Pentoxifylline improves survival following hemorrhagic shock

Pentoxifylline is an agent which improves microcirculatory blood flow, but its use as therapy for shock has not been reported. We performed this study to determine if pentoxifylline improves survival following experimental hemorrhagic shock. Anesthetized Sprague-Dawley rats were studied; the animals were subjected to hemorrhage and then resuscitated using lactated Ringer's solution, with either placebo or pentoxifylline added by random selecting. Animals were then observed for 3 days. There was significantly increased survival in pentoxifylline-treated animals (p less than .05). In additional experiments, animals received more aggressive fluid resuscitation; improved survival in the pentoxifylline group was noted almost immediately and persisted through the 72-h period. This was significant at the p less than .01 level. We conclude that pentoxifylline improves survival from hemorrhagic shock in this experimental model, and has additive survival value to fluid resuscitation.     

Glutamine attenuates lung injury and improves survival after sepsis: role of enhanced heat shock protein expression

OBJECTIVE: Heat shock protein (HSP) expression is vital to cellular and tissue protection after stress or injury. However, application of this powerful tool in human disease has been limited, as known enhancers of HSPs are toxic and not clinically relevant. Glutamine (GLN) can enhance HSP expression in non-clinically relevant animal injury models. The aim of this study was to assess the ability of GLN to enhance pulmonary HSP expression, attenuate lung injury, and improve survival after sepsis in the rat. DESIGN: Prospective, randomized, controlled animal trial. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: We utilized a rat model of cecal ligation and puncture to induce sepsis. GLN or saline was administered 1 hr after initiation of sepsis via single tail-vein injection. We analyzed heat shock factor-1 phosphorylation, HSP-70, and HSP-25 via Western blot. Tissue metabolism was assayed by magnetic resonance spectroscopy. Occurrence of lung injury was determined via histopathologic examination. An inhibitor of HSP expression, quercetin, was utilized to assess role of HSP expression in prevention of sepsis-related mortality. MEASUREMENTS AND MAIN RESULTS: GLN, given after initiation of sepsis, enhanced pulmonary heat shock factor-1 phosphorylation, HSP-70, HSP-25, and attenuated lung injury after sepsis. Further, GLN improved indices of lung tissue metabolic function (adenosine 5-triphosphate/adenosine 5-diphosphate ratio, nicotinamide adenine dinucleotide) after sepsis. No significant effect of GLN on lung tissue-reduced glutathione was observed. GLN treatment led to a significant decrease in mortality (33% [6 of 18] GLN-treated rats vs. 78% [14 of 17] saline-treated rats). Administration of the HSP inhibitor quercetin blocked GLN-mediated enhancement of HSP expression and abrogated GLN's survival benefit. CONCLUSIONS: GLN has been safely administered to critically ill patients and shown to improve outcome without clear understanding of the protective mechanism. Our results indicate GLN may prevent the occurrence of lung injury, lung tissue metabolic dysfunction, and mortality after sepsis via enhancement of deficient lung heat shock factor-1 phosphorylation/activation and HSP expression.     

Oral administration of geranylgeranylacetone improves survival rate in a rat endotoxin shock model: administration timing and heat shock protein 70 induction

The present study was performed to determine whether oral pretreatment with geranylgeranylacetone (GGA) inhibits proinflammatory cytokine liberation and nitric oxide (NO) production in lipopolysaccharide (LPS)-treated rats and protects rats against death from LPS-induced endotoxin shock, and whether such protection by GGA is related to heat shock protein (HSP) 70 induction in multiple organs of rats. GGA (200 mg/kg) was given orally to rats. LPS (20 mg/kg) was administered intraperitoneally 4, 8, 16, or 24 h after GGA administration. The survival of rats was monitored over 24 h after LPS administration. GGA treatment at 8 or 16 h before LPS dramatically improved the survival rate of LPS-treated rats. Plasma levels of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and NO 6 h after LPS administration in these GGA-pretreated rats were less than one-half of those in rats treated with LPS alone. A GGA challenge 8 or 16 h before LPS administration enhanced HSP70 expression in rat organs after LPS. Treatment with GGA 8 h before LPS minimized hepatic and renal damage. Furthermore, the protective effect of GGA on mortality in LPS-treated rats was inhibited with quercetin, known as an HSP70 inhibitor. These results suggest that oral administration of GGA at an optimal time before LPS injection induces and enhances HSP70 expression in several organs, inhibits proinflammatory cytokine and NO production, and prevents organ damage, resulting in an improved survival rate.     

