To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all <0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all <0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(<0.001),occludin(<0.05),Claudin-1(<0.01)and E-cadherin(<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Hypoxia ; Cell Line ; Claudin-1 ; metabolism ; Colon ; cytology ; Humans ; Occludin ; metabolism ; Receptors, Calcitriol ; metabolism ; Tight Junctions ; Vitamin D ; pharmacology ; Zonula Occludens-1 Protein ; metabolism

BACKGROUNDDisrupted Ca2+ homeostasis contributes to the development of colonic dysmotility in ulcerative colitis (UC), but the underlying mechanisms are unknown. This study aimed to examine the alteration of colonic smooth muscle (SM) Ca2+ signaling and Ca2+ handling proteins in a rat model of dextran sulfate sodium (DSS)-induced UC.

METHODSMale Sprague-Dawley rats were randomly divided into control (n = 18) and DSS (n = 17) groups. Acute colitis was induced by 5% DSS in the drinking water for 7 days. Contractility of colonic SM strips (controls, n = 8 and DSS, n = 7) was measured in an organ bath. Cytosolic resting Ca2+ levels (n = 3 in each group) and Ca2+ transients (n = 3 in each group) were measured in single colonic SM cells. Ca2+ handling protein expression was determined by Western blotting (n = 4 in each group). Differences between control and DSS groups were analyzed by a two-sample independent t-test.

RESULTSAverage tension and amplitude of spontaneous contractions of colonic muscle strips were significantly enhanced in DSS-treated rats compared with controls (1.25 ± 0.08 g vs. 0.96 ± 0.05 g, P= 0.007; and 2.67 ± 0.62 g vs. 0.52 ± 0.10 g, P= 0.013). Average tensions of carbachol-evoked contractions were much weaker in the DSS group (1.08 ± 0.10 g vs. 1.80 ± 0.19 g, P= 0.006). Spontaneous Ca2+ transients were observed in more SM cells from DSS-treated rats (15/30 cells) than from controls (5/36 cells). Peak caffeine-induced intracellular Ca2+ release was lower in SM cells of DSS-treated rats than controls (0.413 ± 0.046 vs. 0.548 ± 0.041, P= 0.033). Finally, several Ca2+ handling proteins in colonic SM were altered by DSS treatment, including sarcoplasmic reticulum calcium-transporting ATPase 2a downregulation and phospholamban and inositol 1,4,5-trisphosphate receptor 1 upregulation.

CONCLUSIONSImpaired intracellular Ca2+ signaling of colonic SM, caused by alteration of Ca2+ handing proteins, contribute to colonic dysmotility in DSS-induced UC.

Animals ; Colitis ; chemically induced ; metabolism ; Colon ; cytology ; metabolism ; Dextran Sulfate ; toxicity ; Male ; Muscle, Smooth ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

No abstract available.
Animals ; Colon/*metabolism ; Constipation/*physiopathology ; Diarrhea/*physiopathology ; Female ; Ganglia, Spinal/*cytology ; Humans ; Irritable Bowel Syndrome/*physiopathology ; Male ; Nociceptors/*physiology ; Receptor, PAR-2/*physiology

OBJECTIVETo observe the influence of Shenqing Recipe (SQR), a kind of Traditional Chinese Medicine, on the morphology and quantity of colonic interstitial cells of Cajal (ICC) in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis, and to investigate the possible mechanism of SQR in regulating intestinal dynamics.

METHODSSixty rats were randomly divided into normal control, model 1, model 2, mesalazine, and high-dose, and low-dose SQR groups with 10 rats in each group. TNBS (10 mg) dissolved in 50% ethanol was instilled into the lumen of the rat colon of the latter five groups to induce colitis. On the 4th day after administration of TNBS, each treatment group was administered one of the following formulations by enteroclysis gavage once a day for 7 days: 600 mg•kg⁻¹•d⁻¹ mesalazine, 2.4 g•kg⁻¹•d⁻¹ SQR, and 1.2 g•kg⁻¹•d⁻¹ SQR. Model 2 rats received normal saline solution. After 7 days colonic samples were collected. While the colonic samples of model 1 group were collected on the 3rd day after TNBS administered. Ultrastructure of ICC in the damaged colonic tissues was observed with transmission electron microscope. Expression of c-kit protein in colonic tissue was determined by immunohistochemical staining and Western blot.

