Abdominal Withdrawal Reflex Scores Research Articles

Da-Jian-Zhong decoction alleviates diarrhea-predominant irritable bowel syndrome via modulation of gut microbiota and Th17/Treg balance

Ethnopharmacological relevanceDa-Jian-Zhong decoction (DJZD) is a herbal formula clinically used for abdominal pain and diarrhea induced by spleen-Yang deficiency syndrome. Recently, treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) with DJZD has received increasing attention, but the underlying mechanism of action remains elusive. Aim of the studyWe aimed to evaluate the therapeutic effect of DJZD on IBS-D rats and to elucidate the underlying mechanisms. Materials and methodsAn IBS-D rats model was constructed using a two-factor superposition method of neonatal maternal separation and Senna folium aqueous extract lavage. Moreover, the effect of DJZD was evaluated based on the body weight, rectal temperature, abdominal withdrawal reflex (AWR), and Bristol stool scale score (BSS). The factors that regulate the DJZD effects on IBS-D were estimated using whole microbial genome, transcriptome sequencing (RNA-Seq), flow cytometry, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses. ResultsWe found that DJZD alleviated the symptoms of IBS-D rats, with the low-dose (2.4 g/kg) as the better ones, as shown by the higher body mass and lower AWR score and BSS. At the phylum level, the relative abundance of Bacteroidetes was obviously increased, and at the genus level, Bacteroides was increased, while that of Firmicutes_bacterium_424 and Ruminococcus gnavus was decreased in DJZD group. The significantly enriched GO terms after treatment with DJZD mainly included the immune response, positive regulation of activated T cell proliferation, and positive regulation of interleukin-17 (IL-17) production. Importantly, flow cytometry analysis further revealed that the T helper cell type 17/regulatory T cell (Th17/Treg) balance contributed to the DJZD-induced alleviation of IBS-D symptoms, as DJZD downregulated Th17/Treg ratio and Th17 cell-related cytokines IL-17 and IL-6 levels in the colon. ConclusionsThese results demonstrate that DJZD has a good therapeutic effect on IBS-D rats, probably by maintaining the homeostasis of gut microbiota and regulating Th17/Treg balance and its related inflammatory factors.

Chang-Kang-Fang alleviates diarrhea predominant irritable bowel syndrome (IBS-D) through inhibiting TLR4/NF-κB/NLRP3 pathway

Ethnopharmacological relevanceChang-Kang-Fang (CKF), originated from traditional Chinese medicine (TCM) formulas, has been utilized to treat diarrhea predominant irritable bowel syndrome (IBS-D) based on clinical experience. However, the underlying mechanism of CKF for treating IBS-D remains unclear and need further clarification. Aim of the studyThe objective of this present investigation was to validate the efficacy of CKF on IBS-D model rats and to uncover its potential mechanism for the treatment of IBS-D. Materials and methodsWe first established the IBS-D rat model through neonatal maternal separation (NMS) in combination with restraint stress (RS) and the administration of senna decoction via gavage. To confirm the therapeutic effect of CKF on treating IBS-D, abdominal withdrawal reflex (AWR) scores, the quantity of fecal pellets, and the fecal water content (FWC) were measured to evaluate the influence of CKF on visceral hypersensitivity and the severity of diarrhea symptom after the intragastric administration of CKF for 14 days. Subsequently, enzyme linked immunosorbent assay (ELISA) was applied to assess the effect of CKF on neuropeptides substance P (SP) and 5-hydroxytryptamine (5-HT), as well as inflammatory cytokines in serum and in intestinal tissues. Further, colonic pathological changes, the amount of colonic mast cells, and the expression level of occludin in rat colon tissues, were investigated by hematoxylin-eosin (HE) staining, toluidine blue staining, and immunohistochemistry, respectively. To explore the underlying mechanisms, alterations in colonic RNA transcriptomics for the normal, model, and CKF treatment groups were assessed using RNA sequencing (RNA-Seq). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays were applied to validate the effect of CKF on predicted pathways in vivo and in vitro. In addition, to elucidate the potential active compounds in CKF, 11 representative components found in CKF were selected, and their anti-inflammation potentials were evaluated using LPS-treated RAW264.7 cell models. ResultsCKF treatment significantly reduced the number of fecal pellets, attenuated visceral hypersensitivity, and decreased 5-HT and SP concentrations in serum and colon tissues, along with a reduction in colonic mast cell counts, correlating with improved symptoms in IBS-D rats. Meanwhile, CKF treatment reduced the colonic inflammatory cell infiltration, lowered the levels of IL-6, TNF-α, and IL-1β in serum and colon tissues, and increased the occludin protein expression in colon tissues to improve inflammatory response and colonic barrier function. RNA-Seq, in conjugation with our previous network pharmacology analysis, indicated that CKF might mitigate the symptoms of IBS-D rats by inhibiting the Toll like receptor 4/Nuclear factor kappa-B/NLR family pyrin domain-containing protein 3 (TLR4/NF-κB/NLRP3) pathway, which was confirmed by WB, IF, and qRT-PCR experiments in vivo and in vitro. Furthermore, coptisine, berberine, hyperoside, epicatechin, and gallic acid present in CKF emerged as potential active components for treating IBS-D, as they demonstrated in vitro anti-inflammatory effects. ConclusionOur findings demonstrate that CKF effectively improves the symptoms of IBS-D rats, potentially through the inhibition of the TLR4/NF-κB/NLRP3 pathway. Moreover, this study unveils the potential bioactive components in CKF that could be applied in the treatment of IBS-D.

