Slow Transit Constipation Group Research Articles

Xiao Cheng Qi Decoction, an Ancient Chinese Herbal Mixture, Relieves Loperamide-Induced Slow-Transit Constipation in Mice: An Action Mediated by Gut Microbiota.

Xiao Cheng Qi (XCQ) decoction, an ancient Chinese herbal mixture, has been used in treating slow-transit constipation (STC) for years. The underlying action mechanism in relieving the clinical symptoms is unclear. Several lines of evidence point to a strong link between constipation and gut microbiota. Short-chain fatty acids (SCFAs) and microbial metabolites have been shown to affect 5-HT synthesis by activating the GPR43 receptor localized on intestinal enterochromaffin cells, since 5-HT receptors are known to influence colonic peristalsis. The objective of this study was to evaluate the efficacy of XCQ in alleviating clinical symptoms in a mouse model of STC induced by loperamide. The application of loperamide leads to a decrease in intestinal transport and fecal water, which is used to establish the animal model of STC. In addition, the relationship between constipation and gut microbiota was determined. The herbal materials, composed of Rhei Radix et Rhizoma (Rhizomes of Rheum palmatum L., Polygonaceae) 55.2 g, Magnoliae Officinalis Cortex (Barks of Magnolia officinalis Rehd. et Wils, Magnoliaceae) 27.6 g, and Aurantii Fructus Immaturus (Fruitlet of Citrus aurantium L., Rutaceae) 36.0 g, were extracted with water to prepare the XCQ decoction. The constipated mice were induced with loperamide (10 mg/kg/day), and then treated with an oral dose of XCQ herbal extract (2.0, 4.0, and 8.0 g/kg/day) two times a day. Mosapride was administered as a positive drug. In loperamide-induced STC mice, the therapeutic parameters of XCQ-treated mice were determined, i.e., (i) symptoms of constipation, composition of gut microbiota, and amount of short-chain fatty acids in feces; (ii) plasma level of 5-HT; and (iii) expressions of the GPR43 and 5-HT4 receptor in colon. XCQ ameliorated the constipation symptoms of loperamide-induced STC mice. In gut microbiota, the treatment of XCQ in STC mice increased the relative abundances of Lactobacillus, Prevotellaceae_UCG_001, Prevotellaceae_NK3B31_group, Muribaculaceae, and Roseburia in feces and decreased the relative abundances of Desulfovibrio, Tuzzerella, and Lachnospiraceae_ NK4A136_group. The levels of SCFAs in stools from the STC group were significantly lower than those the control group, and were greatly elevated via treatment with XCQ. Compared with the STC group, XCQ increased the plasma level of 5-HT and the colonic expressions of the GPR43 and 5-HT4 receptor, significantly. The underlying mechanism of XCQ in anti-constipation could be related to the modulation of gut microbiota, the increase in SCFAs, the increase in plasma 5-HT, and the colonic expressions of the GPR43 and 5-HT4 receptor. Our results indicate that XCQ is a potent natural product that could be a therapeutic strategy for constipation.

Effect of miR-129-3p on autophagy of interstitial cells of Cajal in slow transit constipation through SCF C-kit signaling pathway.

To explore the mechanism by which miR-129-3p affected the autophagy of interstitial cells of Cajal (ICCs) in slow transit constipation tissues through the SCF C-kit signaling pathway. Colon samples from 20 Slow transit constipation (STC) patients who underwent total colectomy plus ileorectal anastomosis or subtotal colon resection plus anti-peristaltic rectal anastomosis were collected in our hospital. The colon of 20 non-STC patients was used as control. The control of this study was 20 patients undergoing radical surgery for colon cancer (left colon cancer) in our hospital. Fifty healthy SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mRNA expression of miR-129-3p in the STC group was lower than that in the control group (CTLR) group (P<0.05). The mRNA expression of miR-129-3p in STC group was lower than that in the NC group (P<0.05), and mRNA expression in STC+miR-129-3p group was higher than that in STC+miR-NC group (P<0.05). In the first week, the weight of dry and wet feces of the STC group was lower than that of the NC mice (P<0.05), and the weight of dry feces and wet feces of the STC group was lower than that of the NC group at the 2, 3, and 4 weeks, STC+miR-129 -3p was higher than that in the STC group (P<0.05). The increased expression of C-kit and SCF regulated by miR-129-3p contributed to the protection of interstitial cells. Knockdown of miR-129-3p expression could inhibit the activation of AKT/mTOR signaling pathway, reduce cell proliferation activity.

Dioscin ameliorates slow transit constipation in mice by up-regulation of the BMP2 secreted by muscularis macrophages.

