Slow Transit Constipation Model Research Articles

Ameliorative effects of the mixed aqueous extract of Aurantii Fructus Immaturus and Magnoliae Officinalis Cortex on loperamide-induced STC mice

Aurantii fructus immaturus (AFI) and Magnoliae Officinalis Cortex (MOC) have been used to treat constipation in China for thousands of years. In this study, a mouse model of slow transit constipation (STC) was established by gavage of loperamide at a dose of 10 mg/kg bw/day for seven days. Seventy-two mice were randomly allocated to six groups (control, STC model, 3 g/kg AFI + MOC, 6 g/kg AFI + MOC, 12 g/kg AFI + MOC, and mosapride). A mixed aqueous extract of AFI and MOC was administered to the STC mice at the corresponding doses from the first day of modelling. Body weight, faecal water content, gastrointestinal transit time, and intestinal propulsion rate were evaluated. Serum levels of neurotransmitters and gastrointestinal hormones, colonic expression of aquaporins (AQP), and interstitial cells of Cajal (ICC) were assessed using ELISA, immunohistochemistry, and Western blot analysis. The abundance and diversity of the gut microbiota were analysed by 16S rRNA gene sequencing. The mixed aqueous extract significantly increased faecal water content and intestinal propulsion rate and shortened gastrointestinal transit time in STC mice. Furthermore, the administration of AFI and MOC significantly decreased serum vasoactive intestinal peptide (VIP), nitric oxide (NO), and somatostatin (SS) levels and increased serum motilin (MTL) levels in STC mice. The protein expression levels of AQP3 and AQP4 in the colon tissue of STC mice significantly decreased following AFI + MOC treatment, whereas those of AQP9 significantly increased. Moreover, the AFI + MOC treatment led to an increase in the number and functionality of ICCs. In addition, the relative abundances of Ruminococcus and Oscillospira increased in response to the administration of AFI + MOC in STC mice. In conclusion, the mixed aqueous extract of AFI and MOC promoted defaecation and increased intestinal mobility in STC mice. Its mechanisms of action involve modulatory effects on neurotransmitters, gastrointestinal hormones, AQPs, and ICCs. AFI + MOC treatment also improved the diversity and abundance of the gut microbiota in STC mice, particularly short-chain fatty acid-producing bacteria, which may play an important role in its beneficial effect on constipation.

Effects and mechanisms of sciadonic acid on colonic transit function through regulating 5-HT4/cAMP/PKA/AQP4 signaling pathway in STC model mice

Torreya grandis (T. grandis) oil has been reported to alleviate symptoms of slow transit constipation (STC). However, the impact of sciadonic acid (SA), a distinctive fatty acid found in T. grandis oil, on the pathological progression of STC remains unclear. This study aimed to evaluate the effect of SA on STC and uncover the underlying mechanisms. The STC model was established by feeding Balb/c mice with loperamide. After 2 weeks of intervention, SA significantly improved weight loss and intestinal motility decline induced by STC, along with enhancing plasma indices and reducing colon pathological damage. SA effectively reversed the STC-induced decrease in the 5-HT4/cAMP/PKA/AQP4 signaling pathway genes and expression. Furthermore, 16S rRNA analysis demonstrated that SA mitigated the imbalance of the intestinal microbiota induced by STC, by reducing the ratio of Firmicutes to Bacteroidetes (F/B) and increasing the abundance of beneficial bacteria such as Akkermansia. In conclusion, SA intervention alleviated colonic dysfunction in STC mice. The activation of the SA-mediated 5-HT4/cAMP/PKA/AQP4 signaling pathway may serve as a potential target for STC treatment. These findings suggest that SA holds promise as a treatment option for STC and could potentially be extended to other related gut diseases for further investigation.

Effects of Liqi Tongbian decoction on gut microbiota, SCFAs production, and 5-HT pathway in STC rats with Qi Stagnation Pattern.

