Zhengwu Sun

NF-kappaB activation and inhibition: a review.

Among transcriptional regulatory proteins described, NF-kappaB seems particularly important in modulating the expression of immunoregulatory genes relevant in critical illness, inflammatory diseases, apoptosis, and cancer. In particular, NF-kappaB plays a central role in regulating the transcription of cytokines, adhesion molecules, and other mediators. The biochemical basis by which diverse stimuli converge to activate or intervene this family of transcription factors is still largely unknown. The NF-kappaB transcription factor family represents an important group of regulators of a broad range of genes involved in cellular responses to inflammatory and other kinds of signals. Knockout mouse studies have also revealed a key role for this family in broad physiological processes, including immune function and metabolism. Overall, specificity seems to exist in the role of each transcriptional complex in gene transcription and physiological function. Each NF-kappaB complex displays distinct affinities for the different DNA-binding sites present in the promoters of NF-kappaB-regulated genes, and this may contribute to some of the specificity exhibited. The identification of specific components of the NF-kappaB signal transduction pathway provides an opportunity to define mechanisms at the biochemical level by which specific members of the NF-kappaB family are activated. Furthermore, this may identify specific targets for selective inhibition or promotion of NF-kappaB functions. Further studies will be required to elucidate mechanisms regulating specificity and selectivity of NF-kappaB function, as well as its role in different diseases, prior to potential clinical application.

The effect of intestinal ischemia and reperfusion injury on ICAM-1 expression, endothelial barrier function, neutrophil tissue influx, and protease inhibitor levels in rats.

Multiple organ dysfunction syndrome (MODS) is mediated by complex mechanisms in which interactions between activated leukocytes and endothelial cells play a central role. ICAM-1 (intercellular adhesion molecule-1) mediates firm adhesion and transendothelial migration of activated leukocytes from postcapillary venules into the tissue. The present study evaluated the ICAM-1 expression in various organs after 40 min of intestinal ischemia and 1, 3, 6, 12 h of reperfusion (I/R) in the rat, using a dual monoclonal antibody technique (n = 36). Endothelial barrier permeability, using the vascular leakage of radiolabeled human serum albumin was also assessed (n = 12). Neutrophil sequestration in the lungs was quantitated by myeloperoxidase activity and plasma protease inhibitor levels were measured with electroimmunoassay. Significant regional differences were found in ICAM-1 expression between organs, both constitutively and after I/R-injury. The highest constitutive levels were observed in the liver and lungs, followed by the kidneys. The constitutive ICAM-1 expression in the intestines and in the heart was about 1/20 compared with that found in the liver and lungs. The brain and muscle had levels of about 1/150 of that in the liver and lungs. After intestinal I/R, significant increases (17–45%) were found in the lungs, intestines, brain, heart, and muscle. Albumin leakage index (ALI) in all examined organs and myeloperoxidase activity in the lungs increased after I/R-injury. Serum levels of albumin and most protease inhibitors decreased significantly after I/R challenge. Intestinal I/R results in an increase of systemic ICAM-1 expression with marked organ variability. The upregulation of ICAM-1 could represent a crucial step in the adherence- and migration process of activated leukocytes and potentially in the development of tissue injury.

The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.

Intestinal ischemia and reperfusion injury (I/R) is probably involved in the pathogenesis of intestinal barrier dysfunction, associated with the concomitant translocation of enteric bacteria and toxins and the potential development of multiple organ failure. The intestinal endothelial and epithelial layers play a major role preventing the entry of toxic substances from the gut, but the influence of protease-antiprotease systemic balance on these barrier functions and the relationship between epithelial DNA synthesis, apoptosis, and endothelial and epithelial barrier macromolecule permeability are not fully investigated. Endothelial and epithelial barrier macromolecular permeability, epithelial DNA synthesis, the endothelial and epithelial plasma membrane system, apoptosis and oncosis, plasma levels of proteinase inhibitors, and proenzymes were measured in rats subjected to 20 and 40 min intestinal ischemia and 1, 3, 6, or 12 h reperfusion. Endothelial permeability increased after both 20 and 40 min intestinal ischemia. Epithelial permeability significantly increased during 1-6 h reperfusion after 20 min ischemia and during 1-12 h reperfusion after 40 min ischemia. Epithelial DNA synthesis increased in animals with 20 min ischemia followed by 12 h reperfusion. Plasma levels of prekallikrein, C1-esterase inhibitor, and alpha1-macroglobulin were significantly lower following both 20 and 40 min ischemia from 3 h reperfusion and on. Apoptotic epithelial cells significantly increased in animals subjected to 20 min ischemia followed by 12 h reperfusion. The severity of reperfusion injury in the intestinal endothelial and epithelial barrier seems to correlate with the period of ischemia and the pathway of cell damage and death, together with proteinase-antiproteinase imbalance.

