内毒素休克家兔脑区、脑脊液、血浆中脑啡肽含量的改变及纳洛酮的抗休克作用

本工作在戊巴比妥钠麻醉的家兔上进行。采用夹闭肠系膜上动脉阻断血流方法建立内毒紫休克的实验模型。用放射免疫分析法测定了休克前后脑区、脑脊液及血浆中脑啡肽含量的变化,并观察了脑室或静脉注射纳洛酮的抗休克效应。结果如下:1.休克时,下丘脑、延脑、桥脑的亮脑啡肽含量显著升高,中脑无明显变化、丘脑、纹状体下降。脑脊液和血浆中亮脑啡肽明显增加。外周静脉血与肾静脉血中无显著差别。2.侧脑室或静脉注射纳洛酮,均可使休克动物的血压回升,延长存活时间。脑室注射的升压数值略大,维持升压时间也较长。实验结果提示:脑啡肽参与内毒素休克过程。断阿片样物质的作用,可能是治疗内毒素休克的一个途径。     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

Interference of the PAF-acether antagonist BN 52021 with endotoxin-induced hypotension in the guinea-pig

Because of the potential role of PAF-acether in the pathogenesis of endotoxin shock, we examined the preventive and curative effects of BN 52021, a new PAF-acether antagonist in guinea-pig challenged with S. Typhimurium endotoxin. A biphasic reduction of mean arterial pressure was elicited by i.v. endotoxin (300 micrograms/kg) in control animals, with a rapid drop of blood pressure (maximal decrease within 10 min), partial recovery at 20 min and a second gradual decrease after 30 min. Treatment with BN 52021 injected 15 min prior to endotoxin reduced the initial rapid drop of blood pressure from 38.5 +/- 5 mmHg in vehicle-treated controls (n = 15) to 17 +/- 3 mmHg (p less than 0.01) in animals treated with 1 mg/kg BN 52021(n = 10) and to 9.5 +/- 8 mmHg (p less than 0.01) in guinea-pigs treated with 6 mg/kg BN 52021 (n = 5). The early hypotensive phase was associated with severe thrombocytopenia-leukopenia; only the thrombocytopenia was reduced by BN 52021. The prolonged secondary phase of hypotension was reduced by BN 52021 pretreatment whereas a small increase of hematocrit persisted. The two phases of the arterial pressure profile during endotoxic shock were not observed in animals previously made thrombopenic by rabbit and anti-platelet serum and only the late hypotensive phase persisted. This late hypotension induced by endotoxin in thrombopenic animals was suppressed by BN 52021 pretreatment suggesting that BN 52021 may act via a platelet-independent mechanism. The intravenous injection of BN 52021 during the prolonged secondary phase of shock was followed by an immediate increase of the depressed blood pressure. This increase of blood pressure was dose-dependent, maximum at 6 mg/kg BN 52021, and observed in normal and thrombopenic animals. The interference of BN 52021 with endotoxin shock may be related to its PAF-acether antagonist properties and suggests that PAF-acether is an important participant in endotoxic shock.     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e6) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e6 demonstrated maximal effects on blood SOD activity.     

Reversal of endotoxin-mediated shock by NG-methyl-L-arginine, an inhibitor of nitric oxide synthesis

Septic shock is a life-threatening condition that results from exposure to bacterial endotoxin. It is manifested by cardiovascular collapse and mediated by the release of cytokines such as tumor necrosis factor. Some of these cytokines cause the release of vasoactive substances. In the present study, administration of 40 microgram/kg of bacterial endotoxin to dogs caused a 33% decrease in peripheral vascular resistance and a 54% fall in mean arterial blood pressure within 30 to 90 minutes. Vascular resistance and systemic arterial pressure returned to normal within 1.5 minutes after intravenous administration of NG-methyl-L-arginine (20 mg/kg), a potent and selective inhibitor of nitric oxide synthesis. L-Arginine reversed the effect of L-NMA and restored the endotoxin-induced hypotension. Although NG-methyl-L-arginine injection increased blood pressure in control dogs, the hypertensive effect was much greater in endotoxemic dogs (24.8 +/- 2.7 mmHg vs 47.8 +/- 6.8 mmHg, p = 0.01, n = 4). NG-Methyl-L-arginine caused only a modest increase in blood pressure in dogs made hypotensive by continuous intravenous infusion of nitroglycerin (17.1 +/- 5.0 mm Hg, n = 3). These findings suggest that nitric oxide overproduction is an important contributor to endotoxic shock. Moreover, our findings demonstrate for the first time, the utility of nitric oxide synthesis inhibitors in endotoxic shock and suggest that such inhibitors may be of therapeutic value in the treatment of septic shock.     

