Anti‐inflammatory effects of melatonin in a rat model of caerulein‐induced acute pancreatitis

The purpose of our study was to evaluate the protective effect of melatonin in a rat model of caerulein‐induced acute pancreatitis. For the induction of experimental acute pancreatitis, four subcutaneous injections of caerulein (20 µg kg^–1 body weight) were given to Wistar rats at 2‐h intervals. Melatonin was injected intraperitoneally (25 mg kg^–1 body weight) 30 min before each caerulein injection. After 12 h, rats were sacrificed by decapitation. Blood and pancreas samples were collected and processed for serological and histopathological studies, respectively. Lipase, α‐amylase, corticosterone, total antioxidant power and cytokines interleukin (IL)‐1β, IL‐4 and tumour necrosis factor (TNF)‐α were determined using commercial kits. ANOVA and Tukey tests (P < 0.05) were performed for the statistical analysis of the results. Results showed that the administration of melatonin reduced histological damage induced by caerulein treatment as well as the hyperamylasemia and hyperlipidemia. Corticosterone and antioxidant total power were also reverted to basal activities. Furthermore, melatonin pre‐treatment reduced pro‐inflammatory cytokines IL‐1β and TNF‐α and increased the serum levels of anti‐inflammatory cytokine IL‐4. In conclusion, the findings suggest that the protective effect of melatonin in caerulein‐induced acute pancreatitis is mediated by the anti‐inflammatory ability of this indolamine. Thus, melatonin may have a protective effect against acute pancreatitis. Copyright © 2013 John Wiley & Sons, Ltd.     

Treatment with antileukinate, a CXCR2 chemokine receptor antagonist, protects mice against acute pancreatitis and associated lung injury

Acute pancreatitis is a common, and as yet incurable, clinical condition, the incidence of which has been increasing over recent years. Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that treatment with a neutralizing antibody against CINC, a CXC chemokine, protects rats against acute pancreatitis-associated lung injury. The hexapeptide antileukinate (Ac-RRWWCR-NH2) is a potent inhibitor of binding of CXC chemokines to the receptors (CXCR2). This study aims to evaluate the effect of treatment with antileukinate on acute pancreatitis and the associated lung injury in mice. Acute pancreatitis was induced in adult male Swiss mice by hourly intra-peritoneal injections of caerulein (50 microg/kg/h) for 10 h. Antileukinate (52.63 mg/kg, s.c.) was administered to mice either 30 min before or 1 h after starting caerulein injections. Severity of acute pancreatitis was determined by measuring plasma amylase, pancreatic water content, pancreatic myeloperoxidase (MPO) activity, pancreatic macrophage inflammatory protein-2 (MIP-2) levels and histological examination of sections of pancreas. A rise in lung MPO activity and histological evidence of lung injury in lung sections was used as criteria for pancreatitis-associated lung injury. Treatment with antileukinate protected mice against acute pancreatitis and associated lung injury, showing thereby that anti-chemokine therapy may be of value in this condition.     

Gut-derived endotoxin translocation is the main aggravating mechanism of acute severe pancreatitis

Severe acute pancreatitis (SAP) is a serious systemic disease. It exacerbates when complicated with multiple organ dysfunction syndrome or failure (MODS or MOF). However, the aggravating mechanism of SAP is still unknown up to now. Study showed that maintaining integrity of intestinal mucosal barrier function by given effective antibiotics, selective digestive decontamination (SDD) and enteral nutrition therapy to the patients with SAP could significantly reduce infection of pancreatic necrotic tissue and improve the patient's outcome. Combining the findings of gut-derived bacteria in animals' pancreas, liver, spleen, mesenteric lymph nodes with increasing concentration of inflammatory cytokines and endotoxin in plasma with SAP, we hypothesize that gut-derived endotoxin translocation is the main aggravating mechanism of SAP. The hypothesis holds potential as a target for therapeutic intervention.     