The Influence of Apoptosis on Intestinal Barrier Integrity in Rats

BACKGROUND Apoptosis is a critical step responsible for maintaining the cellular balance between proliferation and death and for controlling tumorigenesis. Although an increase in intestinal apoptotic cells has been considered to be associated with the pathogenesis of gastrointestinal injury, little is understood concerning the role of apoptosis in the development of intestinal barrier dysfunction. METHODS Apoptosis induced by intraperitoneal injection of doxorubicin in rats was evaluated by transmission electron microscopy and the TUNEL histochemistry method. Treatment with deoxy-D-glucose (a glycolytic pathway inhibitor) or cycloheximide (a protein synthesis inhibitor) was performed after doxorubicin challenge. Passage of human serum albumin from blood to the intestinal interstitium and the intestinal lumen or from the intestine to the intestinal interstitium and blood was evaluated by means of albumin clearance. RESULTS A significant increase in gut water content, albumin flux, and bidirectional clearance of albumin accompanied by apoptotic epithelial cell increase was noted in doxorubicin-challenged rats treated with saline. The increase in endothelial and epithelial permeability and the increase of apoptosis could partly be prevented by treatment with deoxy-D-glucose or cycloheximide. CONCLUSION Doxorubicin-increased epithelial apoptosis within the intestine occurs simultaneously with increased bidirectional permeability of the intestinal barrier, probably associated with both glycolytic and protein synthesis pathways. Apoptosis may thus play a role in the pathogenesis of intestinal barrier dysfunction.

Effects of inhibition of PAF, ICAM-1, and PECAM-1 on gut barrier failure caused by intestinal ischemia and reperfusion.

Background: The role of cell adhesion molecules and transmigration of PMNs through the endothelial barrier is probably essential in intestinal ischemia and reperfusion (I/R)-induced gut barrier dysfunction. Although cytokines are released in I/R, it is unclear whether cytokines directly increase permeability or if this phenomenon requires both expression of cell adhesion molecules and PMN adhesion-activation. Endothelial barrier dysfunction plays an important role in the pathogenesis of multiple organ dysfunction syndrome, inducing gut barrier failure, but the mechanisms are not fully understood. The purpose of this study was to evaluate the potential therapeutic value of inhibition of platelet activating factor (PAF), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM1) in gut barrier dysfunction induced by intestinal I/R. Methods:BACKGROUND The role of cell adhesion molecules and transmigration of PMNs through the endothelial barrier is probably essential in intestinal ischemia and reperfusion (I/R)-induced gut barrier dysfunction. Although cytokines are released in I/R, it is unclear whether cytokines directly increase permeability or if this phenomenon requires both expression of cell adhesion molecules and PMN adhesion-activation. Endothelial barrier dysfunction plays an important role in the pathogenesis of multiple organ dysfunction syndrome, inducing gut barrier failure, but the mechanisms are not fully understood. The purpose of this study was to evaluate the potential therapeutic value of inhibition of platelet activating factor (PAF), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) in gut barrier dysfunction induced by intestinal I/R. METHODS A PAF antagonist (lexipafant, BB-882) and monoclonal antibodies against rat ICAM-1 (anti-ICAM-1-MAb) and PECAM-1 (anti-PECAM-1-MAb) were used in a model of gut barrier dysfunction caused by intestinal ischemia for 40 min and concomitant reperfusion for 12 h in the rat, and endothelial permeability, myeloperoxidase activity, interleukin-1beta and protease inhibitor levels were evaluated. RESULTS The endothelial permeability and tissue leukocyte recruitment in the distal small intestine significantly increased in rats with I/R treated with saline. Proteolytic activity in plasma was evident by low levels of the three measured plasma protease inhibitors. These changes were, to different degrees, reduced by treatment with lexipafant, anti-ICAM-1-MAb, or anti-PECAM-1-MAb. Alterations in systemic levels of interleukin-1beta paralleled the changes found in gut barrier permeability and leukocyte trapping. CONCLUSIONS Our results suggest that treatment with the PAF inhibitor lexipafant and monoclonal antibodies against ICAM-1 or, seemingly most efficient, PECAM-1 reduces the severity of I/R-associated intestinal dysfunction, associated with a decrease in systemic concentrations of IL-1beta local leukocyte recruitment, and partly restoring plasma protease inhibitor levels.

