Abdominal Withdrawal Reflex Scores Research Articles

Changes in intestinal barrier protein expression and intestinal flora in a rat model of visceral hypersensitivity.

Destruction of the intestinal mucosal barrier and visceral hypersensitivity are main pathogenesis of irritable bowel syndrome (IBS). The study aimed to establish a rat model of visceral hypersensitivity and explore mechanisms involved the changes of the intestinal barrier protein expression and intestinal flora. A rat model of visceral hypersensitivity was established and evaluated using abdominal withdrawal reflex (AWR) scores, colonic paracellular permeability, and gastrointestinal motility. The expression of tight junction proteins, aquaporin proteins (AQPs), phosphorylated ERK, and proteinase-activated receptor-2 (PAR-2) was determined. The intestinal microflora was evaluated by high-throughput sequencing of the 16S rRNA gene. In model rats, AWR score and fecal water content were significantly increased, gastrointestinal motilities were disorder and characterized by an inhibition of gastric motility and an enhancement of small intestinal and colonic movement. The expressions of colonic occludin, ZO-1, AQP3, and AQP8 were decreased but claudin-2 and claudin-4 were markedly increased. Imbalance of intestinal flora appeared and showed an obvious decrease of Lactobacillus and an increase of Clostridiales_bacterium. Additionally, the total serine protease activity in feces, the expressions of PAR2 and phosphorylated ERK in the colon tissues were increased significantly. The model rats of visceral hypersensitivity possess the decreased expression of occludin, ZO-1, AQP3, AQP8, and the increased expression of claudin-2 and claudin-4, meanwhile develop an imbalance of intestinal flora which probably increase serine protease activity, thereby activating the PAR2/ERK signaling and causing the intestinal barrier disorder.

Manual Acupuncture at ST37 Modulates TRPV1 in Rats with Acute Visceral Hyperalgesia via Phosphatidylinositol 3-Kinase/Akt Pathway.

Acupuncture can significantly ameliorate inflammatory pain in acute visceral hyperalgesia. Hyperalgesia is attenuated by inflammatory mediators that activate transient receptor potential vanilloid 1 (TRPV1), and TRPV1 is regulated by nerve growth factor (NGF)-induced phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, it is unknown whether NGF-induced PI3K/Akt pathway is associated with manual acupuncture (MA). In this study, the effect and mechanism of MA at Shangjuxu (ST37) and Quchi (LI11) were examined using an acetic acid-induced rat model with visceral hyperalgesia. We demonstrated that MA at ST37 significantly decreased abdominal withdrawal reflex (AWR) scores, proinflammatory cytokine expression (TNF-α, IL-1β, and IL-6), and TRPV1 protein and mRNA expression in rats with acute visceral hyperalgesia compared with the untreated controls, while MA at LI11 showed no effect. The effects of MA at ST37 were reversed after treatment with the PI3K agonist IGF-1 30 min before MA. In rats with visceral hyperalgesia, the upregulation of NGF, tropomyosin-receptor-kinase A (TrkA), PI3K, and phosphorylation-Akt (p-Akt) was decreased by MA at ST37, indicating that TRPV1 regulation via the NGF-induced PI3K/Akt pathway plays a vital role in the effects of MA-mediated amelioration of acute visceral hyperalgesia.

Electroacupuncture and Moxibustion Modulate the BDNF and TrkB Expression in the Colon and Dorsal Root Ganglia of IBS Rats with Visceral Hypersensitivity.

Objective To evaluate the effects of electroacupuncture and moxibustion on brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase receptor B (TrkB) protein and mRNA expressions in the colon and dorsal root ganglia of IBS rats with visceral hypersensitivity and to explore their underlying therapeutic mechanisms. Method Forty Sprague Dawley rats were randomly divided into normal, model, model + mild moxibustion (MM), model + electroacupuncture (EA), and model + pinaverium bromide (PB) groups, with eight rats in each group. Chronic visceral hypersensitive IBS rat models were established by colorectal distension (CRD) with mustard oil clyster. Rats in the MM and EA groups, respectively, received moxibustion and electroacupuncture treatments on the Tianshu (ST25) and Shangjuxu (ST37) acupoints once daily for 7 days, and rats in the PB group received pinaverium bromide by oral gavage once daily for 7 consecutive days. After treatment, rats underwent abdominal withdrawal reflex (AWR) scoring under CRD and colon histopathological examination. Immunohistochemistry and real-time quantitative PCR (RT-qPCR) were used to study the protein and mRNA expressions of BDNF and TrkB in the rat colon and dorsal root ganglia. Results Compared with the normal group, AWR scores and body weight were clearly increased in the model group rats (both P < 0.01). The body weights were significantly elevated (P < 0.01, P < 0.05), but the AWR scores were reduced (P < 0.05, P < 0.01), after electroacupuncture and mild moxibustion treatment. Compared with levels in normal rats, BDNF and TrkB protein and mRNA expressions were significantly elevated in the IBS model rats (P < 0.01) but were downregulated after mild moxibustion, electroacupuncture, and Western medicine treatment (P < 0.01). Conclusion Electroacupuncture and moxibustion improved visceral hypersensitivity of IBS rats possibly by reducing BDNF and TrkB protein and mRNA expressions in the colon and dorsal root ganglia.

