(−)-Epicatechin mitigates radiation-induced intestinal injury and promotes intestinal regeneration via suppressing oxidative stress

Abstract

The high radiosensitivity of the intestinal epithelium limits the survival of victims by nuclear accidents or terrorism and limits effective radiotherapy against abdominal malignancies. Recently, we reported that (?)-epicatechin (EC) modulates oxidative stress and exerts neuroprotection. Here, we investigate the protective effects of EC against intestinal damage induced by radiation. The established model is acute moderate but reversible intestinal injury damage. We also set up the injured model of “minigut” ex vivo, which mimic the process of intestinal regeneration in vivo. We found that EC can repress oxidative stress by regulating SOD and MDA levels in serum and intestine tissue. Correspondingly, EC can decrease apoptosis of crypt cells in Lgr5-EGFP-IRES-creERT2 mice after radiation. Further studies demonstrated that EC can promote Nrf2 translocation from cytoplasm to nuclear and then activate the expression of HO1 and NQO1. Interestingly, EC can enhance the activity of intestine stem cells labelled by Lgr5 and promote intestinal epithelium regeneration determined by HE and immunofluorescence staining in vivo and in vitro. We also found that EC can activate the Wnt/β-catenin signal pathway confirmed by TCF/LEF luciferase reporter assay. Together, EC can provide the protective effect on intestine and promote intestinal regeneration after radiation through Nrf2 and Wnt/β-catenin signal pathway.     

The application of the “Lojda” method for intestinal lactase in intestinal biopsies from jaundiced newborn infants

Summary A new use of the histochemical method for intestinal lactase activity is described. Peroral intestinal biopsies from newborn light-treated infants with diarrhoea were investigated for brush-border lactase. The lactase activity found in these infants by the histochemical method correlated well with the infants ability to hydrolyze lactose judged by lactose tolerance test.     

Does the intestinal microflora synthesize pyrroloquinoline quinone?

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and glucose dehydrogenase. When chemically-defined diets without PQQ are fed to animals, lathyritic changes are observed. In previous studies, it was assumed that PQQ was produced by the intestinal microflora; consequently, antibiotics were routinely added to diets. In the present study this assumption is tested further in mice by: (i) examining the effects of dietary antibiotics on fecal PQQ excretion, (ii) isolating the intestinal flora to identify bacteria known to synthesize PQQ and (iii) determining in vitro if the intestinal microflora synthesizes PQQ from radio-chemically labeled precursors. The results of these experiments indicate that little if any PQQ is synthesized by the intestinal microflora. Rather, when PQQ is present in the intestine, the diet is a more obvious source.     

Apoptotic process of porcine intestinal M cells

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer’s patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18⁺/PCNA⁺ cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18⁺/alkaline phosphatase⁺ cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex. This investigation was supported by a Grant-in-Aid for Scientific Research (16658107) from the Ministry of Education, Culture, Sports, Science and Technology, by two grants (Prion Project and Secure and Healthy Livestock Farming Project) from the Ministry of Agriculture, Forestry and Fisheries, and by a grant from the Naito Foundation.     

Do intestinal nematodes affect productivity in adulthood?

Intestinal nematode infections have been associated with many physical and mental developmental insults. These include anaemia, wasting, stunting, cognitive impairment and lowered educational achievement, all of which have in turn been shown to interfere with productivity and wage-earning capacity in adults. Although there is no direct evidence for an effect of intestinal nematodes on productivity, circumstantial evidence suggests such an effect. Here, Helen Guyatt reviews the indirect evidence for an effect of intestinal nematodes on productivity in adults through current infection and associated morbidity, and on early ill-health in children, which might affect productivity later in life.     

Enzymic hydrolysis of the carbon–fluorine bond of α-d-glucosyl fluoride by rat intestinal mucosa: Localization of intestinal maltase