Activation of ASIC3/ERK pathway by paeoniflorin improves intestinal fluid metabolism and visceral sensitivity in slow transit constipated rats.

Slow transit constipation (STC) is one of the most common gastrointestinal disorders in children and adults worldwide. Paeoniflorin (PF), a monoterpene glycoside compound extracted from the dried root of Paeonia lactiflora, has been found to alleviate STC, but the mechanisms of its effect remain unclear. The present study aimed to investigate the effects and mechanisms of PF on intestinal fluid metabolism and visceral sensitization in rats with compound diphenoxylate-induced STC. Based on the evaluation of the laxative effect, the abdominal withdrawal reflex test, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were used to detect the visceral sensitivity, fluid metabolism-related proteins, and acid-sensitive ion channel 3/extracellular signal-regulated kinase (ASIC3/ERK) pathway-related molecules. PF treatment not only attenuated compound diphenoxylate-induced constipation symptoms and colonic pathological damage in rats but also ameliorated colonic fluid metabolic disorders and visceral sensitization abnormalities, as manifested by increased colonic goblet cell counts and mucin2 protein expression, decreased aquaporin3 protein expression, improved abdominal withdrawal reflex scores, reduced visceral pain threshold, upregulated serum 5-hydroxytryptamine, and downregulated vasoactive intestinal peptide levels. Furthermore, PF activated the colonic ASIC3/ERK pathway in STC rats, and ASIC3 inhibition partially counteracted PF's modulatory effects on intestinal fluid and visceral sensation. In conclusion, PF alleviated impaired intestinal fluid metabolism and abnormal visceral sensitization in STC rats and thus relieved their symptoms through activation of the ASIC3/ERK pathway.

Paeoniflorin Alleviates Anxiety and Visceral Hypersensitivity via HPA Axis and BDNF/TrkB/PLCγ1 Pathway in Maternal Separation-induced IBS-like Rats.

Irritable Bowel Syndrome (IBS) is a prevalent gastrointestinal disorder that significantly diminishes the quality of life for affected individuals. The pathophysiology of IBS remains poorly understood, and available therapeutic options for IBS are limited. The crucial roles of brain-gut interaction, which is mediated by the Hypothalamic-Pituitary-Adrenocortical (HPA) axis and the autonomic nervous system in IBS, have attracted increasing attention. The objective of this study was to examine the impact of paeoniflorin (PF) on anxiety and visceral hypersensitivity in maternal separation-induced IBS-like rats. The IBS-like rat model was established through the implementation of Maternal Separation (MS) and subsequently subjected to various doses of PF administered via oral gavage for 14 days. Anxiety-like behavior was evaluated using the Open Field Test (OFT) and Elevated Plus Maze (EPM) test. The assessment of visceral sensitivity involved the utilization of the Abdominal Withdrawal Reflex (AWR) score and electromyographic (EMG) responses of the external oblique muscle in response to colorectal distention. The levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), and corticotrophin-releasing hormone (CRH) were examined by ELISA. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were employed to detect the expressions of CRH receptors 1 (CRHR1) and 2 (CRHR2). Glucocorticoid receptors (GR), mineralocorticoid receptor (MR), brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB), and phospholipase C γ1 (PLCγ1) were examined by Western blot. The results showed that MS induced anxiety-like behavior and visceral hypersensitivity, while PF treatment attenuated these changes. Furthermore, the HPA axis hyperactivity in MS rats was attenuated by PF treatment, indicated by reduced serum ACTH, CORT, and CRH levels and recovered hippocampal CRHR1 and GR expressions. In addition, PF inhibited BDNF/TrkB signaling by downregulating the protein levels of BDNF, TrkB, and phospho-PLCγ1 in the colon. These findings suggest that PF alleviated anxiety and visceral hypersensitivity in MS-induced IBS-like rats, which may be the modulation of HPA axis activity and BDNF/TrkB/PLCγ1 signaling pathway.

Electroacupuncture Improving Intestinal Barrier Function in Rats with Irritable Bowel Syndrome Through Regulating Aquaporins.