Short-term effects of thalidomide analogs on hepatic glycogen and nitric oxide in endotoxin-challenged rats

Hepatic glycogen metabolism is altered by nitric oxide (NO) during endotoxic shock. Thalidomide analogs immunomodulate the endotoxin-induced cytokines which regulate the NO release. We analyzed the short-term effects of some thalidomide analogs on the hepatic glycogen store and on the plasma and hepatic NO in an acute model of endotoxic challenge in rat. An endotoxin dose selection was performed. Rats received vehicle, thalidomide or analogs orally and, two hours after last dose, they were injected with endotoxin (5 mg/kg). Animals were sacrificed 2 h after challenge. Liver glycogen was quantified by the anthrone technique. Plasma and hepatic NO was determined by Griess reagent and HPLC. Hepatic interferon-gamma, a NO co-inducer, was measured by ELISA. Endotoxin caused inverse dose-dependent effects on plasma NO and on glycogen.Thalidomide analogs showed short-term regulatory effects on glycogen, some of them increased it. Plasma NO was almost unaffected by analogs but hepatic NO was strikingly modulated. Analogs slightly up-regulated the liver interferon-gamma and two of them increased it significantly. Thalidomide analogs may be used as a pharmacological tool due to their short-term regulatory effects on glycogen and NO during endotoxic shock. Drugs that increase glycogen may improve liver injury in early sepsis.     

Changes in the arterial pressure and evoked potential in the sensorimotor cortex induced by nalorphine injected in the hypothalamus during painful electric shock in rabbits

During the hypotensive phase of electronocuous shock in rabbits, injection of nalorphine to the hypothalamus provoked elevation in the blood pressure (BP), recovery of the evoked potential (EP) of the sensorimotor brain cortex in response to electrodermal stimulation (EDS) of the hind limb. Injection of the drug to the central gray of the midbrain resulted in an insignificant short-term elevation of the BP followed by its drop. It is concluded that the endogenous opioid system of the hypothalamus is of importance in the genesis of the hypotensive response and suppression of the EP of the rabbit sensorimotor brain cortex in response to EDS during electronocuous shock.     

低压复苏对非控制出血性休克抢救效果的探讨

目的建立非控制性脾大部损伤出血性休克模型,比较低压复苏与常压复苏抢救的效果。方法脾部分切除模拟人失血低压状态下,建立类似人休克的模型后,将动物随机分成4组。1组,假手术组;2组,休克未处理组;3组,正常血压复苏组;4组,低压复苏组。观察其成活率及对内脏的损伤程度。结果动物失血低压抢救比常压抢救存活时间长。结论低压可改善组织代谢,提高生存时间,是更为理想的复苏方法。低压复苏非控制性脾大部损伤出血性休克模型的建立,为临床急性大出血提供新的抢救方法。     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e ₆) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e ₆ demonstrated maximal effects on blood SOD activity.     

Modulation of murine macrophage function by N-acetylcysteine in a model of endotoxic shock

In previous studies we have observed changes in several functions of peritoneal macrophages from BALB/c mice with irreversible endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. Since antioxidants, such as N-acetylcysteine (NAC), are free radical scavengers that improve the immune response, in the present work we have studied different functions of peritoneal macrophages from BALB/c mice suffering the endotoxic shock above indicated and administered N-acetylcysteine (150 mg/kg i.p.) at 30 minutes after LPS injection. In the peritoneal macrophages obtained at 2, 4, 12 and 24 h after LPS injection, the following functions were studied: adherence to substrate, mobility, ingestion of particles, and production of superoxide anion and tumour necrosis factor (TNF alpha). The increase in adherence, ingestion and superoxide anion and TNF alpha production shown by macrophages from animals with endotoxic shock was counteracted by NAC injection. Moreover, the survival time of mice with endotoxic shock was increased in the presence of NAC. These data suggest that NAC, administered intraperitoneally, may be useful for the treatment of irreversible endotoxic shock by modulation of the function of macrophages with decreased superoxide anion and TNF alpha production and concomitant increase of survival time.     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)     

Comparative study of several lymphocyte functions in two strains of mice with different models of endotoxic shock

Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFalpha production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFalpha released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFalpha release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.     

