Pathogenesis Of Slow Transit Constipation Research Articles

Electroacupuncture Improves Intestinal Motility through Exosomal miR-34c-5p Targeting SCF/c-Kit Signaling Pathway in Slow Transit Constipation Model Rats

Background The pathogenesis of slow transit constipation (STC) is associated with exosomal miR-34c-5p. Electroacupuncture (EA) improves gastrointestinal motility in gastrointestinal disorders, especially STC. Our study aimed to explore the mechanism by which EA improves intestinal motility by modulating the release of exosomes and the transmission of exosomal miR-34c-5p. Methods Fifty rats were randomly divided into five groups. STC model rats were induced, and GW4869, the exosome release inhibitor, was used to inhibit the release of exosome. The serum exosomes were authenticated under a transmission electron microscope and nanoparticle tracking analysis. RT-qPCR detected the expression of miR-34c-5p in serum exosomes and colonic tissues. The fecal number in 24 hours, Bristol scores, and intestinal transit rates were used to assess intestinal motility. Subsequently, hematoxylin and eosin (H&E) staining was used to examine the colonic mucosal histology. Finally, the expression of stem cell factor (SCF) and receptor tyrosine kinase (c-Kit) protein was measured using immunohistochemistry staining. Results We found that EA upregulated exosomal miR-34c-5p in serum and downregulated miR-34c-5p in colonic tissues (P < 0.01). EA improved fecal numbers in 24 hours, Bristol scores, and intestinal transit rates in STC rats (P < 0.01). EA recovered the colonic histological structure and enhanced the expression of SCF and c-Kit protein (P < 0.01). The therapeutic effect of EA was attenuated after inhibiting the release of the exosome. Conclusion Our results indicated that EA improves intestinal motility in STC rats by transporting of exosomal miR-34c-5p targeting the SCF/c-Kit signaling pathway.

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.

M1 macrophages-derived exosomes miR-34c-5p regulates interstitial cells of Cajal through targeting SCF

Slow transit constipation (STC) is a gastrointestinal disorder characterized by abnormal prolonged colonic transit time, which affects the life quality of many people. The decrease number of interstitial cells of Cajal (ICCs) is involved in the pathogenesis of STC. However, the molecular mechanism of loss of ICCs in STC remains unclear, making it difficult to develop new agents for the disease. In this study, we investigated the mechanism of decreasing ICCs in the pathogenesis of STC. We constructed the STC model rats by using atropine and diphenoxylate. A series of methods were used including immunofluorescence and immunochemistry staining, western blot, qRT-PCR, exosomes extraction and exosomes labeling. The results indicate that ICCs decreased in the STC rats accompanied with the macrophages activation. Further studies suggested that macrophages decreased the cell viability of ICCs by secretion exosomes containing miR-34c-5p. miR-34c5p targeted the 3Ꞌ -UTR of stem cell factor(SCF) mRNA and regulated the expression of SCF negatively. In conclusion, we demonstrated a novel regulatory mechanism of ICCs cell viability in STC. We found that exosome miR-34c-5p mediate macrophage-ICCs cross-talk. M1 macrophages derived exosomes miR-34c-5p decreased ICCs cell viability by directly targeting SCF.

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.

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.

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

The Relationship Between Colonic Macrophages and MicroRNA-128 in the Pathogenesis of Slow Transit Constipation.

Recent evidence suggests that colonic macrophages and microRNAs play important roles in motor activity in the gastrointestinal tract. However, there are almost no data concerning colonic macrophages and microRNAs in slow transit constipation. The purpose of this study was to investigate colonic macrophages and microRNA-128 expression in the pathogenesis of slow transit constipation in colon tissues. Full-thickness colonic specimens from patients undergoing surgery for slow transit constipation, due to refractoriness to other therapeutic interventions (n=25), were compared to controls (n=25), and the number of colonic macrophages (as evaluated by specific monoclonal antibodies) was counted. Gene expression analysis of microRNA-128 was performed by microRNA microarray and qRT-PCR. Lastly, bioinformatics analysis, coupled with luciferase reporter assays, was used to investigate the mRNA transcript(s) targeted by microRNA-128. Compared to controls, 20 of 25 slow transit constipation patients (80%) had significantly higher numbers of macrophages in colonic specimens, coupled with down-regulation of microRNA-128. Linear regression analyses showed a significant negative correlation between macrophage number and microRNA-128 expression level. Among 83 bioinformatically predicated candidates, mitogen-activated protein kinase 14 (p38α) was validated to be a direct target of microRNA-128 in human intestinal epithelial cells. This study presents evidence for the negative correlation of macrophage number and microRNA-128 expression, in slow transit constipation patients, representing a possible mechanism of impaired gastrointestinal motility.