1. alpha-d-Glucosyl fluoride was hydrolysed by an extract of rat intestinal mucosa. The pH optimum was 6.6 and the K(m) 0.4mm at 20 degrees . Activity was assayed by release of either glucose or fluoride. 2. The alpha-d-glucosyl fluoride-hydrolase activity of the extract was associated with both mutarotase and alpha-d-glucosidase activities. 3. Tris (5mm) inhibited both the alpha-d-glucosidase and alpha-d-glucosyl fluoride-hydrolase activities by 55% but did not inhibit mutarotase. The K(i) of tris for both enzyme activities was 2mm. 4. The extract did not hydrolyse melibiose and lactose. Mutarotase used both alpha-d-glucose and beta-l-arabinose as substrates but the glucosyl fluoride-hydrolase activity did not extend to beta-l-arabinosyl fluoride. 5. The thermal stability of alpha-d-glucosidase and alpha-d-glucosyl fluoride hydrolase was identical. Mutarotase was more thermolabile. 6. A preparation of the brush border of intestinal epithelial cells contained both alpha-d-glucosyl fluoride-hydrolase and alpha-d-glucosidase activities. In each precipitate and washing the ratio of the two activities was the same. All the mutarotase activity was in the first supernatant. 7. Agidex, a fungal amyloglucosidase, cleaved glucosyl fluoride in addition to maltose. Tris inhibited both activities and in each case the K(i) was 3mm. 8. The probable identity of alpha-d-glucosyl fluoride hydrolase with alpha-d-glucosidase is discussed and a possible mechanism for the reaction suggested. 9. Incubation of intestinal slices with alpha-d-glucosyl fluoride led to complete hydrolysis in 30min. The glucose rapidly entered the cell and was metabolized, leaving the fluoride in the incubation medium. This constitutes a further proof that the intestinal alpha-d-glucosidase, although on the brush border, is located outside the site of active transport of sugars.     

Does the intestinal bifidobacterial colonisation affect bacterial translocation?

The aim of this work was to investigate the possible role of the intestinal anaerobic flora (especially bifidobacteria) in regulating bacterial translocation (BT) which can be defined as the passage of intestinal microbes through the mucosa to internal organs. Default in BT regulation concurs with pathogenesis of sepsis in various human conditions, such as acute pancreatitis, cirrhosis, necrotising enterocolitis or multiple organ failure. The intestinal flora was studied in human flora associated mice (HF mice) and BT was quantified in Peyer's patches (PP), blood, spleen, liver and lungs. HF mice displayed a heterogenic intestinal colonisation with bifidobacteria. High colonisation of both caecum and colon by bifidobacteria led to a poorer bacterial contamination of blood, liver and lungs. Moreover, ileal, caecal and colonic bifidobacterial counts negatively correlated with the bacterial dissemination (number of contaminated organs per mouse). In contrast, Bacteroides fragilis group counts positively correlated with bacteraemia, lungs contamination or bacterial dissemination. Additionally, clostridia localised in the colon affected bacterial uptake by PP and lungs contamination as indicated by positive correlations between bacterial populations in these respective locations. These results indicate that bifidobacteria, when established in high counts, reduced BT to liver, blood and lungs, whereas B. fragilis group favoured the bacterial passage. Clostridia established in the distal ileum also seemed to favour BT to lungs. The manipulation of the bacterial flora to optimise the regulatory effect on BT should therefore focus on the selective promotion of bifidobacteria and avoid an increase in potentially detrimental populations such as B. fragilis group and clostridia.     

PPAR-γ agonist protects against intestinal injury during necrotizing enterocolitis

Necrotizing enterocolitis (NEC) remains a lethal condition for many premature infants. Peroxisome proliferator-activated receptor-γ (PPAR-γ), a member of the nuclear hormone receptor family, has been shown to play a protective role in cellular inflammatory responses; however, its role in NEC is not clearly defined. We sought to examine the expression of PPAR-γ in the intestine using an ischemia-reperfusion (I/R) model of NEC, and to assess whether PPAR-γ agonist treatment would ameliorate I/R-induced gut injury. Swiss-Webster mice were randomized to receive sham (control) or I/R injury to the gut induced by transient occlusion of superior mesenteric artery for 45 min with variable periods of reperfusion. I/R injury resulted in early induction of PPAR-γ expression and activation of NF-κB in small intestine. Pretreatment with PPAR-γ agonist, 15d-PGJ₂, attenuated intestinal NF-κB response and I/R-induced gut injury. Activation of PPAR-γ demonstrated a protective effect on small bowel during I/R-induced gut injury.     

