IBS rats show a upregulated expression of MHC-Ⅱ, which may be related to the activation of intracellular ERK1/2 pathway.

Tong Xie Yao Fang relieves irritable bowel syndrome in rats via mechanisms involving regulation of 5-hydroxytryptamine and substance P.

To investigate whether the Chinese medicine Tong Xie Yao Fang (TXYF) improves dysfunction in an irritable bowel syndrome (IBS) rat model. Thirty baby rats for IBS modeling were separated from mother rats (1 h per day) from days 8 to 21, and the rectum was expanded by angioplasty from days 8 to 12. Ten normal rats were used as normal controls. We examined the effects of TXYF on defection frequency, colonic transit function and smooth muscle contraction, and the expression of 5-hydroxytryptamine (5-HT) and substance P (SP) in colonic and hypothalamus tissues by Western blot and RT-PCT techniques in both normal rats and IBS model rats with characterized visceral hypersensitivity. Defecation frequency was 1.8 ± 1.03 in normal rats and 4.5 ± 1.58 in IBS model rats (P < 0.001). However, the defecation frequency was significantly decreased (3.0 ± 1.25 vs 4.5 ± 1.58, P < 0.05), while the time (in seconds) of colon transit function was significantly increased (256.88 ± 20.32 vs 93.36 ± 17.28, P < 0.001) in IBS + TXYF group rats than in IBS group rats. Increased colonic smooth muscle tension and contract frequency in IBS model rats were significantly decreased by administration of TXYF. Exogenous agonist stimulants increased spontaneous activity and elicited contractions of colon smooth muscle in IBS model rats, and all of these actions were significantly reduced by TXYF involving 5-HT and SP down-regulation. TXYF can modulate the activity of the enteric nervous system and alter 5-HT and SP activities, which may contribute to the symptoms of IBS.

Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction

To determine the molecular mechanisms of Shugan decoction (SGD) in the regulation of colonic motility and visceral hyperalgesia (VHL) in irritable bowel syndrome (IBS). The chemical compounds contained in SGD were measured by high-performance liquid chromatography. A rat model of IBS was induced by chronic water avoidance stress (WAS). The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension. Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining. The contents of tumor necrosis factor (TNF)-α in colonic tissue and calcitonin-gene-related peptide (CGRP) in serum were measured by ELISA. The protein expression of serotonin [5-hydroxytryptamide (5-HT)], serotonin transporter (SERT), chromogranin A (CgA) and CGRP in colon tissue was measured by immunohistochemistry. SGD inhibited colonic motility dysfunction and VHL in rats with IBS. Blockers of transient receptor potential (TRP) vanilloid 1 (TRPV1) (Ruthenium Red) and TRP ankyrin-1 (TRPA1) (HC-030031) and activator of protease-activated receptor (PAR)4 increased the pain pressure threshold, whereas activators of PAR2 and TRPV4 decreased the pain pressure threshold in rats with IBS. The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1 (capsaicin), TRPV4 (RN1747), TRPA1 (Polygodial) and PAR2 (AC55541). In addition, CGRP levels in serum and colonic tissue were both increased in these rats. TNF-α level in colonic tissue was also significantly upregulated. However, the levels of 5-HT, SERT and CgA in colonic tissue were decreased. All these pathological changes in rats with IBS were attenuated by SGD. SGD alleviated VHL and attenuated colon motility in IBS, partly by regulating TRPV1, TRPV4, TRPA1, PAR2, 5-HT, CgA and SERT, and reducing CGRP and TNF-α level.

