Role of tight junction derangement in the endothelial dysfunction elicited by exogenous and endogenous peroxynitrite and poly(ADP-ribose) synthetase

DNA single-strand breakage and activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) triggers an energy consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Here, we investigated whether peroxynitrite and PARS activation are involved in tight junctions (tight junction) derangement in the endothelial dysfunction in cells exposed to peroxynitrite and in vascular rings of animals subjected to zymosan non-septic shock. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, as measured by the mitochondrial-dependent conversion of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan. Moreover, peroxynitrite caused activation of PARS. Inhibition of PARS by 3-aminobenzamide (3-AB; 1 mM) reduced the peroxynitrite-induced suppression of mitochondrial respiration in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. Peroxynitrite incubation also caused a significant derangement of zonula occludens (ZO)-1, which was significantly affected by pharmacological inhibition of PARS. 3-AB ameliorated the development of this peroxynitrite-induced endothelial dysfunction. In vascular rings obtained from the zymosan-treated rats, there was a marked suppression of the endothelium-dependent relaxation ex vivo, which was reduced by in vivo 3-AB treatment. A significant derangement of ZO-1 was observed in vascular rings from zymosan-treated rats. Tight junction alteration was significantly reduced by in vivo 3-AB treatment. Thus, activation of PARS by exogenous and endogenous peroxynitrite may be involved in the tight junction derangement associated with endothelial dysfunction. Inhibition of PARS may be a novel pharmacological approach to preserve endothelial tight junction function in shock and inflammation.     

Intestinal ischemic preconditioning protects the intestine and reduces bacterial translocation

Ischemic preconditioning (IPC) was first demonstrated in the heart, but this protective effect has been also recently described in the intestine. The aim of this study was to determine the effects of intestinal ischemic preconditioning on the morphology of intestine and bacterial translocation. Twenty-four male Wistar rats weighting 250 to 300 g were randomized into three groups. A control group of rats (n = 8) were subjected laparotomy. In an ischemic group (n = 8), laparotomy was performed and the superior mesenteric artery was occluded by an atraumatic clamp for 30 min. In the preconditioned group (n = 8), before the ischemia-reperfusion (I/R) period (as in ischemic group), rats were subjected to an initial 10 min of intestinal ischemia and 10 min of reperfusion. Twenty-four hours later, to evaluate whether the I/R induced intestinal injury and bacterial translocation (BT), tissue and blood samples were collected, and liver, spleen, and mesenteric lymph node specimens were obtained under sterile conditions for microbiological analysis. Samples of ileum were removed for both biochemical and histopathological evaluation. In the I/R group, the incidence of bacteria-isolated mesenteric lymph nodes, spleen, liver, and blood was significantly higher than other groups (P < 0.05). IPC prevented I/R-induced BT and it significantly reduced the I/R-induced intestinal injury (P < 0.05). Increased inducible nitric oxide (NO) synthase (iNOS) expression observed on the ileal specimens of the I/R group was found to be prevented by IPC. Our data suggest IPC as a key factor that reduces BT and iNOS activation in intestinal I/R. This is the first study showing that intestinal IPC blocks the cascade of events that causes BT and intestinal injury that may lead to sepsis.     

Effect of timing of glutamine-enriched enteral nutrition on intestinal damage caused by irradiation

Intestinal mucosal damage and bacterial translocation are clinical problems that may be caused by the use of ionizing radiation. Glutamine (Gln) support reduces the mucosal barrier in several ways. This study was undertaken to investigate the effect of timing of Gln-enriched enteral nutrition (EN) on bacterial translocation and mucosal damage due to radiotherapy (RT). A rat model of whole body irradiation was designed in which a single dose of 485 cGy was given. A total of 50 rats were randomly assigned to the following 5 groups, each of which comprised 10 rats: (1) balanced rat chow given for 8 days without RT (group 1); (2) balanced rat chow given 4 days before and 4 days after RT (group 2); (3) Gln-enriched EN given 4 days before RT (group 3); (4) Gln-enriched EN given 4 days after RT (group 4); and (5) Gln-enriched EN given 4 days before and 4 days after RT (group 5). Mesenteric lymph node and ileum samples were removed for evaluation of bacterial translocation (BT) and histopathologic investigation, respectively. BT and intestinal mucosal injury scores in all rats that received RT were higher than in rats without RT. No difference was seen in parameters between groups 3 and 4 (P>.05, P>.016, respectively); BT and intestinal mucosal injury scores of group 5 were significantly lower than those of groups 3 and 4 (P<.05, P<.016, respectively). Meanwhile, the BT and mesenteric injury scores of group 5 were significantly lower than those of group 2 (P<.05, P<.016, respectively). As a result, intestinal injury due to RT was significantly decreased by Gln-enriched EN support given before and after whole body RT.     

Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion

Studies indicate that endotoxin (LPS) causes intestinal injury, increases inducible nitric oxide synthase (iNOS) activity, leads to increased NO production, and promotes bacterial translocation (BT). To investigate the mechanism by which LPS causes gut injury and to test the hypothesis that NO produced by enterocytes promotes gut injury in an autocrine fashion, rat intestinal epithelial cell (IEC-6) monolayers were tested. IEC-6 monolayers grown in a bicameral system were incubated with media or with LPS (25 microg/mL) and tested for permeability to phenol red, BT, and nitrate/nitrite (NO2/NO3) production. To determine the direct effect of NO on permeability, monolayers were incubated with the NO donor S-nitroso-acetylpenicillinamide (SNAP; 1 mM) and tested for permeability. Next, the protective effects of two NOS inhibitors (L-NMMA and L-NIL) were tested. Finally, to determine if LPS-induced permeability occurs via a poly (ADP-ribose) synthetase- (PARS) dependent pathway, monolayers incubated with LPS alone or with the PARS inhibitor, INH2BP (100 microM) were tested. LPS significantly increased IEC-6 permeability to phenol red, as well as increased NO2/NO3 by 20-fold (P < 0.001) and increased BT 10-fold (P < 0.001). SNAP mimicked the effect of LPS and significantly increased both permeability to phenol red and BT. Inhibition of iNOS significantly decreased the LPS-induced increase in monolayer permeability and BT (P < 0.05). Monolayers incubated with INH2BP had significantly decreased permeability to phenol red and BT, suggesting that LPS-induced NO production increases monolayer permeability at least in part via a PARS-dependent mechanism. In summary, LPS-induced disruption of monolayer barrier function appears to be related, at least in part, to enterocyte produced NO. This supports the hypothesis that NO produced by LPS-stimulated enterocytes promotes injury in an autocrine fashion and highlights the fact that enterocytes can be a target as well as a producer of NO.     

双歧杆菌对烫伤大鼠肠道黏膜机械及生物屏障的改善作用

目的 :探讨严重烫伤后补充外源性双歧杆菌对肠黏膜机械及生物屏障的保护作用及其对肠道细菌或内毒素移位的影响。方法 :70只 Wistar大鼠按随机数字表法分为烫伤对照组 ( BC)、烫伤治疗组 ( BT)和假伤对照组 ( NC)。BT组伤后灌饲双歧杆菌悬液 ( 5× 10 1 2 CFU/ L) 1.5 ml,2次 / d。观察细菌或内毒素移位、肠黏膜菌群和肠黏膜损伤情况等。结果 :1伤后 3 d,BC、BT组细菌移位率分别为 42 %和 16 % ( P=0 .0 0 40 ) ,5 d分别为30 %和 6 % ( P=0 .0 0 2 0 ) ;伤后 1d,BC组内毒素水平显著高于 BT组。 2 BC组回盲部黏膜菌群中双歧杆菌量仅为 NC组的 1/ 10～ 1/ 6 0 ,BT组双歧杆菌量明显增多 ;BC组大肠杆菌量在 1、3 d增加约 2 0～ 30倍 ,但 3 d后BT组大肠杆菌量显著减少并接近正常水平。 3回肠黏膜损伤以 1、3d最为明显 ,BC组伤后 5 d损伤评分仍明显高于 NC组 ( P<0 .0 5 ) ;BT组 3d时已明显修复 ,5 d已与 NC组无明显差异。结论 :补充外源性双歧杆菌能促进烫伤后受损肠黏膜屏障和生物屏障修复 ,有效防治肠道细菌或内毒素移位。     

Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite.

DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) contribute to peroxynitrite-induced cellular injury. We investigated the role of PARS activation in the pathogenesis of endothelial dysfunction. In human umbilical vein endothelial cells (HUVEC), DNA strand breakage (alkaline unwinding assay), PARS activation (incorporation or radiolabeled NAD+ into proteins), mitochondrial respiration [conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan] and apoptotic index (cytoplasmatic release of histones) were measured. Endotoxin shock was induced in rats by bacterial lipopolysaccharide. Vascular reactivity of thoracic aortic rings were measured in organ chambers. In HUVEC, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, induced DNA strand breakage and caused an activation of PARS. Pharmacological inhibition of PARS reduced the acute and delayed suppression of mitochondrial respiration when cells were exposed to intermediate, but not high doses of peroxynitrite. Similarly, protection against the intermediate, but not high doses of peroxynitrite was seen in fibroblasts from the PARS-/- mice, when compared to wild-type controls. These data suggest that PARS plays a role in peroxynitrite-induced cytotoxicity, but at very high levels of oxidant exposure, PARS-independent cytotoxic mechanisms become predominant. Peroxynitrite-induced apoptosis was not affected by PARS inhibition. Vascular rings exposed to peroxynitrite and rings taken from rats subjected to endotoxic shock exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. The development of this endothelial dysfunction was ameliorated by the PARS inhibitor 3-aminobenzamide. Activation of PARS by peroxynitrite, therefore, may be involved in the development of endothelial dysfunction in endotoxemia.     

Inhibition of poly (ADP-ribose) Synthetase Attenuates Neutrophil Recruitment and Exerts Antiinflammatory Effects

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10–20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS^−/− mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.     

脾动脉结扎对大鼠肝大部分切除术后细菌移位的影响

目的研究脾动脉结扎对大鼠肝大部分切除术后细菌移位的影响。方法将54只SD大鼠按随机数字表法分为3组：假手术组、三分之二肝切除组、肝切除加脾动脉结扎组,每组18只。54只大鼠中24只用于测定门静脉压力,30只用于分析细菌移位、血浆内毒素。结果三分之二肝切除组门静脉压力（1.31±0.06）kPa显著高于假手术组（0.75±0.04）kPa（P〈0.01）和肝切除加脾动脉结扎组（0.98±0.05）kPa（P〈0.01）。此外,三分之二肝切除组的细菌移位率高于假手术组（60.0%vs 10.0%,P=0.001）,而脾动脉结扎则能显著的减少肝大部分切除术大鼠细菌移位的发生（30.0%vs 10.0%,P=0.011）。三分之二肝切除组血浆内毒素（4.05pg.mL-1）与假手术组（0.00pg.mL-1）、肝切除加脾动脉结扎组（1.75pg.mL-1）相比,大鼠内毒素显著降低,差异有统计学意义（P〈0.01）。结论脾动脉结扎能降低大鼠肝大部分切除术后门静脉压力,增强肠黏膜屏障功能,减少内毒素和细菌移位的发生,所以脾动脉结扎可以预防肝大部分切除后感染的发生。     

Effects of octreotide acetate and Saccharomyces boulardii on bacterial translocation in an experimental intestinal loop obstruction model of rats

Intestinal obstruction (IO) induces bacterial translocation (BT) due to mucosal disruption, motility dysfunction, and increased intestinal volume, leading to bacterial overgrowth. This study was conducted to investigate the effects of octreotide acetate (OA) and Saccharomyces boulardii (SB) on the BT and intestinal integrity in an animal model of intestinal loop obstruction (LO). Forty adult male Sprague-Dawley rats (250-300 g) were randomized into 4 groups containing 10 rats each. Complete IO was created in the distal ileum of rats by a single 3-0 silk suture (LO). Group Sham: Sham (Laparotomy only was performed in this group); group LO: LO; group OA: LO plus OA (100 microg/kg, at 0, 12 hours of obstruction); group (SB): LO plus SB (800 mg/kg/day, via orogastric and preoperative for 3 days). After 24 hours, samples of mesenteric lymph nodes (MLN), liver, spleen and blood were obtained and cultured. The terminal ileum specimens were examined histopathologically. There were no BT in group Sham, but BT was noticed totally in 31 (77.5%) cultures in group LO. This rate was reduced to 30% (n = 12), 10% (n = 4) in the groups OA and SB respectively. Bacterial translocations of MLN and the liver in group LO were significantly higher than those of groups OA and SB. Bacterial translocations of the both spleen and blood in group LO were significantly higher than those of groups OA and SB. The mean bacterial counts, colony-forming units per gram tissue (cfu/g), in the MLN, liver and spleen of group LO were found significantly higher than those of groups OA and SB. The mean villus height in group OA was significantly higher than that of group LO and it in the group SB significantly higher than those of groups LO and OA. The present experimental study has demonstrated that OA and SB may have protective effects against BT in mechanical bowel obstruction and additionally SB preserves intestinal mucosal integrity.     