Intestinal mucosal barrier dysfunction plays a crucial role in the pathogenesis of irritable bowel syndrome with diarrhea (IBS-D). In order to explore the mechanism of electroacupuncture (EA) treatment on intestinal mucosal barrier, this study observed the effect of EA on aquaporins (AQPs), tight junctions (TJs), NF-κB pathway and the gut microbiota in IBS-D rats. The IBS-D model was established by acetic acid enema combined with chronic restraint method. The effects of EA on the treatment of IBS-D were examined by the abdominal withdrawal reflex score, Bristol's fecal character score, fecal water content, small intestine propulsion rate and HE staining. AQPs, TJs and inflammation-related molecular mechanisms were explored. The fecal samples were applied for 16S rRNA sequencing to assess the effect of EA intervention to the intestinal bacterial abundance. EA reduced intestinal sensitization, restored intestinal motility and improved inflammatory cell infiltration. Furthermore, EA improved intestinal inflammation and flora environment significantly, inhibited NF-κB signaling and inflammatory factors (IL-1β and TNF-α). It can also increase the gene and protein expression of AQPs (AQP1, AQP3, and AQP8) and the gene levels of TJs (ZO-1 and Occludin). EA has an inhibitory effect on the NF-κB signaling pathway, and regulates the proteins of AQP1, AQP3, AQP8, and TJs to restore the balance of water metabolism and intestinal permeability in IBS-D, which also restored the function of the intestinal mucosa by regulating the intestinal flora.

Ameliorating effects of transcutaneous auricular vagus nerve stimulation on a mouse model of constipation-predominant irritable bowel syndrome

Limited treatment options have been shown to alter the natural course of constipation-predominant irritable bowel syndrome (IBS-C). Therefore, safer and more effective approaches are urgently needed. We investigated the effects of transcutaneous auricular vagus nerve stimulation (taVNS) in a mouse model of IBS-C. In the current study, C57BL/6 mice were randomly divided into normal control, IBS-C model control, sham-electrostimulation (sham-ES), taVNS, and drug treatment groups. The effects of taVNS on fecal pellet number, fecal water content, and gastrointestinal transit were evaluated in IBS-C model mice. We assessed the effect of taVNS on visceral hypersensitivity using the colorectal distention test. 16S rRNA sequencing was used to analyze the fecal microbiota of the experimental groups. First, we found that taVNS increased fecal pellet number, fecal water content, and gastrointestinal transit in IBS-C model mice compared with the sham-ES group. Second, taVNS significantly decreased the abdominal withdrawal reflex (AWR) score compared with the sham-ES group, thus relieving visceral hyperalgesia. Third, the gut microbiota outcomes showed that taVNS restored Lactobacillus abundance while increasing Bifidobacterium probiotic abundance at the genus level. Notably, taVNS increased the number of c-kit-positive interstitial cells of Cajal (ICC) in the myenteric plexus region in IBS-C mice compared with the sham-ES group. Therefore, our study indicated that taVNS effectively ameliorated IBS-C in the gut microbiota and ICC.

Chronic Sacral Nerve Stimulation Inhibits Visceral Hypersensitivity in Diarrhea–Predominant Irritable Bowel Syndrome Rats Model

ObjectiveSacral nerve stimulation (SNS) is emerging as a novel treatment for irritable bowel syndrome (IBS). However, its effects are limited, and the underlying mechanisms remain largely unknown. Materials and MethodsIn this study, rats were divided into three groups (n = 12 rats per group): 1) the SNS group; 2) the sham SNS group (the sham group for short); and 3) the control group. The SNS and sham groups were exposed to chronic and acute stress to establish an IBS model. Electrode implantation surgery was performed in rats with the IBS model. The SNS group received electrical stimulation for 30 minutes every day for seven days. Abdominal withdrawal reflex (AWR) was used to evaluate the effect of SNS on visceral sensitivity in diarrhea-predominant IBS (IBS-D) rats. The frequency domain of heart rate variability (HRV) was analyzed to assess the effect of SNS on regulating the autonomic function. The expression of transient receptor potential vanilloid 1 (TRPV1) in the colon, spinal cord, and hippocampus was detected by immunohistochemistry to explore the mechanism of SNS in IBS-D rats. ResultsCompared with the sham group, AWR scores were significantly decreased under different gas volumes of stimulation of 0.4, 0.6, and 0.8 ml for rectal distention in the SNS group (all p < 0.05). However, there was no significant difference <1.0 ml between the two groups (p > 0.05). Compared with the sham group, the frequency domain indexes of HRV were significantly altered. Normalized low-frequency power and low frequency-to-high frequency ratio were significantly decreased, and normalized high-frequency power was significantly increased in the SNS group (all p < 0.05). Moreover, the expression of TRPV1 in the spinal cord and colon in the SNS group was significantly decreased compared with the sham group (both p < 0.05). These results suggested that chronic SNS not only improved the visceral sensitivity and autonomic dysfunction but also decreased the expression of TRPV1 in the spinal cord-gut tissue in IBS-D rats. ConclusionChronic SNS was found to have an inhibitory effect on visceral hypersensitivity in IBS-D rats, providing experimental evidence for its potential clinical application in IBS.