Modulation of enteric neurons by interleukin‐6 and corticotropin‐releasing factor contributes to visceral hypersensitivity and altered colonic motility in a rat model of irritable bowel syndrome

The search for effective therapeutic strategies for irritable bowel syndrome (IBS) is hampered by an incomplete understanding of its underlying pathophysiology. Stress and altered plasma cytokine profiles indicative of immune activation are characteristic of the disorder. The neuromodulatory effects of interleukin-6 (IL-6) and corticotropin-releasing factor receptor (CRFR) 1 in visceral pain and stress-induced defecation in the Wistar Kyoto (WKY) rat model of IBS were investigated. Sprague Dawley and WKY rats were administered anti-IL-6 receptor antibodies (xIL-6R, 0.5mgkg(-1) i.p) with or without the CRFR1 antagonist antalarmin (10mgkg(-1) i.p). Post-intervention, the pain threshold to colorectal distension and stress-induced faecal output were compared and changes in colonic mucosal protein expression were investigated. The neuro-stimulatory effects of IBS plasma on the myenteric plexus is mediated by IL-6, IL-8 and CRF. The stimulatory effects of these soluble factors on myenteric neuron excitability and colonic contractility were additive. Moreover, inhibition of IL-6 and CRF1 receptors in vivo in the WKY IBS rat model normalized stress-induced defecation (P<0.01) and visceral pain sensitivity (P<0.001) with associated changes in protein expression of the tight junction proteins occludin and claudin 2, the visceral pain-associated T-type calcium channel CaV3.2 and intracellular signalling molecules STAT3, SOCS3 and ERK1/2. These studies demonstrate the additive effects of immune and stress factors on myenteric neuronal excitability. Moreover, combined targeting of peripheral IL-6 and CRF1 receptors is effective in alleviating IBS-like symptoms in the WKY rat. Thus, crosstalk between stress and immune factors during IBS flares may underlie symptom exacerbation.

Inhibitory effects of SKI3246, the rhizome extract of Atractylodes japonica, on visceral hypersensitivity in experimental irritable bowel syndrome rat models.

We evaluated the effect of SKI3246, the 50% ethanol extract of the rhizome of Atractylodes japonica, on visceral hypersensitivity, which is a major characteristic feature of IBS. We used various rat models of visceral hypersensitivity to assess the visceral pain responses to colorectal distension (CRD) in comparison with conventional IBS treatments. Oral administration of SKI3246 dose-dependently and significantly attenuated the abdominal withdrawal reflex (AWR) score in a model of acetic acid-induced visceral hypersensitivity. We also found that it reduced the number of abdominal contractions in response to CRD in a model of 2,4,6-trinitrobenzenesulfonic acid-induced visceral hypersensitivity, which was comparable to ramosetron or alosetron. Furthermore, treatment with SKI3246 also increased the pain threshold and abolished the elevated AWR scores to CRD in a rat model of neonatal maternal separation. We presumed that the modulation of the NK2 receptor is involved in the inhibitory activity of SKI3246 on the basis that it significantly inhibited the contraction of the distal colonic muscle induced by neurokinin A, the NK2 receptor agonist. The present results indicate that SKI3246 has the potential to be an effective therapeutic agent for IBS, especially insofar as it can relieve visceral hypersensitivity.

Su2048 Fecal Microbiota in Patients With Irritable Bowel Syndrome Compared to Healthy Controls by Real-Time PCR: An Evidence of Dysbiosis

Background and aims: Irritable bowel syndrome (IBS) is a common gastrointestinal dysfunction and affecting 5-22% population. Brain-gut interactions are increasingly recognized as underlying pathomechanisms of IBS. Curcumin is a biologically active phytochemical substance presented in turmeric and it has been proved to make an effective role in treating patients with depression and anxiety through adjusting related neurotransimitters. The aim of this study was to determine the efficacy of curcumin in treating IBS rat model. Methods: We induced IBS in male Sprague-Dawly(SD) rats via chronic and acute stress(CAS). Depressionand anxiety-like behaviors were tested using forced swimming test and marble-burying test. Colorectal distension (CRD) was given and abdominal withdrawal reflex (AWR) was utilized to determine the visceral hypersensitivity. Serotonin, Brain-derived neurotrophic factor(BDNF) and cAMP response element-binding protein(CREB) were detected via western blotting. Results: IBS rats demonstrated depressionand anxiety-like behaviors associated

Differential activation of the prefrontal cortex and amygdala following psychological stress and colorectal distension in the maternally separated rat