Effects of transcutaneous auricular vagus nerve stimulation on autonomic nervous function in rats with functional dyspepsia

To observe the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on the autonomic nerve function in a rat model of functional dyspepsia (FD), so as to explore the mechanism of taVNS underlying regulation of FD. SD rats were randomly divided into normal control group(n=8) and FD model group(n=26).The FD model was replicated with iodoacetamide gavage. The FD model rats were randomly divided into model, taVNS, sham-taVNS and Zusanli(ST36) groups, with 6 rats in each group. Rats in the taVNS group received electrical stimulation of auricular concha,while the sham-taVNS group received no electrical stimulation and rats in the ST36 group received stimulation at ST36 for 30 min once daily for 14 consecutive days. Cervical trapezius electromyography score and abdominal withdrawal reflex (AWR) score were used to evaluate gastric sensitivity. Histopathological changes of the gastric antrum tissue were observed under microscope after H.E. staining. Autonomic nerve function in rats was recorded and assessed by heart rate variability(HRV). The content of acetylcholine (Ach) and the expression of Ach receptor M3R in gastric antrum was detect by ELISA and Western blot, separately. Compared with the normal control group, the cervical trapezius electromyography and AWR scores of the model group increased (P<0.01, P<0.001), and there was no erosion in the gastric antral mucosa and muscle layer. The high-frequency power (HF) in HRV decreased (P<0.05), the ratio of low-frequency power/high-frequency power (LF/HF) increased (P<0.001), and the Ach content and its receptor M3R expression in gastric antrum tissue decreased (P<0.05). Following interventions, the cervical trapezius electromyography and AWR scores decreased (P<0.01,P<0.001, P<0.05), HF in HRV increased and LF/HF decreased(P<0.01，P<0.05,P<0.001), and the content of Ach in gastric antrum tissue and the expression of its receptor M3R increased (P<0.01, P<0.05) in both taVNS and ST36 groups relevant to the model group. taVNS can increase the activity of the vagus nerve and regulate the balance of the autonomic nerve function, which may be one of the mechanisms of taVNS in reducing the gastric sensitivity of rats with FD. In regulating the vagus nerve function, taVNS and acupuncture at ST36 acupoint have the similar effects.

Optimized Tibial Nerve Stimulation Partially Reduces Visceral Hypersensitivity in Rats Mediated via Autonomic and Opioid Mechanisms

The aim of this study was to investigate the therapeutic effect and possible mechanisms of tibial nerve stimulation (TNS) on visceral hypersensitivity in rats. 1) The effects of TNS with five sets of parameters on visceral sensitivity in normal rats were evaluated by the assessment of abdominal electromyogram (EMG) and abdominal withdrawal reflex (AWR). 2) The effects and mechanisms of TNS with a special set of parameters (14Hz, 330 μsec, and 40% motor threshold) were evaluated in acute visceral hypersensitivity rats induced by restraint stress and colonic hypersensitized rats induced by acetic acid during the neonatal stage assessed by the EMG, AWR, and the spectral analysis of heart rate variability derived from the electrocardiogram. 1) In normal rats, TNS did not show any effect on the visceromotor reflex. 2) In rats with restraint stress-induced hypersensitivity, TNS with the special set of parameters reduced AWR scores and EMG responses to rectal distention at a pressure of 20-60 mmHg (p < 0.05, vs. baseline for both AWR and EMG). Concurrently, TNS increased vagal activity and decreased sympathetic activity (p < 0.03 for both). 3) Similar effects were noted on the EMG (p < 0.05, vs. baseline) and AWR (p < 0.05 vs. baseline) with acute and chronic TNS in rats with chronic colonic hypersensitivity and the effects were blocked by naloxone. TNS with parameters of 14Hz, 330 μsec, and 40% motor threshold is effective in improving visceral hypersensitivity in rodent models of colonic hypersensitivity via the modulation of autonomic and opioid mechanisms.

Clostridium butyricum relieve the visceral hypersensitivity in mice induced by Citrobacter rodentium infection with chronic stress.