Slow transit constipation (STC) is a common and debilitating condition characterized by delayed colonic transit and difficulty in fecal expulsion, significantly impacting patients' physical and mental wellbeing as well as their overall quality of life. This study investigates the therapeutic potential of Liqi Tongbian Decoction (LTD) in the treatment of STC, especially in cases involving the context of Qi stagnation, through a multifaceted approach involving the modulation of intestinal flora and short-chain fatty acids (SCFAs). We employed a rat model of STC with Qi Stagnation Pattern, established using the "loperamide + tail-clamping provocation method," to explore the effects of LTD on fecal characteristics, intestinal motility, and colonic pathology. Importantly, LTD exhibited the ability to increase the richness, diversity, and homogeneity of intestinal flora while also modulating the composition of microorganisms. It significantly increased the production of SCFAs, especially butyric acid. Moreover, LTD exerted a substantial influence on the synthesis of serotonin (5-HT) by modulating the expression of tryptophan hydroxylase (TPH) and interacting with the 5-HT4 receptor (5-HT4R), resulting in enhanced colonic motility. Correlation analyses revealed a positive correlation between certain bacterial genera, such as Lachnospiraceae_NK4A136 spp. and Clostridiales spp. and the concentrations of butyric acid and 5-HT. These results suggest a mechanistic link between microbiome composition, SCFAs production, and 5-HT synthesis. These findings highlight the potential of LTD to alleviate STC by facilitating a beneficial interplay among intestinal flora, SCFAs production, and 5-HT-mediated colonic motility, providing novel insights into the management of STC with Qi Stagnation Pattern.

Single-cell RNA sequencing reveals cellular dynamics and therapeutic effects of astragaloside IV in slow transit constipation.

The cellular characteristics of intestinal cells involved in the therapeutic effects of astragaloside IV (AS-IV) for treating slow transit constipation (STC) remain unclear. This study aimed to determine the dynamics of colon tissue cells in the STC model and investigate the effects of AS-IV treatment by single-cell RNA sequencing (scRNA-seq). STC mouse models were developed using loperamide, with subsequent treatment using AS-IV. Colon tissues and feces were collected for scRNA-seq and targeted short-chain fatty acid quantification. We integrated scRNA-seq data with network pharmacology to analyze the effect of AS-IV on constipation. AS-IV showed improvement in defecation for STC mice induced by loperamide. Notably, in STC mice, epithelial cells, T cells, B cells, and fibroblasts demonstrated alterations in cell proportions and aberrant functions, which AS-IV partially rectified. AS-IV has the potential to modulate the metabolic pathway of epithelial cells through its interaction with peroxisome proliferator-activated receptor gamma (PPARγ). AS-IV reinstated fecal butyrate levels and improved energy metabolism in epithelial cells. The proportion of naïve CD4+T cells is elevated in STC, and the differentiation of these cells into regulatory T cells (Treg) is regulated by B cells and fibroblasts through the interaction of ligand-receptor pairs. AS-IV treatment can partially alleviate this trend. The status of fibroblasts in STC undergoes alterations, and the FB_C4_Adamdec1 subset, associated with angiogenesis and the Wingless-related integration (Wnt) pathway, emerges. Our comprehensive analysis identifies perturbations of epithelial cells and tissue microenvironment cells in STC and elucidates mechanisms underlying the therapeutic efficacy of AS-IV.

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.

Effects of electroacupuncture at "Zhongliao" and "Xialiao" on serotonin signaling system and short-chain fatty acids in slow transit constipation rats