失血性休克对大鼠肠道细菌易位和血浆内毒素水平的影响及其意义

目的 探讨失血性休克对肠道细菌易位和血浆内毒素水平的影响。方法 采用甲基－^3胸腺嘧啶核苷（[^3H]-TdR）标记致病人肠杆菌，大失血性休克前注入大鼠的胃肠道，然后于失血性休克的不同阶段，通过检测^3H的放射活性和应用放射自显影的方法进行示踪，同时检测血浆内毒素的含量。结果 失血性休克早期即可发生肠源性内毒素血症；当休克发展到一定严重程度时，肠道细菌可进入血液循环，并侵及多个脏器。结论 失血性休克可致肠源性内毒素血症和肠道细菌易位性感染。     

Poly (ADP-ribose) synthetase mediates intestinal mucosal barrier dysfunction after mesenteric ischemia

Peroxynitrite-mediated DNA strand breaks trigger poly (ADP-ribose) synthetase (PARS) activation, resulting in intracellular energetic failure and organ dysfunction. We investigated the role of PARS activation on the inflammatory and functional response of the intestine to mesenteric ischemia-reperfusion injury. Anesthetized rats exposed to 15 min occlusion of the superior mesenteric artery showed an increased mucosal PARS activity (ex vivo incorporation of radiolabelled NAD+ in gut mucosal scrapings) as soon as 10 min after reperfusion. During the first 30 min of reperfusion, significant mucosal damage developed, as well as mucosal hyperpermeability to a 4000 MW fluorescent dextran (FD4). These alterations were significantly reduced by treatment with the NO synthase inhibitor L-NMA, which blocks the production of peroxynitrite, as well as with the PARS inhibitors 3-aminobenzamide and nicotinamide, whereas they were markedly enhanced by the glutathione depletor L-buthionine-(S,R)-sulfoximine. Also, PARS inhibition significantly reduced ileal neutrophil infiltration (myeloperoxidase activity) at 3 h reperfusion. In a second set of experiments, the effects of 15 or 30 min ischemia followed by 3 h reperfusion were evaluated in PARS knockout and wild-type mice. Significant protection against histological damage, neutrophil infiltration, and mucosal barrier failure (evaluated by the mucosal-to-serosal FD4 clearance of everted ileal sacs incubated ex vivo) was noted in PARS knockout mice, who also showed reduced alterations in remote organs, as shown by lesser lipid peroxidation (malondialdehyde formation) and neutrophil infiltration in the lung and liver. In conclusion, PARS plays a crucial role in mediating intestinal injury and dysfunction in the early and late phases of mesenteric reperfusion. Pharmacological inhibition of PARS may be a novel approach to protect tissues from reperfusion-related damage.     

Peritoneal lavage with oxygenated perfluorochemical preserves intestinal mucosal barrier function after ischemia-reperfusion and ameliorates lung injury

OBJECTIVE: To evaluate the effect of peritoneal lavage with an oxygenated perfluorochemical (PFC) on intestinal ischemia-reperfusion injury (IIR), we assessed intestinal barrier function in terms of bacterial translocation and endotoxemia, morphologic changes, and changes of intestinal luminal pH in rats subjected to IIR. We also examined lung injury after IIR to test the effect of oxygenated PFC lavage on remote organ failure. DESIGN: Prospective, randomized, and controlled animal study. SETTING: Laboratory of a university hospital. SUBJECT: Male Sprague-Dawley rats. INTERVENTIONS: Rats were subjected to ischemia by clipping the superior mesenteric artery. Reperfusion was achieved by release of the clip. Lavage of the abdominal cavity was performed by inflow and outflow of oxygenated PFC solution during ischemia. RESULTS: Rats undergoing peritoneal lavage with oxygenated PFC (PFC group) showed significantly better survival after IIR. The frequency of bacterial translocation and the endotoxin concentration in superior mesenteric venous blood were significantly lower in the PFC group. Luminal acidosis also was alleviated in the PFC group. Furthermore, PFC lavage preserved the intestinal mucosal architecture and inhibited interstitial edema and infiltration of inflammatory cells in the lungs. CONCLUSION: We conclude that peritoneal lavage with oxygenated PFC protects the intestinal mucosa and maintains mucosal barrier function after IIR. Preservation of the intestinal mucosa ameliorates lung injury after IIR.     

