大鼠肝缺血再灌注损伤对心脏能量代谢和结构的影响及其机制

目的：探讨肝缺血再灌注损伤对心脏能量代谢和结构的影响及其可能的发生机制。方法：健康雄性Wistar大鼠48只，随机分为对照组、缺血30 min组（I组）及缺血30 min再灌注即刻组、2 h组、4 h组和6 h组（I/R组、I/R 2 h组、I/R 4 h组和I/R 6 h组），每组8只。用偶氮显色法测定血清中的内毒素，用放射免疫法测定心肌组织胰岛素和胰岛素抗体，取心肌制备组织匀浆测丙二醛（MDA）、髓过氧化物酶（MPO），乳酸含量。 结果：在肝缺血再灌注损伤过程中，内毒素在I组和I/R组达到高峰，随着再灌注时间的延长逐渐下降，但仍高于对照组（P<0.05）。I组及I/R各组MDA的含量明显高于对照组，在I/R 2 h组、I/R 4 h组、I/R 6 h组差别更为明显（P＜0.05）；再灌注各组MPO活性明显高于对照组、I组（P＜0.05）；随着再灌注时间的延长，心肌组织中乳酸含量明显增加（P＜0.05），但在I/R 6 h组呈下降趋势（P＜0.05）；胰岛素的含量在I/R 4 h组和I/R 6 h组明显下降（P＜0.05）；而胰岛素抗体在各组间无显著差异（P＞0.05）。结论：肝缺血再灌注损伤过程中，肠源性内毒素吸收入血及肝脏解毒功能的降低所致的内毒素血症可能是引起心脏能量代谢和结构改变的始动环节。     

肾上腺素对内毒素致大鼠炎症性肝损害的保护作用

目的 探讨肾上腺素（Epi）对内毒素（脂多糖，LPS）致大鼠炎症性肝损害的保护作用及其作用机制。方法 50只SD大鼠随机分为5组（每组各10只）：对照组：静脉滴注生理盐水2．4mL·kg^-1·h^-1；LPS组：静脉注射LPS6mg·kg^-1后，静脉滴注生理盐水2．4mL·kg^-1·h^-1；低、中和高剂量Epi组：静脉注射LPS6mg·kg^-1后，分别静脉滴注Epi0．12、0．3和0．6μg·kg^-1·min^-1。在LPS注射前、注射后2和6h3个时点取血，检测血清ALT、AST、TNF-α、IL-1β和IL-10水平，并在6h时点观察肝脏的组织病理学改变。结果 LPS组注射LPS后2、6h血清AST和ALT水平较对照组显著升高。同时血清TNF-α、IL-1β和IL-10水平亦较对照组显著升高（P〈0．05）。病理检查结果示：LPS组肝窦扩张、充血，局灶性肝细胞坏死。高剂量Epi可显著降低血清AST和ALT水平，减轻肝脏病理损伤，并显著可降低TNF-α水平和升高IL-10水平（1）8LPS组，P均〈0．05），但对IL-1β水平无影响。中、低剂量Epi对LPS致炎症性肝损害无明显保护作用。结论 Epi可通过抗炎作用减轻LPS诱导的炎症性肝损害。     

The role of Kupffer cells in hepatic diseases

Kupffer cells (KCs) constitute 80–90% of the tissue macrophages present in the body. Essential to innate and adaptive immunity, KCs are responsible for the swift containment and clearance of exogenous particulates and immunoreactive materials which are perceived as foreign and harmful to the body. Similar to other macrophages, KCs also sense endogenous molecular signals that may result from perturbed homeostasis of the host. KCs have been implicated in host defense and the pathogenesis of various hepatic diseases, including endotoxin tolerance, liver transplantation, nonalcoholic fatty liver disease, and alcoholic liver disease. In this review, we summarized some novel findings associated with the role of KCs in hepatic diseases, such as the origin and mechanisms KCs polarization, molecular basis for caspase-1 activation called “non-canonical inflammasome pathway” involving the cleavage of Gsdmd by caspase-11, the important role of microRNA in liver transplantation, and so on. A better understanding of KCs biological characteristics and immunologic function in liver homeostasis and pathology may pave the way to investigate new diagnostic and therapeutic approaches for hepatic diseases.     

