Effect of melatonin and misoprostol on bacterial translocation in portal hypertensive rats

Background and Aim: Portal hypertension is the main complication of cirrhosis and it is responsible for its most common complications. Bacterial translocation increases the morbidity and mortality rates in patients with portal hypertension. We aimed to investigate the effects of melatonin and misoprostol on bacterial translocation induced by portal hypertension. Methods: We established four groups, each containing eight rats. Except for the control and sham groups, the animals in the other groups (treatment groups) received misoprostol or melatonin for 3 days after the first operation. In the sham group, a laparotomy was carried out and only the portal vein was dissected. Calibrated portal vein ligation was carried out in the other groups. All animals were given 10¹⁰Escherichia coli by orogastric intubation 12 h before sampling. Seventy‐two hours after the first operation, mesenteric lymph node and blood samples were obtained and cultured. Two cc blood samples were obtained for a polymerase chain reaction study. A piece of terminal ileum was also sampled for histopathologic examination. Results: Mesenteric lymph node and blood cultures of all control animals were positive for microbiological growth, and polymerase chain reaction results were positive in seven of the eight rats. Histopathologically, edema, vasodilatation and inflammatory cell infiltration were found to be less in the other groups in comparison to the control group. The incidence of bacterial translocation was decreased in all treatment groups as compared to the control group. Conclusions: In this study, bacterial translocation occurred in portal hypertension. Melatonin and misoprostol reduced the incidence of bacterial translocation in portal hypertensive rats.     

Tempol 对肝硬化大鼠肝门阻断后肠道菌群移位及内毒素血症的作用

目的：探讨4-羟基-2,2,6,6-四甲基哌啶（Tempol）对肝硬化大鼠肝门阻断后肠道细菌移位和内毒素血症的作用。方法选择SD大鼠30只,采用四氯化碳诱导行肝硬化建模,将建模大鼠随机均分为假手术组（不阻断肝门）、对照组（阻断肝门）、实验组（阻断肝门前后静注Tempol ）。3组均取门静脉、腔静脉血检测血清内毒素,取肠系膜淋巴结、肝组织、肺组织行细菌培养,以及小肠黏膜形态与细胞凋亡检测。结果与假手术组及对照组比较,实验组可明显减少肝门阻断后的肠道细菌移位及血清内毒素水平（ P均＜0．05）。结论 Tempo1可有效地预防肝硬化大鼠肝门阻断后的肠道细菌移位和内毒素血症。     

The Effect of Melatonin on TNBS-Induced Colitis

Ulcerative colitis is a multifactorial inflammatory disease of the colon and rectum with an unknown etiology. The present study was undertaken to investigate the effect of melatonin administration on oxidative damage and apoptosis in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Rats were divided into four groups as follows: Group 1 (n=8)—TNBS colitis; Group 2 (n=8)—melatonin, 10 mg/kg/day ip, for 15 days in addition to TNBS; Group 3 (n=8)—melatonin alone, 10 mg/kg/day ip, for 15 days; and Group 4 (n=8)—isotonic saline solution, 1ml/rat ip, for 15 days (sham control group). Colonic myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels, and glutathione (GSH) levels are indicators of oxidative damage, while caspase-3 activities reveal the degree of apoptosis of the colonic tissue. In all TNBS-treated rats, colonic MPO activity and MDA levels were found to be increased significantly compared to those in the sham group. Colonic MPO activity and MDA levels were significantly lower in the melatonin treatment group compared to TNBS-treated rats. GSH levels of colonic tissues were found to be significantly lower in TNBS-treated rats compared to the sham group. Treatment with melatonin significantly increased GSH levels compared to those in TNBS-treated rats. Caspas-3 activity of colonic tissues was found to be significantly higher in TNBS-treated rats compared to the sham group. Treatment with melatonin significantly decreased caspase-3 activity compared to that in TNBS-treated rats. These results imply a reduction in mucosal damage due to anti-inflammatory and anti-apoptotic effects of melatonin.     

