Inhibition of intestinal ischemia/repurfusion induced apoptosis and necrosis via down-regulation of the NF-kB,c-Jun and caspace-3 expression by epigallocatechin-3-gallate administration

Intestinal ischemia/reperfusion (I/R) produces reactive oxygen species (ROS) activating signal transduction and apoptosis. The aim of this study was to evaluate the effect of (?)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kB, c-Jun and caspace-3 in intestinal I/R. Thirty male wistar rats were used. Group A sham operation, B I/R, C I/R-EGCG 50 mg/kg ip. Intestinal ischemia was induced for 60 min by clamping the superior mesenteric artery. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, Fragment End Labelling of DNA (TUNEL), immunocytochemistry for NF-kB, c-Jun and caspace-3 analysis in intestinal specimens were performed 120 min after reperfusion. Apoptosis as indicated by TUNEL and Caspace-3, NF-kB and c-Jun was widely expressed in I/R group but only slightly expressed in EGCG treated groups. MDA and MPO showed a marked increase in the I/R group and a significant decrease in the EGCG treated group. Light histology showed preservation of architecture in the EGCG treated group. In conclusion, EGCG pre-treatment is likely to inhibit intestinal I/R-induced apoptosis by down-regulating the expression of NF-kB, c-Jun and caspase-3.     

Über die Mikrovillibildung im fetalen Rattendünndarm

Zusammenfassung Die Frage, auf welche Weise die Enterocyten des fetalen Rattendünndarms das für die Mikrovillibildung benötigte Membranmaterial liefern, wurde elektronenmikroskopisch untersucht. Es wird angenommen, daß hufeisenförmige Strukturen, die aus mit elektronendichtem Material bedeckten Elementarmembranen bestehen und möglicherweise Längsschnitten durch kappenförmige Gebilde entsprechen, in das apicale Plasmalemm eingebaut werden und für die Bildung der Mikrovillispitzen verantwortlich sind. Diese Annahme gründet sich in erster Linie auf die Feststellung eines nahezu identischen Durchmessers von Hufeisen und Mikrovilli, auf die Lokalisation der Hufeisen im Terminalgespinst und ihr zahlenmäßiges Verhalten während der Mikrovillibildung. Die Hufeisen entstehen im Golgi-Apparat.

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The formation of microvilli in the fetal rat small intestine

Summary The origin of membranes required for the formation of microvilli has been investigated electronmicroscopically in enterocytes of fetal rat small intestine. It is assumed that horseshoe-like structures consisting of unit membranes covered with electron-dense material, which probably represent longitudinal sections through cap-like structures, are incorporated into the apical cell membrane and give rise to the tips of microvilli. This assumption is based chiefly on the almost identical diameters of horseshoes and microvilli, the localization of horseshoes in the terminal web, and the time of appearance and disappearance of horseshoes with regard to development of microvilli. There are indications that the horseshoes originate in the Golgi apparatus.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Effects of starvation on valine and alanine transport across the intestinal mucosal border in sea bass,Dicentrarchus labrax

Summary kinetics of intestinal transport of l-alanine and l-valine (substrates of the A-system and the L-system, respectively, in mammals) across the brush-border membrane in sea bass, Dicentrarchus labrax, were studied on intact mucosa using a short-term uptake technique. When fish were starved for 4–8 weeks, total influx (mucosa-to-cell) of valine fell owing to disappearance or modification of the diffusion component. The maximum influx rate of saturable component increased but its affinity (reflected by the Michaelis constant) decreased. Alanine transport by Na⁺-dependent and diffusion pathways was unchanged after starvation Fasting also induced an almost 20% decrease in the length of intestinal microvilli.Abbreviations K _d diffusional constant - K _m Michaelis constant - V _max maximum influx rate     

Immunolocalization of EGF receptor (EGFr) in intestinal epithelium: recognition of apoptotic cells