Visceral hypersensitivity is a hallmark of many clinical conditions and remains an ongoing medical challenge. Although the central neural mechanisms that regulate visceral hypersensitivity are incompletely understood, it has been suggested that stress and anxiety often act as initiating or exacerbating factors. Dysfunctional corticolimbic structures have been implicated in disorders of visceral hypersensitivity such as irritable bowel syndrome (IBS). Moreover, the pattern of altered physiological responses to psychological and visceral stressors reported in IBS patients is also observed in the maternally separated (MS) rat model of IBS. However, the relative contribution of various divisions within the cortex to the altered stress responsivity of MS rats remains unknown. The aim of this study was to analyze the cellular activation pattern of the prefrontal cortex and amygdala in response to an acute psychological stressor (open field) and colorectal distension (CRD) using c-fos immunohistochemistry. Several corticoamygdalar structures were analyzed for the presence of c-fos-positive immunoreactivity including the prelimbic cortex, infralimbic cortex, the anterior cingulate cortex (both rostral and caudal) and the amygdala. Our data demonstrate distinct activation patterns within these corticoamygdalar regions including differential activation in basolateral versus central amygdala following exposure to CRD but not the open field stress. The identification of this neuronal activation pattern may provide further insight into the neurochemical pathways through which therapeutic strategies for IBS could be derived.

Mild moxibustion decreases the expression of prokineticin 2 and prokineticin receptor 2 in the colon and spinal cord of rats with irritable bowel syndrome.

It has been proven that prokineticin 2 (PK2) and its receptor PKR2 play an important role in hyperalgesia, while mild moxibustion can relieve visceral hypersensitivity in a rat model of irritable bowel syndrome (IBS). The goal of the present study was to determine the effects of mild moxibustion on the expression of PK2 and PKR2 in colon and spinal cord in IBS rat model, which was induced by colorectal distension using inflatable balloons. After mild moxibustion treatment, abdominal withdrawal reflex (AWR) scores were assessed by colorectal distension; protein and mRNA expression of PK2 and PKR2 in rat colon and spinal cord was determined by immunohistochemistry and fluorescence quantitative PCR. Compared with normal rats, the AWR scores of rats and the expressions of PK2/PKR2 proteins and mRNAs in colon and spinal cord tissue were significantly increased in the model group; compared with the model group, the AWR scores of rats and the expressions of PK2/PKR2 proteins and mRNAs in colon and spinal cord tissue were significantly decreased in the mild moxibustion group. These findings suggest that the analgesia effect of mild moxibustion may be associated with the reduction of the abnormally increased expression of the PK2/PKR2 proteins and mRNAs in the colon and spinal cord.

Abnormal dendritic cells mediated immune response in a rat model of visceral hypersensitivity

To explore the role of altered characteristics of intestinal dendritic cell (DC) in the induction of visceral hyperalgesia through the activation of mast cells in a rat model of irritable bowel syndrome (IBS). A total of 20 Sprague-Dawley rats were divided into modeling and control groups (n = 10 each). The IBS rat model was established by combining colorectal distention with restraint stress. Abdominal withdrawal reflex (AWR) was employed to evaluate visceral sensitivity. The surface marker of intestinal DC was analyzed by immunohistochemistry. Toluidine blue staining was used to determine the number of mast cells (MC). The expressions of interleukin (IL) -4 and IL-9 in colonic mucosa were measured by enzyme-linked immunosorbent assay (ELISA) and the level of proteinase-activated receptor-2 (PAR-2) was measured by Western blot. Mesenteric lymph node (MLN) DC and splenic CD4(+)/CD8(+)T cells were isolated and purified by magnetic label-based technique. Cytokine production of MLN DC co-culturing with CD4(+) or CD8(+)T cells was determined. The number of colonic MC in modeling group was more than that in control group ((2.73 ± 0.21) vs (1.13 ± 0.10), P = 0.000). The expressions of PAR-2, IL-4 and IL-9 in colonic mucosa were all higher than those in control group (2.13 ± 0.81 vs 0.42 ± 0.29, (7.2 ± 1.2) vs (3.3 ± 1.0) pg/ml, (7.3 ± 1.3) vs (5.2 ± 0.6) pg/ml, P = 0.026, 0.000, 0.001). Co-cultured MLN DC with CD4(+) T cells showed a predominant IL-4 secretion in the modeling group ((1.22 ± 0.33) vs (0.80 ± 0.48) pg/ml, P = 0.000). Increased colonic DC may stimulate CD4(+) T cells to secrete a high level of IL-4 to cause the degranulation of mast cells and the generation of visceral hypersensitivity in IBS rats.