Ameliorative effects of bombesin and neurotensin on trinitrobenzene sulphonic acid-induced colitis, oxidative damage and apoptosis in rats

AIM： TO investigate the effects of bombesin （BBS） and neurotensin （NTS） on apoptosis and colitis in an ulcerative colitis model.
METHODS： In this study, a total of 50 rats were divided equally into 5 groups. In the control group, no colitis induction or drug administration was performed. Colitis was induced in all other groups. Following the induction of colitis, BBS, NTS or both were applied to three groups of rats. The remaining group （colitis group） received no treatment. On the 11th d after induction of colitis and drug treatment, blood samples were collected for TNF-α and IL-6 level studies. Malondialdehyde （MDA）, carbonyl, myeloperoxidase （MPO） and caspase-3 activities, as well as histopathological findings, evaluated in colonic tissues.
RESULTS： According to the macroscopic and microscopic findings, the study groups treated with BBS, NTS and BBS ＋ NTS showed significantly lower damage and inflammation compared with the colitis group （macroscopic score, 2.1 ± 0.87, 3.7 ± 0.94 and 2.1 ± 0.87 vs 7.3 ± 0.94; microscopic score, 2.0 ± 0.66, 3.3 ± 0.82 and 1.8 ± 0.63 vs 5.2 ± 0.78, P 〈 0.01）. TNF-α and IL-6 levels were increased significantly in all groups
compared with the control group. These increases were significantly smaller in the BBS, NTS and BBS ＋ NTS groups compared with the colitis group （TNF-α levels, 169.69 ± 53.56, 245.86 ± 64.85 and 175.54 ± 42.19 vs 556.44 ± 49.82; IL-6 levels, 443.30 ± 53.99, 612.80 ± 70.39 and 396.80 ± 78.43 vs 1505.90 ± 222.23, P 〈 0.05）. The colonic MPO and MDA levels were significantly lower in control, BBS, NTS and BBS ＋ NTS groups than in the colitis group （MPO levels, 24.36 ± 8.10, 40.51 ± 8.67 and 25.83 ± 6.43 vs 161.47 ± 38.24; MDA levels, 4.70 ± 1.41, 6.55 ± 1.12 and 4.51 ± 0.54 vs 15.60 ± 1.88, P 〈 0.05）. Carbonyl content and caspase-3 levels were higher in the colitis and NTS groups than in control, BBS and BBS ＋ NTS groups （carbonyl levels, 553.99 ± 59.58 and 336.26 ± 35.72 vs 209.7     

Hyperbaric oxygen ameliorates bacterial translocation in rats with mechanical intestinal obstruction

PURPOSE: The aim of this study was to demonstrate bacterial translocation after experimentally induced intestinal obstruction as well as investigate the preventive effects of hyperbaric oxygen on obstruction-induced bacterial translocation in rats. METHODS: Forty Wistar-albino male and female rats were used. Although no procedure was done in the control group (n = 8), hyperbaric oxygen treatment under 2.5 atm absolute for 90 minutes daily was applied for two days in the hyperbaric oxygen group (n = 8). In the sham group (n = 8), after laparotomy the small bowel was only handled gently, and tissue sampling was done 48 hours later. In the obstruction group (n = 8) the ileum was ligated by 5-0 polypropylene just 5 cm proximal to the ileocecal valve. In the obstruction and hyperbaric oxygen group (n = 8), after obstruction hyperbaric oxygen treatment was applied. Forty-eight hours after the procedures, tissue samples from small bowel, mesenteric lymph nodes, spleen, and liver were taken and 1 ml of blood from the portal vein was withdrawn. All samples were cultured for microbiologic examination. RESULTS: Hyperbaric oxygen treatment significantly reduced the endogenous bacterial overgrowth in the small intestine of normal rats. Endogenous bacteria in the small intestine were significantly increased in the obstruction group, and the presence of bacterial overgrowth was proven by bacterial presence on mesenteric lymph nodes, spleen, liver, and blood. Hyperbaric oxygen treatment significantly reduced the endogenous bacterial overgrowth in the small intestine and prevented the bacterial translocation almost completely in obstruction-induced rats. CONCLUSIONS: Intestinal obstruction causes bacterial overgrowth and translocation. Hyperbaric oxygen treatment prevents the bacterial translocation effectively.     

Intestinal bacterial translocation and tight junction structure in acute porcine pancreatitis

