Luteolin Attenuates Oxidative Stress and Colonic Hypermobility in Water Avoidance Stress Rats by Activating the Nrf2 Signaling Pathway.

SCOPE: Irritable bowel syndrome (IBS) is an intestinal disorder, whose symptoms can be alleviated by certain dietary phytochemicals. This study explores the role and potential mechanisms of a natural flavonoid luteolin (LUT) in alleviating the excessive motility of colonic smooth muscles and reducing oxidative stress in IBS with diarrhea (IBS-D) rats. METHODS AND RESULTS: LUT reduces excessive intestinal motility and lowers reactive oxygen species (ROS) levels in a water avoidance stress (WAS) rat model. Moreover, LUT increases the protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), activates the nuclear translocation of Nrf2, and greatly reduces the hydrogen peroxide (H2 O2 )-induced oxidative damage in intestinal epithelial cells. CONCLUSIONS: LUT, a phyto-active component, protects against excessive intestinal motility and diarrhea by regulating the Nrf2 signaling pathway and effectively reduces oxidative stress damage in the colon.

Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.

Apigenin attenuates visceral hypersensitivity in water avoidance stress rats by modulating the microbiota-gut-brain axis and inhibiting mast cell activation.

Visceral hypersensitivity (VH) and gut microbiota dysbiosis significantly contribute to the occurrence and development of irritable bowel syndrome (IBS), exacerbated by stress. Apigenin, a natural flavonoid derived from plants, possesses a range of beneficial properties. However, additional research is necessary to investigate its potential in alleviating symptoms of IBS and elucidating its underlying mechanisms of action. Our study confirms that apigenin effectively reverses mast cell and microglial activation, regulates the composition and abundance of the gut microbiota, improves intestinal barrier function in rats induced with water-avoidance stress, and mitigates VH and colonic hypermotility. Furthermore, in vitro studies suggest a potential role of dysbiotic gut microbiota in activating mast cells at the cellular level. Notably, apigenin inhibits mast cell degranulation through the toll-like receptor 4 (TLR4) / myeloid differentiation primary response gene 88 (MyD88) / nuclear factor-kappa B (NF-κB) pathway. In conclusion, this study discusses the potential therapeutic effects of apigenin in alleviating VH and modulating the gut-brain axis in water-avoidance stress rats, providing a novel or alternative treatment approach for IBS.

Two fragments of HBV DNA integrated into chrX: 11009033 and its genetic regulation in HepG2.2.15.

Hepatitis B virus (HBV) integration into human genome causes hepatocellular carcinoma (HCC). The present study used inverse nested PCR; the full sequence of HBV DNA fragments of the chrX: 111009033 integration site was detected (987 bp), containing two fragments of double­stranded linear DNA with the same orientation (1,744­1,094 and 1,565­1,228 nt). By reverse transcription­quantitative PCR, HBV­cell fusion transcript was observed in HepG2.2.15 cells. The mean copy number of this site in cells with H2O2 treatment (8.73x10­2±1.65x10­2 copies/cell) was significantly higher than that in the cells without H2O2 treatment (3.02x10­2±2.33x10­2 copies/cell; P<0.0001). The mean levels of P21­activated kinase 3 (PAK3) were 15.67±5.65 copies/cell in HepG2.2.15 cells with H2O2 treatment, significantly higher than in the cells without H2O2 treatment (11.34±4.58 copies/cell, P=0.0076) and in HepG2 cells (5.92±1.54 copies/cell, P<0.0001). Significant difference of PAK3 levels was also found between HepG2.2.15 cells without H2O2 treatment and HepG2 cells (11.34±4.58 vs. 5.92±1.54 copies/cell, P<0.0001). The average copy numbers of the integration site chrX: 111009033 were positively correlated with the average levels of PAK3 (P=0.0013). The overall trend of PAK3 expression was significantly increased in HepG2.2.15 cells with H2O2 treatment compared with that in HepG2.2.15 cells without H2O2 treatment (37.63±8.16 and 31.38±7.94, P=0.008) and HepG2 cells (21.67±7.88, P<0.0001). In summary, the chrX: 11009033 integration site may originate from primary human hepatocytes, occurrence and clonal expansion of which may upregulate PAK3 expression, which may contribute to hepatocarcinogenesis.

Targeting Vascular Smooth Muscle Cell Senescence: A Novel Strategy for Vascular Diseases.