NGF/PI3K/TRPV1 pathway mediates the regulation of visceral pain by acupuncture at "Shangjuxu" (ST37) in irritable bowel syndrome rats with chronic visceral hyperalgesia.

To investigate the effect of manual acupuncture at "Shangjuxu"(ST37) on nerve growth factor(NGF)/phosphatidylinositol 3-kinase(PI3K)/transient receptor potential vanilloid 1(TRPV1) signaling pathway in rats with chronic visceral hyperalgesia of irritable bowel syndrome (IBS), so as to explore its underlying mechanism in treating IBS chronic visceral hyperalgesia. IBS chronic visceral hyperalgesia model was established by colorectal dilation stimulation for 2 weeks for SD pups at 8 d after birth, which were fed until 8-week age after the stimulation. Then the verified successfully modeled adult rats were randomly divided into model, Shangjuxu, and non-acupoint groups, with 6 rats in each group, and 6 unmodeled rats were selected as normal group. On the next day of model evaluation, rats in the Shangjuxu group received acupuncture at right ST37 while rats in the non-acupoint group received acupuncture at the non-meridian and non-acupoint point in the right hypochondrium, both for 15 min, with manual twisting of mild reinforcing and reducing performed for 30 s at an interval of 5 min, once a day, for a total of 7 d. Abdominal withdrawal reflex(AWR) score was used to evaluate the degree of chronic visceral pain in rats. Western blot and real-time fluorescence quantitative PCR were used to detect the colonic protein and mRNA expressions of NGF, tropomyosin receptor kinase A (TrkA), PI3K and TRPV1. The positive expressions of PI3K and TRPV1 proteins in the colon of rats were detected by immunohistochemistry method. Compared with the normal group, AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1 in colon tissue, and positive expressions of PI3K and TRPV1 in colon tissue were significantly increased(P<0.05) in the model group. After intervention, compared with the model group, rats in the Shangjuxu group had reduced AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, lower colonic mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1, and decreased positive expressions of PI3K and TRPV1 in colon tissue(P<0.05), while there were no significant differences in the above indexes of the non-acupoint group. Manual acupuncture at ST37 can alleviate IBS chronic visceral hyperalgesia in rat and its analgesic effect may be related to regulating NGF/PI3K/TRPV1 signaling pathway.

Linderae Radix water extract treats diarrhea-predominant irritable bowel syndrome in rats: a serum metabolomics study

This study aims to investigate the mechanism of Linderae Radix water extract(LRWE) in the prevention and treatment of diarrhea-predominant irritable bowel syndrome(IBS-D) based on serum metabolomics. Eighteen 2-week-old male SD rats were randomized into control, IBS-D model, and LRWE groups. The rats in other groups except the control group received gavage of senna concentrate combined with restraint stress for the modeling of IBS-D. The rats in the LRWE group were administrated with LRWE(5.4 g·kg~(-1)) by gavage, and those in the control and IBS-D model groups with an equal volume of distilled water for a total of 14 days. The visceral sensitivity was evaluated by the abdominal withdrawal reflex(AWR) score, and the degree of diarrhea was assessed by the fecal water content(FWC). The morphological changes of the colon and the morphology and number of goblet cells were observed by hematoxylin-eosin(HE) and periodic acid-schiff(PAS) staining, respectively. Ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was used for the screening of the potential biomarkers in the rat serum and their related metabolic pathways. The results showed that LRWE reduced the AWR score, decreased FWC, and alleviated visceral sensitivity and diarrhea symptoms in IBS-D rats. HE and PAS staining showed that LRWE mitigated low-grade intestinal inflammation and increased the number of mature secretory goblet cells in the colonic epithelium of IBS-D rats. A total of 25 potential biomarkers of LRWE in treating IBS-D were screened out in this study, which were mainly involved in riboflavin, tryptophan, glycine, serine and threonine metabolism, glyoxylate and dicarboxylate metabolism, and cysteine and methionine metabolism. The regulatory effects were the most significant on the riboflavin and tryptophan metabolism pathways. LRWE may alleviate the visceral hypersensitivity by promoting energy metabolism and amino acid metabolism, enhancing intestinal barrier function, and improving intestinal immune function in IBS-D rats.

How Tongxie-Yaofang Regulates Intestinal Synaptic Plasticity by Activating Enteric Glial Cells and NGF/TrkA Pathway in Diarrhea-Predominant Irritable Bowel Syndrome Rats.