Background/Aims: To examine whether intestinal bacterial translocation occurs early in acute mild and severe pancreatitis and whether the intestinal expression of tight junction proteins (claudins-2, -3, -4, -5, -7), apoptosis or proliferation would explain the possible translocation. Methodology: Fifteen pigs were randomized to controls (n=5) or to develop mild edematous pancreatitis (n=5, saline infusion to pancreatic duct) or severe necrotic pancreatitis (n=5, taurocholic acid infusion). Translocation was studied by measuring bacterial cultures from portal vein blood and mesenteric lymph nodes. Immunohistochemical expression of the tight junction proteins, apoptosis rate (TUNEL) and Ki-67 were analyzed quantitatively from the epithelium of the jejunum and colon. Results: There was no bacterial translocation during the 6 hours followup, nor changes in the expression of tight junction proteins claudins-2 and -5 in jejunum or colon. Saturation and proportional area of claudin-3 staining decreased in the colon, as did claudins-4 and -7 staining in the jejunum of the necrotic pancreatitis group. Increased apoptosis was found in all samples from controls and the edematous pancreatitis group but not in jejunum in the necrotic pancreatitis group. Ki-67 activity tended to increase in the upper half of the villus in edematous and necrotic pancreatitis. There were no changes in the basic histology. Conclusions: The major finding of this study was that bacterial translocation from the gut is not present at the beginning of acute pancreatitis. Tight junction proteins claudin-2 and -5 do not become altered in the early stages of pancreatitis. Claudin-3 decreases in the colon and claudins-4 and -7 in the jejunum in necrotic pancreatitis. Laparotomy itself causes increased apoptosis in the colon and the jejunum.     

Modulation of the Effect of Dextran Sulfate Sodium-Induced Acute Colitis by the Administration of Different Probiotic Strains of Lactobacillus and Bifidobacterium

The pathogenic mechanism of inflammatory bowel diseases is not fully understood but colonic microflora including Lactobacillus and Bifidobacterium species may affect the induction of colonic inflammation. In this study the relative efficacy of different probiotic organisms in the prevention of colitis was compared in an induced rat colitis model. Three Lactobacillus strains and two Bifidobacterium strains were fed to Sprague-Dawley rats for 7 days prior to offering the rats 5% dextran sulfate sodium (DSS) in their drinking water to induce colitis and the administration of the probiotics continued for 7 days with the DSS. Colitis severity was assessed daily using a disease activity index (DAI). Samples were collected 7 days after colitis induction for intestinal bacterial flora and bacterial translocation. The DAI decreased significantly on days 4, 5, 6, and 7 in the Lactobacillus plantarum DSM 9843, Bifidobacterium sp. 3B1, and Bifidobacterium infantis DSM 15158 groups compared to the colitis control. It decreased significantly on days 5, 6, and 7 in the Bifidobacterium infantis DSM 15158 group compared to the Lactobacillus paracaesi DSM 13434 and Lactobacillus gasseri 5B3 groups. It also decreased significantly on day 7 in the L. plantarum DSM 9843 group compared to the L. gasseri 5B3 group. Bacterial translocation to the mesenteric lymph nodes decreased significantly in all treatment groups compared to the colitis control. Enterobacteriaceae bacterial translocation to the liver decreased in all treatment groups compared to the colitis control. Administration of certain strains of Lactobacillus and Bifidobacterium significantly improves the DAI and reduces bacterial translocation, and L. plantarum DSM 9843, Bifidobacterium sp. 3B1, and Bifidobacterium infantis DSM 15158 seem to have the best effect.     

Inhibition of Nitric Oxide Modulates the Effect of Oral Arginine Supplementation in Acute Liver Injury

Background: Arginine possesses numerous unique and advantageous biochemical and pharmacologic properties. We have previously shown that arginine supplementation in an acute liver injury model reduces both the extent of the liver injury and bacterial translocation. We therefore studied the role of nitric oxide on the effects of oral arginine supplementation in acute liver injury, bacterial translocation, ileal and cecal mucosal nucleotides, and RNA and DNA, to investigate pathogenetic mechanisms. Methods: Sprague-Dawley rats were divided into normal, liver injury control, N-nitro-L-arginine methyl ester (L-NAME), arginine, and L-NAME + arginine supplementation groups. Oral supplementation was performed daily through a nasogastric tube for 8 days. Acute liver injury was induced on the 8th day by intraperitoneal injection of D-galactosamine (1.1 g/kg body weight). Twenty-four hours after the liver injury, liver function tests, bacterial translocation, and ileal and cecal mucosal nucleotides, RNA, and DNA were evaluated. Results: Bilirubin and liver enzymes increased significantly in the L-NAME group compared with the arginine group, whereas the liver enzymes increased significantly compared wim the liver injury control group. In the L-NAME group the number of bacteria translocated to the portal and arterial blood increased significantly compared with all groups. In the arginine group the bacteria translocated to the liver and mesenteric lymph nodes decreased significantly compared with the liver injury control and L-NAME groups. The ileal and cecal mucosal nucleotides, RNA, and DNA in the arginine group increased significantly compared with the normal, liver injury, and L-NAME groups. Conclusions: Nitric oxide plays a role in the beneficial effect of the arginine supplementation in acute liver injury. It significantly improves the liver damage indicated by the increase of liver enzymes when its production was inhibited by L-NAME. Nitric oxide has a role in bacterial translocation since the number of bacteria significantly increased in arterial and portal blood when L-NAME was used to inhibit its production. Furthermore, arginine supplementation improved mucosal nucleotides, RNA, and DNA in ileum and colon.     