Objective(s): The loss of enteric neurons has been shown to be a major cause of slow transit constipation (STC). Gut microbiota and muscularis macrophages (MMs) are associated with the enteric nervous system (ENS) development and gastrointestinal (GI) motility. This study aimed to investigate whether Dioscin (DIO) increased GI motility and inhibited neuron loss by modulating gut microbiota profile, improving inflammation in the ENS microenvironment.Materials and Methods:The STC model was established by loperamide. The alteration of the gut microbiota was analyzed by 16S rDNA sequencing. The longitudinal muscle and myenteric plexus (LMMP) from the colon were prepared for flow cytometry, immunofluorescence, western blot, and qRT-PCR. Results:DIO increased the stool number, stool water content and shortened whole gut transit time, helped to recover the gut microbial diversity and microbiota community structure, and increased the abundance of Muribaculaceae in STC mice. Compared with the STC group, the number of MMs and the level of the iNOS, IL-6, and TNFα genes were significantly decreased following DIO treatment. Moreover, DIO may increase the number of HuC/D+ neurons per ganglion by up-regulating the BMP2 secreted by MMs and activating the BMP2/p-Smad1/5/9 signaling pathway. Furthermore, the level of excitatory neurotransmitter AchE in colon tissues exhibited a substantial increase in the DIO group. However, the level of inhibitory neurotransmitter VIP was markedly decreased. Conclusion:Our results provide that DIO increases GI motility and inhibits neuron loss by modulating gut microbiota profile, improving inflammation in the ENS microenvironment and up-regulating the BMP2 secreted by MMs.

Abnormal bile acid metabolism is an important feature of gut microbiota and fecal metabolites in patients with slow transit constipation.

Destructions in the intestinal ecosystem are implicated with changes in slow transit constipation (STC), which is a kind of intractable constipation characterized by colonic motility disorder. In order to deepen the understanding of the structure of the STC gut microbiota and the relationship between the gut microbiota and fecal metabolites, we first used 16S rRNA amplicon sequencing to evaluate the gut microbiota in 30 STC patients and 30 healthy subjects. The α-diversity of the STC group was changed to a certain degree, and the β-diversity was significantly different, which indicated that the composition of the gut microbiota of STC patients was inconsistent with healthy subjects. Among them, Bacteroides, Parabacteroides, Desulfovibrionaceae, and Ruminiclostridium were significantly upregulated, while Subdoligranulum was significantly downregulated. The metabolomics showed that different metabolites between the STC and the control group were involved in the process of bile acids and lipid metabolism, including taurocholate, taurochenodeoxycholate, taurine, deoxycholic acid, cyclohexylsulfamate, cholic acid, chenodeoxycholate, arachidonic acid, and 4-pyridoxic acid. We found that the colon histomorphology of STC patients was significantly disrupted, and TGR5 and FXR were significantly downregulated. The differences in metabolites were related to changes in the abundance of specific bacteria and patients’ intestinal dysfunction. Analysis of the fecal genomics and metabolomics enabled separation of the STC from controls based on random forest model prediction [STC vs. control (14 gut microbiota and metabolite biomarkers)—Sensitivity: 1, Specificity: 0.877]. This study provided a perspective for the diagnosis and intervention of STC related with abnormal bile acid metabolism.

Characterization of Circular RNA Expression Profiles in Colon Specimens of Patients with Slow Transit Constipation.

Background Slow transit constipation (STC) is a clinical syndrome characterized by a decreased urge to defecate and delayed colonic transit. Circular RNAs (circRNAs) are a recently discovered class of regulatory RNAs that have emerged as critical biomarkers and regulators of various diseases. However, the expression profiles and mechanisms underlying circRNA regulation in human STC tissues have not been explored. Methods High-throughput RNA sequencing technology was used to compare the differences in circRNA expression profiles in colon samples taken from patients with STC or controls. Bioinformatics analyses were performed on the host genes of the differentially expressed circRNAs (DE-circRNAs), a competing endogenous RNA network was constructed, and the expression levels of some DE-circRNAs were verified using quantitative real-time polymerase chain reactions (qRT-PCR). Results There were 190 DE-circRNAs identified in the STC group. Bioinformatics analysis predicted that the DE-circRNAs were enriched in the relaxation of smooth muscle, actin binding, actin cytoskeleton organization, dilated cardiomyopathy, and cardiac muscle contraction. These results suggest that muscle diseases may be related to the pathogenesis of STC. The expression levels of the 12 most differentially expressed circRNAs were verified using qRT-PCR. In addition, circRNA–microRNA–mRNA regulatory networks were constructed using the 8 most significant circRNAs. Some mRNAs predicted to be closely related to smooth muscle function were found in these networks. Conclusions This study provides a helpful blueprint for researchers to select candidate circRNAs for further study of the pathogenesis of STC and screen potential biomarkers or targets for use in the diagnosis and treatment of STC.