Intra-pancreatic release of bradykinin during the course experimental pancreatitis in rat

Kallikrein/Kininogn activation is an important pathophysiological event in acute pancreatitis, leading to microcirculatory changes within the gland. Hitherto, only indirect measurements of pancreatic bradykinin formation have been performed, monitoring the peptide in the circulation and in the peritoneal exudate. In the present study, intra-pancreatic bradykinin release was assessed using microdialysis during experimental acute pancreatitis in rat. In mild, oedematous pancreatitis, induced by caerulein hyperstimulation, the levels of bradykinin within the gland were not elevated compared with those of control rats. However, in necrotic pancreatitis, induced by retrograde injection of taurocholate into the pancreatic duct, significantly elevated levels of intraglandular bradykinin were seen. Several rats in this group died whilst in a state of circulatory shock.     

Gut-associated lymphoid T cell suppression enhances bacterial translocation in alcohol and burn injury

The mechanism of alcohol-mediated increased infection in burn patients remains unknown. With the use of a rat model of acute alcohol and burn injury, the present study ascertained whether acute alcohol exposure before thermal injury enhances gut bacterial translocation. On day 2 postinjury, we found a severalfold increase in gut bacterial translocation in rats receiving both alcohol and burn injury compared with the animals receiving either injury alone. Whereas there were no demonstrable changes in intestinal morphology in any group of animals, a significant increase in intestinal permeability was observed in ethanol- and burn-injured rats compared with the rats receiving either injury alone. We further examined the role of intestinal immune defense by determining the gut-associated lymphoid (Peyer's patches and mesenteric lymph nodes) T cell effector responses 2 days after alcohol and burn injury. Although there was a decrease in the proliferation and interferon-gamma by gut lymphoid T cells after burn injury alone; the suppression was maximum in the group of rats receiving both alcohol and burn injuries. Furthermore, the depletion of CD3(+) cells in healthy rats resulted in bacterial accumulation in mesenteric lymph nodes; such CD3(+) cell depletion in alcohol- and burn-injured rats furthered the spread of bacteria to spleen and circulation. In conclusion, our data suggest that the increased intestinal permeability and a suppression of intestinal immune defense in rats receiving alcohol and burn injury may cause an increase in bacterial translocation and their spread to extraintestinal sites.     

Induction of heat shock protein and prevention of caerulein-induced pancreatitis by water-immersion stress in rats

Water-immersion stress is known to be involved in the development of hemorrhagic pancreatitis in caerulein-induced pancreatitis, when the stress is given following caerulein injection. The effects of pre-treatment with water-immersion to caerulein-induced pancreatitis were investigated in this study.

• 1.1. A 60-kDa heat shock protein was induced by pre-treatment with water-immersion stress in the pancreas.
• 2.2. Intra-peritoneal injection of caerulein (40 μg/kg) induced acute pancreatitis in rats without pre-treatment with water-immersion. However, when the rats were pre-treated with water-immersion, acute pancreatitis was not developed and no change of serum amylase levels was observed by i.p. injection of caerulein.

     

Protective effect of CR 1409 (cholecystokinin antagonist) on experimental pancreatitis in rats and mice

CR 1409, a glutaramic acid derivative with competitive cholecystokinin-antagonistic activity, was administered IP and evaluated in comparison with proglumide (the model CCK-receptor antagonist), gabexate (protease inhibitor) and PGE₂ (cytoprotective) on two different models of experimental pancreatitis. Acute pancreatitis was induced in mice by six IP injections of 50 μg/kg caerulein at hourly intervals. The drugs were administered 30 minutes before each caerulein administration. Blood samples and pancreata were collected 3 hours after the last caerulein injection. In the second experiment, pancreatitis was induced in rats by injecting 0.3 ml 6% sodium taurocholate interstitially into the pancreas. The drugs were administered twice, 30 minutes before and 3 hours after taurocholate. The animals were killed 6 hours after laparotomy and blood samples and pancreata were collected. CR 1409 exhibited on both pancreatitis models a protective effect in a dose range of 0.3–10 mg/kg. Proglumide exhibited a protective activity at higher doses (200–400 mg/kg). Gabexate and PGE₂ were effective only in pancreatitis induced by taurocholate in a dose range of 30–60 mg/kg and 60–130 μg/kg respectively. These results, showing a high protective effect of CR 1409 on different models of acute pancreatitis, suggest an important role of CCK in the pathogenesis of pancreatitis.     