BackgroundVisceral hypersensitivity is a common symptom in patients with post-infectious irritable bowel syndrome (PI-IBS), and change of the microbiota is a vital etiological factor of it. Clostridium butyricum (C. butyricum) is one of the probiotics which is reported as the active components in the treatment of IBS, especially IBS with diarrhea. Citrobacter rodentium (C. rodentium) is an enteropathogenic bacteria which can produce self-limiting colitis in mice, which have been used to produce a PI-IBS-like mice model. Whether C. butyricum could influence the visceral hypersensitivity and gut microbiota of PI-IBS is still unknown. Our study aimed to examine whether the intervention of C. butyricum or antibiotics could affect the etiology of visceral hypersensitivity.MethodsC57BL/6 male mice were gavaged with the C. rodentium to induce a infective colitis. The C. butyricum and antibiotic compound were used to intervene the infected mice 3 days later. A 9-day chronic water avoidance stress (WAS) process was implemented to help induce the visceral hypersensitivity. The abdominal withdrawal reflex (AWR) score was assayed to indicate the visceral hypersensitivity of different groups. On the 7th, 14th, and 30th days after infection, mice feces were collected and high-throughput sequencing was carried out to analyze their gut microbiota.ResultsCombined, the C. rodentium infection plus chronic stress (WAS) could induce the visceral hypersensitivity in mice. Treatment of the C. butyricum after C. rodentium infection could relieve visceral hypersensitivity of mice, while no difference was observed in the antibiotic treatment group. The gut microbiota diversity of C. rodentium infected mice was similar to the uninfected mice, while there were different microbial communities structure between them. The Shannon and Chao indexes significantly decreased in the antibiotic treatment group compared to other groups at 7th, 14th, and 30th days post-infection, while treatment of C. butyricum could maintain the indexes within normal range. At day 14 after infection, the structure of microbiota headed towards normality after the C. butyricum treatment. After the WAS, the Shannon and Chao indexes of the control group decreased and the structure of microbiota changed. The C. butyricum treatment could prevent these changes of the gut microbiota induced by WAS.ConclusionC. butyricum could relieve the visceral hypersensitivity in mice induced by C. rodentium infection plus chronic stress. It could also remodel the microbiota change caused by the infection and chronic stress. It may be a more effective treatment strategy for PI-IBS than antibiotics.

Tongxie Anchang Decoction Relieves Visceral Hypersensitivity in Diarrhea-Predominant Irritable Bowel Syndrome Rats by Regulating the NGF/TrkA Signaling Pathway.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disease characterized by visceral hypersensitivity-related abdominal pain, in which diarrhea-predominant IBS (IBS-D) is the main subtype and has a high clinical incidence. Tongxie Anchang Decoction (TXACD) has been proved to significantly improve abdominal pain in patients with IBS-D, but its underlying therapeutic mechanism still remains unclear. In the present study, IBS-D model rats were induced by neonatal maternal separation (NMS) combined with restraint stress (RS). The therapeutic effect of TXACD was evaluated by fecal characteristics and abdominal withdrawal reflex (AWR) scores. After 14 days of intragastric administration, the colonic tissues of rats were collected to detect the protein and gene level of the NGF, TrkA, and TRPV1 using Western blotting and real-time polymerase chain reaction, respectively, and detect mast cells infiltration using toluidine blue staining. The abdominal aorta blood centrifuged was collected for detecting serum levels of SP, 5-HT, and CGRP with ELISA. The results revealed that TXACD could significantly improve visceral hypersensitivity in IBS-D rats, reflected in the decrease of AWR score and the serum levels of SP, 5-HT, and CGRP. In addition, TXACD treatment could alleviate mast cells infiltration. Moreover, the expression levels of the NGF, TrkA, and TRPV1 were repressed by TXACD. The findings of the present study indicated that the therapeutic effect of TXACD on visceral hypersensitivity might be closely related to the downregulation of the NGF/TrkA signaling pathway, the reversal of TRPV1 expression and mast cells infiltration, and the decreased release of neuroendocrine factors SP, 5-HT, and CGRP.

Beneficial effect of Bletilla striata extract solution on zymosan-induced interstitial cystitis in rat.

Interstitial cystitis (IC) is a chronic pain syndrome that is characterized by suprapubic pain upon bladder filling. Bletilla striata, a well-known traditional Chinese herb with established efficacy in wound healing and anti-inflammation, was hypothesized to improve the symptoms of IC possibly though forming a physical barrier that could isolate the bladder tissue from irritants. This study was conducted to evaluate the beneficial effects of intravesical treatment with B. striata extract solution (BSES) on visceral pain and bladder function of rats with zymosan-induced IC. Thirty female rats were randomly divided into control group, zymosan-induced cystitis rats treated with normal saline (Z + NS), and zymosan-induced cystitis rats treated with BSES (Z + BSES). All rats underwent evaluation for abdominal withdrawal reflex (AWR) scores to assess visceral hypersensitivity, cystometrography, and electromyogram (EMG) of both external urethral sphincter and bladder detrusor. Data were analyzed by one way analysis of variance. The Z + NS group had an increased visceral hypersensitivity as compared to control group. Rats treated with BSES (Z + BSES group) had decreased AWR scores and amplitude of bladder detrusor-EMG. Besides, BSES treatment improved overactive bladder with significant effects on the extend of micturition interval and increase of storage of urine. Intravesical instillation of BSES can significantly alleviate zymosan-induced visceral hypersensitivity and bladder overactivity associated with IC. This study suggested that intravesical instillation with BSES might be a promising treatment for IC.