RESULTSThe ultrastructure of colonic ICC in the rat model of TNBS-induced colitis showed a severe injury, and administration of SQR or mesalazine reduced the severity of injury. Similarly, the expression of c-kit protein of TNBS-induced colitis rat model was significantly decreased compared with the normal control group (P < 0.05). Treatment with SQR or mesalazine significantly increased the expression of c-kit protein compared with the administration of control formulations (P < 0.05), especially the high-dose SQR group.

CONCLUSIONSQR could alleviate and repair the injured ICC, and improve its quantity, which might be involved in regulating intestinal motility.

Animals ; Colitis ; chemically induced ; drug therapy ; pathology ; Colon ; cytology ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interstitial Cells of Cajal ; drug effects ; pathology ; ultrastructure ; Male ; Medicine, Chinese Traditional ; Mesalamine ; therapeutic use ; Peroxidase ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Trinitrobenzenesulfonic Acid ; adverse effects

PURPOSE: Postinfectiously irritable bowel syndrome (PI-IBS) develops in 3-30% of individuals with bacterial gastroenteritis. Recent studies demonstrated increases in inflammatory components in gut mucosa of PI-IBS patients even after complete resolution of infection. We aimed to investigate histological changes in colon and rectum of PI-IBS subjects after long term period of infection. MATERIALS AND METHODS: We recruited PI-IBS subjects who had been diagnosed IBS after complete resolution of enteritis caused by shigellosis outbreak 3 years earlier. We compared unmatched four groups, PI-IBS (n = 4), non PI-IBS (n = 7), D-IBS (n = 7, diarrhea predominant type) and healthy controls (n = 10). All of them underwent colonoscopic biopsy at three areas, including descending colon (DC), sigmoid colon (SC) and rectum, which were assessed for 5-hydroxytryptamine (5-HT)/peptide YY (PYY)-containing enterochromaffin (EC) cell, intraepithelial (IEL) and lamina propria T lymphocyte (CD3), CD8 lymphocytes, mast cells and CD68/calprotectin+ macrophages. RESULTS: All subjects had no structural or gross abnormalities at colonoscopy. In PI-IBS, 5-HT containing EC cells, PYY containing EC cells, IELs, CD3 lymphocytes, CD8 lymphocytes, mast cells, and CD68 + macrophages were increased compared to control (p < 0.05). In D-IBS, PYY containing EC cells, IELs, and CD3 lymphocytes were increased compared to control (p < 0.05). In PI-IBS, 5-HT containing EC cells tended to increase and PYY containing EC cells, CD8 lymphocytes, mast cells, and CD68+ macrophages were increased compared to non PI-IBS (p < 0.05). Calprotectin + marcrophages were decreased in PI-IBS, non PI-IBS and IBS compared to control. CONCLUSION: The immunoendocrine cells were sporadically increased in PI-IBS, non PI-IBS and D-IBS compared with control. Our findings in a very small number of patients suggest that mucosal inflammation may play a role in long-term PI-IBS, and that other sub-groups of IBS and larger scale studies are needed to confirm this observation.
Adult ; Antigens, CD/metabolism ; Antigens, Differentiation, Myelomonocytic/metabolism ; CD8-Positive T-Lymphocytes/cytology ; Case-Control Studies ; Colon, Descending/pathology ; Colon, Sigmoid/pathology ; Colonoscopy ; Dysentery, Bacillary/*complications ; Enterochromaffin Cells/cytology ; Female ; Humans ; Immunohistochemistry ; Intestinal Mucosa/*pathology ; Irritable Bowel Syndrome/metabolism/*pathology ; Macrophages/cytology ; Male ; Mast Cells/cytology ; Peptide YY/metabolism ; Rectum/pathology ; Serotonin/metabolism

OBJECTIVETo study the influence of serum from scalded rats on the cytoskeleton of colonic smooth muscle cells (CSMC) of rats cultured in vitro, and to probe the possible mechanism of gastrointestinal motility disorder after burn.

