Short-chain fatty acids and water in the hindgut contents and feces of rats after hindgut bypass surgery

Omnivorous experimental animals with different levels of short-chain fatty acids in hindgut contents or in feces were established by surgical bypass of the colon and/or the cecum. Levels of short-chain fatty acids, amounts of luminal contents, and the water contents in these animals were compared. Regional variations in water absorption and retention of luminal contents are likely to be major factors in the regulation of hindgut functions.     

The effects of glutamine on intestinal epithelial cell proliferation in parenterally fed rats

BACKGROUND: Several papers have indicated that glutamine is a preferred fuel for the enterocyte and that it can increase intestinal epithelial cell proliferation. AIMS: To investigate the effects of glutamine on intestinal epithelial cell proliferation in the parenterally fed rat. METHODS: Five groups of six rats were fed parenterally; a group of orally fed rats was also studied. Crypt cell proliferation was studied after six days using native mitoses in microdissected crypts and bromodeoxyuridine labelling. RESULTS: No effect of treatment was seen on intestinal weight; however, the weights of the small intestine, caecum, and colon were all significantly heavier in the orally fed group than in the total parenteral nutrition groups (p<0.001). There was no effect of any of the glutamine treatments on mitotic activity in the small intestine. In the colon there was a small increase in native mitoses with glutamine (p=0.03). There was also an indication of increased proliferative activity in the first fifth of the small intestine and colon with glutamine. Little effect of glutamine on bromodeoxyuridine labelling in either site was observed, but there was a small but significant reduction in growth fraction of the colon of the glutamine treated group. The labelling distribution curve from sections and the mitotic distribution curve obtained from crypt squashes showed a good correlation. CONCLUSION: Glutamine has a small, but significant effect on mitotic activity but only in the colon. Modest effects on the distribution of labelled cells were also seen.     

Lipopolysaccharide- and proinflammatory cytokine-induced energy production in intestinal and colonic epithelial cell lines

BACKGROUND: Although epithelial cells in ulcerative colitis may be metabolically deficient, it remains unknown whether epithelial cells modulate energy metabolism in inflamed mucosa. The purpose of the present study is to investigate whether inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) alter energy metabolism in epithelial cells. METHODS: Adenosine 5'-triphosphate (ATP) levels in HT29 cells cultured with LPS, IL-1beta, IL-6, or TNF-alpha were measured with high-performance liquid chromatography, using a reversed-phase chromatography column. Cellular and mitochondrial (antimycin A-sensitive) respiration rates were determined polarographically, using a Clark-type oxygen electrode. RESULTS: When the cells were cultured with LPS, IL-6, and TNF-alpha but not IL-1beta, ATP levels increased significantly at 6 h, followed by a decrease at 24 h. Enhancement of oxygen consumption, which was completely blocked by antimycin A, was also shown at 3 h by the exposure to these substrates. CONCLUSION: LPS and proinflammatory cytokines induced cellular ATP generated by mitochondrial phosphorylation. An active energy production in epithelial cells on the exposure to inflammatory mediators may be critical for escape from chronic mucosal inflammation.     

Chronic low vitamin intake potentiates cisplatin-induced intestinal epithelial cell apoptosis in WNIN rats