Effects of Dexmedetomidine on Rats with Functional Chronic Visceral Pain Based on Protein Expressions in the Extracellular Signal-Regulated Kinase 1/Cyclic Adenosine Monophosphate Response Element-Binding Protein Signaling Pathway

To evaluate the protective effects of dexmedetomidine on the colon and spinal cord of rats with functional chronic visceral pain and its regulatory mechanism on extracellular signal-regulated kinase 1-cyclic adenosine monophosphate response element-binding protein signaling pathway. A rat model of irritable bowel syndrome was established by colorectal distention stimulation. Animals were categorized into normal group, model group, experimental group and control group. No treatment was given in normal group, while colorectal distention stimulation was utilized in model group, experimental group and control group. After successful modeling, the animals in experimental group were injected intraperitoneally with 5 μg/kg of dexmedetomidine hydrochloride injection daily, those in control group were injected intraperitoneally with 5 μg/kg pinaverium bromide daily and those in normal group and model group were injected intraperitoneally daily with the same volume of normal saline, for 14 consecutive days. Abdominal withdrawal reflex score and pain threshold of rats were measured. Cell apoptosis in the spinal cord of rats was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. The messenger ribonucleic acid expressions of extracellular signal-regulated kinase 1 and cyclic adenosine monophosphate response element-binding protein in the spinal cord were detected by realtime quantitative polymerase chain reaction and phosphorylated-extracellular signal-regulated kinase 1 and phosphorylated-cyclic adenosine monophosphate response element-binding protein expressions in the spinal cord of rats were tested by Western blotting. Compared with normal group, abdominal withdrawal reflex score, cell apoptosis rate, messenger ribonucleic acid expressions of extracellular signal-regulated kinase 1 and cyclic adenosine monophosphate response element-binding protein, phosphorylatedextracellular signal-regulated kinase 1 and phosphorylated-cyclic adenosine monophosphate response element-binding protein expressions were significantly higher (p<0.05) and pain threshold value was significantly lower (p<0.05) in model group, experimental group and control group at 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg pressures. Abdominal withdrawal reflex score, cell apoptosis rate, messenger ribonucleic acid expressions of extracellular signal-regulated kinase 1 and cyclic adenosine monophosphate response element-binding protein, phosphorylated-extracellular signal-regulated kinase 1 and phosphorylated-cyclic adenosine monophosphate response element-binding protein expressions were significantly lower (p<0.05) and pain threshold value was significantly higher (p<0.05) in experimental group and control group than those in model group at 20 mmHg, 40 mmHg, 60 mmHg and 80 mmHg pressures. Dexmedetomidine can prominently ameliorate the clinical symptoms of rats with functional chronic visceral pain and may protect the colon and spinal cord by inhibiting the extracellular signalregulated kinase 1/cyclic adenosine monophosphate response element-binding protein signaling pathway.

The Role of Intestinal Microbiota and Mast Cell in a Rat Model of Visceral Hypersensitivity.

Background/AimsTo explore the role of intestinal flora and mast cells in visceral hypersensitivity (VH).MethodsThe experimental animals were divided into 4 groups control group, VH group, VH + VSL#3 group, and VH + ketotifen group. Stool samples were collected from each group (n = 3) for a further analysis using 16S ribosomal DNA gene sequence. Visceral sensitivity was evaluated by abdominal withdrawal reflex (AWR) score. Colon tissues of rats were obtained from each group. Mast cells were detected by toluidine blue staining. The degranulation of mast cells was assessed by transmission electron microscopy.ResultsVH rat model could successfully be induced by acetic acid enema combined with partial limb restraint method. Compared with rats in the control group, AWR score, number of mast cells, and degranulation of mast cells were increased in the VH rats, which could be reduced by administration of ketotifen or probiotic VSL#3. Clostridium sensu stricto 1 abundance was higher in the VH group compared to the control group, which could be restored by application of probiotic VSL#3.ConclusionsProbiotic VSL#3 decreases visceral sensitivity in VH rats. The mechanism may be related to mast cell and intestinal flora. Change of Clostridium sensu stricto 1 abundance may be a basis for VH observed in irritable bowel syndrome and may be prevented by specific probiotic administration.

Ginger relieves intestinal hypersensitivity of diarrhea predominant irritable bowel syndrome by inhibiting proinflammatory reaction