Role of glucagon-like peptide-1 in the pathogenesis of experimental irritable bowel syndrome rat models

Alterations in gut motility and visceral hypersensitivity are two major features of irritable bowel syndrome (IBS). The aim of this study was to investigate the roles of glucagon-like peptide-1 (GLP-1) in the pathogenesis of experimental IBS. Rat models of constipation-predominant IBS (IBS-C) and diarrhea-predominant IBS (IBS-D) were established. Fecal water content and behavioral responses to colorectal distention (CRD), using electromyography (EMG), were measured. The expression of glucagon-like peptide-1 receptor (GLP-1R) in the colon was detected by immunohistochemistry, and the serum concentration of GLP-1 was measured by ELISA assay. The movement of circular and longitudinal colonic muscle was detected using an organ bath recording technique. Compared to controls, the fecal water contents were lower in the IBS-C group, while they were higher in the IBS-D group (P<0.05). EMG response to CRD in the experimental IBS groups was increased compared with their respective controls (P<0.05). GLP-1R was localized in the mucosa layer, circular muscle and myenteric nerve plexus of the colon. Notably, the expression of GLP-1R in the IBS-C group was higher, but in the IBS-D group, it was lower compared with controls. The serum levels of GLP-1 in the IBS-C group were higher compared to those in the IBS-D group (P<0.05). In addition, administration of exogenous GLP-1 and exendin-4 inhibited colonic circular muscle contraction, particularly in the IBS-C group, while there was no significant effect on longitudinal muscle contraction. In conclusion, these results indicated that GLP-1 and GLP-1R are implicated in the pathogenesis of IBS-C and IBS-D.

Warming Moxibustion Relieves Chronic Visceral Hyperalgesia in Rats: Relations to Spinal Dynorphin and Orphanin-FQ System

As a twin therapy of acupuncture in traditional Chinese medicine, moxibustion has shown its effects in relieving abdominal pain in irritable bowel syndrome (IBS) patients and IBS rat models, but its mechanisms are largely unknown. In this paper, we determined the role of spinal dynorphin and orphanin-FQ system in analgesic effect of warming moxibustion (WM) on chronic visceral hyperalgesia (CVH) in IBS-like rat model. Here, we show that (1) repeated WM at bilateral ST25 and ST37 acupoints markedly attenuated the abdominal withdrawal reflex scores in CVH rats; (2) intrathecal administration of κ receptor antagonist prior to WM significantly attenuated the WM analgesia and dynorphinA (1-17) enhanced the WM analgesia. WM significantly reinforced the upregulation of spinal dynorphin mRNA/protein and κ receptor mRNA levels in CVH rats; (3) intrathecal administration of orphanin-FQ receptor antagonist prior to WM significantly attenuated the WM analgesia and orphanin-FQ enhanced the WM analgesia. WM reinforced the upregulation of spinal orphanin-FQ mRNA/protein and orphanin-FQ receptor mRNA levels in CVH rats. These results suggest that moxibustion may relieve CVH at least in part by activating spinal dynorphin and orphanin-FQ system.