Diarrhea-predominant irritable bowel syndrome (D-IBS) is a frequent functional gastrointestinal disease that affects health and quality of life owing to its high incidence and recurrence rate. Tongxie-Yaofang (TXYF) is a traditional Chinese medicine prescribed for D-IBS. However, the therapeutic mechanism of TXYF has not been fully elucidated. This study aimed to investigate the effects of TXYF on visceral hypersensitivity in stress-induced D-IBS rats and the underlying mechanisms. Electromyographic (EMG) activity of the external oblique muscles and the abdominal withdrawal reflex (AWR) score captured by Barostat were used to quantify the effect of TXYF on visceral sensitivity. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the enteric nervous system (ENS). For molecular detection, the colonic expression of enteric glial cell's (EGC's) activation markers, glial fibrillary acidic protein (GFAP) and calcium-binding protein S100β, NGF, TrkA, synaptic plasticity-related factors, synaptophysin (SYN) and postsynaptic density-95 (PSD-95), glutamate, glutamate receptors α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR), and N-methyl-D-aspartate receptor (NMDAR) were detected by immunohistochemistry, enzyme-linked immunosorbent assay, and real-time PCR. An ex vivo experiment was conducted to measure the EGC-induced NGF release. TXYF decreased the EMG activity and AWR scores in rats with D-IBS. Under TEM, TXYF improved the dense and irregular nerve arrangement, narrowed the synaptic cleft, and decreased the number of synaptic vesicles in D-IBS rats. In addition, TXYF decreased the expression of GFAP, S100β, SYN, and PSD-95; down-regulated the levels of NGF, TrkA, and glutamate; and reduced the mRNA expression of AMPAR1, NMDAR1, and NMDAR2B. In an ex vivo experiment, TXYF decreased NGF release in D-IBS rats, and this trend disappeared under EGC inhibition. TXYF alleviated visceral hypersensitivity in D-IBS rats possibly by improving synaptic plasticity through inhibiting the activity of EGCs and the NGF/TrkA signaling pathway in the colon.

Effect of acupuncture-moxibustion stimulation of combined "Biao-Ben" acupoints on autonomic nervous activity and related factors in rats with irritable bowel syndrome diarrhea

To observe the effect of acupuncture and moxibustion of the combined "Biao-Ben" acupoints on autonomic nervous function and related factors in rats with irritable bowel syndrome diarrhea (IBS-D). Female SD rats were randomly divided into normal control, IBS-D model, "Biao-Ben" acupoint combination, and conventional acupoint combination groups, with 10 rats in each group. The IBS-D model was established by repeated chronic stress stimulation (water or food deprivation, painful tail pinching, exposure to a 43 ℃ environment, forced swimming in 4 ℃ water, day-night inversion and horizontal vibration) for 28 d, and followed by acute restraint stress (wrapping of shoulders, forelimbs and trunk) for 1 h and gavage of senna fluid, once daily for 28 d. For rats of the "Biao-Ben" acupoint combination group, acupuncture and moxibustion were applied to "Guanyuan" (CV4), bilateral "Zusanli" (ST36), and "Neiguan" (PC6), and for rats of the conventional acupoint combination group, acupuncture and moxibustion were applied to bilateral ST36, "Tianshu"(ST25), and "Shangjuxu"(ST37). Both acupuncture and suspension moxibustion treatment were conduced for 15 min, once daily for 21 days. The fecal water content was calculated, and the spontaneous activity behaviors (total distance of crossing and the number of squares crossed in 5 min) were evaluated by open-field tests. The abdominal withdrawal reflex (AWR) was detected. H.E. staining was used to observe the histopathological changes of colon tissue. The domains of heart rate variability (HRV) including low frequency (LF), high frequency (HF), and LF/HF were analyzed using an electrophysiological recorder. The contents of calcitonin gene-related peptide (CGRP) in serum and noradrenaline (NE) in plasma were detected using ELISA. The expression level and immunoactivity of 5-hydroxytryptamine (5-HT) in colon tissues were detected by Western blot and immunofluorescence histochemistry, separately. Compared with the normal group, the model group had a striking increase in fecal water content, AWR scores at 20, 40, 60, and 80 mm Hg, NE and CGRP contents, LF, LF/HF, and 5-HT protein expression and immunoactivity (P<0.01), and an obvious decrease in the total distance of crossing, the number of squares crossed, and HF of HRV (P<0.01). In comparison with the model group, the fecal water content, AWR scores at 20, 40, 60, and 80 mm Hg, NE and CGRP contents, LF, LF/HF, and 5-HT protein expression and immunoactivity were significantly decreased (P<0.01,P<0.05), while the total distance of cros-sing, number of squares crossed, HF of HRV were considerably increased (P<0.01, P<0.05) in both "Biao-Ben" acupoint combination and conventional acupoint combination groups. The effects of the "Biao-Ben" acupoint combination were apparently superior to those of conventional acupoint combination in down-regulating the fecal water content, AWR score at 20, 40, 60 and 80 mm Hg, NE and CGRP contents, LF, LF/HF, and 5-HT expression and immunoactivity, and in increasing the number of squares crossed, and HF of HRV (P<0.01, P<0.05). HE staining showed no pathological changes in colonic mucosa in all groups. Acupuncture and moxibustion stimulation of combined "Biao-Ben" acupoints can effectively improve the symptoms (spontaneous activities, visceral hypersensitivity) of IBS-D model rats, which may be related to its functions in regulating autonomic nervous activities, and down-regulating blood NE and CGRP contents and colonic 5-HT protein expression and immunoactivity, and the effects of "Biao-Ben" acupoint combination are superior to those of conventional acupoint combination.