BackgroundGinger or ginger extracts have been used in traditional medicine relieve pain caused by diarrhea predominant irritable bowel syndrome (IBS-D), but few data exists about its effectiveness. This present study was to validate the effect of ginger on visceral pain, and to further explore the possible underlying mechanism by which ginger is used to relieve IBS-D intestinal hypersensitivity.MethodsFirst, the IBS-D rat model was established by chemical stimulation and acute and chronic pressure stimulation. Then, different dose of ginger were administrated to IBS-D rats and evaluate the defecation frequency, fecal water content (FWC) and abdominal withdrawal reflex (AWR) scores in IBS-D rats. Further, the IBS-D rats were sacrificed to collecte the colonic tissues to evaluate the effect of ginger administration on its pathology and changes of pro-inflammatory factors, and changes of NF-κB pathway. Second, the ginger was taken to HPLC analysis and 6-gingerol was choosen to further experiment. Then, IBS-D rats were treated with different dose of 6-gingerol, and the behavioral evaluation were to evaluate the effect of 6-gingerol on IBS-D rats. Further, colonic epithelial cells (CECs) were collectted and to evaluate the effect of 6-gingerol on the expression of inflammatory factors and changes of NF-κB pathway.ResultsThe IBS-D rat model was successfully established by chemical stimulation and acute and chronic pressure stimulation. And ginger treatment significantly reduced the defecation frequency, fecal water content and AWR scores in IBS-D rats. Histopathological analysis showed that ginger treatment can significantly reduce colonic edema and promote the recovery of inflammation in IBS-D rats, and the effect is equivalent to rifaximin. Elisa and RT-qPCR showed that ginger inhibited the expression of proinflammatory factors (TNF-α, IL-6, iNOS) in IBS-D rats. Western blot showed IkBα was up-regulated while p-p65 was inhibited under ginger treatment. HPLC analysis showed that 6-gingerol was the main component of ginger, which could improve clinical symptoms in IBS-D rats. Western blot and RT-qPCR showed that 6-gingerol inhibited the expression of proinflammatory factors (TNF-α, IL-6, iNOS) in CECs, and inhibition of IκBα degradation and phosphorylation of p65 involved in NF-κB pathway.ConclusionGinger and ginger extract could relieve intestinal hypersensitivity of IBS-D by inhibiting proinflammatory response.

Effect of water extracts from Cynanchum thesioides (Freyn) K. Schum. on visceral hypersensitivity and gut microbiota profile in maternally separated rats

Ethnopharmacological relevanceIrritable bowel syndrome (IBS) is a chronic, stress-related, functional gastrointestinal disorder characterized by abdominal discomfort and altered bowel habits; the manipulation of the microbiota is emerging as a promising therapeutic option for IBS. Cynanchum thesioides (CT) is an herb of traditional Mongolian medicine that has been employed in treating abdominal pain and diarrhea for hundreds of years. Phytochemical studies of this plant showed the presence of various flavonoids with antibacterial and anti-inflammatory activities. We hypothesized that Cynanchum thesioides manipulates the gut mycobiome and reverses visceral hypersensitivity in IBS rat model. Purpose of the studyThe aims of this study were to prove the in vivo efficacy of Cynanchum thesioides on improving visceral hypersensitivity in IBS rat model and to examine its effect on gut bacterial communities, focusing on the potential interrelationships among microbiota and visceral hypersensitivity. Materials and methodsWe induced visceral hypersensitivity rat models by maternal separation (MS) of Sprague-Dawley rats, and administered CT water extracts to MS rats for 10 consecutive days. The abdominal withdrawal reflex score and threshold of colorectal distention were employed to assess visceral sensitivity. We then used the Illumina HiSeq platform to analyze bacterial 16S rRNA gene. ResultsTreatment with CT improved visceral hypersensitivity in MS rats, and this was accompanied by alterations in the structure and composition of the gut microbiota. The extent of the stability of the gut microbiota was improved after treatment with CT. The genera Pseudomonas, Lachnospiracea_incertae_sedis, and Clostridium XlVa (which were more prevalent in MS rats) were significantly decreased, whereas the abundance of some genera were less prevalent in MS rats—for example, Clostridium IV, Elusimicrobium, Clostridium_sensu_stricto, and Acetatifactor were significantly enriched after treatment with CT. ConclusionWater-extracted CT was beneficial against visceral hypersensitivity in IBS and favorably affected the structure, composition, and functionality of gut microbiota. CT is therefore a promising agent in therapy of IBS.

Protective effect of microinjection of glutamate into hypothalamus paraventricular nucleus on chronic visceral hypersensitivity in rats

Chronic visceral hypersensitivity (CVH) is a major pathophysiological feature of patients experiencing in irritable bowel syndrome (IBS) and other disorders with visceral pain. However, little is known about its regulation of the central nucleus. In this research, we investigated the protective effect of microinjection of glutamate into hypothalamus paraventricular nucleus (PVN) on CVH and its possible regulatory mechanism in rats. Visceral sensitivity was assessed by pain threshold, abdominal withdrawal reflex (AWR) score, and the abdominal external oblique muscle electromyography (EMG) amplitude. Pathological changes in colorectal mucosa were assessed using immunohistochemical, biochemical analysis and Western blot. Results showed that microinjection of different doses of glutamate into PVN reduced the visceral sensitivity in a dose-dependent manner. This effect can be reversed after chemical ablation of PVN or nucleus tractus solitarius (NTS) or pretreatment with the arginine vasopressin (AVP)-V1 receptor antagonist ([Deamino-pen1,val4,D-Arg8]-vasopressin) DPVDAV into NTS. The vagus discharge frequency was significantly reduced after the glutamate microinjection into PVN. Additionally, oxidation, proliferation and apoptosis in colorectal mucosa were related to the CVH regulations. These findings suggested that PVN and NTS are involved in the regulatory process of CVH and exert the protective effect on CVH, providing new ideas and therapeutic targets for CVH research.