Vascular diseases are a major threat to human health, characterized by high rates of morbidity, mortality, and disability. VSMC senescence contributes to dramatic changes in vascular morphology, structure, and function. A growing number of studies suggest that VSMC senescence is an important pathophysiological mechanism for the development of vascular diseases, including pulmonary hypertension, atherosclerosis, aneurysm, and hypertension. This review summarizes the important role of VSMC senescence and senescence-associated secretory phenotype (SASP) secreted by senescent VSMCs in the pathophysiological process of vascular diseases. Meanwhile, it concludes the progress of antisenescence therapy targeting VSMC senescence or SASP, which provides new strategies for the prevention and treatment of vascular diseases.

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

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

Nano-encapsulation of halofuginone hydrobromide enhances anticoccidial activity against Eimeria tenella in chickens.

Coccidiosis is a worldwide epidemic intestinal disease with high incidence, which causes huge economic losses. Halofuginone hydrobromide (HF) is widely applied as an effective anticoccidial drug in the poultry industry. However, its therapeutic efficacy is severely restrained due to toxic effects, poor aqueous solubility and low permeability. Nanotechnology can improve the biological effect of drugs, and thus, reduce administered doses and toxic effects. The objective of this study was to investigate the therapeutic and preventive potential of novel HF-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles (HTPM) for preventing coccidiosis in chickens. The HTPM were approximately spherical with a hydrodynamic diameter of 12.65 ± 0.089 nm, a zeta potential of 8.03 ± 0.242 mV, a drug loading of 14.04 ± 0.12%, and an encapsulation efficiency of 71.1 ± 4.15%. HF was encapsulated in the polymer micelles through interactions with TPGS, as characterized by X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Cellular take up assays showed that TPGS polymer micelles could enhance drug internalization to alleviate intestinal apoptosis induced by coccidiosis and promote the necrosis of second-generation merozoites of E. tenella. Notably, clinical trials proved that 1.5 mg L-1 HTPM had a stronger anticoccidial effect on E. tenella than that of 3 mg kg-1 HF premix. Amplicon sequencing identified that HTPM could alleviate coccidiosis by restoring the structure of the gut microbiome. These findings indicated that the anticoccidial efficacy of HF was significantly enhanced after being encapsulated in polymer micelles, and further demonstrated the potential protective application of nano-encapsulating anticoccidial drugs as a promising approach to control coccidiosis in poultry. In summary, HTPM hold huge potential as an effective therapeutic agent for coccidiosis.

Dysregulated cystathionine-ß-synthase/hydrogen sulfide signaling promotes chronic stress-induced colonic hypermotility in rats.

BACKGROUND: Hydrogen sulfide (H2 S), an important endogenous gasotransmitter, is involved in the modulation of gastrointestinal motility, but whether it mediates the intestinal dysmotility in irritable bowel syndrome (IBS) is not known. This study explored the significance of cystathionine-ß-synthase (CBS)/H2 S signaling in stress-induced colonic dysmotility. METHODS: A rat model of IBS was established using chronic water avoidance stress (WAS). Colonic pathological alterations were detected histologically. Intestinal motility was determined by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was assessed using the visceromotor response (VMR) to colorectal distension (CRD). Real-time PCR, Western blotting, and immunostaining were performed to identify the expression of CBS in the colon. The contractions of distal colon were studied in an organ bath system and H2 S content was measured by ELISA. The effects of SAM, a selective CBS activator, on colonic dysmotility were examined. MEK1 was tested as a potential upstream effector of CBS/H2 S loss. KEY RESULTS: After 10 days of WAS, the ITT was decreased and FWC was increased, and the VMR magnitude in response to CRD was enhanced. The colonic CBS expression and H2 S levels were significantly declined in WAS-exposed rats, and the density of CBS-positive enteric neurons in the myenteric plexus in WAS-treated rats was lower than that in controls. SAM treatment relieved WAS-induced colonic hypermotility via increased H2 S production. AZD6244, a selective inhibitor of MEK1, partially reversed CBS downregulation and colonic hypermotility in WAS-treated rats. CONCLUSIONS & INFERENCES: Decreased CBS/H2 S signaling through increased MEK1 signaling might be important in the pathogenesis of chronic stress-induced colonic hypermotility. SAM could be administered for disorders associated with intestinal hypermotility.

An interpretable artificial intelligence system for detecting risk factors of gastroesophageal variceal bleeding.