Influences of enteral nutrition combined with probiotics on gut microflora and barrier function of rats with abdominal infection

AIM: To investigate the influences of enteral, parenteral nutrition and probiotics delivered by gut on intestinal microecology, epithelial tight junctions, immune and barrier function of rats with abdominal infection. METHODS: Rat abdominal infection models established with cecal ligation and perforation method, were divided into three groups: parenteral nutrition (PN group, n = 7), PN enteral nutrition (EN group, n = 7) and PN EN probiotics (probiotics group, n = 7) via the needle jejunostomy and neck vein for five days. The total nutritional supplement of the three groups was isonitrogenic and isocaloric. Probiotics was delivered by jejunostomy 10 mL/d (1×108 cfu/mL). The rats were killed on the sixth day. The feces in the cecum were cultured for anaerobic bacterial growth and analyzed with bacterial group DNA fingerprint profile with random amplified polymorphic DNA. The transmembrane binding proteins (occludin) and IgA level in plasma cells of intestine epithelium in colon and terminal ileum were measured by an immunohistochemistry method. The ultrastructure of intestinal epithelial tight junctions in colon and small intestine was observed by electron-microscopy. Vena cava blood and the homogenated tissue of liver, lung and mesenteric lymph nodes were cultured to determine the bacterial translocations, and endotoxin in the blood from portal vein was detected. RESULTS: (1) The amount of bacteria of gut species in EN group and probiotic group was higher than that in PN group. The DIMA-profiles in EN group and probiotic group were similar to that of normal rats. The number of DNA-profiles in probiotics group was much more than that in PN group and EN group. Moreover, there were strange stripes in PN group. (2) The expression of occludin and IgA in the small and large intestine in EN group (2.309±0.336, 15.440±2.383) and probiotic group (2.938±0.515, 16.230±3.183) was improved as compared with PN group (1.207±0.587, P < 0.05, 11.189±2.108, P < 0.01). The expression of occludin in probiotic group (intestine: 2.93±0.515; cecum: 3.40±0.617) was higher than that in EN group (intestine: 2.309±0.336; cecum: 2.076±0.670; P < 0.05). The expression of IgA, especially in EN group (intestine: 15.440±2.383) and probiotic EN group (large intestine: 12.516±1.542) significantly increased as compared with PN group (intestine: 11.189±2.108; cecum: 10.160±1.643; P < 0.01). The intestinal epithelial tight junctions and microvilli of the probiotic group were more intact than those in the PN group. (3) The bacterial translocations in blood, liver, lung and mesenteric lymph nodes, and the levels of endotoxin were significantly reduced in probiotic (0.082±0.029) and EN (0.125±0.040) groups as compared with PN group (0.403±0.181, P < 0.05). CONCLUSION: Application of EN combined with probiotics could improve the expression of transmembrane binding proteins (occludin) and IgA, correct the intestinal flora disturbance, maintain gut barrier functions and tight junctions, and reduce the occurrence of gut bacterial translocation.     

Obstructive jaundice promotes bacterial translocation in humans.

BACKGROUND/AIMS: Significant bacterial translocation was demonstrated following experimental biliary obstruction, however very little is known about the importance and the prevalence of gut-origin sepsis in obstructive jaundice patients. Therefore, the aim of this study was to investigate the concept of gut-origin sepsis in obstructive jaundiced patients and its clinical importance. METHODOLOGY: Twenty-one patients requiring laparotomy for obstructive jaundice (group I) and thirty patients operated on electively mainly for chronic cholecystitis (group II) were studied. Peritoneal swab, mesenteric lymph node, portal venous blood, liver wedge biopsy and bile were sampled for culture immediately after opening the peritoneum. Additionally, peripheral blood samples were taken pre- and post-operatively from all patients. Post-operatively, patients were monitored for infectious complications. RESULTS: The mean serum bilirubin concentration, gamma glutamyl transferase and alkaline phosphatase levels in jaundiced patients before therapeutic intervention were significantly higher than in control patients. Five patients demonstrated bacterial translocation in group I (24%), whereas only one did so in group II (3.5%, p < 0.05). Septic complications were detected in three patients, but only in two with bacterial translocation in group I. There was one patient with bacterial translocation who had septic complication in group II. CONCLUSIONS: The present study demonstrated that obstructive jaundice significantly promotes bacterial translocation in humans, however, its clinical importance has yet to be defined.     