AIM:To investigate if cisplatin alters vitamin status andif VR modulates cisplatin induced intestinal apoptosis andoxidative stress in Wistar/NIN(WNIN)male rats.METHODS:Weanling,WNIN male rats(n=12 pergroup)received adlibitum for 17 wk:control diet(20%protein)or the same with 50% vitamin restriction.Theywere then sub-divided into two groups of six rats eachand administered cisplatin(2.61 mg/kg bodyweight)once a week for three wk or PBS(vehicle control).Intestinal epithelial cell(IEC)apoptosis was monitoredby morphometry,Annexin-V binding,M30 cytodeathassay and DNA fragmentation.Structural and functionalintegrity of the villus were assessed by villus height/crypt depth ratio and activities of alkaline phosphatase,lys,ala-dipeptidyl amino-peptidase,respectively.Toassess the probable mechanism(s)of altered apoptosis,oxidative stress parameters,caspase-3 activity,andexpression of Bcl-2 and Bax were determined.RESULTS:Cisplatin per se decreased plasma vitaminlevels and they were the lowest in VR animals treatedwith cisplatin.As expected VR increased only villusapoptosis,whereas cisplatin increased stem cellapoptosis in the crypt.However,cisplatin treatmentof VR rats increased apoptosis both in villus and cryptregions and was associated with higher levels of TBARS,protein carbonyls and caspase-3 activity,but lower GSHconcentrations.VR induced decrease in Bcl-2 expression was further lowered by cispiatin.Bax expression,unaffected by VR was increased on cisplatin treatment.Mucosal functional integrity was severely compromisedin cisplatin treated VR-rats.CONCLUSION:Low intake of vitamins increases thesensitivity of rats to cisplatin and promotes intestinalepithelial cell apoptosis.     

Does dietary fibre stimulate intestinal epithelial cell proliferation in germ free rats?

The aim of the present experiment was to investigate the role of hind gut fermentation in the proliferative response of the intestinal epithelium to dietary fibre. We have previously shown that refeeding starved rats with an elemental diet supplemented with fermentable dietary fibre (but not inert bulk) is capable of stimulating intestinal epithelial cell proliferation throughout the gastrointestinal tract. Three groups of 10 germ free (GF) rats and three groups of 10 conventional (CV) rats, were used. All groups were starved for three days and then refed for two days with either an elemental diet (Flexical); Flexical plus 30% kaolin; or Flexical plus 30% of a fibre mixture. Cell production was determined by the accumulation of vincristine arrested metaphases in microdissected crypts. There was no significant difference between refeeding the rats with an elemental diet alone or with kaolin supplementation, however, the addition of fibre in CV rats was associated with a significant increase in intestinal crypt cell production rate in both the small intestine (p less than 0.01) and the colon (p less than 0.001). This marked proliferative effects of fibre was abolished in the GF rats. It can be concluded that it is the products of hind gut fermentation, not fibre per se that stimulate intestinal epithelial cell proliferation in the colon and small intestine.     

Reduced intestinal epithelial cell brush border membrane calcium transport in spontaneously hypertensive rats.

OBJECTIVE: The present study aimed to determine whether there were alterations in intestinal calcium homeostasis in the spontaneously hypertensive rats (SHR) and to identify at which interface of the intestinal epithelial cell (brush border or basolateral) this occurs. DESIGN: Controversy exists as to whether intestinal calcium transport is altered in association with hypertension. Studies using perfused duodenal segments of the SHR have shed little light on the problem; other studies have only measured calcium transport in brush border membrane vesicles. This study allows specific focus on calcium transport mechanisms at both the brush border and basolateral membrane using simultaneously prepared membrane vesicles. METHODS: Calcium transport was studied by measuring radiolabelled calcium (45Ca) uptake in isolated brush border and basolateral membrane vesicles, prepared from the small intestines of SHR and Wistar-Kyoto (WKY) rats. Calcium uptake was measured when vesicles were incubated in solutions containing different concentrations of ATP and calcium. Orientation and membrane marker assays were used to confirm the phenotypes of the two membrane vesicle preparations. RESULTS: ATP-dependent calcium efflux was only observed in the basolateral membrane, which contains the Ca2+ -ATPase pump. SHR brush border membrane vesicles displayed no significant increase in calcium incorporation, whereas WKY brush border vesicles showed a 500% increase in uptake (ANOVA, P<0.05, n = 7). CONCLUSIONS: This study indicates that deficiencies exist in SHR intestinal calcium transport at the brush border membrane of intestinal epithelial cells. While further studies are required to ascertain the exact mechanisms involved, postulated deficiencies in the actions of calcium regulating hormones at this membrane suggest the need for concurrent intake of a calcitrophic agent to assist calcium uptake at the brush border membrane.     