Based on Network Pharmacology and Gut Microbiota Analysis to Investigate the Mechanism of the Laxative Effect of Pterostilbene on Loperamide-Induced Slow Transit Constipation in Mice

Background: Pterostilbene (PTE) is a natural polyphenol compound that has been proven to improve intestinal inflammation, but its laxative effect on slow transit constipation (STC) has never been studied. This study aims to investigate the laxative effect of PTE on loperamide (LOP)-induced STC mice and its influence on intestinal microbes through a combination of network pharmacological analysis and experimental verification. Material and Methods: PTE was used to treat LOP-exposed mice, and the laxative effect of PTE was evaluated by the total intestinal transit time and stool parameters. The apoptosis of Cajal interstitial cells (ICCs) was detected by immunofluorescence. The mechanism of PTE’s laxative effect was predicted by network pharmacology analysis. We used western blot technology to verify the predicted hub genes and pathways. Malondialdehyde (MDA) and GSH-Px were tested to reflect oxidative stress levels and the changes of gut microbiota were detected by 16S rDNA high-throughput sequencing. Results: PTE treatment could significantly improve the intestinal motility disorder caused by LOP. Apoptosis of ICCs increased in the STC group, but decreased significantly in the PTE intervention group. Through network pharmacological analysis, PTE might reduce the apoptosis of ICCs by enhancing PI3K/AKT and Nrf2/HO-1 signaling, and improve constipation caused by LOP. In colon tissues, PTE improved the Nrf2/HO-1 pathway and upregulated the phosphorylation of AKT. The level of MDA increased and GSH-Px decreased in the STC group, while the level of oxidative stress was significantly reduced in the PTE treatment groups. PTE also promoted the secretion of intestinal hormone and restored the microbial diversity caused by LOP. Conclusion: Pterostilbene ameliorated the intestinal motility disorder induced by LOP, this effect might be achieved by inhibiting oxidative stress-induced apoptosis of ICCs through the PI3K/AKT/Nrf2 signaling pathway.

MOTILITY OF THE UNPREPARED INTESTINAL TRACT IN HEALTHY HUMANS AND PATIENTS WITH SLOW TRANSIT CONSTIPATION REVEALED BY USING MICRO-ELECTRONIC CAPSULE

Slow transit constipation (STC) is usually accompanied by intestinal motility abnormalities. Although conventional anorectal manometry could record the pressure in the colon, most patients need preparation of intestinal tract. The intervention of catheter for monitoring the intestinal pressure also affects the clinical measurement. The pressure data collected by the conventional anorectal manometry cannot fully characterize the dynamic characteristics of the intestine. Thus, we aimed to obtain colonic pressure data under normal physiological conditions. Utilizing these data, we analyze the difference of colonic motility parameters between healthy control and patients with STC. A micro-electronic capsule made by ourselves was used to gather the subjects’ intestinal pressure in their daily life. Several intestinal motility parameters were calculated from the pressure profile. The average energy of colonic pressure data in the STC group is higher than the healthy control group (HC: 259.95 vs. STC: 821.28). But the STC group has a lower average complexity of colonic motility (HC: 0.80 vs. STC: 0.64). About 81.25% of the colonic data from patients with STC could be identified by using slow transit constipation (SVM) classifier. Compared with health control, most colonic parameters of patients with STC are higher under the normal physiological conditions, but the complexity of colonic motility is lower in the STC group. The correct rate of colonic pressure recognition in the STC group is more than 80% by using SVM classifier.

MiR-128 Participates in the Pathogenesis of Slow Transit Constipation by Regulating the P38α/M-CSF Inflammatory Signaling Pathway