重构型天冬氨酸特异性半胱氨酸蛋白酶3表达载体诱导人肺腺癌细胞凋亡的意义

目的构建天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)表达载体,观察Caspase3表达载体对人肺腺癌A549细胞凋亡的影响。方法应用基因重组方法,建立重构型Caspase3基因的真核表达系统pcDNA3.1revCaspase3质粒和野生型pcDNA3.1Caspase3质粒。将实验细胞分为3组转染pcDNA3.1revCaspase3质粒组,转染pcDNA3.1Caspase3质粒组,转染pcDNA3.1质粒空白对照组。前2组用Caspase3抑制剂DEVDfmk干预。应用Caspase3酶活性分析、流式细胞仪及甲基偶氮唑蓝(MTT)比色法检测A549细胞Caspase3酶活性变化及细胞凋亡和增殖情况。结果(1)pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组的A549细胞Caspase3酶活性分别是(11.87±0.92)%、(5.34±0.38)%(t=16.02,P<0.01);用Caspase3抑制剂DEVDfmk干预后,pcDNA3.1revCaspase3质粒组酶活性为(7.04±0.48)%,仍明显高于野生型pcDNA3.1Caspase3质粒组的(4.51±0.20)%(t=11.86,P<0.01)。(2)流式细胞分析结果显示,pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组和pcDNA3.1质粒组的A549细胞凋亡率分别是(20.1±3.5)%、(7.8±2.8)%、(1.4±0.3)%,3组差异有统计学意义(F=44.01,P<0.01)。(3)MTT比色检测显示pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组和pcDNA3.1质粒组的A549细胞生存率分别是(35.7±1.1)%、(72.8±2.9)%、(85.4±4.8)%,转染pcDNA3.1revCaspase3质粒组生存率明显低于其他两组(F=375.07,P<0.01)。结论pcDNA3.1revCaspase3在A549细胞内有较强的自身活化能力,对Caspase3抑制剂抵抗作用较强,可明显诱导A549细胞凋亡并抑制A549细胞生长。     

Norepinephrine induces apoptosis in neonatal rat endothelial cells via down-regulation of Bcl-2 and activation of beta-adrenergic and caspase-2 pathways

OBJECTIVES: Heart failure is associated with increased plasma norepinephrine (NE) and endothelial apoptosis. Recent reports have suggested that endothelial dysfunction is an important target for future therapies of heart failure. However, whether NE can induce endothelial apoptosis and its mechanism remains unknown. METHODS: Endothelial cells from neonatal rat heart were treated with various concentrations of NE for different durations. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and DNA fragmentation assays. Caspase activity was measured using specific fluorogenic substrates. Proteins of Bcl-2 family and cytochrome c were assayed by Western blotting. RESULTS: NE induced endothelial apoptosis in a dose- and time-dependent manner. After treatment for 48 h, increasing NE concentration from 5, 10, 50, 100 to 200 microM resulted in 6+/-3%, 14+/-5%, 43+/-4%, 66+/-5%, and 89+/-6% apoptotic cells, respectively. The apoptosis was accompanied by down-regulation of Bcl-2 protein synthesis but not by cytosolic cytochrome c translocation. Caspase-2, -3, -6 and -9 were activated during apoptosis and caspase-2 inhibitor (Z-VDVAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) significantly attenuated the apoptosis. Overexpression of Bcl-2 inhibited caspase activity and decreased the apoptosis. Moreover, the NE-mediated apoptotic effect was attenuated by beta- (beta2>beta>beta1) adrenergic antagonists (ICI-118,551>propranolol>atenolol) but was not affected by alpha1- or alpha2-adrenergic antagonists (prazosin or yohimbine). CONCLUSION: Our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells mainly through down-regulation of Bcl-2 protein and activation of the beta-adrenergic (beta2>beta1) and caspase-2 pathways. beta-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.     