Triptolide attenuates irritable bowel syndrome via inhibiting ODC1

BackgroundIrritable bowel syndrome (IBS) is a chronic disorder of the gut-brain axis with significant morbidity. Triptolide, an active compound extracted from Tripterygium wilfordii Hook F (TwHF), has been widely used as a major medicinal herb in the treatment of inflammatory disease.MethodsThe chronic-acute combined stress (CAS) stimulation was used to establish IBS rat model. The model rats were then gavaged with triptolide. Forced swimming, marble-burying, fecal weight and abdominal withdrawal reflex (AWR) score were recorded. Pathologic changes in the ileal and colonic tissues were validated by hematoxylin and eosin staining. The inflammatory cytokines and Ornithine Decarboxylase-1 (ODC1) in the ileal and colonic tissues were performed by ELISA and WB.ResultsTriptolide didn’t have antidepressant- and antianxiety- effects in rats caused by CAS, but decreased fecal weight and AWR score. In addition, Triptolide reduced the release of IL-1, IL-6, and TNF-α and the expression of ODC1 in the ileum and colon.ConclusionThe therapeutic efficacy of triptolide for IBS induced by CAS was revealed in this study, which may be related to the reduction of ODC1.

Effects of moxibustion on behaviors and tryptophan metabolism-related products in mice with irritable bowel syndrome

ObjectiveTo observe the effect of moxibustion on behaviors and related products of tryptophan (Trp) metabolism in the colon of mice with irritable bowel syndrome (IBS), and to explore the mechanism of moxibustion in the IBS treatment.MethodsTwenty-four mice were randomly divided into a normal group, a model group, a moxibustion group, and a probiotic group, with 6 mice in each group. The visceral pain model of IBS was established by enema with 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution. Mice in the moxibustion group were treated with mild moxibustion at bilateral Zusanli (ST36), and those in the probiotic group were treated with probiotics such as Bifidobacterium by gavage. Abdominal withdrawal reflex (AWR) test, elevated plus-maze (EPM) test, and forced swimming test (FST) were performed after treatment. The expression levels of 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (TPH1) in the colon were detected by immunofluorescence, and the expression levels of Trp, kynurenine (Kyn), and indole-2,3-oxygenase (IDO) in the colon were detected by enzyme-linked immunosorbent assay.ResultsCompared with the normal group, the AWR scores were increased significantly in the model group under different pressure values (P<0.01), the open-arm staying time and open-arm entries in the EPM test were decreased significantly (P<0.01, P<0.05), the motionless time in the FST was increased significantly (P<0.01), and the expression levels of colonic Trp, TPH1, IDO, 5-HT, and Kyn were increased significantly (P<0.01) in the models. Compared with the model group, the AWR scores were differently decreased (P<0.05 or P<0.01), the open-arm entries in the EPM test were increased (P<0.05), the motionless times in the FST were decreased (P<0.05), and the colonic expression levels of Trp, TPH1, IDO, and 5-HT were decreased (P<0.01 or P<0.05) in the moxibustion and probiotic groups; the open-arm staying time was significantly increased in the moxibustion group (P<0.01), and the colonic expression level of Kyn was significantly decreased in the probiotic group (P<0.01).ConclusionMoxibustion at Zusanli (ST36) improves visceral pain and pain mood and down-regulates the expression levels of colonic TPH1, IDO, Trp, 5-HT, and Kyn in IBS mice.

Low FODMAP Diet Relieves Visceral Hypersensitivity and Is Associated with Changes in Colonic Microcirculation in Water Avoidance Mice Model.

(1) Background: Irritable bowel syndrome (IBS) is a global public health problem, the pathogenesis of which has not been fully explored. Limiting fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) can relieve symptoms in some patients with IBS. Studies have shown that normal microcirculation perfusion is necessary to maintain the primary function of the gastrointestinal system. Here, we hypothesized that IBS pathogenesis might be related to abnormalities in colonic microcirculation. A low-FODMAP diet could alleviate visceral hypersensitivity (VH) by improving colonic microcirculation; (2) Methods: C57BL/6 mice were raised to establish an IBS-like rodent model using water avoidance (WA) stress or SHAM-WA as a control, one hour per day for ten days. The mice in the WA group were administered different levels of the FODMAP diet: 2.1% regular FODMAP (WA-RF), 10% high FODMAP diet (WA-HF), 5% medium FODMAP diet (WA-MF), and 0% low FODMAP diet (WA-LF) for the following 14 days. The body weight and food consumption of the mice were recorded. Visceral sensitivity was measured as colorectal distention (CRD) using the abdominal withdrawal reflex (AWR) score. Colonic microcirculation was assessed using laser speckle contrast imaging (LCSI). Vascular endothelial-derived growth factor (VEGF) was detected using immunofluorescence staining; (3) Results: The threshold values of CRD pressure in the WA-RF, WA-HF, and WA-MF groups were significantly lower than those in the SHAM-WA group. Moreover, we observed that colonic microcirculation perfusion decreased, and the expression of VEGF protein increased in these three groups of mice. Interestingly, a low-FODMAP dietary intervention could reverse this situation. Specifically, a low-FODMAP diet increased colonic microcirculation perfusion, reduced VEGF protein expression in mice, and increased the threshold of VH. There was a significant positive correlation between colonic microcirculation and threshold for VH; (4) Conclusions: These results demonstrate that a low-FODMAP diet can alter VH by affecting colonic microcirculation. Changes in intestinal microcirculation may be related to VEGF expression.