LncRNA XIST modulates 5-hydroxytrytophan-induced visceral hypersensitivity by epigenetic silencing of the SERT gene in mice with diarrhea-predominant IBS

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a prevalent gastrointestinal disorder with a high incidence in children. The role of long non-coding RNAs (lncRNAs) in gastrointestinal diseases has been previously highlighted. Nevertheless, the underlying regulatory mechanism of lncRNA X inactivate-specific transcript (XIST) in IBS-D requires further studies. Thus, the present study was conducted with the main objective of elucidating the underlying mechanism of lncRNA XIST in visceral hypersensitivity in IBS-D. An in vivo mouse model of IBS-D was constructed via rectal perfusion of acetic acid. Next, in order to evaluate the effect of lncRNA XIST on the development of visceral hypersensitivity in IBS-D, different vector plasmids were injected into mice along with rectal mucosal epithelial cells, followed by the measurement of abdominal withdrawal reflex (AWR) score, counts of peristaltic wave, abdominal wall contraction and defecation particles. Furthermore, luciferase reporter assay, FISH, RIP and ChIP assays were conducted to determine the interactions between lncRNA XIST and SERT. Subsequently, MS-PCR was adopted to test the methylation level of SERT promoter. 5-hydroxytrytophan (HT) content in rectal tissues was detected using immunohistochemistry. The IBS-D mouse models presented with a high expression of lncRNA XIST along with low expression of SERT. LncRNA XIST was observed to recruit methylase DNMT1, DNMT3A and DNMT3B to promote SERT promoter methylation, reducing its expression. Restoration of lncRNA XIST resulted in increased AWR score, counts of peristaltic wave, abdominal wall contraction and defecation particles along with stimulated 5-HT expression and SERT methylation level, while downregulation of lncRNA XIST reversed these effects. In conclusion, the key findings from our study indicated that lncRNA XIST acts as a regulator in 5-HT-induced visceral hypersensitivity in mice with IBS-D, providing a new insight into the regulatory effect of lncRNA XIST and its epigenetic diagnostic and therapeutic properties in IBS-D.

Electroacupuncture Inhibits the Activity of Astrocytes in Spinal Cord in Rats with Visceral Hypersensitivity by Inhibiting P2Y1 Receptor-Mediated MAPK/ERK Signaling Pathway.

Background Irritable bowel syndrome (IBS) is a chronic functional bowel disease characterized by abdominal pain and changes in bowel habits in the absence of organic disease. Electroacupuncture (EA) has been shown to alleviate visceral hypersensitivity (VH) in IBS rat models by inhibiting the activation of astrocytes in the spinal cord. However, the underlying molecular mechanisms mediated by P2Y1 receptor of this effect of electroacupuncture remain unclear. Aim To explore whether EA inhibits the activity of astrocytes in the spinal cord dorsal horn of rat with visceral hypersensitivity by inhibiting P2Y1 receptor and its downstream mitogen activated protein kinase/extracellular regulated kinase 1 (MAPK/ERK) pathway. Methods Ten-day-old Sprague-Dawley (SD) male rats were given an intracolonic injection of 0.2 ml of 0.5% acetic acid (AA) to establish a visceral hypersensitivity model. EA was performed at Zusanli (ST 36) and Shangjuxu (ST 37) at 100 Hz for 1.05 s and 2 Hz for 2.85 s alternately, pulse width for 0.1 ms, 1 mA, 30 min/d, once a day, for 1 week. Cytokines IL-6, IL-1β, and TNF-α were analyzed by ELISA. The expressions of the P2Y1 receptor and pERK1/2 were analyzed by Western Blot and real-time PCR in the model and EA treated animals to explore the molecular mechanism of EA in inhibiting the activity of spinal cord dorsal horn (L6-S2 segment) astrocytes in rats with IBS visceral hypersensitivity. Results EA significantly reduced the behavioral abdominal withdrawal reflex score (AWRs) of IBS rats with visceral hypersensitivity induced by AA. For comparison, intrathecal injection of astrocytes activity inhibitor fluorocitrate (FCA) also reduced visceral hypersensitivity in IBS rats. EA at Zusanli and Shangjuxu inhibited the mRNA and protein expression of the glial fibrillary acidic protein (GFAP) and in rat spinal cord and reduced the release of inflammatory cytokines IL-6, IL-1, and TNF-α were analyzed by ELISA. The expressions of the P2Y1 receptor and pERK1/2 were analyzed by Western Blot and real-time PCR in the model and EA treated animals to explore the molecular mechanism of EA in inhibiting the activity of spinal cord dorsal horn (L6-S2 segment) astrocytes in rats with IBS visceral hypersensitivity. β, and TNF-μg, 10 μg, 10 Conclusion EA inhibited astrocyte activity in the spinal cord dorsal horn of rat with IBS visceral hypersensitivity by inhibiting the P2Y1 receptor and its downstream, PKC, and MAPK/ERK1/2 pathways.