Background: Slow transit constipation (STC) is a gastrointestinal disorder that adversely affects the quality of life. MicroRNAs are involved in the pathogenesis of functional gastrointestinal disorders. This study aims to investigate the molecular mechanism of miR-128 in STC. Methods: A murine model of STC was constructed based on morphine and rhubarb. MiRNA array, bioinformatic analysis and experimental validation were applied to confirm the miR-128 and its target signaling pathway. The expression of miR-128 and downstream genes analysis were performed in the models. Inflammatory cytokine release in response to miR-128 regulation was evaluated in vitro. Findings: The expression of stem cell factor (SCF) and neuron-specific enolase (NSE) were low in the STC model. In addition, STC group showed a significant downregulation of miR-128 and upregulation of p38α and macrophage colony stimulating factors (M-CSF). Moreover, we observed elevated inflammatory cytokine and decreased anti-inflammatory cytokine levels in colonic tissues. Furthermore, co-culture assays indicated that regulating expression of miR-128 in colonic epithelial cells induced the secretion of IL-6 and TNF-α by macrophages. Interpretation: Our study demonstrated that miR-128 regulated the p38α/M-CSF signaling pathway to promote chronic inflammatory responses and changes in the immune microenvironment of the colon, thereby offering potential insights into the pathogenesis of STC and therapeutic targets for its treatment. Funding Information: This work was supported by grants from the National Natural Science Foundation of China (No.81570492, No.81500505), Science and Technology Innovation Fostering Foundation of Zhongnan Hospital, Wuhan University (cxpy2017018), Medical Science and Technology Innovation Platform of Health Commission of Hubei Province /Zhongnan Hospital of Wuhan University (No.PTXM2019011), Health Commission of Hubei Province (No. WJ2019M206), Science and Technology Department of Hubei Province (No.2018CKB913), and the Clinical Research Special Fund of Wu Jieping Medical Foundation (No.320.6750.18467, No.320.6750.19089-14). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: Experiments were performed under a project license (No.2018039) granted by the ethics committee of Zhongnan Hospital of Wuhan University, Wuhan, China, in compliance with Animal Research Center of Wuhan University guidelines for the care and use of animals.

CircORC2 is involved in the pathogenesis of slow transit constipation via modulating the signalling of miR-19a and neurotensin/motilin.

In this study, we aimed to investigate the role of circORC2 in modulating miR‐19a and its downstream signalling during the pathogenesis of STC. In this study, three groups of patients, that is healthy control (HC) group, normal transit constipation (NTC) group (N = 42) and slow transit constipation (STC) group, were, respectively, recruited. RT‐PCR and Western blot analysis were exploited to investigate the changes in the expression levels of miR‐19a and circORC2 in these patients, so as to establish a circORC2/miR‐19a signalling pathway. The basic information of the patients showed no significant differences among different patient groups. Compared with the HC group, concentrations of neurotensin (NST) and motilin (MLN) were both significantly reduced in the NTC and STC groups, especially in the STC group. Also, miR‐19a level was highest, whereas circORC2 level was lowest in the STC group. Furthermore, circORC2 was validated to sponge the expression of miR‐19a, and the transfection of circORC2 reduced the expression of miR‐19a. Meanwhile, MLN and NST mRNAs were both targeted by miR‐19a, and the transfection of circORC2 dramatically up‐regulated the expression of MLN and NST. On the contrary, the transfection of circORC2 siRNA into SMCs and VSMCs exhibited the opposite effect of circORC2. Collectively, the results of this study established a regulatory relationship among circORC2, miR‐19a and neurotensin/motilin, which indicated that the overexpression of circORC2 could up‐regulate the levels of neurotensin and motilin, thus exerting a beneficial effect during the treatment of STC.

Abnormal Bile Acid Metabolism is an Important Feature of Gut Microbiota and Fecal Metabolites in Patients with Slow Transit Constipation

Background: Destructions in the intestinal ecosystem are implicated with changes in slow transit constipation (STC), which is a kind of intractable constipation characterized by colonic motility disorder. However, the role of gut microbes in the pathophysiology of STC and deep relationships between the gut microbiota and fecal metabolites have not been understood. Methods: Fecal samples of 30 patients suffering from STC and 30 healthy subjects were performed 16S rDNA amplicon sequencing and metabolomics to obtain evidence of distinct phenotypes of fecal microorganisms and metabolites. The relationship between fecal microorganisms, metabolites, and clinical manifestations was explored according to the correlation analysis. Findings: The α-diversity of the STC group was changed to a certain degree, and the β-diversity was significantly different, which indicated that the composition of the gut microbiota of STC patients was inconsistent with healthy subjects. Different metabolites between STC and control group were involved in the process of bile acids and lipid metabolism. The colon histomorphology of STC patients was significantly disrupted, and TGR5 and FXR were significantly down-regulated. The differences in metabolites were related to changes in the abundance of specific bacteria and patients' intestinal dysfunction. Analysis of the fecal genomics and metabolomics enabled separation of the STC from controls based on random forest model prediction (STC vs control (14 gut microbiota and metabolite biomarker s) – Sensitivity: 1, Specificity: 0.877) Interpretation: Abnorma fecal microorganisms and metabolites related to bile acid metabolism is characteristic of STC, which provided a perspective for the diagnosis and intervention of STC. Funding: This work was supported by the National Key R&D Program of China (2018YFC1706506), Foundation of Tianjin Municipal Health Commission (No. ZC20097), Foundation of Tianjin Union Medical Center (No. 2020YJ017, 2017YJZD005), and National Natural Science Foundation of China (No. 82174374). Declaration of Interest: All authors declare that they do not have any competing interests. Ethical Approval: This study was conducted in Tianjin Union Medical Center, Tianjin, China. The samples and clinical data used in this study were obtained with approval of institutional review boards and under conditions of informed consent (China Clinical Trial Registry, ChiCTR2000033227).