To observe the effect of electroacupuncture (EA) at "Zhongliao" (BL33) and "Xialiao" (BL34) on the 5-hydroxytryptamine (5-HT) signaling system in colon tissue and short-chain fatty acids in feces of rats with slow transit constipation (STC), so as to explore the underlying mechanisms of EA in the treatment of STC. A total of 32 SD rats were randomly divided into normal, model, drug control and EA groups, with 8 rats in each group. The STC model was established by intragastric administration of loperamide for 14 days. The EA stimulation (2 Hz/15 Hz) was performed at bilateral BL33 and BL34 for 30 min, once a day for 14 days. The first black stool de-fecation time and fecal water content were detected after treatment. The expressions of 5-hydroxytryptamine 4 receptor (5-HT4R), tryptophan hydroxylase 1 (TPH1) and 5-HT transporter (SERT) in colon tissues were detected by Western blot. The contents of substance P (SP) and vasoactive intestinal peptide (VIP) in serum were detected by ELISA. The contents of 5-HT in colon tissue and short chain fatty acid (SCFA) in feces were detected by mass spectrometry. Compared with the normal group, the fecal water content, the expressions of 5-HT, 5-HT4R, TPH1 and SERT in colon tissue, the content of serum SP were significantly decreased (P<0.05), the first black stool de-fecation time, and the content of serum VIP was significantly increased (P<0.05), the contents of SCFA in feces were significantly decreased except isobutyric acid (P<0.05) in the model group. Compared with the model group, the fecal water content, the expressions of 5-HT, 5-HT4R, TPH1 and SERT in colon tissues, the contents of acetic acid and butyrate in feces were significantly increased (P<0.05) in the EA and drug control groups, the first black stool defecation time was decreased (P<0.05) in the EA and drug control groups, and the content of serum SP was increased and the content of serum VIP was decreased (P<0.05) in the EA group. Compared with the drug control group, the content of serum VIP was significantly decreased (P<0.05), and the expressions of TPH1 and SERT in colon tissue were significantly increased (P<0.05) in the EA group. EA at BL33 and BL34 can promote intestinal motility by intervening multiple links of 5-HT signaling system in treating STC.

Zhizhu decoction alleviates slow transit constipation by regulating aryl hydrocarbon receptor through gut microbiota

Context Slow transit constipation (STC), the most common type of constipation, seriously affects the life of patients. Zhizhu decoction (ZZD), a traditional Chinese medicine compound, has is effective against functional constipation, but the mechanism is still unclear. Objective This research explores the mechanism of ZZD on STC from the perspective of metabolomics and gut microbiota. Materials and methods Fifty-four C57BL/6 mice were randomly divided into six groups (n = 9): control (control); STC (model); positive control (positive); low-dose (5 g/kg; L-ZZD), medium-dose (10 g/kg; M-ZZD), and high-dose (20 g/kg; H-ZZD) ZZD treatment. Following treatment of mice with ZZD for two weeks, the changes in intestinal motility, colon histology, intestinal neurotransmitters, and aryl hydrocarbon receptor (AHR) pathway determined the effects of ZZD on the pathophysiology of STC. LC-MS targeting serum metabolomics was used to analyze the regulation of ZZD on neurotransmitters, and 16S rRNA high-throughput sequencing was used to detect the regulation of the gut microbiome. Results ZZD had the highest content of naringin (6348.1 mg/L), and could significantly increase the 24 h defecations (1.10- to 1.42-fold), fecal moisture (1.14-fold) and intestinal transport rate (1.28-fold) of STC mice, increased the thickness of the mucosal and muscular tissue (1.18- to 2.16-fold) and regulated the neurotransmitters in the colon of STC mice. Moreover, ZZD significantly activated the AHR signaling pathway, and also affected the composition of gut microbiota in STC mice. Discussion and conclusions The beneficial effect and the possible mechanism of ZZD on STC could provide a theoretical basis for the broader clinical application of ZZD.

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.

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.

Zhizhu Decoction Alleviates Intestinal Barrier Damage via Regulating SIRT1/FoxO1 Signaling Pathway in Slow Transit Constipation Model Mice.