Effect of HSP70 induced by warm ischemia to the liver on liver function after partial hepatectomy

BACKGROUND/AIMS: The purpose of this study was to determine if induction of HSP70 (heat shock protein 70), a stress protein which plays a cytoprotective role in response to various stimuli, protects hepatocytes from damage caused by partial hepatectomy and, if so, to elucidate the mechanism of such protection. METHODOLOGY: One hundred and eight male F344 rats weighing 190-220 g were randomly assigned to two groups with or without the presence of preconditioning. Fifteen-minute warm ischemia was applied to the liver of rats to induce HSP70, and 70% hepatectomy was performed 48 hours after the induction of HSP70 (ischemia group; n = 72). The rats in the nonischemia group did not undergo 15-min warm ischemia prior to 70% hepatectomy (nonischemia group; n = 36). Six rats, selected randomly from each group, were sacrificed at each measurement point to obtain blood and liver tissue samples. The levels of HSP70 in the liver, serum nitric oxide, levels of catalase and superoxide dismutase activity in the liver as antioxidative enzymes, and levels of Bcl-xL and Bax proteins and caspase-3-like activity in the liver as indices of apoptosis, were measured. RESULTS: The mean +/- SD level of HSP70 in the ischemia group (100 +/- 42 arbitrary unit (au)) was significantly higher than that of the nonischemia group (2 +/- 0.7 au) immediately before hepatectomy (P < 0.05). The ischemic preconditioning attenuated the liver damage caused by the subsequent partial hepatectomy. The levels of superoxide dismutase and catalase activity, serum nitric oxide level, and Bax protein level of the ischemia and nonischemia groups showed no significant differences after the partial hepatectomy. In contrast, the mean +/- SD level of Bcl-xL in the liver of the ischemia group (261 +/- 52 au) was significantly higher than that in the nonischemia group (114 +/- 33 au) 12 hours after the hepatectomy (P < 0.01). Furthermore, the mean +/- SD level of caspase-3-like activity in the liver of the ischemia group (18.1 +/- 4.6 au) was significantly lower than that of the nonischemia group (26.0 +/- 4.8 au) at 12 hours after the hepatectomy (P < 0.05). CONCLUSIONS: HSP70 induced by ischemic preconditioning prior to the partial hepatectomy was considered to protect the liver itself. In addition, the induced HSP70 may affect the Bcl-xL level after partial hepatectomy. Therefore, Bcl-xL seems to be involved in the reduction of liver damage after partial hepatectomy along with HSP.     

P-selectin mediates leukocyte rolling in concanavalin-A-induced hepatitis.

Concanavalin-A (Con-A)-induced hepatitis is an experimental model of human autoimmune hepatitis characterized by leukocyte activation and infiltration of the liver. The aim of the present study was to evaluate the role of P-selectin on leukocyte-endothelial interactions within the hepatic microvasculature in response to Con-A. METHODS: The study was performed in P-selectin-deficient mice and wild-type mice pretreated with anti-P-selectin blocking monoclonal antibody (mAb) or vehicle. After 2 h of Con-A (20 mg/kg i.v.) or PBS administration, leukocyte rolling and adhesion and the index of sinusoidal perfusion were evaluated using the intravital microscopy technique in the liver. Apoptosis was determined by flow cytometry analysis of caspase-3 activity assayed on freshly isolated hepatocytes. RESULTS: Con-A induced a significant increase in leukocyte rolling, mainly located at the central venule (2.1+/-0.4 vs 0.6+/-0.2 cells/min in wild-type mice treated with vehicle) and less marked, but still significant, in portal venules. This was associated with a significant increase in leukocyte adhesion. In P-selectin-deficient mice treated with Con-A, leukocyte rolling in portal and central venules was markedly reduced. However, leukocyte adhesion was only partially attenuated. A few sinusoids were perfused in wild-type mice treated with Con-A (26%). The percentage of perfused sinusoids was significantly higher in P-selectin-deficient mice (45%; P<0.05 vs wild-type). Similar effects were noted after the simultaneous injection of Con-A and anti-P-selecting mAb in wild-type mice. After Con-A treatment, apoptosis was markedly reduced in isolated hepatocytes of P-selectin-deficent mice (37+/-7% vs 75+/-5% in wild type). CONCLUSION: The results of this intravital microscopy study clearly demonstrate that P-selectin is involved in the initial leukocyte rolling that leads to the development of Con-A-induced liver injury.     