The influence of probiotics and probiotic product on respiration of mitochondria and intracellular calcium signal in cells of cardiovascular system

The influence of lactobacilli and new probiotic product on mitochondrial energetics of rat heart mitochondria and on dynamics of intracellular calcium concentration ([Ca²⁺]_i) of cardiomyocytes and rat aortic smooth muscle cells was investigated. Respiration of mitochondra was estimated polarographically. [Ca²⁺]_i was measured using fluorescent calcium indicator Fura 2 AM and calcium imaging system. The application of lactobacilli (5 × 10⁷ CFU/mL) was shown to increase [Ca²⁺]_i in cardiomyocytes, thereby increasing myocardial contractility. On the other hand, application of lactobacilli reduced thapsigargin-induced calcium influx in smooth rat aortic muscle, thus exhibiting some hypotensive effect. It was shown that probiotic product stimulated mitochondria respiration and exerted a mild uncoupling effect on electronic transport and oxidative phosphorylation in mitochondria. In cardiomyocytes and in smooth muscles probiotic product increased [Ca²⁺]_i and consequent increase in contractility of blood vessels and myocardium. It is supposed that the probiotic product can be effectively applied at the endotoxic shock, when contractility of blood vessels in response to vasoconstrictor agents is suppressed.     

Development of the sympathetic nervous system response to endotoxicosis in the rat: importance of non-baroreflex mechanisms in pre-weanlings and adults.

The sympathetic nervous system response to endotoxicosis was studied in the rat at ages before (11-12 days) and after (19-20 days) maturation of the baroreflex and in adults by recording preganglionic impulses during i.v. infusions of endotoxin (S. enteriditis). At all ages, the discharge rate increased before there was any decrease in arterial blood pressure. The magnitude of the increase (65%) was the same in 11-12 days-old and adult rats while 19-20 days-old rats were hyperactive (278% increase). Subsequently, in the hypotensive phase of the endotoxicosis (diastolic pressure decrease 50-60%) there was an additional increase in discharge in the 19-20 days- old rats (43% and in adults (70%) but not in 11-12 days-old rats. The hypotensive discharge rate of the 11-12 days-old rat reached only 20% of the maximum; it reached 80% in the hyperactive 19-20 days-old rat and 65% in adults. At all ages, the elevated hypotensive discharge rate persisted after steady state blood pressure was raised by infusing dextran. The discharge rate was diminished transiently during pressor responses to accelerated infusion or bolus injections of dextran. The conclusions are: (i) endotoxic stimulation of sympathetic outflow is initiated by a non-baroreflex mechanism in adult as well as in pre-weaning rats; (ii) there is added stimulation during hypotension after the baroreflex is mature, but (iii) the non-baroreflex stimulation continues to excite the preganglionic neurons and obtunds baroreflex feedback inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

脂多糖诱导小鼠脏器中胞间粘附分子-1的表达

为研究脂多糖（lipopolysaccharide,LPS)诱导的内毒素休克小鼠多种脏器中胞间粘附分子-1（intercellu-lar adhesion molecule-1,ICAM-1)表达的差异。用5mg/kgLPS腹腔注射小鼠后,分别采用Western blotting和RT-PCR法检测组织中ICAM-1蛋白和mRNA的表达情况,结果显示,在正常小鼠,ICAM-1蛋白和mRNA的表达在肺中最多,其次是脾脏,在肾脏和肠有少量表达,在肝脏和心脏中未能检出,LPS腹腔注射后6h可诱导小鼠发生内毒素休克,此时,ICAM-1蛋白表达仍以在肺中最多,在肝、脾、心、肾和肠依次减少;其中在肺,肾和脾分别比正常时增加4.5、3.0和1.5倍,而且在正常时不能检出的肝和心中呈现阳性,但在肠中则变化不大,脏器中ICAM-1mRNA亦相应显著增加,上述结果表明,在LPS诱导的内毒素休克小鼠的多种脏器中ICAM-1蛋白和mRNA表达显著增加,脏器间ICAM-1表达上调的差异可能带来内毒素休克时脏器的不同易伤性,抑制ICAM-1的表达可能对内毒素休克的防治有重要的意义。     

A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock

The regulation of monocyte function and the inhibition of TNF-alpha production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-alpha found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-alpha Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-alpha production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-kappa B nuclear translocation resulting in greater TNF-alpha production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-alpha secretion and attenuating endotoxic shock.     