Bleeding risk factors for gastroesophageal varices (GEV) detected by endoscopy in cirrhotic patients determine the prophylactical treatment patients will undergo in the following 2 years. We propose a methodology for measuring the risk factors. We create an artificial intelligence system (ENDOANGEL-GEV) containing six models to segment GEV and to classify the grades (grades 1-3) and red color signs (RC, RC0-RC3) of varices. It also summarizes changes in the above results with region in real time. ENDOANGEL-GEV is trained using 6034 images from 1156 cirrhotic patients across three hospitals (dataset 1) and validated on multicenter datasets with 11009 images from 141 videos (dataset 2) and in a prospective study recruiting 161 cirrhotic patients from Renmin Hospital of Wuhan University (dataset 3). In dataset 1, ENDOANGEL-GEV achieves intersection over union values of 0.8087 for segmenting esophageal varices and 0.8141 for gastric varices. In dataset 2, the system maintains fairly accuracy across images from three hospitals. In dataset 3, ENDOANGEL-GEV surpasses attended endoscopists in detecting RC of GEV and classifying grades (p < 0.001). When ranking the risk of patients combined with the Child‒Pugh score, ENDOANGEL-GEV outperforms endoscopists for esophageal varices (p < 0.001) and shows comparable performance for gastric varices (p = 0.152). Compared with endoscopists, ENDOANGEL-GEV may help 12.31% (16/130) more patients receive the right intervention. We establish an interpretable system for the endoscopic diagnosis and risk stratification of GEV. It will assist in detecting the first bleeding risk factors accurately and expanding the scope of quantitative measurement of diseases.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion.

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.

Optimization of Maduramicin Ammonium-Loaded Nanostructured Lipid Carriers Using Box-Behnken Design for Enhanced Anticoccidial Effect against Eimeria tenella in Broiler Chickens.

Maduramicin ammonium (MAD) is one of the most frequently used anticoccidial agents in broiler chickens. However, the high toxicity and low solubility of MAD limit its clinical application. In this study, MAD-loaded nanostructured lipid carriers (MAD-NLCs) were prepared to overcome the defects of MAD by using highly soluble nanostructured lipid carriers (NLCs). The formulation was optimized via a three-level, three-factor Box-Behnken response surface method. Then, the optimal MAD-NLCs were evaluated according to their hydrodynamic diameter (HD), zeta potential (ZP), crystal structure, encapsulation efficiency (EE), drug loading (DL), in vitro release, and anticoccidial effect. The optimal MAD-NLCs had an HD of 153.6 ± 3.044 nm and a ZP of -41.4 ± 1.10 mV. The X-ray diffraction and Fourier-transform infrared spectroscopy results indicated that the MAD was encapsulated in the NLCs in an amorphous state. The EE and DL were 90.49 ± 1.05% and 2.34 ± 0.04%, respectively, which indicated that the MAD was efficiently encapsulated in the NLCs. In the in vitro study, the MAD-NLCs demonstrated a slow and sustained drug release behavior. Notably, MAD-NLCs had an excellent anticoccidial effect against Eimeria tenella in broiler chickens. In summary, MAD-NLCs have huge potential to form a new preparation administered via drinking water with a powerful anticoccidial effect.

Activation of cannabinoid 2 receptor relieves colonic hypermotility in a rat model of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. The endocannabinoid system is emerging as an important modulator of gastrointestinal (GI) motility in multiple diseases, but its involvement in IBS is unknown. We aimed to determine whether cannabinoid 2 (CB2) receptor modulates intestinal motility associated with stress-induced IBS. METHODS: A rat IBS model was established by chronic water avoidance stress (WAS). Colonic pathological alterations were detected histologically and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). Real-time PCR, western blot, and immunostaining were performed to identify colonic CB2 receptor expression. Colonic muscle strip contractility was studied by isometric transducers and nitric oxide (NO) was detected by the Griess test. The effects of AM1241, a selective agonist of CB2 receptors, on colonic motility were examined. KEY RESULTS: After 10 days of WAS exposure, ITT was decreased and FWC elevated while VMR magnitude in response to CRD was significantly enhanced. Colon CB2 protein and mRNA levels increased and density of CB2-positive macrophages in the mucosa and enteric neurons in the myenteric plexus was higher than in controls. Pharmacological enhancement of CB2 activity by AM1241 relieved colonic hypermotility in WAS rats in a concentration-dependent manner via inhibition of p38 phosphorylation and elevation of NO production. CONCLUSION: CB2 receptor may exert an important inhibitory effect in stress-induced colonic hypermotility by modulating NO synthesis through p38 mitogen-activated protein kinase signaling. AM1241 could be used as a potential drug to treat disorders with colonic hypermotility.