Lack of endotoxin tolerance with respect to TNF alpha production in the subarachnoid space

To study endotoxin tolerance in the subarachnoid space 0.1 mg of endotoxin derived from Neisseria meningitidis was injected intracisternally into rabbits on 2 consecutive days. On day 1 the maximum peak level of TNF alpha was 7 ng/ml 2 h after injection, whereas on day 2 the highest levels were 3.6 ng/ml and 3.7 ng/ml, respectively, 1 and 2 h after injection. Pretreatment with intravenous endotoxin 5 or 21 h before consecutive intracisternal endotoxin did not affect the cerebrospinal fluid (CSF) levels of TNF alpha. In contrast, there was a marked endotoxin tolerance with respect to TNF alpha in the systemic circulation. Cells appearing in the CSF 5, 12 and 20 h after intracisternal injection of endotoxin were harvested, cultured, and then stimulated with 0.1 mg/ml of endotoxin. In 10 experiments a marked TNF alpha production in the range 10-70 ng/ml was detected in the supernatants, whereas unstimulated cells did not produce TNF alpha. We conclude that tolerance to endotoxin does not develop in the subarachnoid space as evaluated by the present experimental design. The pattern of TNF alpha production and endotoxin tolerance is distinctly different in the subarachnoid space and systemic circulation.     

家兔法与内毒素法检查生物材料热原的适用性研究

药品与生物材料在生物安全性评价方法上有着很大的区别。临床上广泛运用内毒素法检查药品热原,然而,运用细菌内毒素法进行部分组成成分较为复杂的生物材料的热原试验是否适当有待明确。本研究在2005版药典的基础上,分别运用内毒素法和家兔法对两种组织工程支架材料进行热原试验的比较研究,实验结果表明运用内毒素法得到的试验结果为阴性,运用家兔法得到的试验结果为阳性。这两种方法分别测定每种材料所得到的热原试验结果不相符合,表明对组成成分复杂的生物材料,含热原的因素较为复杂,用家兔法进行试验检测热原可能更加灵敏。     

Inhibition of nitric oxide synthase delays the development of tolerance to LPS in rats

The role of nitric oxide (NO) was investigated in endotoxin (lipopolysaccharide, LPS) tolerance in freely moving biotelemetered rats. We monitored changes in febrile response and feeding behavior (food intake, water intake) during the development of tolerance to repeated intraperitoneal injections of LPS (50 microg/kg) along with injections of N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg), an inhibitor of NO synthase. Rats were treated with LPS and L-NAME for three consecutive days. On the fourth day, all rats were injected with LPS alone. Control rats were injected with saline along with saline or with L-NAME for four consecutive days. Rats repeatedly injected with LPS became tolerant to pyrogenic and hypophagic/cachexic effects of LPS as early as on the second day of experiment. The treatment with L-NAME prevented the attenuation of febrile response following the second LPS injection. Moreover, the depressive effects of LPS on body weight as well as on water and food intake were prolonged in rats treated with a combination of L-NAME and LPS. Injection of LPS caused a 3.5-fold increase in plasma nitrite within 3 h and nitrite levels remained significantly elevated 6 and 24 h after LPS. Rats injected secondly with LPS did have still 2.5- to 3-fold increase in plasma nitrite levels 3 and 6 h, but not 24 h, after injection. Third injection of LPS did not elevate nitrite level in plasma. Taken together, presented data provide clear evidence that NO formation is involved in mechanisms responsible for development of early-stage tolerance to endotoxin.     