Short-Chain Inulin-Like Fructans Reduce Endotoxin and Bacterial Translocations and Attenuate Development of TNBS-Induced Colitis in Rats

Anti-inflammatory effects of short-chain inulin-like fructans (SCF) on trinitrobenzene sulfonic acid (TNBS)-induced colitis were investigated in rats, focusing specifically on endotoxin and bacterial translocations. SCF with degrees of polymerization (DP) of 4 and 8 were used. Rats were fed either control diet or diets including 60 g DP4 or DP8 per kilogram for 7 days, and then received intracolonic TNBS and were fed the respective diets for a further 10 days. DP4 and DP8 significantly reduced colonic injuries as assessed by damage score, but the reduction of colonic myeloperoxidase activity was manifest solely with DP8. At 3 days after colitis induction, bacterial translocation to the mesenteric lymph node was significantly lower in the DP4 and DP8 groups, but significant reduction in the portal endotoxin concentration was achieved solely in the DP8 group. Immediately prior to colitis induction, cecal immunoglobulin A and mucin concentrations were higher in the DP4 and DP8 groups, but these changes were abolished at 10 days post colitis induction. The data suggest that SCF exert prophylactic effects against TNBS colitis, presumably as a result of inhibitory effects on endotoxin and bacterial translocations.     

肿瘤坏死因子α及caspase-3表达与暴发性肝衰竭细胞凋亡

目的　研究肿瘤坏死因子α(TNFα)与caspase 3表达在实验性暴发性肝衰竭 (FHF)中对肝细胞凋亡的作用。方法　用脂多糖和D 氨基半乳糖制备FHF小鼠模型 ;ELISA和逆转录PCR法检测血清TNFα水平及肝组织TNFαmRNA表达 ;原位杂交法检测肝组织内caspase 3表达 ;DNA琼脂糖凝胶电泳和末端转移酶介导的dUTP缺口末端标记 (TUNEL)检测肝细胞凋亡。结果　用药后 2h开始 ,TNFαmRNA表达增加 ( 0 91± 0 75 ,正常值 0 32± 0 10 ) ,血清TNFα水平升高 [( 32 0 5 0± 86 5 7)ng/L ,正常值 ( 16 6 6± 7 0 1)ng/L],caspase 3少量表达 ;8h后 ,血清ALT和总胆红素 (TBil)水平显著增加 [分别为 ( 5 6 0 6 6± 6 0 2 0 )U/L和 ( 16 3 6 6± 34 5 1) μmol/L ,正常值为 ( 2 3 5 6± 8 0 3)U/L和 ( 14 90± 4 80 ) μmol/L],caspase 3表达至最高峰 ,并出现典型的肝细胞凋亡改变 ;12h后 ,血清ALT和TBil水平达最高峰 ,caspase 3表达较 8h减少 ,肝细胞坏死和凋亡同时存在。抗TNFα单抗阻断试验后 ,肝细胞凋亡和坏死明显减轻 ,TUNEL和caspase 3表达显著减少。 结论　TNFα有促进肝细胞凋亡与坏死作用 ,肝细胞凋亡与caspase 3的激活有关 ,在时间上肝细胞凋亡先于坏死。     

Melatonin reduces disseminate neuronal death after mild focal ischemia in mice via inhibition of caspase-3 and is suitable as an add-on treatment to tissue-plasminogen activator

The effects of i.p. melatonin (4 + 4 mg/kg, after induction of ischemia and at reperfusion onset) administered either alone or in combination with the thrombolytic tissue-plasminogen activator (t-PA, 10 mg/kg), on cerebral laser Doppler flow (LDF) and ischemic injury were studied after 30 min of middle cerebral artery (MCA) thread occlusion in male C57BL/6 mice. Thread occlusions resulted in reproducible focal ischemias, followed by hyperperfusion reactions immediately after thread withdrawal, as revealed by LDF measurements. Compared with animals receiving normal saline (peak LDF after reperfusion: 172.0 +/- 24.2%), postischemic LDF was significantly attenuated in animals treated with melatonin (105.1 +/- 6.7%, P < 0.05). Delivery of t-PA (132.8 +/- 22.3%) or t-PA plus melatonin (164.7 +/- 36.7%), on the contrary, did not influence postischemic LDF recordings. Twenty-four hours after reperfusion, melatonin treated mice had significantly increased neuronal survival and decreased disseminate cell injury in the ischemia-vulnerable striatum, as investigated by cresyl violet and terminal transferase biotinylated-dUTP nick end labeling stainings. The protective effects were associated with inhibition of caspase-3 activity. Melatonin administration also increased neuronal survival after 30 min MCA occlusion in animals treated with t-PA, although t-PA itself already decreased the degree of injury in a significant manner. Our data demonstrate that melatonin reduces disseminated neuronal injury in the striatum after mild focal ischemia. Brain protection is independent of hemodynamic changes and involves inhibition of caspase-3.     