Role of Intestinal Permeability in Monitoring Mucosal Barrier Function

The intestinal barrier function is considered to play an important role in protecting the penetration of luminal antigens, associated with the development of secondary infection and sepsis and the initiation of the multiple organ dysfunction syndrome. The intestinal mucosal barrier against luminal macromolecules and microorganisms consists of both non-immunological and immunological defence mechanisms. The main constituents of the intestinal barrier are the endothelial and epithelial barriers. The epithelial barrier selectively restricts micromolecular permeation and almost completely restricts macromolecular permeation, while the endothelial barrier has a very limited restriction to micromolecules and only partly to macromolecules. Maintenance of the barrier depends on the integrity of cellular plasma membranes and tight junctions, as well as the elaboration of endothelial and epithelial secretory products. Focal denudation of the barrier results in permeation of potentially threatening luminal compounds including antigens, proteases, H+, bacteria and endotoxin, and also other factors chemotactic for inflammatory cells. By initiating inflammation and thus acting on subepithelial tissues, such factors can further influence endothelial and epithelial transport and barrier function. The repair of endothelial and epithelial injury is also complex, and both restitution and enhanced endothelial and epithelial cell proliferation are likely to be important. At present, however, mechanisms for intestinal epithelial and endothelial permeation, their alterations in disease and potential ways to prevent or repair injury, are still not fully elucidated.

Effect of a Platelet-Activating Factor Antagonist on Pancreatitis-Associated Gut Barrier Dysfunction in Rats

Platelet-activating factor (PAF) may play a critical and primary role in the pathogenesis of acute pancreatitis and pancreatitis-associated distant organ injury. The present study evaluated the effect of a PAF antagonist, lexipafant (an (S)-4-methyl-2 (methyl-imidazo {4,5-c} pyridin-l-ylmethyl)-benzenesulphonyl]-amino} pentanoic acid ethyl ester, BB-882; British Biotech Ltd.), on the potential prevention of gut barrier dysfunction, by measuring gut origin sepsis, bidirectional permeability of the intestinal barrier, and pancreatic capillary endothelial barrier integrity, in acute pancreatitis induced by intraductal infusion of 5% sodium taurodeoxycholate. Pancreatic endothelial permeability significantly increased in animals with acute pancreatitis, whereas pretreatment with lexipafant had a preventive effect (p <0.05 vs. pancreatitis with saline). Similarly, alterations noted in hematocrit and plasma levels of lipase and calcium were counteracted by the PAF antagonist. It also prevented the increase in albumin leakage from blood to the mucosal interstitium and from blood to the intestinal lumen in acute pancreatitis. Albumin passage from the gut lumen to blood in animals with pancreatitis pretreated with saline increased from 3 h and on, and lexipafant prevented alterations in mucosal epithelial permeability. Bacterial translocation was commonly seen in pancreatitis, whereas only a few positive cultures were observed in pancreatitis animals given lexipafant. Microthrombosis in intestinal villi seemed less frequent after lexipafant pretreatment. We conclude that (a) PAF may play a role in the pathogenesis of pancreatitis-associated intestional dysfunction, (b) PAF may be involved in the development of distant organ dysfunction by triggering endothelial barrier dysfunction, and (c) PAF antagonists may provide potential agents for preventing pancreatitis-associated gut barrier dysfunction.

Phagocytic and intestinal endothelial and epithelial barrier function during the early stage of small intestinal ischemia and reperfusion injury.