Cell cooperation in development of eosinophil-predominant inflammation in airways

A large body of research supports a pathogenic role of Th2 cells in allergic diseases such as asthma. These disorders are characterized by recruitment to selected peripheral tissues of a mixed leukocyte inflammatory infiltrate including a predominant e osinophil component. The development of this inflammatory response is dependent on accumulation of Th2 cellsin the affected tissues. Our studies aim to define the mechanisms that control the development of this tissue inflammatory response, focusing particularly on the mechanisms that recruit Th2 cells to the lung and airway. We have found that Th2 cells are their own poorly competent for antigen-induced recruitment to the lung. By contrast, Th1 cells are avidly recruited to the lungs in response to airway antigen challenge. More important, recruitment of Th1 cells to the lung resulted in enhanced recruitment of Th2 cells to this tissue. The increased. Th1 cell-induced recruitment of Th2 cells was associated with upregulation of endothelial vascular cell adhesion molecule-1 (VCAM-1) expression in airway-associated endothelial cells and could be largely blocked by systemic treatment with a monoclonal anti-VCAM-1 antibody. Systemic blocking of tumor necrosis factor (TNF) also blunted the airway inflammatory response. The prominent roles of TNF and VCAM-1 in recruitment of Th2 cells suggested that an inflammatory microenvironment was essential for the recruitment of Th2 cells. Infact, recruitment of Th2 cells to the airway could be induced in an antigen-independent fashion by proinflammatory stimuli such as intranasal in stillation of endotoxin. This antigen nonspecificity of the Th2 cell recruitment suggested a model in which Th2 cell recruitment is in response to general inflammatory signals rather than to antigen itself. This model provides an explanation for the clinical observation that bacterial or viral respiratory tract infections are associated with disease exacerbations in allergic asthmatics. More generally, these data imply that Th2 cells, like other leukocytes, are recruited efficiently to site of tissue inflammation, and that these nonspecifically recruited Th2 cells have substantial potential to modulate local inflammatory processes.     

Thymulin,A Thymic Peptide,Prevents the Overproduction of Pro-Inflammatory Cytokines and Heat Shock Protein Hsp70 in Inflammation-Bearing Mice

The effects of synthetic analogue of peptide hormone thymulin, which is normally produced by thymic epithelial cells, on immune cells activity and blood cytokine profile had been studied in male NMRI mice with acute inflammation induced by injection of lipopolysaccharide from gram-negative bacteria (LPS, 250 μg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1β, IL-2, IL-6, TNF-α, interferon-γ, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 μg/100 g body weight, prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also prevented LPS-induced up-regulation of production of several cytokines by spleen lymphocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of TNF-α production in macrophages cultivated with LPS. In addition, thymulin lowered the peak of Hsp70 production induced by LPS treatment. The results indicate that thymulin having significant anti-inflammatory effect may be promising in clinical application.     

Superantigen and endotoxin synergize in the induction of lethal shock

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-γ (IFN-γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D -galactosamine (D -GalN). Opposed to D -GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.     

rAAV2-BF增强两品系小鼠抗致死剂量细菌感染

 目的 通过观测rAAV2-BPI1-199-Fc 1重组病毒（rAAV-BF）感染对不同品系小鼠的抗细菌感染保护作用。方法 采用Dot-blot、Western blot、免疫组化、改良ELISA方法和内毒素检测试剂盒,检测重组BPI1-199-Fc 1（rBF）蛋白在CHO细胞和小鼠骨骼肌细胞中的表达及血清中内毒素含量的变化;建立最小致死量(MLD)E.coli致死模型,检测rBF基因转染小鼠对MLD E.coli感染的抵抗作用。结果（1）rBF蛋白在CHO细胞和两品系小鼠骨骼肌细胞中成功表达,血清中rBF蛋白具有中和内毒素和杀伤E.coli 的作用;（2）在MLD E.coli腹腔感染后,两品系小鼠rAAV-BF感染组存活率均显著高于空载体对照组小鼠;基因转染组小鼠血清内毒素含量明显低于空载体对照组;rAAV-BF感染与头孢呋辛钠联合应用具有协同抗菌作用,两品系小鼠存活率明显高于单纯rAAV-BF感染组。结论rAAV-BF感染组小鼠对致死性E.coli感染具有抵抗作用。