Does the activation of poly (ADP-ribose) synthetase mediate tissue injury in the sepsis induced by cecal ligation and puncture?

Poly (ADP-ribose) synthetase (PARS) is a DNA protective enzyme activated by single-strand breakage. It is suspected that exaggerated PARS activation related to biochemical stress by reactive oxygen and nitrogen species contributes to cellular injury in sepsis. The main hypothesis is that PARS activation leads to massive ATP and NAD consumption and consequent cellular energy depletion. The PARS inhibitor 3-amino-benzamide (3AB) is protective in rodents challenged with either endotoxin or intraperitoneal zymozan. The present experiment was designed to test the effect of 3AB in a more clinically relevant model of sepsis, namely polymicrobial sepsis induced by cecal ligature and puncture (CLP). Adult male Wistar rats were anesthetized, instrumented with catheters in the jugular vein and in the carotid artery, and then randomized into three groups: Sham (no laparotomy, n = 13), CLP (n = 15), and CLP/3AB (n = 18). All animals were allowed to recover and they received a continuous intravenous infusion of saline (20 mL/kg/h) and fentanyl (20 microg/kg/h). 3AB was administered to the CLP/3AB group as an intravenous bolus (10 mg/kg) followed by a continuous intravenous infusion (10 mg/kg/h). After 24 h, blood was drawn for the determination of biological indicators of organ injury. Rats were then anesthetized and biopsies of the liver were quickly frozen into liquid nitrogen for the subsequent determination of NAD and ATP levels. Further organ samples were collected for the assay of myeloperoxidase (MPO) to indicate tissue infiltration by leukocytes, and nitrotyrosine to indicate the level of biochemical stress by reactive nitrogen species. Twenty-four-hour mortality was 0/13 (Sham), 1/15 (CLP), and 5/18 (CLP/3AB; p = NS). In the surviving rats, CLP induced a clear elevation of liver enzymes, bilirubin, and pancreatic lipase, but not creatinine in the plasma, as well as a marked increase of MPO activity in liver, jejunum, and lung, but not kidney or heart. None of these variables was affected by treatment with 3AB. Furthermore, CLP did not cause depletion of NAD or ATP in the liver, nor any change in the nitrotyrosine content of any organ. These data argue against a general role of PARS activation in the pathogenesis of sepsis-induced tissue injury.     

烫伤后肠壁组织T淋巴细胞和浆细胞数量的变化

目的：了解烧伤后肠壁组织T淋巴细胞和IgA浆细胞数量的变化与伤后肠道细菌易位及肠源性脓毒症发生、发展的关系。方法：Wistar大鼠50只，随机分成对照组（l只）和烫伤组（40只）。烫伤组动物背部脱毛，将背部浸于沸水中12秒钟，造成40％体表面积Ⅲ度烫伤，分别于伤后第1、3、7和10日处死，留取标本；对照组动物不烫伤，于处理后第1日处死，留取标本。采用免疫组化方法分别测定回肠固有层和粘膜层CD3 、CD4 和CD8 淋巴细胞以及IgA浆细胞数量，并测定肠道细菌IgA包被率和肠道细菌易位率。结果：伤后肠壁组织中CD3 、CD4 T淋巴细胞和lgA浆细胞的数量明显减少，CD4 ／CD8 比值和肠道细菌IgA包被率亦明显降低，而肠道细菌易位率则明显升高。结论：肠壁组织中T淋巴细胞和IgA浆细胞数量减少、肠道细菌IgA包被率降低与肠道细菌易位率升高有密切关系，其在烧伤后肠源性脓毒症的发生和发展中可能具有重要意义。     

Effects of propofol on hemodynamic and inflammatory responses to endotoxemia in rats