Electroacupuncture Improves IBS Visceral Hypersensitivity by Inhibiting the Activation of Astrocytes in the Medial Thalamus and Anterior Cingulate Cortex

Objective To explore whether the effect of electroacupuncture (EA) on visceral hypersensitivity (VH) in rats with irritable bowel syndrome (IBS) is related to the changes of astrocyte activation in the medial thalamus (MT) and anterior cingulate cortex (ACC). Method Male Sprague-Dawley rats were randomly divided into the normal control (NC) group, model control (MC) group, electroacupuncture (EA) group, and fluorocitrate (FCA) group. A model of visceral hypersensitivity was established by neonatal colorectal irritation. In the EA group, needles were inserted into the skin at the Tianshu (ST25) and Shangjuxu (ST37) acupoints, once a day for 7 days. The FCA group received intrathecal injection of FCA on the 1st, 4th, and 7th days. Visceral hypersensitivity was evaluated by the abdominal withdrawal reflex (AWR), and glial fibrillary acidic protein (GFAP) mRNA and protein levels in the MT and ACC were detected by real-time PCR, immunohistochemistry, and western blots. Results The AWR score in the MC group was significantly higher than in the NC group, and EA and FCA reduced the AWR score of VH rats. GFAP mRNA and protein levels in the MT and ACC of rats in the MC group were significantly increased compared with the NC group. After either electroacupuncture or fluorocitrate, GFAP mRNA and protein levels in the MT and ACC were both clearly reduced. Conclusion Electroacupuncture alleviates IBS visceral hypersensitivity by inhibiting the activation of astrocytes in the MT and ACC.

Electroacupuncture Relieves Irritable Bowel Syndrome by Regulating IL-18 and Gut Microbial Dysbiosis in a Trinitrobenzene Sulfonic Acid-Induced Post-Inflammatory Animal Model.

Post inflammatory irritable bowel syndrome (PI-IBS), a subset of IBS, is characterized by symptoms of visceral pain, bloating, and changed bowel habits that occur post initial episode of intestinal infection. Gut microbial dysbiosis or inflammation plays a key role in the pathogenesis of abdominal hypersensitivity of PI-IBS. Electroacupuncture (EA) stimulation results in an alleviated PI-IBS-associated symptom. This study investigated the effect of EA on IL-18 and gut microbial dysbiosis in one visceral hypersensitive rat models with PI-IBS. A trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity rat model was developed. EA stimulation was applied to the ST25 and ST36 acupoints. Animals were assessed using abdominal withdrawal reflex (AWR) scores to determine the development of colonic visceral hypersensitivity. The 16S rRNA was used to correlate microbial diversity. IL-18 expression in colon was quantified by quantitative real-time PCR and western blotting. We identified that model rats had an increased visceral hypersensitivity to colorectal distention at different distention pressures compared with the normal group. Sensitivity to colorectal distention decreased after EA stimulation. The composition of the fecal microbiota was different between groups. Specifically, in the model group Empedobacter, Psychrobacter, Enterococcus, Butyricimonas, Vampirovibrio, Kurthia, Intestinimonas, Neisseria, Falsiporphyromonas, Bilophila, Fusobacterium, Alistipes, Veillonella, Flavonifractor, Clostridium XlVa weremore abundantaffected genera, whereas Lactobacillus was enriched in normal rats. EA stimulation was correlated with significant decrease in the phyla of Fusobacteria. The mRNA and protein levels of IL-18 were higher in the model group. Meanwhile, EA stimulation attenuated this response. In a word, our findings suggest that PI-IBS is associated with significant increase in IL-18 levels as well as an alteration in microbiome diversity. These changes can be reversed with EA treatment. EA stimulation has a positive effect in alleviating symptoms of visceral hypersensitivity and protecting the gastrointestinal tract.

Effects of Deoxyschisandrin on Visceral Sensitivity of Mice with Inflammatory Bowel Disease.

The aims of this study were to build an IBD mouse model and further to observe the effects of deoxyschisandrin on IBD and visceral sensitivity and to evaluate the relevance of brain-derived neurotrophic factor (BDNF) to intestinal hypersensitivity of IBD mice. The results showed that deoxyschisandrin could depress the contraction of isolated smooth muscle, modulate gastrointestinal function, and efficiently decrease the disease activity index (DAI) of IBD mice, which proved that deoxyschisandrin had antidiarrheal effects on the animals. In the colorectal distention (CRD) experiment, visceral sensibility was increased in the model group. However, abdominal withdrawal reflex (AWR) scores were decreased after deoxyschisandrin intervention, indicating that deoxyschisandrin could reduce the visceral hypersensitivity of IBD mice. Both IHC observation and western blotting analysis showed that BDNF protein expression increased evidently in colon of IBD mice. After the intervention of deoxyschisandrin, colon mucosa BDNF protein expression in IBD mice decreased, indicating that deoxyschisandrin could decrease mouse intestinal sensitivity by reducing colon mucosa BDNF expression. In conclusion, deoxyschisandrin possessed antidiarrheal effects and visceral hypersensitivity inhibitory effects in the mice with IBD induced by TNBS, which was related to the reduction in BDNF expression in the colon.