Peripheral blood and portal tract lymphocyte populations in primary sclerosing cholangitis

The relative distribution of lymphocyte subpopulations in the blood and liver of patients with primary sclerosing cholangitis (PSC) and related diseases has been studied using immunoenzyme techniques. The peripheral blood CD4/CD8 T lymphocyte ratio was significantly higher in active ulcerative colitis (UC) and in PSC with inactive UC than in inactive UC alone. In contrast, no relationship with disease activity was seen in Crohn's disease. The portal tract t lymphocyte count per high power field (mean +/- S.D.) was higher in pre-cirrhotic PSC (173 +/- 105) and primary biliary cirrhosis (PBC: 210 +/- 110) than in histologically normal liver (42 +/- 27). However, the overall portal tract CD4/CD8 ratio was similar in PSC (1.49), PBC (1.89) and normal controls (1.63). The results are consistent with immunological involvement in the pathogenesis of PSC, but argue against the hypothesis that changes in the peripheral blood T cell subsets are due to sequestration at the site of tissue inflammation.     

Protective effect of low dose of melatonin against cholestatic oxidative stress after common bile duct ligation in rats

AIM: To investigate the role of oxidative injury and the effect of exogenous melatonin administration on liver damage induced by bile duct ligation (BDL), and second, to evaluate the role of nitric oxide (NO), a free oxygen radical, in oxidative injury. METHODS: Thirty-two Sprague-Dawley rats were assigned to four groups: sham operation (SO), BDL, BDL+melatonin, and BDL+vehicle. Cholestasis was achieved by double ligature of the common bile duct. Melatonin was injected intraperitoneally 500 μg/(kg·d) for 8 d. Hepatic oxidative stress markers were evaluated by changes in the amount of lipid peroxides, measured as malondialdehyde (MDA), and reduced GSH. Total nitrite (NOx) concentrations were determined in hepatic homogenates. Histopathological examination was performed using a histological scoring system. RESULTS: The histopathological changes including portal inflammation, necrosis,apoptosis, focal inflammation and fibrosis were severe in the BDL and BDL+vehicle groups. There were numerous large areas of coagulation necrosis. Histological Activity Index scores of these groups were significantly higher than that of the SO group. Treatment with melatonin reduced these alterations significantly. The degree of necro-inflammation and fibrosis showed significant difference between the BDL and BDL+melatonin groups. BDL was accompanied by a significant increase in MDA and NOx, and a significant decrease in GSH levels. Mean±SE values of MDA, GSH and NOx levels of SO group were 147.47±6.69, 0.88±0.33 μmol/g and 180.70±6.58 nm/g, respectively. The values of BDL group were 200.14±21.30, 0.65±0.02 μmol/g, and 400.46±48.89 nm/g, respectively, whereas the values of BDL+melatonin group were 115.93±6.8,0.74±0.02 μmol/g, and 290.38±32.32 nm/g, respectively. Melatonin treatment was associated with a significant recovery of MDA, GSH and NOx levels. CONCLUSION: We have concluded that oxidative stress is associated with the pathogenesis of cholestatic liver damage and NO contributes to oxidative damage. Melatonin, even at low dose, is an efficient agent in reducing negative parameters of cholestasis.     

Allopurinol and glutamine attenuate bacterial translocation in chronic portal hypertensive and common bile duct ligated growing rats.

BACKGROUND: Spontaneous bacterial infections and septicaemia result in morbidity and mortality in patients with portal hypertension and obstructive jaundice. AIM: The aim of this study in rats was to investigate the incidence of bacterial translocation in portal hypertension and obstructive jaundice, and to evaluate the effects of allopurinol and glutamine. METHODS: Rats were subjected to sham laparotomy (SL), portal hypertension (PH) by calibrated stenosis of the portal vein, and common bile duct ligation (CBDL). Animals of each group were either treated with allopurinol (50 mg/kg twice a week), glutamine (1 g/kg/d), and allopurinol and glutamine. RESULTS: After four weeks, significant bacterial translocation in the untreated PH and CBDL rats occurred. Intestinal mucosal malondialdehyde concentrations (MDA), as an indicator for lipid peroxidation, and myeloperoxidase activity (MPO) released from activated neutrophils were also significantly increased (p < 0.01). Allopurinol and glutamine in PH and CBDL rats improved bacterial translocation, and decreased MDA and MPO values (p < 0.01). CONCLUSION: In PH and CBDL rats significant bacterial translocation, ileal mucosal lipid peroxidation, and neutrophil derived MPO activity occurred. Allopurinol and glutamine significantly reduced bacterial translocation, as well as ileal mucosal MDA and MPO activities.