Dietary α-ketoglutarate supplementation ameliorates intestinal injury in lipopolysaccharide-challenged piglets

Neonates are at increased risk for inflammatory bowel disease, but effective prevention and treatments are currently limited. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of dietary supplementation with α-ketoglutarate (AKG) on the intestinal morphology and function. Eighteen 24-day-old pigs (weaned at 21 days of age) were assigned randomly to control, LPS, and LPS + AKG groups. The piglets in the control and LPS groups were fed a corn- and soybean meal-based diet, whereas the LPS + AKG group was fed the basal diet supplemented with 1% AKG. On days 10, 12, 14, and 16, piglets in the LPS and LPS + AKG groups received intraperitoneal administration of LPS (80 μg/kg BW), whereas piglets in the control group received the same volume of saline. On day 16, d-xylose was orally administrated to all pigs at the dose of 0.1 g/kg BW, 2 h after LPS or saline injection, and blood samples were collected 3 h thereafter. Twenty-four hours post-administration of LPS or saline, pigs were killed to obtain intestinal mucosae for analysis. Compared with the control group, LPS challenge reduced (P < 0.05) protein levels, the ratio of villus height to crypt depth, and the ratio of phosphorylated mTOR to total mTOR in duodenal, jejunal, and ileal mucosa. These adverse effects of LPS were attenuated (P < 0.05) by AKG supplementation. Moreover, AKG prevented the LPS-induced increase in intestinal HSP70 expression. Collectively, these novel results indicate that dietary supplementation with 1% AKG activates the mTOR signaling, alleviates the mucosal damage, and improves the absorptive function of the small intestine in LPS-challenged piglets. The findings not only help understand the mode of AKGs actions in the neonatal gut but also have important implications for infant nutrition under inflammatory conditions.     

Where intestinal epithelial stem cells are localized? About molecular markers

Using stem cells as an example the review considers a new history and methodology of search for stem cells (SC), found in tissues of adult Homo sapiens and Drosophila melanogaster organisms. These studies of SC resulted in several original hypotheses explaining their unusual features. Impressive progress recently achieved in this direction (2008–2010) is associated with employment of new methods of somatic recombination for long-term registration of various strains of differentiated cells, early and distant SC progeny. 1) Although anatomic localization of intestinal epithelium cells lacking marked morphological and biochemical differentiation markers (the lower third of intestinal and colon crypts) is known for about 40 years results of their experimental identification, isolation and detection of their functional characteristics still represent the subject for discussions. Particularly, it remains unclear, which SC are involved in crypt regeneration: the same as those involved into homeostatic renewal or their various subpopulations or early SC progenies acquired stem features by reprogramming? 2) In addition, most detected biochemical markers of potential SC are common for SC from other tissues of embryonic and mature organisms so it is possible to apply method developed for intestinal epithelium for their isolation. 3) Data on induction of intestinal epithelium polyps and neoplasias by mutations in genes encoding SC markers and identification of biochemical characteristics of potential SC in these tumors support the hypothesis of stem tumor cell origination from normal SC or their earliest progeny. In general, facts considered in this review may be useful for both development of optimal methods for the use of SC in cell therapy (as the source of humoral factors), regenerative medicine (as the source of differentiated cells for restoration of injured tissue), and also for targeted search of antitumor drugs (SC as the target) and preparations modifying genetic and epigenetic reactions of SC to genotoxic and stress treatments.     

Histochemical demonstration of the intestinal hetero-β-galactosidase (glucosidase)