Moxibustion Eases Chronic Inflammatory Visceral Pain In Rats Via MAPK Signaling Pathway In The Spinal Cord.

PurposeThe purpose of this study was to explore the central analgesia mechanism of moxibustion for chronic inflammatory visceral pain (CIVP).MethodsA CIVP rat model was established by 2,4,6-trinitrobenzene sulfonic acid (TNBS) plus 50% ethanol via enema. The analgesic effect of moxibustion was evaluated using the abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). The expression profile of phosphorylated proteins of the mitogen-activated protein kinase (MAPK) signaling pathway in the spinal cord was assayed by protein microarray. The differentially expressed proteins were examined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional clusters and corresponding signaling pathways.ResultsMoxibustion exerted a significant analgesic effect for CIVP rats, mainly presenting as a decrease in the AWR score (all P<0.01) under different levels of distending pressure and an increase in MWT and TWL thresholds (all P<0.05). Compared with the normal group, 76 proteins were upregulated while 15 were downregulated, and MAPK signaling pathway was activated in the model group. Compared with the model group, there were 53 downregulated and 38 upregulated proteins in the moxibustion group, and MAPK signaling pathway was inhibited. Fold change (FC)>1.3 or <0.77 was taken as the screening standard to define the differentially expressed proteins. Fifteen differentially expressed proteins upregulated in the model group were downregulated in the moxibustion group. GO analysis showed that the differentially expressed proteins mainly controlled cellular metabolism regulation, transportation, and stress reactions. KEGG analysis revealed that these differentially expressed proteins were mostly involved in the ERK, JNK, and p38 pathways, and the ERK pathway was predominant.ConclusionMoxibustion mitigates CIVP in rats and inhibits the phosphorylation of proteins in the spinal MAPK signaling pathway. The analgesic effect of moxibustion may be associated with the regulation of the spinal MAPK signaling pathway.

Effects and mechanism of injection of glutamate into hypothalamic paraventricular nucleus on visceral pain in chronic visceral hypersensitivity rats

Objective To investigate the effects of glutamate (Glu) injected into hypothalamic paraventricular nucleus (PVN) on visceral pain of chronic visceral hypersensitivity (CVH) rats and its possible mechanism. Methods Newborn SD rats were given CVH rat model by colorectal distension (CRD) on the 8th, 10th and 12th day after birth. Thirty rats with successful CVH model were randomly divided into CVH model group (CVH group), CVH + injection of saline into PVN group (NS group), CVH+ injection of Glu into PVN (3, 6, and 12 μg Glu, namely G3, G6, and G12, respectively), 6 rats in each group, and 6 SD rats with matching body mass were taken as sham operation group (Sham group). The pain behavior of the rats was evaluated by pain threshold, abdominal withdrawal reflex (AWR) score, and abdominal external oblique muscle electromyography (EMG). The expression of arginine vasopressin (AVP) and the proliferation of colon tissue were detected by immunohistochemical staining. The apoptosis of colon tissue was detected by TUNEL. Results Compared with the NS group, the pain thresholds of the G3, G6 and G12 groups increased, and the AWR scores and EMG amplitudes decreased. The differences were statistically significant(Pain threshold: t=7.65, 16.31, 24.78, both P<0.05; AWR scores: t=-2.98, -4.77, -7.29, both P<0.05; EMG amplitudes: t=-3.06, -5.75, -8.92, both P<0.05). Compared with the Sham group, the expression of AVP in PVN of the CVH group and NS group decreased ((42.63±5.20) %, (18.67±2.94) %, (17.53±2.47) %; t=6.95, t=7.56, both P<0.05). The expression of AVP was increased after different doses of Glu into PVN, and the AVP level in G12 group ((18.15±6.49)%) was higher than that of NS group, the difference was statistically significant (t=-4.21, P<0.05). Compared with the Sham group, the expression of PCNA in colonic mucosal cells of the CVH group and NS group decreased ((65.48±1.68) %, (18.39±1.67) %, (17.69±1.68) %; t=34.35, t=34.80, both P<0.05). The expression of PCNA was increased after different doses of Glu injected into PVN, and the PCNA level in G12 group ((59.91±5.63)%) was higher than that of NS group, the difference was statistically significant (t=-12.44, P<0.05). Compared with the Sham group, the expression of apoptotic cells in colonic mucosal cells of the CVH group and NS group increased ((23.38±11.40)%, (83.79±3.57)%, (80.91±2.47)%; t=-8.77, t=-8.54, both P<0.05). The expression of apoptotic cells was decreased after different doses of Glu into PVN, and the G12 group was ((18.15±6.49) %). Compared with NS group, the difference was statistically significant (t=15.65, P<0.05). Conclusion Injection of Glu into hypothalamic PVN can alleviate the visceral pain behaviors in CVH rats, and its mechanism may be related to arginine vasopressin. Key words: Chronic visceral hypersensitivity; Hypothalamic paraventricular nucleus; Glutamate; Arginine vasopressin; Pain; Rat