Activation of the High-Affinity Choline Transporter 1 in the Spinal Cord Relieves Stress-Induced Hyperalgesia.

The mechanism underlying irritable bowel syndrome (IBS), a common disease with hyperalgesia, remains elusive. The spinal cholinergic system is involved in pain modulation, but its role in IBS is unknown. To determine whether high-affinity choline transporter 1 (CHT1, a major determinant of the cholinergic signaling capacity), is implicated in spinal modulation of stress-induced hyperalgesia. A rat IBS model was established by water avoidance stress (WAS). Visceral sensations were detected by abdominal withdrawal reflex (AWR) and visceromotor response (VMR) to colorectal distension (CRD). Abdominal mechanical sensitivity was determined by von Frey filaments (VFFs) test. RT-PCR, Western blot, and immunostaining were performed for spinal CHT1 expression. Spinal acetylcholine (ACh) was measured by ELISA; the influence of spinal CHT1 on hyperalgesia were evaluated by intrathecal administration of MKC-231 (a choline uptake enhancer) and hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Minocycline treatment was used to explore the role of spinal microglia in hyperalgesia. After 10days of WAS, AWR scores and VMR magnitude to CRD, and the number of withdrawal events in VFF test were increased. Double-labeling showed that CHT1 in the dorsal horn was expressed in most of the neurons and almost all the microglia. The CHT1 expression and ACh levels in the spinal cord and the density of CHT1-positive cell in the spinal dorsal horn were enhanced in WAS-exposed rats. HC-3 enhanced pain responses in WAS rats; MKC-231 alleviated pain in WAS rats by upregulating CHT1 expression and increasing ACh production in the spinal cord. Furthermore, microglial activation in the spinal dorsal horn promoted the stress-induced hyperalgesia, and MKC-231 achieved analgesic effects byinhibiting the spinal microglial activation. CHT1 exerts antinociceptive effects in spinal modulation of chronic stress-induced hyperalgesia by increasing ACh synthesis and suppressing microglial activation. MKC-231 has potential for treating disorders accompanied by hyperalgesia.

Modulation of the microbiota across different intestinal segments by Rifaximin in PI-IBS mice

BackgroundRifaximin has been increasingly applied in irritable bowel syndrome (IBS) treatment. Whether there were differences in the effects of rifaximin on microbiota from different intestinal segments, especially the small intestine where rifaximin predominantly acted, has not been confirmed.MethodsIn this study, we used Trichinella spiralis infection to induce post infectious irritable bowel syndrome (PI-IBS) and measured visceral sensitivity of mice by means of abdominal withdrawal reflex (AWR) tests to colorectal distention (CRD). We compared the effects of rifaximin on the composition of ileal, colonic mucosal and fecal microbiota in PI-IBS mice.ResultsRifaximin significantly reduced AWR scores and increased pain threshold in PI-IBS mice, and this effect was associated with the change in the relative abundance of ileal mucosal microbiota. Rifaximin could obviously decrease ileum mucosal microbiota alpha diversity assessed by Shannon microbial diversity index. Meanwhile, the analysis of beta diversity and relative abundance of microbiota at phylum, family and genus levels showed that rifaximin could improve the microbiota structure of ileal mucosa. However, for colonic mucosal and fecal microbiota, this effect of rifaximin was not obvious. Rifaximin could reshape the correlation of genera between different intestinal segments.ConclusionRifaximin improved visceral hypersensitivity in PI-IBS mice. Rifaximin mainly affected ileal mucosal microbiota, and its improvement effect on IBS might be closely related to the improvement of ileal microbiota structure.

Therapeutic effects of paeoniflorin on irritable bowel syndrome in rats.