Analysis of Gut Microbiome and Metabolite Characteristics in Patients with Slow Transit Constipation.

Slow transit constipation (STC) is a type of functional constipation in which colon transit time is extended as a result of a reduction in the high amplitude of colon contraction activity. The utility of gut microbiome and metabolite characteristics in patients with STC is rarely studied. Short-chain fatty acids (SCFAs) enhance colonic fluid and sodium absorption and thus may aggravate the symptoms of STC. However, the content and role of SCFAs in constipation patients are not clear. We speculate that gut microbiome and SCFAs in the colon of STC patients may be abnormal and linked to the underlying mechanism of STC. This observational study is registered at ClinicalTrials.gov (NCT02984969). The high-throughput sequencing was used to analyze the diversity and composition of fecal microbial communities. Gas chromatography-mass spectrometry (GC-MS) was used to determine the properties and concentrations of the SCFAs in the two groups. The Shannon diversity and Simpson diversity of the gut microbiome were significantly greater in the STC group than the control group. The two groups also showed significant differences in the species composition of the gut microbiome at different classification levels. The results of GC-MS showed that the acetate concentrations in the STC group were significantly reduced compared with the control group, but the other five types of SCFAs and total SCFAs showed no significant difference between groups. ROC curve analyses revealed that the AUC of Acetate (AUC = 0.758) was higher than Propionate (AUC = 0.660). The largest AUC of gut microbiome for predicting STC was Prevotella (AUC = 0.807). Correlation analysis showed a positive correlation between the concentration of Ruminococcus and Disease history (rs = 0.519). Meanwhile, a positive correlation between the concentration of Roseburia and Acetate (rs = 0.606) or Butyrate (rs = 0.543) was found. We found significant differences between the STC and control groups in the main components of the gut microbiome, with greater diversity in the STC group and differences between the groups in species composition at different classification levels. These different microbiome and metabolite may be valuable biomarkers for STC.

Role of Tenascin-X in regulating TGF-β/Smad signaling pathway in pathogenesis of slow transit constipation.

BACKGROUNDChronic constipation is a gastrointestinal functional disease that seriously harms physical and mental health and impacts the quality of life of patients. Its incidence rate is 2%-27%. Slow transit constipation (STC) is a common type of chronic functional constipation, accounting for 10.3%-45.5% of such cases. Scholars have performed many studies on the pathogenesis of STC. These studies have indicated that the occurrence of STC may be related to multiple factors, such as dysfunction of the enteric nervous system, interstitial cells of Cajal (ICC) damage, and changes in neurotransmitters regulating intestinal peristalsis.AIMTo investigate the role of Tenascin-X (TNX) in regulating the TGF-β/Smad signaling pathway in the pathogenesis of STC.METHODSThis study included an experimental group and a control group. The experimental group included 28 patients with severe colonic STC, and the control group included 18 patients with normal colon tissues. Immunohistochemistry (IHC) was used to detect c-Kit, a specific marker of the ICC. Western blot, immunofluorescence, and IHC were used to detect the localization and expression of TNX and TGF-β/Smad.RESULTSIHC showed that the number of ICC with positive c-Kit expression was significantly reduced in the colon of STC patients (22.17 ± 3.28 vs 28.69 ± 3.53, P < 0.05) and that the distribution was abnormal. Western blot results showed that c-Kit and Smad7 levels were significantly decreased in the colon of STC patients (c-kit: 0.462 ± 0.099 vs 0.783 ± 0.178, P < 0.01; Smad7: 0.626 ± 0.058 vs 0.799 ± 0.03, P < 0.01) and that TNX and Smad2/3 levels were higher in the STC group (TNX: 0.868 ± 0.028 vs 0.482 ± 0.032, P < 0.01). There was no significant difference in TGF-β between the two groups (0.476 ± 0.028 vs 0.511 ± 0.044, P = 0.272). Pearson correlation analysis showed that the TNX protein exhibited a strong correlation with Smad2/3 and Smad7 (P < 0.05, |R| > 0.8) and TGF-β (P < 0.05, |R| = 0.7).CONCLUSIONThe extracellular matrix protein TNX may activate the TGF-β/Smad signaling pathway by upregulating the Smad 2/3 signaling protein and thereby induce slight or complete epithelial stromal cell transformation, leading to an abnormal distribution and dysfunction of ICC in the diseased colon, which promotes the occurrence and development of STC.