To explore the possible effects and mechanism of Zhizhu Decoction (ZZD) on the pathophysiology of slow transit constipation (STC). A total of 54 C57BL/6 mice was randomly divided into the following 6 groups by a random number table, including control, STC model (model), positive control, and low-, medium- and high-doses ZZD treatment groups (5, 10, 20 g/kg, namely L, M-, and H-ZZD, respectively), 9 mice in each group. Following 2-week treatment, intestinal transport rate (ITR) and fecal water content were determined, and blood and colon tissue samples were collected. Hematoxylin-eosin and periodic acid-Schiff staining were performed to evaluate the morphology of colon tissues and calculate the number of goblet cells. To determine intestinal permeability, serum levels of lipopolysaccharide (LPS), low-density lipoprotein (LDL) and mannose were measured using enzyme-linked immunosorbent assay (ELISA). Western blot analysis was carried out to detect the expression levels of intestinal tight junction proteins zona-occludens-1 (ZO-1), claudin-1, occludin and recombinant mucin 2 (MUC2). The mRNA expression levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-4, IL-10 and IL-22 were determined using reverse transcription-quantitative reverse transcription reaction. Colon indexes of oxidative stress were measured by ELISA, and protein expression levels of colon silent information regulator 1/forkhead box O transcription factor 1 (SIRT1/FoxO1) antioxidant signaling pathway were detected by Western blot. Compared with the model group, ITR and fecal moisture were significantly enhanced in STC mice in the M-ZZD and H-ZZD groups (P<0.01). Additionally, ZZD treatment notably increased the thickness of mucosal and muscular tissue, elevated the number of goblet cells in the colon of STC mice, reduced the secretion levels of LPS, LDL and mannose, and upregulated ZO-1, claudin-1, occludin and MUC2 expressions in the colon in a dose-dependent manner, compared with the model group (P<0.05 or P<0.01). In addition, ZZD significantly attenuated intestinal inflammation and oxidative stress and activated the SIRT1/FoxO1 signaling pathway (P<0.05 or P<0.01). ZZD exhibited beneficial effects on the intestinal system of STC mice and alleviated intestinal inflammation and oxidative stress via activating SIRT1/FoxO1 antioxidant signaling pathway in the colon.

Effect of electroacupuncture of "Tianshu"(ST25) at different frequencies on electromyography and immunoactivity of SP and VIP of colon in rats with slow transit constipation

To observe the effect of electroacupuncture (EA) of bilateral "Tianshu"(ST25) at different frequencies on the first black stool discharge time, colonic electromyography (EMG) and immunoactivity of vasoactive intestinal peptide (VIP) and substance P (SP) in the colon tissue in rats with slow transit constipation (STC), so as to choose a better stimulating frequency in the treatment of STC. A total of 50 male Wistar rats were randomized into control, model, 2 Hz-EA, 100 Hz- EA, and 2 Hz/100 Hz-EA groups, with 10 rats in each group. The STC model was established by intraperitoneal injection of compound diphenoxylate suspension fluid (10 mg/kg). EA was applied to bilateral ST25 for 30 min, once a day for 14 consecutive days. The discharge time for the first black stool was recorded after gavage of mixed suspension fluid of active carbon (2 mL) for assessing the gastrointestinal motility. The colonic EMG was recorded by using a pair of silver electrodes and bioelectric amplifier. The expression of SP and VIP in the colonic tissue was detected by immunohistochemistry. Following mode-ling, the colonic EMG amplitude, the discharge time for the first black stool and VIP immunoactivity were significantly increased (P<0.01), and the EMG frequency and SP immunoactivity were significantly decreased （P<0.01） in the model group compared with the control group. Compared with the model group, the increase of the discharge time for the first black stool, EMG amplitude and VIP immunoactivity, the decrease of EMG frequency and SP immunoactivity were reversed in the 3 EA groups (P<0.01, P<0.05). The therapeutic effect of 100 Hz-EA was notably weaker than that of both 2 Hz-EA and 2 Hz/100 Hz-EA in up-regulating EMG frequency and SP immunoactivity and in down-regulating the discharge time for the first black stool, EMG amplitude and VIP immunoactivity (P<0.05, P<0.01). There was no significant difference between the 2 Hz/100 Hz-EA and 2 Hz-EA groups (P>0.05). EA can accelerate colonic EMG activities, which may be associated with its functions in down-regulating VIP immunoactivity and up-regulating SP immunoactivity in the colonic tissues. The therapeutic effects of 2 Hz/100 Hz-EA and 2 Hz-EA are better than that of 100 Hz-EA.

Effects of Maren Pills on the Intestinal Microflora and Short-Chain Fatty Acid Profile in Drug-Induced Slow Transit Constipation Model Rats.