Effect of L-arginine on endothelial injury and hemostasis in rabbit endotoxin shock.

To investigate whether impaired endothelial function was related to alteration of nitric oxide (NO) formation during endotoxic shock, we studied the effects of supplementation of L-arginine (L-Arg), D-arginine (D-Arg), and N(G)-nitro-L-arginine methyl ester (L-NAME), on endothelial function and structure in a rabbit model. Endotoxic shock was induced by a single lipopolysaccharide bolus (0.5 mg/kg i.v., Escherichia coli endotoxin). Coagulation factors and expression of monocyte tissue factor were determined by functional assays. Endothelium-dependent vascular relaxation was assessed by in vitro vascular reactivity. Immunohistochemical staining (CD31) was performed to assess damaged endothelial cell surface of the abdominal aorta. These parameters were studied 5 days after the onset of endotoxic shock and were compared under three conditions: in absence of treatment, with L-Arg or D-Arg supplementation, or with L-NAME. Both L-Arg and D-Arg significantly improved endothelium-dependent relaxation and endothelial morphological injury. L-NAME did not alter endothelial histological injury induced by lipopolysaccharide. These data indicate that arginine supplementation nonspecifically prevents endothelial dysfunction and histological injury in rabbit endotoxic shock. Moreover, L-Arg has no effect on coagulation activation and expression of monocyte tissue factor induced by endotoxic shock.     

Effect of phorbol ester on contractile response of aorta from endotoxic rats

The influence of phorbol ester on the isometric contractile response of aorta from endotoxic rats was examined. In endotoxic rat aorta, the contractile responses to KCl and phorbol 12,13-dibutyrate (PDBu) were both remarkably diminished, compared to those in control rat aorta. Preincubation with PDBu augmented the aortic contractile response to KCl in both control and endotoxic rats. This augmentative effect of PDBu was significantly more pronounced in endotoxic rats than in controls. When the contractile response to 80 mM KCl reached a plateau after PDBu pretreatment, addition of 5 mM CaCl2 (final concentration) to the organ bath completely reversed the diminished contractile response of endotoxic rat aorta to the control level. These results suggest that the hyporesponsiveness of endotoxic rat aorta to KCl may be caused by decreases in both protein kinase C mediated response and calcium sensitivity of vascular smooth muscle cells.     

Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures

Endotoxins (lipopolysaccharide, LPS) from Gram-negative bacteria are considered as the agents responsible for the induction of endotoxic shock, producing severe cellular metabolic dishomeostasis. Cytotoxic lesions, as well as functional and metabolic disturbances, occur mainly in the liver, which is one of the target organs and exerts an LPS clearance function. In an attempt to approach the molecular basis of endotoxic shock, and to propose an experimental model, we have focused this study on cytoskeleton (microtubules and microfilaments) alterations induced by different doses of endotoxin in different target liver cells. Microfilaments and microtubules were studied by immunofluorescence and different microscopy techniques (optic fluorescence microscopy and confocal laser scanning microscopy) in order to improve the cytoskeleton study resolution. Parenchymal and sinusoidal cell morphology, severely damaged by the LPS action, is related to a disturbance on the cytoskeletal organisation, even more evident in a particular proliferating rat liver cell culture. The most relevant changes are seen in the microtubule patterns in all liver cells tested, which could be related, depending on cell type, either to a direct LPS action or to [Ca+2]i dishomeostasis as well as free radical and cytokine (IL-1beta and TNF-alpha) production. Due to their features, proliferating rat liver cell cultures are used as a sensitive cell model to understand the effect of LPS on cytoskeleton organisation.     

Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock

Endotoxic shock is a life-threatening condition caused by exposure to bacterial LPS. LPS triggers the release of acute phase, proinflammatory, and Th1 cytokines that facilitate the development of endotoxic shock. Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited by a variety of immune stimuli. The current results demonstrate that suppressive ODN protect mice from LPS-induced endotoxic shock. Underlying this protective effect is the ability of suppressive ODN to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade mediated by LPS-induced IFN-beta and IL-12. These findings suggest that suppressive ODN might be of use in the treatment of endotoxic shock.