Effects of electroacupuncture on expression of c-fos protein in the spinal dorsal horn of rats with chronic visceral hyperalgesia

Acupuncture is widely used in clinics to suppress chronic visceral pain in patients with irritable bowel syndrome (IBS); however, the exact neurobiological mechanisms for its therapeutic effects need further exploration. The aim of this study was to investigate the possible involvement of spinal neurons in the effects of electroacupuncture (EA) in relieving chronic visceral hyperalgesia in a rat model of IBS. Colon mechanical irritation was applied to male neonatal Sprague-Dawley rats to establish the IBS model. Behavioral test of the abdominal withdraw reflex (AWR) response to colorectal distention stimuli was conducted to judge the degree of colorectal sensitivity. EA at acupoints Zusanli (ST36) and Shangjuxu (ST37) was applied bilaterally in a total of four times every other day, while sham-EA at similar acupoints was done by inserting needles without electrical stimulation. Immunohistochemical methods were used to display the expression of proto-oncogene protein c-fos in the spinal dorsal horn. It was found that AWR scores were significantly increased in the IBS model rats (P<0.01), accompanied with significant increase in the expression of c-fos protein in the superficial laminae (SDH, laminae I and II) and nucleus proprius (NP, laminae III and IV), the neck of the dorsal horn (NECK, laminae V and VI) at lumbosacral (L6-S2) spinal level, and in NECK at thoracolumbar (T13-L2) spinal level, when compared with normal rats (P<0.05). After EA treatment, AWR scores and the expression of c-fos protein in SDH, NP and NECK at similar spinal levels were significantly decreased in the IBS model rats (P<0.05). No such effects on either AWR scores or the expression of c-fos protein were observed in IBS model rats after sham-EA treatment. The abnormally high neuronal excitability in the spinal dorsal horn may be an important reason underlying the visceral hyperalgesia in IBS model rats. EA treatment can relieve the chronic visceral hyperalgesia in IBS rats by suppressing the abnormal neuronal excitability in the spinal dorsal horn.

Schisandra chinensis reverses visceral hypersensitivity in a neonatal–maternal separated rat model

Visceral hypersensitivity is an important characteristic feature of functional gastrointestinal disorders, such as irritable bowel syndrome (IBS). This study evaluated the effect of Schisandra chinensis on visceral hyperalgesia induced by neonatal maternal separation (NMS) in an IBS rat model. The visceromotor responses to colorectal balloon distension (CRD) were measured by abdominal withdrawal reflex (AWR) and electromyographic (EMG) activities. NMS control rats (receiving vehicle) underwent aggravated visceral pain in response to CRD as compared to normal rats, evidenced by the reduced pain threshold, enhanced AWR scores and EMG responses. Treatment with a 70% ethanol extract of S. chinensis (0.3g/kg and 1.5g/kg/day) for 7 days resulted in an increase in the pain threshold (NMS control: 19.1±1.0mmHg vs low-dose: 24.8±1.3mmHg and high-dose: 25.2±1.8mmHg, p<0.01), and abolished the elevated AWR and EMG responses to CRD in NMS rats (AUC values of EMG response curve were: 1952±202 in NMS control group vs 1074±90 in low-dose group and 1145±92 in high-dose group, p<0.001), indicating that S. chinensis could reverse the visceral hypersensitivity induced by early-life stress event. The result of ELSA measurement shows that the elevated serotonin (5-HT) level in the distal colon of NMS rats returned to normal level after treatment with S. chinensis. Moreover, the increase in pain threshold in rats treated with S. chinensis was associated with a decline of the mRNA level of 5-HT3 receptor in the distal colon. All available results demonstrate that S. chinensis can reverse visceral hypersensitivity induced by neonatal–maternal separation, and the effect may be mediated through colonic 5-HT pathway in the rat.

Chronic treatment with otilonium bromide affects the tachykinergic and nitrergic systems in the rat colon