Background: Slow transit constipation (STC) is becoming a common and frequently occurring disease in today’s society, and it is necessary to explore the safe and effective treatment of STC. Method: Our study aimed to investigate whether the laxative effect of Maren pills (MRW) is associated with the regulation of intestinal microflora and intestinal metabolism in the colon. Loperamide hydrochloride-induced STC rats received MRW intragastrically for two consecutive weeks to evaluate the laxative effect of MRW involving the regulation of intestinal microflora, intestinal metabolism, and 5-HT signaling pathway. Intestinal microflora was detected by 16s rDNA sequencing, intestinal metabolism of short-chain fatty acids (SCFAs) was detected by HPLC, and the 5-HT signaling pathway was detected by WB, ELISA, immunofluorescence, and immunohistochemical analysis. Results: Our results revealed that the treatments with MRW increased not only the body weight, 24-h fecal number, 24-h wet fecal weight, 24-h dry fecal weight, fecal water content, and the intestinal propulsion rate but also the colonic goblet cell number, colonic Muc-2 protein expression, and colonic mucus layer thickness in the STC model rats. Moreover, MRW activated the 5-HT pathway by increasing the levels of 5-HT, 5-HIAA, 5-HT4R, CFTR, cAMP, and PKA in the colon tissue of STC rats. The 16S rDNA sequencing results showed that MRW improved the colonic microflora structure in colonic contents of STC rats, mainly by increasing Lactobacillus and decreasing Prevotella. Finally, we found that MRW regulated the SCFA metabolism in the colonic contents of the STC rats, mainly by increasing the contents of acetic acid, propionic acid, and butyric acid; the relative abundance of Lactobacillus was positively correlated with either contents of acetic acid, propionic acid, and butyric acid, and the relative abundance of Clostridium was negatively correlated. Conclusion: Our study further showed that MRW could improve constipation in STC rats, and the mechanism may be by regulating the intestinal microflora structure and improving the metabolism of SCFAs.

An Experimental Study on Intestine Transmission Function in SD Rats With Slow Transit Constipation After Electro-acupuncture Treatment in BL32 Acupoints

Objective: This study aims to use loperamide by gavage in SD rats to establish the model of STC, and evaluate the degree of similarity between the model and clinical diseases. Furthermore, the effect of electro-acupuncture when stimulating BL32 acupoints in SD rats with slow transit constipation were observed to determine changes in intestinal transmission function. Methods: Thirty SD rats were selected and divided randomly into three groups. Rats in the model group were given 2 mg/kg/d of loperamide tablets by gavage for 15 days. The morphology of the feces of rats was observed, and the dry weight of the feces and the time of first black feces movement with black ink by gavage after molding were measured. After 20 days of electro-acupuncture treatment at the BL32 acupoint, the time of first black feces movement, the ratio of black ink length of the entire length of the intestine, and contraction amplitude and frequency of contraction were measured. Results: The time of black feces movement in the two model groups was significantly prolonged (P &lt;0.05). Furthermore, fecal grains, dry weight and moisture content were significantly reduced (P &lt;0.05). On the 10th and 20th day of treatment, the time of black feces movement in the treatment group significantly decreased (P &lt;0.05). The ratio of black ink length of the entire length of the intestine in the electro-acupuncture group was significantly higher than that in the model group (P &lt;0.05). Moreover, contraction amplitude was significantly higher than in the model control group (P &lt;0.05). Conclusion: Through loperamide gavage, the model of slow transit constipation in SD rats was successfully established in a relatively short period of time. Electro-acupuncture treatment had a rapid onset of action, and its mechanism might have been caused through strengthening the ability of intestinal smooth muscle contraction, and are independent with increased smooth muscle contraction frequency.

Effect of electroacupuncture on gastrointestinal motility in rats with slow transit constipation based on GDNF methylation modification