Neutrophils injure gallbladder interstitial Cajal-like cells in a guinea pig model of acute cholecystitis.

Acute cholecystitis is a common disease with gallbladder dysmotility. Disease pathogenesis involves immune cell infiltration as well as changes in gallbladder interstitial Cajal-like cells (ICLCs). However, it remains unclear if or how the immune cells affect ICLC morphology, density, distribution, and function in gallbladder tissue during acute cholecystitis. In this study, we explored the acute cholecystitis-related alterations in gallbladder ICLCs in a guinea pig model, focusing on the effects of neighboring neutrophils. Adult guinea pigs were randomly divided into four groups (control, 24 hr common bile duct ligation [CBDL], 48-hr CBDL, and antipolymorphonuclear neutrophil [PMN] treated) and analyzed using methylene blue staining and immunofluorescence. Gallbladder contractility was also monitored. To culture gallbladder ICLCs, collagenase digestion was performed on tissue from 10- to 15-day-old guinea pigs. Neutrophils isolated from the peripheral blood of experimental animals 48-hr postsurgery were also cocultured with the gallbladder ICLCs. Intracellular calcium was detected with Fluo-4 AM dye. Our results showed that gallbladder ICLC density significantly declined during acute cholecystitis and was accompanied by shortening of the cellular processes and damage to their network-like structure. However, pretreatment with anti-PMN partially prevented these changes. Gallbladder contraction was also significantly decreased during acute cholecystitis, and this appeared to be mediated by the neutrophils. Moreover, ICLCs cocultured with neutrophils also had shortened and reduced processes and impaired network-like structure formation. Intracellular calcium transient was less sensitive to contraction agonists and inhibitors when cocultured with neutrophils. Taken together, neutrophils greatly affect gallbladder ICLCs and dysmotility during acute cholecystitis.

Upregulation of the high-affinity choline transporter in colon relieves stress-induced hyperalgesia.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disease with hyperalgesia, the mechanisms of which remain elusive. The cholinergic system is known to be involved in pain inhibitory pathways in multiple diseases, and its involvement in IBS is unknown. OBJECTIVE: We aimed to determine whether high-affinity choline transporter CHT1, a major determinant of the cholinergic signaling capacity, is involved in regulating intestinal sensations associated with stress-induced visceral pain. MATERIALS AND METHODS: An IBS rat model was established by chronic water avoidance stress (WAS). Colonic pathologic alterations were detected by H&E staining. Visceral sensations were determined by scoring the abdominal withdrawal reflex (AWR) and visceromotor response (VMR) magnitude of the electromyogram in response to colorectal distension (CRD). Abdominal mechanical hyperalgesia was assessed by counting the number of withdrawal events evoked by applying von Frey filaments. Real-time PCR, Western blot, and immunostaining were performed to identify CHT1 expression in the colon. Acetylcholine (ACh) secretion was determined by ELISA. Effects of MKC-231, a choline uptake enhancer, on visceral pain were examined. RESULTS: After 10 days of WAS exposure, AWR score and VMR magnitude in response to CRD were significantly enhanced and the number of withdrawal events was elevated. Protein and mRNA levels of CHT1 were considerably increased in the colon after WAS. CHT1 upregulation in the WAS-exposed group was largely abolished by ammonium pyrrolidinedithiocarbamate. The density of CHT1-positive intramuscular cells and enteric neurons in the myenteric plexus was enhanced in WAS-exposed rats. Pharmacologic enhancement of CHT1 activity by MKC-231 gavage could relieve the visceral pain of WAS rats by upregulating CHT1 protein expression and enhancing ACh production. CONCLUSION: CHT1 may exert an antinociceptive effect in stress-induced visceral pain by modulating ACh synthesis through nuclear factor kappa B signaling. MKC-231 could be used as a potential drug to treat disorders with hyperalgesia.