酒精通过抑制肠上皮干细胞增殖损伤肠上皮的更新修复能力

目的 研究酒精对肠上皮干细胞(ISC)和肠上皮更新修复能力的影响。方法 将18只C57BL/6小鼠随机分为对照组(n=9)和酒精处理组(n=9)。采用Gao-Binge法制备慢性酒精中毒模型。在造模成功后,腹腔注射5-溴-2-脱氧脲苷(BrdU),分别在注射后2 h、24 h和72 h取小肠组织,采用免疫组化法检测BrdU阳性细胞和ISC特异性标志物Lgr5表达。结果 与对照组小鼠比,酒精处理组小鼠小肠绒毛高度显著缩短、萎缩;酒精处理组小鼠ISC细胞Lgr5表达显著弱于对照组;酒精处理组小鼠每个肠隐窝BrdU阳性细胞数量为(3.50±0.65)个/肠隐窝,显著少于对照组【(7.90±1.08)个/肠隐窝,P＜0.05】;在注射BrdU 后2 h、24 h和72 h,酒精处理组小鼠小肠BrdU阳性细胞迁移距离分别为(66.67±1.60)μm、(219.40±12.11)μm和(313.90±9.76)μm,显著短于对照组【分别为(111.10±1.60)μm、(319.00±10.04)μm和(625.90±3.34)μm,P＜0.05】。结论 酒精通过抑制ISC引起肠上皮细胞增殖和迁移能力下降,从而损伤肠上皮的更新修复能力,导致肠上皮屏障功能障碍。     

Crosstalk between intestinal epithelial cell and adaptive immune cell in intestinal mucosal immunity

Constantly challenged by luminal bacteria, intestinal epithelium forms both a physical and biochemical defense against pathogens. Besides, intestinal epithelium senses dynamic and continuous changes in luminal environment and transmits signals to subjacent immune cells accordingly. It has been long accepted that adaptive immune cells fulfill their roles partly by modulating function of intestinal epithelial cells. Recent studies have brought up the proposal that intestinal epithelial cells also actively participate in the regulation of adaptive immunity, especially CD4+ adaptive T cells, which indicates that there is reciprocal crosstalk between intestinal epithelial cells and adaptive immune cells, and the crosstalk may play important role in intestinal mucosal immunity. This Review makes a comprehensive summary about crosstalk between intestinal epithelial cells and CD4+ adaptive T cells in intestinal immunity. Special attention would be given to their implications in inflammatory bowel disease pathogenesis and potential therapeutic targets.     

Hemolysis results in impaired intestinal microcirculation and intestinal epithelial cell injury

AIM:To study the effect of circulating cell-free oxy-hemoglobin(FHb) on intestinal microcirculation and intestinal epithelial injury in a rat model. METHODS:To induce elevated intravascular circulating FHb,male Sprague-Dawley rats received water or FHb infusion.Microcirculatory changes in jejunum,ileum and colon were evaluated using fluorescent microspheres.Intestinal injury was quantified as plasmatic release of ileal lipid binding protein(iLBP) and verified by histological analysis of the ileum. RESULTS:W...     

Adipose-derived mesenchymal stem cells alleviate TNBS-induced colitis in rats by influencing intestinal epithelial cell regeneration,Wnt signaling,and T cell immunity