Changes of intestinal microbiota in slow transit constipation patients and significance

Objective To explore the correlation between intestinal microbiota and the pathology of slow transit constipation (STC) through an exploratory research of the intestinal micro-ecology transformation in STC patients. Methods The illumina high throughput sequencing method was applied to detect the 16SrDNAV4 in the fecal bacteria. The difference in composition and diversity of the intestinal micro-ecological flora between control group and STC group was analyzed. Results No significant differences on the diversity of the fecal bacteria were discovered. The result indicated that Bacteroidete and firmicutes were the most abundant phylum in control group and STC group, Bacteroidete took 50.84% and 48.22% respected, firmicutes account for 35.68% and 37.63%. Compared to control group, fusobacteria in STC group were significantly lower (P=0.009) and actinobacteria (P=0.020) were significantly higher. Euryarchaeota only could be found in STC group. At the level of genus, the number of the bifidobacterium, coprococcus, escherichia, phascolarctobacterium and ruminococcus in STC group were significant more than the control group. Conclusion The diversity of the fecal bacteria in STC group and control group showed consistency without significant difference, but there was significant difference in the species abundance. Key words: Slow transit constipation; Intestinal microbiota

Effect of bisacodyl on rats with slow transit constipation.

The effect of bisacodyl on the treatment of rats with slow transit constipation (STC) was studied. Forty-five female Wister rats were divided into control group, STC group, and STC bisacodyl group. The immunohistochemical method was used to determine interstitial cells of Cajal (ICC) and the expression of c-Kit protein. Body mass and the number of defecations were significantly decreased in the STC group compared with the control group on the 100th day after diphenoxylate administration, while dry weight of feces was significantly increased and the intestinal transit time was prolonged. There were significant differences in the number of defecations, dry weight of feces, and intestinal transit time among the three groups. The number of defecations was higher, dry weight of feces was lower, and intestinal transit time was shorter in the STC bisacodyl group compared to the STC group. In addition, ICC basement membrane dissolution occurred in the colon wall of the STC group. The connection between ICC and surrounding cells was destroyed, and the nucleus shrunken to different degrees. Moreover, c-Kit expression in the STC group was significantly lower than the control group. The connection between ICC and surrounding cells in the STC bisacodyl group was significantly stronger than the STC group, and the number of ICC and the expression of c-Kit were increased. Bisacodyl could reduce the severity of STC in rats by increasing the number of ICC and the expression of c-Kit.

Effects of glial cell derived neurotrophic factor on colon glial cells in rats with slow transit constipation

Objective To investigate the effects of exogenous glial cell derived neurotrophic factor (GDNF) on colon glial cells in slow transit constipation (STC) rats, and to explore the optimal concentration of GDNF in order to provide evidence for intestinal neurotrophic therapy in the treatment of STC. Methods A total of 132 SD rats were divided into STC group and control group, 66 rats in each group. STC rats were established by feeding with rhubarb. Six rats were randomly selected from either groups to verify whether STC model was successfully established.And the left 120 rats of two groups were randomly divided into six subgroups: STC group one to group six and control group one to group six, ten rats in each group, which were untreated, injected through tail vein with saline, and 0.001, 0.010, 0.050, 0.100 μg/L GDNF 2 mL respectively for one week. The expression of Sox-8 at protein level of either group were detected by Western blotting. Independent sample t test was performed for statistical analysis. Results After treated with 0.001 μg/L GDNF (STC group three), there was no significant difference in expression level of Sox-8 between STC group three and STC group one (13.38±0.70 vs 13.39±0.45, t=0.042, P=0.969). After treated with 0.010 μg/L GDNF (STC group four), the difference in expression level of Sox-8 between STC group four and STC group three was significant (21.11±2.56 vs 13.38±0.70, t=5.040, P<0.01). After treated with 0.050 μg/mL GDNF (STC group five), the expression level of Sox-8 was higher than that in STC group four (31.86±1.57 vs 21.11±2.56, t=-6.198, P<0.01). The Sox-8 expression of untreated, saline treated, 0.001 and 0.050 μg/L GDNF treated STC rats (STC group one, two, three and five) were lower than those of the corresponding control groups (t=3.394, 12.103, 10.302, -6.120, all P<0.05). Conclusion Exogenous GDNF could increase Sox-8 expression in colon tissue of STC rats, an increase in the number of colon glial cells could repair enteric nervous system, and 0.050 μg/L was the optimal concentration. Key words: Slow transit constipation; Glial cell line-derived neurotrophic factor; Colon glial cell; Sox-8; Rats