Role of High-affinity Choline Transporter 1 in Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS) is a common disease characterized by intestinal dysmotility, the mechanism of which remains elusive. We aim to determine whether the high-affinity choline transporter 1 (CHT1), a determinant of cholinergic signaling capacity, modulates intestinal motility associated with stress-induced IBS. METHODS: A rat IBS model was established using chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically and intestinal motility was assessed by intestinal transit time and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response to colorectal distension. RT-PCR, western blotting, and immunostaining were performed to identify colonic CHT1 expression. Contractility of colonic muscle strips was measured using isometric transducers. enzyme-linked immunosorbent assay was used to measure acetylcholine (ACh). We examined the effects of MKC-231, a choline uptake enhancer, on colonic motility. RESULTS: After 10 days of WAS, intestinal transit time was decreased and fecal water content increased. Visceromotor response magnitude in WAS rats in response to colorectal distension was significantly enhanced. Protein and mRNA CHT1 levels in the colon were markedly elevated after WAS. The density of CHT1-positive intramuscular interstitial cells of Cajal and myenteric plexus neurons in WAS rats was higher than in controls. Ammonium pyrrolidine dithiocarbamate partly reversed CHT1 upregulation and alleviated colonic hypermotility in WAS rats. Pharmacological enhancement of CHT1 activity by MKC-231 enhanced colonic motility in control rats via upregulation of CHT1 and elevation of ACh production. CONCLUSION: Upregulation of CHT1 in intramuscular interstitial cells of Cajal and myenteric plexus neurons is implicated in chronic stress-induced colonic hypermotility by modulation of ACh synthesis via nuclear factor-kappa B signaling.

Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome Is Associated with Upregulation of TMEM16A in Myenteric Plexus.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. TMEM16A is a calcium-activated chloride channel (CaCC) involved in intestinal slow-wave generation. AIMS: To investigate whether TMEM16A is involved in colonic dysmotility in IBS. METHODS: A rat model of IBS was established by chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically, and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). TMEM16A expression was evaluated by RT-PCR, Western blot, and immunofluorescence. Colonic muscle strip contractility was measured by isometric transducers, and the effect of niflumic acid (NFA), a CaCC antagonist, on colonic motility was examined. RESULTS: After 10 days of WAS exposure, ITT was decreased and FWC was elevated. Furthermore, VMR magnitude of WAS rats in response to CRD was significantly enhanced. Protein and mRNA levels of TMEM16A in colon were considerably increased after WAS. The percentage of TMEM16A-positive neurons in myenteric plexus (MP) of WAS rats was significantly higher than controls. Pharmacological blockade of TMEM16A activity by NFA considerably enhanced ITT, with concentration-dependent declines in FWC and VMR magnitude in NFA-treated rats. Further, spontaneous contraction of colonic strips of NFA-treated rats was significantly ameliorated in a concentration-dependent manner in vitro. CONCLUSIONS: Upregulation of TMEM16A in MP neurons may play an important role in chronic stress-induced colonic hypermotility, making CaCC-blocking drugs a putatively effective treatment method for colonic hypermotility in IBS.

Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress.

BACKGROUND: Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1) is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs) and the role of CHT1 in visceral hypersensitivity. METHODS: Repetitive water avoidance stress (WAS) was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD) was determined, and the abdominal withdrawal reflex (AWR) and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3), the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. RESULTS: Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. CONCLUSION: Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity.

The Role of Interstitial Cells of Cajal in Acute Cholecystitis in Guinea Pig Gallbladder.

BACKGROUND/AIMS: Acute cholecystitis is common in gallbladder motility disorder. Interstitial cells of Cajal (ICCs) in the gallbladder are involved in the regulation of gallbladder motility. The aim of this study was to explore the change of gallbladder ICCs in acute cholecystitis. METHODS: Thirty adult guinea pigs were randomly divided into 3 groups: a sham-operated group (healthy controls) and 2 study groups. The animals in the study group were subjected to bile duct ligation and then to laparotomy and cholecystectomy at 24 and 48 hours after surgery. Immunohistochemistry, immunohistofluorescence, and laser confocal microscopy were performed to observe the shape, size, morphology, and density of gallbladder ICCs. Western blot and real-time PCR were performed to detect stem cell factor and c-kit protein and mRNA expression, respectively. RESULTS: There were no differences in the shape, size, and morphology of the gallbladder ICCs in the control and the two acute cholecystitis groups. Density of gallbladder ICCs, SCF level, and c-kit protein and mRNA expression all decreased in the acute cholecystitis groups. Further, SCF level and c-kit protein and mRNA expression decreased with progress of acute cholecystitis (all P < 0.05). CONCLUSION: Acute cholecystitis can decrease ICCs through repression of SCF and c-kit expression and that ICCs loss play a role in acute cholecystitis.