ABSTRACT The effects of intestinal ischemia and reperfusion (I/R) on small intestinal mucosal endothelial and epithelial barrier integrity and phagocytic function were assessed in rats subjected to 20‐ or 40‐min mesenteric ischemia and a 3‐h reperfusion. The results showed that human serum albumin (125I‐HSA) flux through the endothelial layer to the interstitial space increased as did 125I‐HSA clearance from blood to the gut lumen and 131I‐HSA flux from the gut lumen to the interstitial space in rats with I/R. E.coli adhering to microvilli, invading and passing into the microvessels, were noted on the small intestinal mucosa in animals subjected to 40‐min ischemia and a 3‐h reperfusion. Phagocytic function increased, especially in the small intestinal wall, lungs, liver, and spleen in the groups with I/R, correlating with the length of ischemia. The results imply that both endothelial and epithelial barrier integrity is impaired in the early phase after I/R and that the epithelial barrier more effectively restricts macromolecular leakage compared with the endothelial barrier. I/R impairs the intestinal barrier not only by causing tissue hypoxia but also by activating the phagocytic system and aggravating barrier damage, which finally may result in bacterial translocation and remote organ dysfunction.

Gut barrier permeability, reticuloendothelial system function and protease inhibitor levels following intestinal ischaemia and reperfusion--effects of pretreatment with N-acetyl-L-cysteine and indomethacin.

BACKGROUND Pathophysiological mechanisms and ways to intervene on intestinal barrier dysfunction following small intestinal ischaemia and prolonged reperfusion are still not fully clarified. AIMS To evaluate the effect of oxygen free radical and prostaglandin inhibition on intestinal barrier injury following intestinal ischaemia/reperfusion. METHODS Endothelial and epithelial barrier permeability was evaluated by clearance of radiolabelled albumin. Parameters included 125I-Escherichia coli uptake rate index, host reticuloendothelial system function and organ distribution, as well as protease inhibitor and proenzyme activities in rats subjected to small intestinal ischaemia for 40 minutes followed by 12 hours reperfusion (ischaemia/reperfusion), pretreated with N-acetyl-L-cysteine or indomethacin. RESULTS Following ischaemia/reperfusion, endothelial and epithelial permeability increased, reticuloendothelial system activation occurred and plasma protease inhibitors were consumed. N-acetyl-L-cysteine pretreatment resulted in improved endothelial and epithelial barrier integrity, a decrease in protease inhibitor consumption and less pronounced reticuloendothelial system activation. Pretreatment with indomethacin was not effective. CONCLUSION Oxygen free radicals seem to play an important role in the development of intestinal barrier impairment following ischaemia/reperfusion. N-acetyl-L-cystine may be a potential agent for preventing ischaemia/reperfusion damage.

Treatment with lexipafant ameliorates the severity of pancreatic microvascular endothelial barrier dysfunction in rats with acute hemorrhagic pancreatitis

SummaryConclusion: Treatment with lexipafant reduced the severity of pancreatitis-associated endothelial barrier compromise, also associated with a decrease in systemic concentrations of interleukin (IL) 1. Thus, the present findings imply that platelet-activating factor (PAF) may play an important role in the pathogenesis of pancreatic endothelial dysfunction by signaling and triggering the production and release of certain cytokines.Background: Pancreatic capillary endothelial barrier dysfunction is an initial and characteristic feature of acute pancreatic injury and pancreatitis. PAF, a proinflammatory mediator and an intercellular signaling substance, has been considered to be involved in the inflammatory reaction and the systemic endothelial dysfunction of acute pancreatitis.Methods: The development of pancreatic capillary endothelial barrier dysfunction was monitored by tissue edema and exudation of plasma albumin into the interstitium, 3 and 12 h after induction of acute pancreatitis by intraductal infusion of 5% sodium taurodeoxycholate in rats. Pancreatic leukocyte recruitment was reflected by measuring myeloperoxidase activity. Serum levels of IL-1β and IL-6 were determined by an enzyme-linked immunosorbent assay (ELISA).Results: Pretreatment with lexipafant, a potent PAF receptor antagonist, significantly reduced the pancreatitis-induced increase in pancreatic endothelial barrier dysfunction, pancreatic leukocyte recruitment, and serum levels of IL-1β, although a difference persisted between animals with sham operation and pancreatitis.