Bone morphogenetic protein 2 regulates the differentiation of nitrergic enteric neurons by modulating Smad1 signaling in slow transit constipation.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor superfamily and have been implicated in chondrogenesis and neuronal differentiation. In order to examine the function of bone morphogenetic protein 2 (BMP-2) on the differentiation of nitrergic enteric neurons in slow transit constipation (STC), the expression of BMP-2 and neuronal nitric oxide synthase (nNOS) was investigated in the myenteric nerve plexus in STC and control tissues by immunohistochemical assays. The present study demonstrated that BMP-2 and nNOS were expressed in the myenteric nerve plexus and their levels were differentially altered in the STC group and control group. In addition, the effect of BMP-2 on primary myenteric neurons was investigated by measuring the neurite length. The results demonstrated that BMP-2 regulated the differentiation of primary enteric neurons and increased the length of neurites compared with the control group. In addition, the effect of BMP-2 on the expression of nNOS was also investigated in primary enteric neurons and the Smad1 signal transduction pathway by western blot analysis, reverse transcription quantitative polymerase chain reaction and immunofluorescence assay. The results suggested that BMP-2 promoted the expression of nNOS in primary myenteric neurons and induced phosphorylation of Smad1. These data indicate a new role for BMP-2 as an important transcriptional cofactor that regulates the differentiation of nitrergic enteric neurons through the Smad1 pathway. Intervention of BMP-2 may be useful for the treatment of STC.

Expression of transit receptor potential ankyrin 1 and 5-hydroxytryptamine in the colonic mucosa of patients with slow transit constipation

Objective To investigate the expression of transit receptor potential ankyrin 1 (TRPA1) and 5-hydroxytryptamine (5-HT) in the colonic mucosa of patients with slow transit constipation (STC). Methods The clinical data of 10 patients with STC and 10 patients with sigmoid cancer who were respectively admitted to the Daping Hospital of the Third Military Medical University from March 2013 to March 2014 and from December 2013 to May 2014 were retrospectively analyzed. Ten and 10 surgical specimens of descending colon from patients with STC and sigmoid cancer (tumor edge measuring in controls >10 cm) were allocated into the STC group and the control group, respectively. The expressions of TRPA1 and 5-HT in the colonic mucosa in the control group and in the 2 groups were detected by immunofluorescence and immunochemistry staining, respectively. The expression of TRPA1 in the colonic mucosa was observed by Western blot test. The measurement data with normal distribution was presented as ±s, and the comparison between groups were analyzed using the t test. Results The results of immunofluorescence showed that TRPA1 and 5-HT were co-expressed in the colonic mucosa of controls. The results of immunochemistry staining showed that the integrated optical density (IOD) of expressions of TRPA1 and 5-HT in the STC group and in the control group were 3 619 ±1 702, 55 721 ±28 613 and 9 894 ± 3 325, 12 949 ± 2 200, respectively, showing significant differences between the 2 groups (t =-5.312, 4.713, P <0.05). The results of Western blot test showed that the relative expressions of TRPA1 in the STC group and in the control group were 0.8 ± 0.3 and 1.5 ± 0.5, showing a significant difference between the 2 groups (t =-2.379, P <0.05). Conclusions TRPA1 is expressed in the enterochromaffin (EC) cells of colonic mucosa. High expression of 5-HT and low expression of TRPA1 in the colonic mucosa are closely related with the pathogenesis of STC. Key words: Slow transit constipation; Colonic mucosa; Transit receptor potential ankyrin 1; 5-hydroxytryptamine

Decreased enteric fatty acid amide hydrolase activity is associated with colonic inertia in slow transit constipation

Constipation is one of the most common chronic digestive complaints. Gastrointestinal transit studies have divided it into three patterns: normal transit, slow transit constipation (STC), and outlet obstruction. It has been demonstrated that STC patients respond poorly to standard therapies, and the etiology of STC remains poorly understood. Animal studies have also shown that fatty acid amide hydrolase (FAAH) controls intestinal motility through its putative receptors or non-receptor-mediated pathways. However, the role of FAAH in STC has not been elaborated. A case series was carried out on thirty-two STC patients fulfilling the Rome II criteria and on 24 controls. All of the subjects underwent a laparotomy in Shengjing Hospital. Colonic specimens were obtained and used for FAAH expression analysis, enzyme activity assay, and cannabinoid detection. FAAH immunoreactivity occurred in the enteric neurons and in the surface epithelial and glands. The expression level and enzyme activity of FAAH in the STC group were both significantly lower than those in the control group (P < 0.05). The amounts of anandamide, 2-arachidonylglycerol, and palmitoylethanolamide, which are negatively correlated with enzyme activity, were significantly higher in the constipation group than that in the control group. In the STC group, cannabinoid receptor type 1 immunoreactivity occurred predominantly in the submucosal and myenteric fibers that were obviously strong and wave-like in their appearance. Enteric ganglions decreased or disappeared. The tone of the enteric cannabinoids system is disturbed in STC, and the decreased enteric FAAH activity contributes to colonic inertia in STC.