Opioid receptors and associated regulator of G protein signaling are involved in the cathartic colon of rats.

A cathartic colon is characteristic of slow transit constipation (STC), which can result following the long-term use of irritant laxatives. In the present study, the involvement of three opioid receptor subtypes (μ, MOR; δ, DOR; and κ, KOR), regulator of G protein signaling 4 (RGS-4) and β-arrestin-2 were investigated in the cathartic colon of rats. A rat model of a cathartic colon was established by feeding the animals with phenolphthalein, while normal rats were used as a control. The mRNA and protein expression levels of the opioid receptors, RGS-4 and β-arrestin-2 were detected in the rat colon using semi-quantitative reverse transcription polymerase chain reaction and western blot analysis, respectively. The rat model of a cathartic colon was successfully established using the phenolphthalein stimulus, and was shown to result in shrunken myenteric neurons and loose muscle fibers in the intestinal wall. The mRNA and protein expression levels of the three opioid receptor subtypes, RGS-4 and β-arrestin-2 were significantly higher in the cathartic colon group when compared with the levels in the normal control group (all P<0.01). With regard to the protein expression levels, MOR protein increased 2.4 fold, DOR expression increased 1.5 fold, KOR levels increased 1.5 fold, RGS-4 protein increased 3.5 fold and β-arrestin-2 expression increased 2.0 fold. Therefore, the expression levels of opioid receptors were found to increase in the cathartic colons of the rats, indicating that opioid receptors and downstream RGS-4 and β-arrestin-2 signaling may play an important role in the pathogenesis of STC.

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.

Expression and activity of fatty acid amide hydrolase in the colon in children with slow transit constipation

To investigate the expression and activity of fatty acid amide hydrolase (FAAH) in the colon and its role in children with slow transit constipation (STC). Patients were divided into constipation group (n=21) and control group (n=15). The constipation group was consistent with the diagnostic criteria for STC. Western blotting, immunohistochemistry and real-time PCR were used to examine the FAAH expression in surgical specimen of colon. The location and distribution of FAAH and cannabinoid receptor type 1 (CB1) were detected by immunofluorescence double staining. The biological activity of colon FAAH was detected by high-performance liquid chromatography. Western blotting revealed that FAAH protein expression in the ascending colon, descending colon and sigmoid colon were significantly decreased in the myenteric neurons and absorption cells in the constipation group as compared with the control group (8.68+/-3.4 vs 10.47+/-3.7, 8.21+/-1.2 vs 9.95+/-6.4, 8.01+/-7.2 vs 9.79+/-3.4, all P<0.05). The same results were found by immunohistochemistry and real-time PCR. The FAAH hydrolysis activity in the ascending colon, descending colon or sigmoid colon decreased significantly in the constipation group, as compared with the control group [(0.51+/-0.23) nmol x min(-1) x mg(-1) vs (0.84+/-0.24) nmol x min(-1) x mg(-1), (0.39+/-0.25) nmol x min(-1) x mg(-1) vs (0.55+/-0.44) nmol x min(-1) x mg(-1), (0.35+/-0.37) nmol x min(-1) x mg(-1) vs (0.58+/-0.48) nmol x min(-1) x mg(-1), all P<0.05]. FAAH expression and hydrolysis activity in the colon decrease in children with STC. FAAH may play a role in the pathogenesis of slow transit constipation in Children.

Proteomic analysis of down-regulated proteins in colonic mucosa of chronic slow transit constipation rats

Objective To investigate the alternations of proteins in the colonic mucosa of chronic slow transit constipation (STC) rats with a 2-DE-based proteomic method and analyze the function of these down-regulated proteins so as to provide theoretical basis for the pathogenesis of intestinal mucosa of chronic STC rats. Methods STC model was established by feeding rats with 8 mg/(kg·d) diphenoxylate for 120 d. An experimental model of chronic STC rat was used for separation of proteomics from colonic mucosa using two-dimensional electrophoresis (2-DE). Proteins altered in expressional level were identified by Image Master 2DElite, mass spectrometry, and bibliometrics were applied to identify the differential protein expression and their clinical significance and function were analyzed. Results Obvious differential protein expression was observed in the pathogenesis of STC, including mast cell protease (A1), non-specific dipeptidase (A2) and chondrosome succinate dehydrogenase precursor (A3). The expressions of A1, A2 and A3 were down-regulated in the gel graph of STC rats. Conclusion The down-regulation of chondrosome succinate dehydrogenase, mast cell protease as well as non-specific dipeptidase in rat colon suggests the functional impairment of the oxidoreduction of mitochondrion is very important in the genesis and development of STC. The immunological reaction of STC rats is weakened, and the function of digesting and absorbing protein may be damaged to some extent.