A key role of neurokinin 1 receptors in acute pancreatitis and associated lung injury

Earlier studies have shown that mice deficient in NK1 receptors or its ligand, substance P, are protected against acute pancreatitis and associated lung injury. In the current study, the protective effect of NK1 receptor blockage against acute pancreatitis and associated lung injury was investigated, using a specific receptor antagonist, CP-96345. Acute pancreatitis was induced in mice by intraperitoneal (i.p.) injections of caerulein. Substance P levels in plasma, pancreas, and lungs were found to be elevated in a caerulein dose-dependent manner. Mice treated with CP-96345, either prophylactically, or therapeutically, were protected against acute pancreatitis and associated lung injury as evident by attenuation in plasma amylase, pancreatic and pulmonary myeloperoxidase activities, and histological evidence of pancreatic and pulmonary injuries. Pulmonary microvascular permeability was also reduced as a result of CP-96345 treatment. These results point to a key role of NK1 receptors in acute pancreatitis and associated lung injury.     

Caerulein-induced acute pancreatitis in the rat. Pancreatic secretory response to cholecystokinin.

The response of pancreatic exocrine secretion to cholecystokinin (CCK), has been studied in experimental acute pancreatitis induced in rats by supramaximal doses of caerulein. Several doses of caerulein were used (4, 20 and 40 micrograms/Kg) and each one was administered by four subcutaneous injections over 3 h at hourly intervals. Pancreatic juice was collected 9 h after the first injection. The caerulein-treated animals showed a statistically significant increase in serum amylase levels. Secretory activity of ductular cells remained unchanged in all the caerulein-treated animals, but total protein and amylase secretion decreased significantly at all the caerulein doses used, both in resting conditions and under stimulation with CCK (1.25 micrograms/Kg/h). Despite this the acinar cells of rats treated with the lowest dose of caerulein retained a certain degree of secretory function since amylase activity in pancreatic juice was greater than in other groups of rats treated with higher doses of caerulein. Moreover, the percentage of increase observed in total protein and amylase in response to CCK respect to basal secretion is similar to that of the untreated animals. At higher doses (20 and 40 micrograms/Kg) the secretory capacity in response to CCK was inhibited. Therefore CCK administration in slight acute pancreatitis could be used as a therapy since it favours the secretion of pancreatic enzymes at percentual levels similar to those of the controls.     

参麦注射液对严重烫伤大鼠肠道屏障功能的保护作用

目的:探索参麦注射液对30% Ⅲ°烫伤早期肠道屏障功能的保护作用,为参麦注射液防治肠源性感染提供实验依据。方法:Wistar大鼠60只,随机分为正常对照组、模型对照组,地塞米松5 mg/kg组、参麦注射液5、10、15 mg/kg组,每组10只,使用烫伤仪建立30% Ⅲ°烫伤动物模型,立即腹腔注射相应的药物,每天1次。烫伤72 h后,检测肝脏、脾脏、肠系膜淋巴结细菌移位量、血浆内毒素、二胺氧化酶(DAO)、肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)水平和肠粘膜分泌型免疫球蛋白A(sIgA)的水平。结果:与正常对照组比较,模型组肝脏、脾脏、肠系膜淋巴结细菌移位量,血浆内毒素、DAO、TNF-α及 IL-6和肠黏膜sIgA水平明显升高(P＜0.01);与模型组比较,地塞米松组和参麦注射液5、10、15 mg/kg组肝脏、脾脏、肠系膜淋巴结细菌移位量,血浆内毒素、DAO、TNF-α及 IL-6和肠黏膜sIgA水平明显降低(P＜0.05或P＜0.01)。结论:参麦注射液可减轻严重烫伤引起的肠粘膜损伤,效果与地塞米松相当,高剂量组效果更好。     