Function of colonic mucosal barrier of patients with colonic slow transit constipation

Objective To explore the function of colonic mucosal barrier of patients with colonic slow transit constipation (STC).Methods From June 2008 to June 2012,a total of 136 patients with STC were enrolled.Among them,course of disease of 55 cases was between one and six years,of 43 cases was between six and 10 years,and of 38 cases was over 10 years.The colonic transit time of 66 cases was between three and five days,of 42 cases was between five and seven days,and of 28 cases was over seven days.Altogether,35 cases received subtotal resection of the colon.At same time,individuals who received partial resection of the colon because of single polypus were set as control group.Fasting blood and urine samples of all subjects were collected.The colonic specimens of STC patients who received surgery and control group were harvested.The urinary lactulose and mannitol ratio (L/M) was detected by high performance liquid chromatography (HPLC).The level of blood Dlactic acid (D-LAC) was tested by enzymatic spectrophotometric.The level of blood diamine oxidase (DAO) was determined by speetrophotometry.The level of endotoxin (ET) was detected by azo chromogenic substrate limulus test.The colonic epithelial cells membrane resistance (TER) and paracellular mannitol permeability (PMP) were measured with Ussing perfusion chamber.t-test was performed for comparison between groups.Results Urinary L/M of STC group and control group was 0.16±0.03 and 0.10±0.02,respectively.The level of blood D-LAC was (1.81±0.19) and (1.04±0.13) mmol/L.The level of blood DAO was (17.07±1.81) and (9.78±1.14) U/L.The level of blood ET was (64.20±6.85) and (51.30±5.90) EU/L.The TER of colonic epithelia cell was (61.23±7.76) and (75.87±9.65) Ω/cm2.The PMP of colonic epithelia cell was (3.17±0.35) ％ and (2.14 ±0.22)％.All the differences were statistically significant (t =3.185,3.378,3.863,3.201,3.125 and 3.543,all P＜0.05).Among patients with disease course between one and six years,six to 10 years and over 10 years,colonic transit time of STC between three and five days,five to seven days and over seven days,urinary L/M,blood D-LAC level and blood DAO level increased along with the disease course and colonic transit time and the differences were statistical significantly compared with control group (urinary L/M:t=1.993,2.311,2.356,2.204,2.347 and 3.673; blood D-LAC level:t=2.023,2.886,4.124,1.999,2.998 and 3.465; blood DAO level:t=1.994,2.995,4.423,2.203,3.673 and 5.211; all P＜0.05).Compared with control group,there were significant differences in blood ET level of course of STC between six and 10 years,over 10 years,colonic transit time of STC between five and seven days and over seven days (t=2.121,4.245,3.241 and 4.657,all P＜0.05).Conclusion The permeability of colonic mucosal barrier increased and which was more significant in longer colonic transit time and long course of disease. Key words: Constipation; Colonic diseases; Intestinal mucosa

Alterations of Cajal cells in the colon of slow transit constipation rats

To investigate the association of expression of c-kit (marker of interstitial cells of Cajal, ICC) in colon with slow transit constipation (STC) in rats. Slow transit constipation (STC) rat model was induced by intragastric administration of compound diphenoxylate. Western blotting was used to measure the expression of c-kit in colon of STC rats (model group) and normal rats (control group). Gray scale ratio of c-kit to β-actin was used as the relative quantity of c-kit. Fecal quantity per day of STC group was (1.3±0.7) g/100 g, significantly lower than that in normal rats [(1.6±0.9) g/100 g, t=10.798, P<0.05]. In model rats, the time of discharge of the first black fecal was (461.6±150.8) min, significantly longer than that in normal rats [(351.3±119.9) min, t=2.291, P<0.05]. Western blotting revealed that the average values of gray scale ratio of c-kit in proximal colon were 0.277±0.077 and 0.576±0.081 (t=10.719, P<0.05), in distal colon were 0.280±0.075 and 0.571±0.079 (t=10.700, P<0.05) in model group and control group respectively. Down-regulation of c-kit expression in proximal colon and distal colon is associated to the pathogenesis of slow transit constipation in rats.