Otilonium bromide (OB), a quaternary ammonium derivative used for the treatment of intestinal motility disorders such as the irritable bowel syndrome (IBS). It exerts several actions, among which the ability to bind to the neurokin-2 receptor (NK2r) inhibiting NK2r mediated contraction and, in the human colon, NK2r internalization in the smooth muscle cells (SMC) (Cipriani et al., 2011). Substance P (SP) is an excitatory neurotransmitter that, interacting mainly with the neurokinin-1 receptor (NK1r), can stimulate bowel motility by SMC direct activation or inhibit it by an indirect action through enteric neural circuits. In an IBS rat model, the increase in NK1rmediated colonic motor response was associated to a decrease in the nitrergic activity. On these basis, we tested whether OB modifies NK1r, NK2r, SP and neuronal nitric oxide synthase (nNOS) expression in rat colon after chronical administration of the drug (2 or 20mg/Kg/daily) for 10 or 30 days. At the end of the treatments, specimens of proximal colon were collected and the expression of NK1r, NK2r, SP and neurogenic and myogenic nNOS were evaluated by immunohistochemistry and Western blot. Our data show that SP expression was significantly decreased in 10 and 30 days treated rats in myenteric ganglia and, in 30 days treated rats, also in the intramuscular nerve fibres. No quantitative change of the two NKr was observed, whereas, after 30 days, the NK1r was concentrated in the SMC cytoplasm. In parallel, the neurogenic nNOS expression increased and reached the significance after 30 days of treatment; the myogenic nNOS expression increased, but these increase reached the significance only at 10 days. Our findings suggest that the main target of the OB chronically administered is the NO-mediated system that is stimulated earlier at the muscular level, later at the neuronal level. We interpret the systemic decrease in the SP expression as consequence of the potentiated NO availability in the ganglia and muscle coat. If true, the late concentration of NK1r in the cytoplasm could represent an attempt of the SMC to overcome the deficit of its main ligand SP.

Interleukin-6 Modulates Colonic Transepithelial Ion Transport in the Stress-Sensitive Wistar Kyoto Rat

Immunological challenge stimulates secretion of the pro-inflammatory cytokine interleukin (IL)-6, resulting in variety of biological responses. In the gastrointestinal tract, IL-6 modulates the excitability of submucosal neurons and stimulates secretion into the colonic lumen. When considered in the context of the functional bowel disorder, irritable bowel syndrome (IBS), where plasma levels of IL-6 are elevated, this may reflect an important molecular mechanism contributing to symptom flares, particularly in the diarrhea-predominant phenotype. In these studies, colonic ion transport, an indicator of absorption and secretion, was assessed in the stress-sensitive Wistar Kyoto (WKY) rat model of IBS. Mucosa-submucosal colonic preparations from WKY and control Sprague Dawley (SD) rats were mounted in Ussing chambers and the basal short circuit current (ISC) was electrophysiologically recorded and compared between the strains. Exposure to IL-6 (1 nM) stimulated a secretory current of greater amplitude in WKY as compared to SD samples. Furthermore, the observed IL-6-mediated potentiation of secretory currents evoked by veratridine and capsaicin in SD rats was blunted in WKY rats. Exposure to IL-6 also stimulated an increase in transepithelial resistance in both SD and WKY colonic tissue. These studies demonstrate that the neuroexcitatory effects of IL-6 on submucosal plexi have functional consequences with alterations in both colonic secretory activity and permeability. The IL-6-induced increase in colonic secretory activity appears to neurally mediated. Thus, local increases in IL-6 levels and subsequent activation of enteric neurons may underlie alterations in absorpto-secretory function in the WKY model of IBS.

Elevated expression of c-fos in central nervous system correlates with visceral hypersensitivity in irritable bowel syndrome (IBS): a new target for IBS treatment

Background and aimsAlthough visceral hypersensitivity is a major pathophysiological feature of irritable bowel syndrome (IBS), its molecular mechanisms are still poorly understood. c-fos is a well-established marker of cell activation. Accumulating evidence demonstrates that norepinephrine (NE) system is dysregulated in IBS; however, very little is known on its mechanism. It is our hypothesis that elevated expression of c-fos in central nervous system (CNS) correlates with visceral hypersensitivity in rat model of IBS. Furthermore, we explored the changes of NE system in IBS patients.MethodsThe rat model of IBS was induced by heterotypic chronic and acute stress. Tissues obtained from rat model were analyzed for c-fos levels in CNS (frontal lobe, hippocampus, cornu dorsale) and colon by immunohistochemistry. Real-time reverse transcription polymerase chain reaction was used to detect tyrosine hydroxylase (TH) in the colonic tissues obtained from IBS patients.ResultsThe rat model of IBS was associated with increased expression of c-fos in different parts of CNS (P = 0.001, P = 0.002, and P = 0.002, respectively), but normal in colon (P = 0.207). The clinical parameters (colonic motility and sensation) of rat model were significantly correlated with elevated c-fos in CNS (P < 0.05). Enterochromaffin cells and serotonin in colon were related to the elevated c-fos in CNS (P < 0.05). The TH messenger ribonucleic acid (mRNA level of IBS-D patients was almost four times as much as that of controls.ConclusionsElevated expression of c-fos in CNS might be one of key mechanisms in etiology of IBS. Therefore, regulation of CNS activation could be a major targeting effect when treating IBS patients.