BACKGROUND Conventional Crohn's disease(CD) treatments are supportive rather than curative and have serious side effects. Adipose-derived mesenchymal stem cells(ADSCs) have been gradually applied to treat various diseases. The therapeutic effect and underlying mechanism of ADSCs on CD are still not clear.AIM To investigate the effect of ADSC administration on CD and explore the potential mechanisms.METHODS Wistar rats were administered with 2,4,6-trinitrobenzene sulfonic acid(TNBS) to establish a rat model of CD, followed by tail injections of green fluorescent protein(GFP)-modified ADSCs. Flow cytometry, qRT-PCR, and Western blot were used to detect changes in the Wnt signaling pathway, T cell subtypes, and their related cytokines.RESULTS The isolated cells showed the characteristics of ADSCs, including spindle-shaped morphology, high expression of CD29, CD44, and CD90, low expression of CD34 and CD45, and osteogenic/adipogenic ability. ADSC therapy markedly reduced disease activity index and ameliorated colitis severity in the TNBS-induced rat model of CD. Furthermore, serum anti-sacchromyces cerevisiae antibody and panti-neutrophil cytoplasmic antibody levels were significantly reduced in ADSCtreated rats. Mechanistically, the GFP-ADSCs were colocalized with intestinal epithelial cells(IECs) in the CD rat model. GFP-ADSC delivery significantly antagonized TNBS-induced increased canonical Wnt pathway expression, decreased noncanonical Wnt signaling pathway expression, and increased apoptosis rates and protein level of cleaved caspase-3 in rats. In addition, ADSCs attenuated TNBS-induced abnormal inflammatory cytokine production, disturbed T cell subtypes, and their related markers in rats.CONCLUSION Successfully isolated ADSCs show therapeutic effects in CD by regulating IEC proliferation, the Wnt signaling pathway, and T cell immunity.     

Polyamines and cytoskeletal proteins in intestinal epithelial cell migration

Abstract Our previous studies have shown that polyamines are essential for early mucosal restitution in vivo and cell migration in vitro. The current study determines whether cytoskeleton is involved in the process requiring polyamines for the stimulation of cell migration. Treatment with α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the intestinal epithelial cells (IEC-6) derived from rat small intestinal crypt cells. Polyamine deficiency resulted in reorganization of F-actin in migrating cells but had no effect on the concentrations of filamentous actin and β-actin mRNA. The actin cortex was greatly increased in density and lamellipodia were less extensive. In contrast, non-muscle myosin I and II levels in DFMO-treated cells were decreased by 70 and 75%, respectively, and stress fibres were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous polyamine not only returned cytoskeleton levels and distribution towards normal but also restored cell migration to control levels. These results indicate that actin and myosins have a role in polyamine-dependent epithelial cell migration and may be part of the mechanism that requires polyamines for early mucosal restitution.     

Uptake of L-carnitine by a human intestinal epithelial cell line, Caco- 2

BACKGROUND & AIMS: The mechanism of intestinal uptake of L-carnitine is controversial. The aim of this study was to clarify the mechanism and regulation of L-carnitine uptake. METHODS: Uptake of [3H]-L-carnitine was measured across the apical membrane of confluent monolayers of Caco- 2 cells. RESULTS: [3H]-L-carnitine uptake was linear and appreciable for up to 7 minutes with minimal metabolic alteration, was temperature- and Na(+)-(but not pH-) dependent, and included a saturable component with an apparent Michaelis constant of 45.5 +/- 6.5 mumol/L and a maximum velocity of 83.5 +/- 5.6 nmol.mg protein-1.5 min-1. Unlabeled L- carnitine and its structurally related analogues significantly (P < 0.01) inhibited [3H]-L-carnitine uptake, whereas unrelated compounds were ineffective. L-Carnitine uptake was also energy-dependent, being significantly (P < 0.01) inhibited by metabolic inhibitors. Our results also suggested that a calmodulin- but not a protein kinase C- or protein kinase A-mediated pathway plays a role in regulating L- carnitine uptake by Caco-2 cells. CONCLUSIONS: L-carnitine uptake by intestinal epithelial cells (Caco-2) involves a carrier-mediated system that is temperature-, Na(+)-, and energy-dependent and seems to be under the regulation of a calmodulin-mediated pathway. (Gastroenterology 1996 Dec;111(6):1534-40)