OBJECTIVE: To document the effects of propofol on the hemodynamic and inflammatory responses to endotoxemia in an animal model. DESIGN: Randomized, prospective laboratory study. SETTING: University experimental laboratory. SUBJECTS: Thirty-two male rats. INTERVENTIONS: The animals were randomly assigned to one of four groups: a) endotoxemia group (n = 8), which received intravenous Escherichia coli endotoxin (15 mg/kg over 2 mins); b) control group (n = 8), which was treated identically to the endotoxemia group except for the substitution of 0.9% saline for endotoxin; c) propofol group (n = 8), which was treated identically to the control group but also received propofol (10 mg/kg bolus, followed by infusion at 10 mg/kg/hr) immediately after the injection of 0.9% saline; and d) propofol-endotoxemia group (n = 8), which was treated identically to the endotoxemia group with the additional administration of propofol (10 mg/kg bolus, followed by infusion at 10 mg/kg/hr) immediately after endotoxin injection. MEASUREMENTS AND MAIN RESULTS: Hemodynamics, arterial blood gases, and acid-base status were recorded and the blood propofol concentrations and plasma cytokine concentrations were measured during the 5-hr observation. Microscopic findings of lung tissue for each group were obtained at necropsy. The systolic arterial pressure and heart rate of the propofol-endotoxemia group were similar to those of the endotoxemia group. The increases in the plasma cytokine (tumor necrosis factor, interleukin-6, and interleukin-10) concentrations, in the base deficit, and in the infiltration of neutrophils in the air space or vessel walls of the lungs were attenuated in the propofol-endotoxemia group compared with the endotoxemia group. CONCLUSIONS: Propofol attenuated cytokine responses, base deficit, and activation of neutrophils to endotoxemia. These findings suggest that propofol may inhibit inflammatory response and prevent the development of metabolic acidosis during endotoxemia.     

Reduction in intestinal leukocyte adherence in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G

OBJECTIVES: To investigate leukocyte adherence in intestinal venules in experimental endotoxemia after treatment with the 21-aminosteroid U-74389G. DESIGN AND SETTING: Prospective, randomized, controlled animal study in an experimental laboratory. SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g. INTERVENTIONS: The rats were divided equally into three groups: (a) control group, (b) endotoxemia (5 mg/kg lipopolysacharide from Escherichia coli O55:B5), and (c) endotoxemia and U-74389G administration 30 min before (3 mg/kg) and 60 min after endotoxin challenge (1.5 mg/ kg). MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined using intravital fluorescence videomicroscopy 2 h after endotoxin challenge. Leukocytes were stained in vivo by means of rhodamine 6G. In the endotoxemic animals we observed a fourfold increase in the count of firmly adherent leukocytes in submucosal post-capillary and collecting venules. Treatment with the 21-aminosteroid U-74389G significantly attenuated the count of sticking leukocytes in the collecting venules (control, 61 +/- 10 cells/mm2; lipopolysaccharide, 237 +/- 42 cells/mm2; U-74389G 125 +/- 9 cells/mm2; p < 0.05). In these venules leukocyte rolling behavior was comparable to that in the control group without endotoxin challenge. CONCLUSIONS: Administration of U-74389G, which has radical scavenging properties, attenuates leukocyte adherence in selected populations of intestinal venules which is found increased during endotoxemia. Thus, 21-aminosteroids may have an impact in the treatment of endotoxin-induced intestinal injury.     

烫伤延迟复苏后肠上皮细胞凋亡诱导肠道内毒素和细菌移位

目的 :探讨烫伤延迟复苏后肠上皮细胞凋亡对肠道内毒素和细菌移位的影响。方法 :Wistar大鼠 110只 ,30 %总体表面积 ( STBA) 度烫伤 ,立即或伤后 6 h进行复苏。观察伤后肠上皮细胞凋亡率 ( ap% )变化 ,并测定了门静脉和体循环内毒素及肠系膜淋巴结 ( ML N)细菌移位率和移位量变化。结果 :电泳、原位末端标记( TU NEL )法和电镜均观察到伤后肠上皮凋亡梯形带和凋亡阳性细胞增多。延迟复苏组肠上皮细胞凋亡发生早且更严重 ;其门静脉内毒素水平及 ML N细菌移位量均显著高于立即复苏组 ( P<0 .0 1) ;门静脉内毒素变化与肠黏膜 ap%成显著正相关 (烫伤后立即复苏组 r=0 .936 ,P<0 .0 1;烫伤后延迟复苏组 r=0 .899,P<0 .0 5 )。结论 :烫伤后延迟复苏使肠黏膜上皮发生病理性凋亡 ,可能导致肠道细菌和内毒素移位增加。     