Chromosomal study of enteric glial cells and neurons by fluorescence in situ hybridization in slow transit constipation

The pathogenesis of slow transit constipation is still elusive. However, a genetic basis may be present. We investigated possible chromosomal abnormalities in enteric neurons and glial cells in patients with slow transit constipation. Colonic specimens from 22 patients with slow transit constipation undergoing surgery for intractable symptoms were obtained, and investigated by fluorescence in situ hybridization (FISH) for chromosomal abnormalities (chromosomes 1, 8, 17 and XY). These specimens were compared with of those obtained in 12 control subjects. Data analysis showed that 45.5% of patients displayed significant (>10%) aneusomy of chromosome 1 in enteric neurons. Aneusomy <10% for the same chromosome, but less than the cutoff suggested (10%), was found in enteric glial cells in 45.4% of the same patients. One patient had <10% aneusomy in enteric neurons for chromosome 8. No other abnormalities were found for the remaining probes, and no abnormalities were found in controls. We concluded that in a subgroup of patients with slow transit constipation a genetic basis may be present.

Is constipation caused by a loss of colonic interstitial cells of Cajal?

Is constipation caused by a loss of colonic interstitial cells of Cajal?

Are colonic regular contractile frequency patterns in slow transit constipation a relevant pathophysiological phenomenon?

Pathogenesis of slow transit constipation still remains elusive. Some studies have shown several colonic motor abnormalities; however, it is not easy to understand the relative importance of the single ones. Since it has been hypothesized that an excess of periodic distal motor activity may be of pathophysiological importance in patients with slow transit constipation, we evaluated regular colonic contractile frequencies in a homogeneous cohort of these patients. A total of 26 female patients (age range 34 to 67 years) fulfilling the Rome II criteria for constipation entered the study. No patient had evidence of secondary forms of constipation and distal obstruction. Twenty-four hour colonic manometric studies were obtained for each patient. Regular contractile patterns (with frequencies ranging from 2 to 8 cycles/min) were calculated for the entire recording period and in single colonic segments. Overall, regular patterns accounted for about 3% of the total colonic motor activity (average 30 min/day per subject), with the 3 cycles/min being the predominant contractile rhythm. Most of this activity was present in the sigmoid colon, accounting for >50% of the total amount of motility, and it was more prevalent than in the descending and transverse colon; no differences were revealed in the descending with respect to the transverse colon. No daily fluctuations of regular contractile activity, nor a cyclic pattern, nor migration between recording points were observed. Regular colonic frequency patterns are probably of minor pathophysiological importance in slow transit constipation, even in the light of the scant amount of such phenomena previously documented in healthy subjects.

Slow-transit constipation: solitary symptom of a systemic gastrointestinal disease.

Autonomic neuropathy is thought to play a role in the pathogenesis of slow-transit constipation, but other gastrointestinal organs may also be involved, even if they are symptom-free. We investigated whether motility in gastrointestinal organs other than the colon was impaired in patients with slow-transit constipation and whether the autonomic nervous system was involved. Twenty-one consecutive patients (18 females; median age, 46 years) with severe chronic constipation (< or = 2 defecations/week and delayed colonic transit time) were studied. Autonomic neuropathy function was tested with esophageal manometry, gastric and gallbladder emptying (fasting and postprandial motility) by ultrasonography, orocecal transit time (H2-breath test), colonic transit time (radiopaque markers), and anorectal volumetric manometry. The integrity of the autonomic nervous system was assessed by a quantitative sweat-spot test for preganglionic and postganglionic fibers, tilt-table test, and Valsalva electrocardiogram R-R ratio. Esophageal manometry showed gastroesophageal reflux or absence of peristalsis in five of the seven patients examined. Gallbladder dysmotility (i.e., increased fasting, postprandial residual volume, or both) was observed in 6 of 14 (43 percent) patients. Gastric emptying was decreased in 13 of 17 (76 percent) patients. Orocecal transit time was delayed in 18 of 20 (90 percent) patients; median transit time was 160 (range, 90-200) minutes. Median colonic transit time was 97 (range, 64-140) hours. Anorectal function showed abnormal rectoanal inhibitory reflex and decreased rectal sensitivity in 11 of 19 (58 percent) patients. Signs of autonomic neuropathy of the sympathetic cholinergic system were found in 14 of 18 (78 percent) patients. Only one of nine patients had vagal abnormalities detected with the Valsalva test and four of five patients with a history of orthostatic hypotension had a positive tilt-table test. Slow-transit constipation may be associated with impaired function of other gastrointestinal organs. More than 70 percent of patients with slow-transit constipation present some degree of autonomic neuropathy. Severe constipation may be the main complaint in patients with a systemic disease involving several organs and possibly involving the autonomic nervous system. This should be considered in the management of such cases.