To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Dachangshu"(BL25) on the intestinal transit function， expression level of glial cell-derived neurotrophic factor (GDNF) and methylation level of GDNF gene promoter region in colon tissue of rats with slow transit constipation (STC)， so as to explore its mechanisms underlying improvement of STC. Male Sprague-Dawley (SD) rats were randomized into control， saline， model and EA groups (n=16 in each group). The STC model was replicated by gavage of compound diphenoxylate suspension (10 mL· kg-1· d-1) for 28 days. Rats of the saline group received the same dose of normal saline via gavage. EA (2 Hz/15 Hz， 0.1-1 mA) was applied to bila-teral ST25 and BL25 for 15 min， once daily for 14 days. The intestinal transmission function (the intestinal propulsion rate) was assessed by recording the first black grain stool discharge time and the number and weight of the discharged stool grains in 30 min after gavage of the activated carbon suspension (1 mL/100 g， 150 g/L). The score of fecal trait and the weight of stool within 24 h were recorded. The ultrastructural changes of Cajal interstitial cells in the colon tissue were observed by transmission electron microscope. The expression levels of GDNF protein and mRNA in the colon tissue were detected by using Western blot and real-time fluorescent quantitative PCR， separately， and changes of methylation level in the promoter region of GDNF gene detected by using Bisulfite sequencing method. Compared with the control group， the time of the 1st black stool grain discharging was obviously prolonged， and the number and weight of the discharged black stool grains were significantly decreased in the mo-del group (P<0.05)， suggesting a success of STC. The weight and trait score of stool in 24 h， intestinal propulsive rate， and the expression levels of GDNF protein and mRNA were significantly lower in the model group than in the control group (P<0.01， P<0.05). After EA， the weight and trait score of stool within 24 h， intestinal propulsive rate，and the expression levels of GDNF protein and mRNA were significantly increased in the EA group in contrast to the model group (P<0.01，P<0.05). The total CpGs methylation level of GDNF gene in colon tissue was considerably higher in the model group than in the control group (P<0.05)， and markedly lower in the EA group than in the model group (P<0.05). No significant differences were found between the control and saline groups in all the above-mentioned indexes (P>0.05). EA of back-shu and front-mu acupoints can effectively improve symptoms of constipation and intestinal transport function in STC rats， which may be related to its function in up-regulating the expression of GDNF and down-regulating the methylation level in the promoter region of GDNF gene in colon tissue.

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.

Comparative Transcriptome Analysis Reveals Relationship among mRNAs, lncRNAs, and circRNAs of Slow Transit Constipation

Background Slow transit constipation (STC) is characterized by persistent, infrequent, or incomplete defecation. Systematic analyses of mRNA, lncRNA, and circRNA expression profiling in STC provide insights to understand the molecular mechanisms of STC pathogenesis. The present study is aimed at observing the interaction of mRNAs, lncRNAs, and circRNAs by RNA sequencing in vivo of STC. Methods A rat model of STC was induced by loperamide. The expression profiles of both mRNAs and miRNAs were performed by RNA sequencing. Enrichment analyses of anomalous expressed mRNAs, lncRNAs, and circRNAs were performed in order to identify the related biological functions and pathologic pathways through the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results In total, 26435 mRNAs, 5703 lncRNAs, and 7708 circRNAs differentially expressed were identified between the two groups. The analyses of GO and KEGG show that (1) upregulated genes were enriched in a positive regulation of GTPase activity, cell migration, and protein binding and lipid binding and (2) GO annotations revealed that most trans-target mRNAs are involved in the regulation process of immune signal together with the proliferation and differentiation of immune cells. Additionally, the protein-protein interaction (PPI) network of differentially expressed (DE) mRNAs was constructed. Interestingly, all of the core lncRNAs and their coexpression mRNAs in this network are downregulated. Moreover, downregulated circRNAs have a set of target mRNAs related to immunoreaction, which was consistent with the overall tendency. Conclusion Our investigation enriches the STC transcriptome database and provides a preliminary exploration of novel candidate genes and avenues expression profiles in vivo. The dysregulation of mRNAs, lncRNAs, and circRNAs might contribute to the pathological processes during STC.

Effect of atractylenolide III on interstitial cells of Cajal and C-kit/SCF pathway of rats with loperamide-induced slow transit constipation