METHODSCSMC isolated from healthy adult Wistar rat were cultured and divided into scald serum group (SS) and normal serum group (NS) according to the random number talbi. Two normal Wistar rats were used, one of which was inflicted with deep partial-thickness scald. Serum was obtained from blood collected from these two rats respectively and diluted to 20% in concentration. Serum from scald and normal rats were respectively added to the culture of CSMC in SS and NS groups. The expression of actin and the relative content of β-tubulin in CSMC was respectively determined with flow cytometry and Western blot at post treatment hour (PTH) 1, 3, 6, and 12 (with 10 samples in each group at each time point). Data were processed with t test.

RESULTSFluorescence intensity of actin in SS group at PTH 1, 3, 6, and 12 was respectively 59 ± 4, 26 ± 6, 39 ± 6, and 42 ± 6, all significantly lower than those in NS group (95 ± 10, 91 ± 10, 102 ± 9, and 97 ± 9, with t value respectively 10.528, 18.069, 18.748, 16.647, P < 0.05 or P < 0.01). In SS group, the fluorescence intensity decreased to the nadir at PTH 3, and then increased persistently at PTH 6 and 12. (2) Relative content of β-tubulin in SS group at PTH 1, 3, 6, and 12 was respectively 14.44 ± 0.26, 8.61 ± 0.19, 11.76 ± 0.31, and 12.13 ± 0.29, all significantly less than those in NS group (22.37 ± 1.15, 21.87 ± 1.79, 23.24 ± 1.55, and 21.99 ± 2.02, with t value respectively 21.176, 23.365, 23.000, 15.273, P values all below 0.01). In SS group, the relative content of β-tubulin decreased to the nadir at PTH 3 and increased slowly at PTH 6 and 12.

CONCLUSIONSThe reduction of CMSC content which has the tendency of increasing later, can be attributed to the influence of scald serum in initial stage. This may be related to the tolerance and adaptation to scald serum and self-repair of CMSC.

Animals ; Burns ; metabolism ; Cells, Cultured ; Colon ; cytology ; Cytoskeleton ; metabolism ; Male ; Microtubules ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Wistar ; Serum

This study was performed in order to assess whether acute stress can increase mast cell and enterochromaffin (EC) cell numbers, and proteinase-activated receptor-2 (PAR2) expression in the rat colon. In addition, we aimed to investigate the involvement of corticotrophin-releasing factor in these stress-related alterations. Eighteen adult rats were divided into 3 experimental groups: 1) a saline-pretreated non-stressed group, 2) a saline-pretreated stressed group, and 3) an astressin-pretreated stressed group. The numbers of mast cells, EC cells, and PAR2-positive cells were counted in 6 high power fields. In proximal colonic segments, mast cell numbers of stressed rats tended to be higher than those of non-stressed rats, and their PAR2-positive cell numbers were significantly higher than those of non-stressed rats. In distal colonic segments, mast cell numbers and PAR2-positive cell numbers of stressed rats were significantly higher than those of non-stressed rats. Mast cell and PAR2-positive cell numbers of astressin-pretreated stressed rats were significantly lower than those of saline-pretreated stressed rats. EC cell numbers did not differ among the three experimental groups. Acute stress in rats increases mast cell numbers and mucosal PAR2 expression in the colon. These stress-related alterations seem to be mediated by release of corticotrophin-releasing factor.
Animals ; Colon/*metabolism ; Corticotropin-Releasing Hormone/antagonists & inhibitors/metabolism/pharmacology/*physiology ; Enterochromaffin Cells/cytology ; Male ; Mast Cells/*cytology/immunology/metabolism ; Peptide Fragments/pharmacology ; Rats ; Rats, Wistar ; Receptor, PAR-2/*metabolism ; Restraint, Physical ; *Stress, Physiological

OBJECTIVETo observe expressions of glucose-regulated protein (GRP78) and caspase-S12 in nervous system-interstitial cells of Cajal (ICC) in smooth muscle in colonic wall in rats with scald injury, as well as their relevant ultrastructural changes, so as to probe the possible mechanisms of dynamic damage in murine colon after a scald injury.