Enhanced Excitability and Down-Regulated Voltage-Gated Potassium Channels in Colonic DRG Neurons from Neonatal Maternal Separation Rats

Irritable bowel syndrome (IBS), characterized mainly by abdominal pain, is a functional bowel disorder. The present study aimed to examine changes in the excitability and the activity of the voltage-gated K+ channel in dorsal root ganglia (DRG) neurons innervating the colon of rats subjected to neonatal maternal separation (NMS). Colonic DRG neurons from NMS rats as identified by FAST DiI™ labeling showed an increased cell size compared with those from nonhandled (NH) rats. Whole cell current-clamp recordings showed that colonic DRG neurons from NMS rats displayed: 1) depolarized resting membrane potential; 2) increased input resistance; 3) a dramatic reduction in rheobase; and 4) a significant increase in the number of action potentials evoked at twice rheobase. Whole cell voltage-clamp recordings revealed that neurons from both groups exhibited transient A-type (IA) and delayed rectifier (IK) K+ currents. Compared with NH rat neurons, the averaged density of IK was significantly reduced in NMS rat neurons. Furthermore, the Kv1.2 expression was significantly decreased in NMS rat colonic DRG neurons. These results suggest that NMS increases the excitability of colonic DRG neurons mainly by suppressing the IK current, which is likely accounted for by the downregulation of the Kv1.2 expression and somal hypertrophy. PerspectiveThis study demonstrates the alteration of delayed rectifier K current and Kv1.2 expression in DRG neurons from IBS model rats, representing a molecular mechanism underlying visceral pain and sensitization in IBS, suggesting the potential of Kv1.2 as a therapeutic target for the treatment of IBS.

Establishment of irritable bowel syndrome rat model by combination of intestinal infection with Trichinella spiralis and acute stress

To establish the irritable bowel syndrome (IBS) rat model by the combination of acute stress and transient intestinal infection with Trichinella spiralis (T.S.). The rat model of acute cold restraint stress post-infection (PI + ACRS) was established as following: the intestinal infection with 1500 T.S. in 1 ml saline to adult male BN rats was performed at Day 0 by gastric lavage. Then a 2-hour stimulus of ACRS was administered at Day 100. Age matched transiently infected without stress rats (PI) and normal rats served as controls (CON) (n = 6, for each). After anesthesia, all the rats underwent colonic manometry in vivo at Day 100. The colonic pressures at 3 different states (baseline for 20 min; 1 ml balloon distension stimulation for 5 min and 2 ml balloon distension stimulation for 20 min) were traced with a 5-minute interval between each two. The following parameters were recorded: (1) Duration (Dur.): total time of contractions during each state. (2) Maximum (Max.): highest amplitude of constructional waves (mm Hg). (3) Area: area under contraction waves. (4) Number (Num.): frequencies of contraction wave during each state. The visceromoter response to colorectal distension (CRD) was analyzed at Day 100 post-infection. And the distension volume of AWR 3 was detected for 5 times with a 20-min interval in each rat. The histological damage of intestine induced by T.S. infection is transient. Although such acute infectious features as epithelial edema, hyperemia and marked eosinophil infiltration appeared at Day 10 PI, the histological changes almost recovered at Day 100 PI in both the PI group and the PI + ACRS group. Both the stimuli of transient infection and the ACRS post-infection induced intestinal dysmotility and visceral hypersensitivity. The ACRS post infection further worsened the transiently induced infection. The parameters of Num, Max and Area in the PI + ACRS group were all significantly higher than those of the PI group [Max: (41 +/- 17) mm Hg vs (22 +/- 6) mm Hg, P = 0.000; Area: (7693 +/- 2822) mm Hg.s vs (5092 +/- 1687) mm Hgxs, P = 0.000; Num: 9.5 +/- 2.6 vs 6.6 +/- 3.1, P = 0.000]; so was the distension volume of AWR3 [(2.25 +/- 0.29) ml vs (2.52 +/- 0.32) ml, P = 0.004]. As compared with the range of normal values from controls, the abnormality rates of motility parameters and visceral threshold in PI + ACRS group also had an larger increment than those of the PI group (PI + ACRS: 50.0% - 87.5% and 100% respectively, PI: 25.0% - 37.5% and 90.0% respectively). The pathophysiological changes in the PI + ACRS rats are consistent with those of IBS. Aggravated by psychological factors, these rats reproduce the symptoms of intestinal dysmotility and visceral hypersensitivity. A proper animal model has been established for the investigation of IBS.