Mathematical Modeling Reveals Kinetics of Lymphocyte Recirculation in the Whole Organism

The kinetics of recirculation of naive lymphocytes in the body has important implications for the speed at which local infections are detected and controlled by immune responses. With a help of a novel mathematical model, we analyze experimental data on migration of ⁵¹Cr-labeled thoracic duct lymphocytes (TDLs) via major lymphoid and nonlymphoid tissues of rats in the absence of systemic antigenic stimulation. We show that at any point of time, 95% of lymphocytes in the blood travel via capillaries in the lung or sinusoids of the liver and only 5% migrate to secondary lymphoid tissues such as lymph nodes, Peyer''s patches, or the spleen. Interestingly, our analysis suggests that lymphocytes travel via lung capillaries and liver sinusoids at an extremely rapid rate with the average residence time in these tissues being less than 1 minute. The model also predicts a relatively short average residence time of TDLs in the spleen (2.5 hours) and a longer average residence time of TDLs in major lymph nodes and Peyer''s patches (10 hours). Surprisingly, we find that the average residence time of lymphocytes is similar in lymph nodes draining the skin (subcutaneous LNs) or the gut (mesenteric LNs) or in Peyer''s patches. Applying our model to an additional dataset on lymphocyte migration via resting and antigen-stimulated lymph nodes we find that enlargement of antigen-stimulated lymph nodes occurs mainly due to increased entrance rate of TDLs into the nodes and not due to decreased exit rate as has been suggested in some studies. Taken together, our analysis for the first time provides a comprehensive, systems view of recirculation kinetics of thoracic duct lymphocytes in the whole organism.     

骶神经电刺激对脊髓损伤大鼠肠黏膜机械屏障的保护作用

目的：观察骶神经电刺激对脊髓损伤大鼠肠黏膜机械屏障的保护作用。方法：56只Wistar大鼠分7组（n=8）：正常组、急性完全性脊髓损伤（SCI）组和骶神经电刺激组（按24、48、72h各8只）。进行内毒素测定;肠系膜淋巴结、肝脏、脾脏菌培养;肠道形态学观察;紧密连接蛋白zo-1的蛋白表达测定。结果：对照组肠黏膜不同程度损伤;肠道上皮细胞及细胞间连接破坏;内毒素血症和细菌移位明显。实验组肠黏膜得到改善,内毒素水平下降且细菌移位减少。ZO-1蛋白表达无统计学差异。对照组ZO-1的分布出现不同程度的散乱、排列不规则,实验组分布得到改善。结论：骶神经电刺激可促肠蠕动、排肠内容物、减少肠道菌群数量,保护肠黏膜上皮细胞及紧密连接的机械屏障,减少细菌移位和内毒素血症。     

The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 microg/kg caerulein, L-arginine used for NO induction and N(omega)-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.     

Trauma hemorrhagic shock-induced lung injury involves a gut-lymph-induced TLR4 pathway in mice

Background

Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.

Methods/Principal Findings

The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan''s blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.

Conclusions/Significance

Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation.     

The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 µg/kg caerulein, L-arginine used for NO induction and N^ω-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.     