METHODSFifty healthy Sprague-Dawley rats were randomly divided into scald (n = 40) and control (n = 10) groups. Rats in scald group were inflicted with 30% TBSA full-thickness scald, and received an intraperitoneally injection of Ringer lactate solution (50 mg/kg) for resuscitation, while those in control group had similar treatment with the exception of scald. Rats in control group and scald group were sacrificed at 3, 6, 12, 24 post scald hour (PSH, 10 rats at each time point) for collection of 4 cm of colonic tissue, 5 cm proximal to the cecum. A segment of colonic wall, 1 cm in length, was obtained from the middle of the harvested segment of colon, and it was fixed with 3% glutaraldehyde or 10% formaldehyde. The samples fixed with glutaraldehyde were used to observe ultrastructural alterations under transmission electron microscope, while that with formaldehyde were used to observe expressions of GRP78 and caspase-12 in colonic wall by immunohistochemical assay.

RESULTSThe colonic smooth muscle cells of rats in control group showed regular arrangement, their organelles were abundant, nucleus centrally located, euchromatin distributed evenly with more abundant mitochondrial cristae and less smooth endoplasmic reticulum, neuronal organelles were abundant in intermuscular plexus, and ICC could be seen in the neighborhood of neurons. The colonic smooth muscle cells appeared in irregular and disordered manner in scald group, perinuclear space was widened, intercellular vacuoles were observed, mitochondria showed vacuolation degeneration with dissolved and condensed cristae, rough endoplasmic reticula were dilated with partial dissolution, and perinuclear cytoplasm of ICC was obviously decreased. The expression of GRP78 was increased in scald group at 3, 6, 12 PSH (4.3 +/- 0.9, 6.0 +/- 0.7, 4.8 +/- 1.1 score) as compared with that in control group (2.4 +/- 0.7 score, P < 0.05). The expression of caspase-12 in scald group at 6, 12, 24 PSH was higher than that in control group (P < 0.05). GRP78 was consistently expressed in cytoplasm in control group, while in scald group, it mainly appeared in mucosa, myenteric plexus, and stromal cells, but only moderately or lightly expressed in smooth muscle cells. The expression of GRP78 was positive in scald group at 3, 6, 12 PSH, strongly positive at 6 PSH, and it was also expressed in cytoplasm in control group. The expression of caspase-12 in scald group was not obviously positive at 3 PSH, and weakly positive at 6, 24 PSH, but strongly positive at 12 PSH, while no expression was shown in control group.

CONCLUSIONSMarked pathological changes are observed in enteric nervous system-interstitial cells of Cajal-smooth muscle in rats with severe scald injury. It may be related with cellular injuries induced by caspase-12 apoptotic pathways through activated endoplasmic reticulum stress.

Animals ; Apoptosis ; Burns ; metabolism ; pathology ; physiopathology ; Caspase 12 ; metabolism ; Colon ; cytology ; Endoplasmic Reticulum ; metabolism ; Female ; Heat-Shock Proteins ; metabolism ; Interstitial Cells of Cajal ; cytology ; pathology ; ultrastructure ; Male ; Rats ; Rats, Sprague-Dawley

To investigate the characteristics of large-conductance calcium-activated potassium channels (BK(Ca)) and the effect of hydrogen peroxide (H2O2) on BK(Ca) in guinea-pig proximal colon smooth muscle cells, single smooth muscle cells of guinea-pig colon were enzymatically isolated in low calcium solution containing papain (3 mg/mL), DTT (2 mg/mL), and bovine serum albumin (BSA, 2 mg/mL). Tissues were incubated at 36 degrees C in enzyme solution for 15 min and were then suspended in enzyme-free low calcium solution. Inside-out single channel recording technique was used to record BK(Ca) current. The intracellular (bath) and microelectrode solution both contained symmetrical high potassium. The BK(Ca) in guinea-pig colon smooth muscle cell possesses: 1) voltage-dependence, 2) high selectivity for potassium ion, 3) large conductance (223.7 pS+/-9.2 pS), 4) dependence of [Ca(2+)](i). Intracellular application of H2O2 decreased the open probability (P(o)) of BK(Ca) at low concentration ( Animals ; Colon ; cytology ; Guinea Pigs ; Hydrogen Peroxide ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; physiology ; Myocytes, Smooth Muscle ; physiology ; Oxidation-Reduction