The EGF-like family of growth factors are known to be involved in the control of the intestinal epithelium. The intracellular events are mediated by the EGF receptor (EGFr), a transmembrane glycoprotein which is overexpressed in many malignancies and also in many radiosensitive cell types. The precise mode of action of the receptor in controlling proliferation and whether the factor is also involved in controlling apoptosis in this tissue is not clear. Using polyclonal antibodies raised against a cytoplasmic region of the receptor distant to the phosphorylation site and one raised against the peptide sequence DVVDADEYLIPQ, which is present in the cytoplasmic tail phosphorylation site of the EGFr, we have examined the immunostaining in normal and irradiated murine intestine. The former antibody labelled the basolateral membranes of the epithelial cells in the proliferative zones of both the small intestine and colon, in both control and irradiated tissue. The latter antibody however, strongly labelled the Goblet cells and the microvilli of the enterocyte apical membrane in control tissue. Following irradiation\ the apical labelling redistributed and was localized in the apical cytoplasm and in a paranuclear region. Furthermore, strong labelling was now seen in many of the apoptotic cells of the small intestinal epithelium. The greatly differing results with the two antibodies indicates that interpretation of such immunostaining must be viewed with caution and may relate to the availability of each particular epitope. These results also suggest that antibodies to DVVDADEYLIPQ may be a useful marker of apoptotic calls and could imply a correlation between high levels of epitope availability, the radiosensitive (frequently p53 expressing) cells of the crypt epithelium and the induction of apoptosis.This work was supported by the Cancer Research Campaign.     

Induction of cell death in rat small intestine by ischemia reperfusion: differential roles of Fas/Fas ligand and Bcl-2/Bax systems depending upon cell types

Although ischemia reperfusion (I/R) induces apoptotic damage of mammalian small intestine, the molecular mechanism is largely unknown. We investigated the appearance of apoptosis at various time-points (0–24 h) of reperfusion after 1-h ischemia and the expression of various apoptosis-related proteins, such as Bcl-2, Bax, Fas, Fas ligand (FasL), activated caspase-3, and cytochrome c, immunohistochemically in rat small intestine. As assessed by TUNEL and electron microscopy, apoptotic cells were increased at 3 h of reperfusion in all intestinal parts (villous epithelium, crypt epithelium, and stroma of intestine). Moreover, the TUNEL-positive cells in the stroma were later identified as T cells. The expression of Fas and FasL as well as activated caspase-3 was markedly increased at 3 h of reperfusion in the stroma. In the villous epithelium, a transient decrease in Bcl-2 expression was found while in the crypt epithelium, Fas expression was induced. Finally, intraperitoneal injection of leupeptin (an SH-protease inhibitor) after I/R resulted in a significant inhibition of the induction of apoptosis in the stroma and crypt epithelium. Our results indicate that the triggering molecules of apoptosis in the I/R rat small intestine may vary depending on cell type and that the use of a broad-spectrum protease inhibitor may reduce intestinal damage.     

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by31P NMR

Summary The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe³¹P NMR. The broad. asymmetric lineshape of the³¹P NMR spectrum of isolated brush-border vesicles demostrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18mm), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, watersoluble fatty acids do not, suggesting a difference in the absorption mechanisms.     

Intestinal epithelium in Anarhichas lupus L., with emphasis on cell renewal

Histomorphological studies on the dynamics of intestinal epithelium in larvae, juveniles and adult stages of common wolffish Anarhichas lupus showed that the major structural development took place during the first 8 weeks after hatching ( c . 320 day degrees). This development preceeded a period of increase in relative growth. The mucosa of the proximal and distal intestine gradually developed from simple primary mucosal folds into a complex structure of primary and secondary folds with regularly distributed crypts. Cell proliferation, based on the observations of the presence of immature cells, mitotic figures and proliferating cell nuclear antigen reacting cells, was limited to the crypts after 8 weeks of age. The observation of bands of epithelial cells showing apoptotic and necrotic changes, suggested the existence of extrusion zones, where effete cells were eliminated by both shedding and phagocytosis. In the proximal intestine of adult common wolffish, increasing amounts of lipids were observed from the upper part of the crypts to the surface epithelium, probably reflecting an increased maturation of epithelial cells.