Distribution of Chromate-labeled Brucella abortus Endoxin in Tolerant Mice

Endotoxin tolerance as manifested by a lesser degree of hypoferremia was demonstrated in mice when both pretreatment (10 mug per injection) and challenge (100 mug) does of Brucella abortus endotoxin were administered intraperitoneally. Qualitative and quantitative studies on the distribution of chromate-labeled endotoxin in normal mice revealed that the endotoxin localized predominately in the liver and hypoferremia could be related to a high uptake of endotoxin by this organ. In tolerant mice, the labeled endotoxin was found mainly in the mesenteric lymph nodes (MLN) with smaller quantities in the blood, spleen, and liver. Experiments with splenectomized mice provided supporting evidence that the liver was the target organ of the hypoferremic response to endotoxin. High localization of endotoxin in the MLN with lower quantities in the blood, livers, and spleens of tolerant mice indicated that tolerance may be the result of a blockage by the MLN, preventing the endotoxin from reaching the liver. This inference was supported by the finding that hypoferremic tolerance did not occur when the hypoferremia-provoking dosage of endotoxin (100 mug) was given intravenously to mice pretreated intraperitoneally. There was less hypoferremia in normal mice injected with a mixture of antiserum and 100 mug of endotoxin than in mice given the same dosage of endotoxin in saline. Distribution studies on endotoxin treated with specific antiserum revealed that the endotoxin localized principally in the MLN, thus preventing most of the endotoxin from reaching the liver, the target organ of the hypoferremic response.     

Platelet activating factor (PAF) inhibitor (TCV-309) reduces caerulein- and PAF-induced pancreatitis. A morphologic and functional study in the rat.

Caerulein-induced acute pancreatitis was studied in rats. Consistent with this type of acute pancreatitis morphological (edema, leukocytic infiltration and acinar cell vaculization) and biochemical (increase in pancreatic protein content. PAF release and serum amylase) changes developed 5 hours after caerulein administration. In addition increase in pancreatic weight and decrease in pancreatic blood flow were noticed. PAF administration caused pancreatic damage similar in some parameters to caerulein-induced pancreatitis, along with reduction of pancreatic blood flow, increase in pancreatic protein content, and serum amylase. TCV-309, a selective PAF antagonist, administered prior to caerulein and/or PAF, reduced caerulein-induced pancreatitis and prevented PAF-induced pancreatitis. Results of our present studies indicate the crucial role of PAF in pathogenesis of experimental acute pancreatitis.     

多器官功能不全综合征时肠道细菌易位及通里攻下法影响的实验研究

肠道是全身感染的起源,细菌易位在ＭＯＤＳ的发生发展中具有重要作用。本实验以放射性同位素３５Ｓ标记致病Ｅｃｏｌｉ作示踪剂,研究不同剂量酵母多糖腹腔注射所致ＭＯＤＳ时肠道细菌易位的途径和程度,并观察通里攻下中药和抗生素对细菌易位的影响。结果表明,酵母多糖腹腔注射能造成肠道屏障损伤引起细菌易位,细菌易位的程度具有剂量依赖性。细菌易位途径主要有两条,低剂量时细菌易位以肠系膜淋巴结途径为主,高剂量时以门静脉途径为主。予先应用新霉素和灭滴灵给肠道脱污染反而加重细菌易位,抗生素治疗对细菌移位效果不明显。以大承气汤为代表的通里攻下中药对控制细菌易位有显著效果     

An outbreak of Yersinia pseudotuberculosis infection in a small indoor breeding colony of red-bellied (Saguinus labiatus) tamarins

A spontaneous outbreak of yersiniosis caused by Yersinia pseudotuberculosis serotype IIB occurred in a small indoor breeding colony of red-bellied tamarins (Saguinus labiatus) during the winter of 1981. Of 35 monkeys at risk 6 died of an acute or subacute infection over a period of 23 days. Clinical signs were anorexia, weakness, listlessness and depression. The disease was characterized by focal necrosis of the liver, spleen, mesenteric lymph nodes, ulcerative enteritis, and the presence of colonies of Gram-negative bacilli in the lesions. Y. pseudotuberculosis was isolated from the liver, spleen, mesenteric lymph nodes and kidney but not from the blood, lung or intestine. Contaminated food was believed to be the source of infection.