Effect of poly(ADP-ribose) polymerase inhibition on outer medullary hypoxic damage

Poly(ADP-ribose) polymerase (PARP) activation after free-radical-induced DNA damage depletes cellular energy stores and participates in ischemia-reflow injury. We studied the potential protective effect of the water-soluble PARP inhibitor 3-aminobenzamide (3-AB) in a rat model of acute renal failure (ARF) from combined administration of radiocontrast, indomethacin and N(omega)-nitro-L-arginine methyl ester. Kidney function at 24 h was better preserved in rats treated with 3-AB as compared to control animals. However, the extent of tubular hypoxic damage was not significantly mitigated. It is concluded that PARP inhibition may attenuate renal dysfunction in this model of ARF with medullary hypoxic tubular injury even while the extent of tubular necrosis is not significantly altered. Further studies of this dyssynchrony of structure and function may provide important insights into the sequence of events that promotes renal failure after medullary injury.     

舱内腹部闭合性爆炸伤对大鼠肠道细菌移位的影响

目的 探讨舱内大鼠腹部爆炸伤对肠道细菌移位的影响. 方法 100只成年SD大鼠随机分为舱内组和舱外组(50只/组),在陆军模拟战斗舱室和舱外开阔地爆炸复制腹部爆炸伤模型,爆炸后3、8、24、48和72 h采集门静脉血、外周血和肠系膜淋巴结、肝脏组织进行普通细菌培养并鉴定细菌菌株,同时采用PCR法检测血液细菌DNA. 结果 组织细菌培养在舱内组大鼠伤后3 h即为阳性,而舱外组在伤后8 h才出现细菌,四种组织细菌培养总阳性率舱内组为42.0%,舱外组为18.5%(χ~2=22.763, P<0.001),经鉴定阳性细菌绝大部分为大肠埃希菌(81.8%).舱内组细菌DNA检出率91.0%,舱外组为64.0%(χ~2 =20.903, P<0.001). 结论舱内爆炸致大鼠肠道细菌移位较舱外早且发生率高,在早期救治战斗舱室内爆炸伤伤员时需采取有效措施预防肠源性感染.     

Pretreatment with high-fat enteral nutrition reduces endotoxin and tumor necrosis factor-alpha and preserves gut barrier function early after hemorrhagic shock

Gram-negative sepsis is a potentially fatal clinical syndrome characterized by a proinflammatory response (tumor necrosis factor-alpha) to bacterial (endo)toxins and gut barrier function loss. Recently, we found that high-fat enteral nutrition protects against late bacterial translocation in a model of hemorrhagic shock in rats. However, the basis for this protection is unknown. We hypothesized that the observed protection is the result of an early inhibition of endotoxin and the subsequent inflammatory response resulting in a preserved gut barrier function. Sprague-Dawley rats were divided into a group that was starved overnight (HS-S), fed with a low-fat enteral diet (HS-LF) or fed wih a high-fat enteral diet (HS-HF), and subsequently subjected to a nonlethal hemorrhagic shock. Ninety minutes after hemorrhage, arterial endotoxin significantly decreased in HS-HF rats (4.0 +/- 0.6 pg/mL) compared with HS-LF rats (10.7 +/- 0.9 pg/mL, P = 0.002) and HS-S rats (15.2 +/- 2.2 pg/mL P = 0.001). Interestingly, arterial tumor necrosis factor-alpha was also decreased in HS-HF rats (17.9 +/- 10.4 pg/mL) compared with HS-LF (83.5 +/- 16.7 pg/mL, P < 0.01) and HS-S rats (180.9 +/- 67.9 pg/mL, P < 0.02). Loss of tight junction structure (ZO-1) observed in ileum and colon of control hemorrhagic shock rats was prevented in HS-HF rats. In parallel, intestinal barrier function was preserved in HS-HF rats, evidenced by a reduced permeability to horseradish peroxidase (P < 0.05), less bacterial invasion, and a 10-fold reduction of bacterial translocation early after hemorrhagic shock. This report describes a new strategy to nutritionally prevent endotoxemia, the subsequent inflammatory response and gut barrier failure following hemorrhagic shock. High-fat enteral nutrition requires further evaluation as an intervention to prevent a potentially fatal systemic inflammatory response in patients at risk for sepsis.