Effect of electro-acupuncture on substance P, its receptorand corticotropin-releasing hormone in rats with irritablebowel syndrome

To investigate the effect and mechanism of electro-acupuncture (EA) at ST25 and ST37 on irritable bowel syndrome (IBS) of rats. A total of 21 male Sprague-Dawley rats were randomly divided into normal group, model group and EA group. A rat model of IBS was established by constraining the limbs and distending the colorectum of rats. Rats in EA group received bilateral EA at ST25 and ST37 with a sparse and intense waveform at a frequency of 2/50 Hz for 15 min, once a day for 7 d as a course. Rats in normal and model groups were stimulated by distending colorectum (CR). An abdominal withdrawal reflex (AWR) scoring system was used to evaluate improvements in visceral hypersensitivity. Toluidine blue-improved method, immunohistochemistry and radioimmunoassay were used to observe mucosal mast cells (MC), changes of substance P (SP) and substance P receptor (SPR) in colon and change of corticotropin-releasing hormone (CRH) in hypothalamus. The threshold of visceral sense was significantly lower in model group than in normal group, and significantly higher in EA group than in model group. The number of mucosal MC was greater in model group than in normal group and significantly smaller in EA group than in model group. The CRH level in hypothalamus of rats was significantly higher in model group than in normal group, which was remarkably decreased after electro-acupuncture treatment. The SP and SPR expression in colon of rats in model group was decreased after electro-acupuncture treatment. EA at ST25 and ST37 can decrease the number of mucosal MC and down-regulate the expression of CRH in hypothalamus, and the expression of SP and SPR in colon of rats with IBS.

RETRACTED: Repeated electro-acupuncture attenuates chronic visceral hypersensitivity and spinal cord NMDA receptor phosphorylation in a rat irritable bowel syndrome model

Acupuncture has been used in clinical trials for the treatment of abdominal pain in patients with irritable bowel syndrome (IBS). However, scientific evidence is still lacking and the underlying mechanism remains largely unexplored. The aim of this study was to examine the effects of repeated administration of electro-acupuncture (EA) on chronic visceral hypersensitivity and on the phosphorylation of spinal cord N-methyl-D-aspartic acid (NMDA) receptors in a rat model of IBS. The results showed that repeated administration of EA at bilateral points of Zu-san-li (ST-36) and Shang-ju-xu (ST-37) significantly attenuated chronic visceral hypersensitivity induced in young adult rats by neonatal colon irritation. Such an effect was not seen in either of the two controls: sham-EA at ST-36 and ST-37 without electrical stimulation and EA at control points (BL-62 and tail). Furthermore, rats with chronic visceral hypersensitivity exhibited high-level expression of phosphorylated NMDA receptor subunit 1 (pNR1) in the spinal cord (L4-L5 segments), which was markedly attenuated by EA treatment. In addition, EA at ST-36 and ST-37 neither altered the pain threshold of normal rats nor affected the expression of pNR1 in the lumbosacral spinal cord. Altogether, these data indicate that the EA-mediated attenuation of chronic visceral hypersensitivity is correlated with the down-regulation of NMDA receptors phosphorylation at the spinal level.