Summary The histochemical demonstration of hetero--galactosidase (glucosidase) has been attempted in sections and zymograms of rabbit, monkey and human intestine and of rat kidney.The leakage of this enzyme from unfixed sections was prevented by the use of cold microtome sections adherent to semipermeable membranes. Methods with -D-glucosides and galactosides of 6-Br-2-naphthol (postincubation azocoupling with Fast Blue B as well as simultaneous azocoupling with hexazonium-p-rosaniline), of -naphthol (simultaneous azocoupling with hexazonium-p-rosaniline) and of 4-Cl-5-Br-3-indolyl (with ferricyanide, phenazonium methosulfate or nitro BT and without any oxidation agent) were used an evaluated concerning the specificity, localization ability and inhibition of enzyme activity. Pretreatment of sections with distilled water or saline and inhibition by p-Cl-mercuribenzoate, glucono- and galactono-lactones were used for the characterization of the demonstrated enzyme activity.6-Br-2-naphthyl--D-glucoside is the most specific substrate for hetero--galactosidase. It is not split by lactase and acid -galactosidase. Only lysosomal -glucosidase can interfere. Because the latter enzyme is membrane-bound the difference in color intensity between untreated and prewashed sections are due to hetero--galactosidase. Only localization on the cellular (not intracellular) level can be achieved, however.The simultaneous azocoupling method with -naphthyl--D-glucoside and hexazonium-p-rosaniline enables a very good localization of hetero--galactosidase in the rabbit intestine. Due to a great inhibition exerted by hexazonium-p-rosaniline on the enzyme activity the method is unsuitable for the detection of hetero--galactosidase in zymograms and in the human intestine. Interference of lactase (or lactase-phlorizine hydrolase complex) is to be considered. The lysosomal -glucosidase does not seem to interfere.Indigogenic methods are not sensitive either. With ferricyanide as an oxidation agent it was not possible to detect the activity of hetero--galactosidase in zymograms and in sections. This is possibly due to overoxidation of indigo. The same holds true for phenazonium methosulfate used for the processing of zymograms. However, it was possible to reveal the activity of hetero--galactosidase in sections of the rabbit and monkey intestine with phenazonium methosulfate as oxidation agent. Nitro BT enhanced the coloration both in zymograms and in sections. In the latter case diffusion artifacts cannot be prevented, however. The interference of lactase, lysosomal -galactosidase and possibly of lysosomal -glucosidase (depending on the glycoside used) is always to be considered.Hetero--galactosidase was localized in the cytoplasm (particularly in the supranuclear region) of differentiated enterocytes covering the villi of the rabbit (the highest activity), monkey and human (the lowest activity) intestine. In crypt enterocytes and in cells of Brunner's glands the activity was lower. The occurrence of a low activity of hetero--galactosidase in the brush border of enterocytes of the rabbit intestine was also demonstrated.A proximodistal gradient was observed in the rabbit and monkey intestine, the upper jejunum displaying the highest activity.In jejunal biopsies of patients with celiac sprue (in the acute stage of the disease) the activity of hetero--galactosidase was lowered. No changes of activity were observed in jejunal biopsies of patients with isolated deficiencies of lactase or sucrase.In the rat kidney the enzyme was demonstrated particularly in the cytoplasm of cells of proximal convoluted tubules.     

Decreased expression of the gut-enriched Krüppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients

Onconase((R)) (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C-terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10-fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity.     

Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.     

Cutting edge: is vasoactive intestinal peptide a type 2 cytokine?

A component of the chemical language shared by the immune and nervous system is the expression of neuropeptides by immune cells. Vasoactive intestinal peptide (VIP) was shown to be produced by T lymphocytes. Here we investigate whether T cell subsets differentially express VIP. Our studies indicate that, upon specific Ag stimulation, Th2 and T2 cells, but not Th1 and T1 cells derived from TCR transgenic (Tg) mice, express VIP mRNA and protein, and secrete VIP. Following immunization with the specific Ag, significant levels of VIP are present in the serum of syngeneic, non-Tg hosts that receive Th2, but not Th1 Tg cells. Th2 Tg cells recovered from the non-Tg hosts immunized with the specific Ag, but not with an irrelevant Ag, express intracellular VIP. Because VIP is produced by Ag-stimulated type 2 T cells, and differentially affects Th1 and Th2 cells, could VIP be viewed as a type 2 cytokine?     

DHA attenuates postprandial hyperlipidemia via activating PPARα in intestinal epithelial cells

It is known that peroxisome proliferator-activated receptor (PPAR)α, whose activation reduces hyperlipidemia, is highly expressed in intestinal epithelial cells. Docosahexaenoic acid (DHA) could improve postprandial hyperlipidemia, however, its relationship with intestinal PPARα activation is not revealed. In this study, we investigated whether DHA can affect postprandial hyperlipidemia by activating intestinal PPARα using Caco-2 cells and C57BL/6 mice. The genes involved in fatty acid (FA) oxidation and oxygen consumption rate were increased, and the secretion of triacylglyceride (TG) and apolipoprotein B (apoB) was decreased in DHA-treated Caco-2 cells. Additionally, intestinal FA oxidation was induced, and TG and apoB secretion from intestinal epithelial cells was reduced, resulting in the attenuation of plasma TG and apoB levels after oral administration of olive oil in DHA-rich oil-fed mice compared with controls. However, no increase in genes involved in FA oxidation was observed in the liver. Furthermore, the effects of DHA on intestinal lipid secretion and postprandial hyperlipidemia were abolished in PPARα knockout mice. In conclusion, the present work suggests that DHA can inhibit the secretion of TG from intestinal epithelial cells via PPARα activation, which attenuates postprandial hyperlipidemia.     

Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-β1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-β1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.