Purpose: To determine the effect of atractylenolide-III (ATL-III) on loperamide-induced slow transit constipation (STC) in a rat STC model, and to elucidate the mechanisms involved.&#x0D; Methods: Male Wistar rats were divided into five groups (n=6 per group): normal control group (NG), model group, and three STC rat groups treated with different doses of ATL-III, viz, 5, 10 and 15 mg/kg. The rats were treated for 15 days. Feed consumption, fecal excretion and intestinal transit rate were determined. Nitric oxide synthase (NOS), somatostatin (SS), serotonin (5-HT), and vasoactive intestinal peptide (VIP) were measured with enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expressions of C-kit, SCF, PKC, and PI-3K were assayed using Western blot analysis and realtime reverse transcription polymerase chain reaction (RT-PCR), respectively.&#x0D; Results: The amount, weight, and moisture content of stool, and water consumption were significantly higher in ATL-III-treated groups than in the untreated (model) group (p &lt; 0.05), whereas no difference was observed in feed intake. Intestinal transit rate was higher in the ATL-III-treated groups (p &lt; 0.05). Decreased NOS, SS and VIP levels and increased 5-HT level were seen in the ATL-III-treated groups (p &lt; 0.05). ATL-III treatment also induced increases in smooth muscle cells, neuronal cells, and mucous layer (p&lt;0.05). Results from RT-PCR and Western blot revealed that ATL-III–treated groups had elevated c-kit, SCF, PKC, as well as PI-3K mRNA and protein expressions (p &lt; 0.05).&#x0D; Conclusion: These results suggest that ATL-III mitigates loperamide-induced STC in rats via stimulation of NOS, SS, VIP, and 5-HT secretions. It also increases smooth muscle cells, neuronal cells, and mucous layer, and regulates the signaling pathways involving PKC, PI3K, SCF, and c-kit.

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.

Therapeutic effect and mechanism of Shouhui Tongbian Capsules on slow transit constipation model mice

Shouhui Tongbian Capsules was used to explore the therapeutic effect and potential mechanism on slow transit constipation model mice induced by loperamide hydrochloride. In the experiment, loperamide hydrochloride-induced ICR mice were used as the model of slow transit constipation. Fifty ICR mice were divided into the blank group, model group and high, medium and low dose groups of Shouhui Tongbian Capsules extract(100, 200 and 400 mg·kg~(-1)). The model group and the administration groups were then modeled using loperamide hydrochloride intragastrically to obtain slow transit constipation. After successful modeling, high, medium and low doses of drugs were given to each drug group by intragastric administration. After 14 days of administration, the first defecation time, 6 h defecation grain number, 6 h defecation wet weight and dry weight, black feces discharged within 6 h and the fecal water content were measured. Intestinal tissues were taken for c-Kit and SCF immunohistochemical sections to detect the expression of c-Kit and SCF in the blank group, model group and high, medium and low dose groups of the medicinal extract of Shouhui Tongbian Capsules. The tissue changes in the intestinal wall of mice were detected by HE staining. At the same time, partial intestinal tissues were taken to test the activity of ATP synthase and isocitrate dehydrogenase in intestinal tissues of mice. RESULTS:: showed that Shouhui Tongbian Capsules effectively improved the symptoms of slow transit constipation in ICR mice and promoted intestinal movement. Shouhui Tongbian Capsules obviously shortened the time of discharging black stool for the first time, improved the intestinal propulsion rate, increased the water content and amount of feces, and improved the constipation symptoms. Mechanism study revealed that Shouhui Tongbian Capsules increased ATP synthase activity and mitochondrial isocitrate dehydrogenase activity in intestinal tissue, and up-regulated c-Kit/SCF signaling pathway to promote interstitial Cajal cells proliferation, intestinal nerve transmission, intestinal motility and transport capacity.

Comparison of the effects of colonic electrical stimulation and prucalopride on gastrointestinal transit and defecation in a canine model of constipation

Objective The aim of this study was to compare the effects of colonic electrical stimulation (CES) and prucalopride on gastrointestinal transit and defecation and to verify the safety of CES in a canine model of constipation. Methods Eight beagles received CES implantation and induction drugs for slow transit constipation (STC). In the STC model, the gastrointestinal transit time (GITT), colonic transit time (CTT), stool frequency and stool consistency were assessed to compare the effects of CES and prucalopride on gastrointestinal transit and defecation. The histocompatibility of the implantable device was evaluated. Results The individualized parameters for CES varied greatly among the animals, and the GITTs were not significantly shortened by CES or prucalopride; however, both the CES and prucalopride treatment significantly accelerated CTT and improved stool consistency compared with sham stimulation. CES treatment also resulted in significantly higher stool frequency than prucalopride treatment, which did not significantly change the stool frequency. No severe inflammation response was detected in the gross and microscopic appearance around the implants. Conclusion CES and prucalopride treatment may yield similar short-term effects for improving gastrointestinal transit and stool consistency, and CES outperformed prucalopride treatment in terms of defecation inducement in the short term. There were ideal levels of endurance and histocompatibility for the animals that underwent CES.