Irritable bowel syndrome (IBS) is a functional bowel disorder (FBD). To assess the therapeutic effects of paeoniflorin (PF) on IBS in rats. Sixty male Sprague-Dawley rats were randomly divided into normal, model, positive drug, low-dose PF, medium-dose PF and high-dose PF groups (n = 10). After gavage for 2 consecutive weeks, the effect of PF on abdominal pain symptoms was assessed based on the abdominal withdrawal reflex (AWR) score, fecal water content and pathological changes in colon tissues. D-lactate, interleukin-1β (IL-1β), transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay, and phosphorylated nuclear factor kappa B (p-NF-κB) p65 was detected by Western blotting. The abundance and diversity changes of intestinal flora were explored using 16S ribosomal RNA sequencing. In PF groups, the mucosal morphology of colon tissues was intact, and the glands were arranged neatly and structured clearly, without obvious inflammatory cell infiltration. Compared with the model group, PF groups had significantly elevated pain threshold, and mRNA and protein levels of zonula occludens-1 (ZO-1) and occludin, decreased AWR score at 20 mmHg pressure, fecal water content, mRNA levels of IL-1β, TGF-β, and TNF-α, protein level of p-NF-κB p65 and level of serum D-lactate, and reduced levels of serum IL-1β, TGF-β, and TNF-α (p < 0.05, p < 0.01). PF groups had higher abundance of Lactobacillus, Akkermansia, Alistipes, and Bacteroides, but lower abundance of Desulfovibrio, Parasutterella, and Enterococcus than those of the model group. PF exerts therapeutic effects on IBS in rats probably by regulating the intestinal flora, and then up-regulating the expressions of ZO-1 and occludin in colon tissue while down-regulating the levels of IL-1β, TGF-β, TNF-α, D-lactate and p-NF-κB p65.

Increased GABAergic projections in the paraventricular nucleus regulate colonic hypersensitivity via oxytocin in a rat model of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is characterized by gastrointestinal dysmotility and visceral hyperalgesia, and the impaired brain-gut axis is accepted as a crucial cause for the onset of IBS. The objective of this study is to investigate the effects of the adaptive changes in the central neural system induced by stress on IBS-like syndromes in rats. Long-term water avoidance stress (WAS) was used to prepare IBS animals. The changes in neuronal excitation and GABA expression were shown by immunohistochemistry. The mRNA and protein expressions of neurotransmitters were detected with Quantitative reverse-transcription PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA). The intestinal transit time, fecal moisture content, and abdominal withdrawal reflex scores of rats were recorded to monitor intestinal motility and visceral hyperalgesia. In the WAS-treated rats with enhanced intestinal motility and visceral hypersensitivity, more GABAergic projections were found in the paraventricular nucleus (PVN) of the hypothalamus, which inhibited the firing rate of neurons and decreased the expression of oxytocin. Exogenous oxytocin improved gut motility and decreased AWR scores. The inhibition of oxytocin by the adaptive GABAergic projection in the PVN might be an important mediator of IBS, which indicates a potential novel therapeutic target.

Effect of Acute Brief Social Isolation on Visceral Pain

The sensitivity of somatic pain could be affected by social isolation; however, few studies have examined the impact of social isolation on visceral pain. In the present study, the effect of acute brief social isolation on visceral pain response was investigated. Adult male rats were either reared individually or grouped for 2 hours or 24 hours. Colorectal distention (CRD)-induced abdominal withdrawal reflex (AWR) score and pain threshold were used to assess visceral pain sensitivity. The amount of fecal bolus was used to determine the stress level. Acute brief isolation rearing for 2 hours significantly increased AWR score and reduced visceral pain threshold in rats when compared to group rearing. Similarly, acute isolation for 24 hours resulted in visceral hypersensitivity, as indicated by an increase in the AWR score and a decrease in the visceral pain threshold. Furthermore, the amount of fecal bolus in acute isolation rearing (2 or 24 hours) rats was considerably higher than in the control group rearing rats. Acute short-term social isolation enhances visceral pain sensitivity, which could be related to an increase in stress levels.

Coptisine attenuates post‑infectious IBS via Nrf2‑dependent inhibition of the NLPR3 inflammasome.

Inhibition of the activation of the NLR family pyrin domain‑containing 3 (NLRP3) inflammasome has previously been reported to confer protection against post‑infectious irritable bowel syndrome (PI‑IBS). Coptisine, the second most abundant isoquinoline alkaloid in Coptischinensis, can inhibit NLRP3 inflammasome activation; however, whether coptisine exhibits protective effects against PI‑IBS remains unclear. In the present study, coptisine significantly reduced gastrointestinal motility and abdominal withdrawal reflex scores in a PI‑IBS rat model that was induced using intragastric administration of Trichinellaspiralis larvae. Coptisine treatment significantly decreased the protein levels of oxidative stress markers, 4‑hydroxynonenal, protein carbonyl and 8‑hydroxy‑2'deoxyguanosine, and proinflammatory cytokines, TNF‑α, IL‑1β and IL‑18 in the colon of PI‑IBS rats. Moreover, coptisine treatment significantly increased nuclear factor erythroid 2‑related factor 2 (Nrf2) nuclear translocation and heme oxygenase‑1 protein expression levels, while significantly downregulating the protein expression levels of NLRP3, apoptosis‑associated speck‑like protein containing a CARD and caspase‑1 in the colons of PI‑IBS rats. It is important to note that the anti‑inflammatory effects of coptisine were blocked by the Nrf2 inhibitor ML385. In summary, the present study indicated that coptisine potentially attenuated PI‑IBS in rats via Nrf2‑dependent inhibition of the NLPR3 inflammasome.