局部微波热疗对热休克蛋白表达的影响

目的探讨骨肉瘤术中热疗对热休克蛋白表达的影响。方法对61例骨肉瘤组织进行免疫组化染色,观察热休克蛋白HSP-60、HSP-70及GP-94的表达。结果骨肉瘤组织中GP-94表达最高,阳性率为100%,其次为HSP-60,阳性率为94.5%,HSP-70弱表达,阳性率41.8%。热疗后HSP均不同程度增多,以HSP-70较明显,阳性率上升到72%。体外骨肉瘤细胞株加热后热休克蛋白也明显增多。结论局部微波热疗后,3种热休克蛋白在骨肉瘤组织均有表达。     

Parenteral glutamine increases serum heat shock protein 70 in critically ill patients

Objective Heat shock protein 70 (HSP-70) is protective against cellular and tissue injury. Increased serum HSP-70 levels are associated with decreased mortality in trauma patients. Glutamine (Gln) administration increases serum and tissue HSP-70 expression in experimental models of sepsis. Gln has been safely administered to critically ill patients and can improve clinical outcomes, but the effect of Gln administration on HSP-70 expression in humans is unknown. We examined whether Gln-supplemented parenteral nutrition (PN) increases serum HSP-70 levels in critically ill patients.Design and setting Randomized, controlled, double-blind study in surgical intensive care units (SICU) in a university hospital.Patients 29 patients admitted to the SICU and requiring PN for more than 7 days.Interventions Patients received either Gln-PN (containing alanyl-glutamine dipeptide; 0.5 g/kg per day; n=15) or standard Gln-free PN (control-PN) that was iso-nitrogenous to Gln-PN (n=14). Serum HSP-70 concentrations were measured at enrollment and at 7 days. Clinical outcome measures were also determined.Results HSP-70 concentrations were unchanged in control-PN subjects from baseline to day 7. In marked contrast, Gln-PN subjects demonstrated significantly higher (3.7-fold) serum HSP-70 concentrations than control subjects. In Gln-PN patients there was a significant correlation between increases in HSP-70 levels over baseline and decrease in ICU length of stay.Conclusions Gln-PN significantly increases serum HSP-70 in critically ill patients. The magnitude of HSP-70 enhancement in Gln-treated patients was correlated with improved clinical outcomes. These data indicate the need for larger, randomized trials of the Gln effect on serum and tissue HSP-70 expression in critical illness and relationship to clinical outcomes.     

早期谷氨酰胺强化肠外营养对危重患者保护机制的研究

目的 探讨早期给予谷氨酰胺(Gln)强化肠外营养对危重患者脏器功能的影响及其与预后的关系,进一步探讨谷氨酰胺对危重患者保护的内在机制.方法 选择本院急诊ICU及脑外科收治的44例危重患者,按随机原则分为常规组和Gln强化组,每组22例.两组患者均行肠内、外营养, Gln强化组另予静脉注射Gln 0.4 g·kg-1·d-1,连续7 d.观察两组患者治疗前后体内热休克蛋白70(HSP70)、Gln水平、机械通气时间、细胞免疫功能状况.结果 治疗前常规组和强化组Gln、HSP70水平比较差异无统计学意义(P均＞0.05).治疗后常规组Gln、HSP70水平较治疗前稍有增加,但差异无统计学意义;而强化组治疗后Gln、HSP70水平均较治疗前显著升高(P均＜0.01),且两组治疗后Gln、HSP70水平比较差异均有统计学意义(P均＜0.01).强化组体内Gln与HSP70存在正相关性(r=0.65,P=0.001).两组机械通气时间比较差异有统计学意义(P均＜0.05).治疗后强化组CD3、 CD4、CD4/CD8与常规组比较差异有统计学意义(P<0.05).结论 危重患者早期肠外补充Gln能有效改善患者的预后,其机制可能与提高患者体内HSP70水平有关.     

谷氨酰胺对COPD外周血单个核细胞HSP70、TNF-α表达的影响

目的探讨谷氨酰胺（Gin）对慢性阻塞性肺疾病患者外周血单个核细胞（PBMC）中热休克蛋白70（HSP70）和肿瘤坏死因-α（TNF-α）表达的影响。方法选择30例COPD急性加重期（AECOPD）患者为研究对象,设为AECOPD组．将治疗10～20d后处于稳定期的上述患者设为SCOPD组,分别以Gin干预,并以健康人为正常对照组。采用Real—timePCR法检测上述5组PBMC中HSP70mRNA和TNF-α mRNA的表达水平。结果AECOPD和SCOPD空白对照组HSP70mRNA、TNF-α mRNA的表达高于正常对照组,差异有统计学意义（t分别=-3．74、-3．57、-3．63、-3．31,P均〈0．05）;AECOPD组和SCOPD组中,用Gin干预的较未用Gln的PBMC中HSP70表达均升高,差异有统计学意义（t分别=4．87、2．81,P均〈0．05）,而TNF-α表达均下降（t分别=-3．42、-2．97,P均〈0．05）。结论Gin可抑制COPD患者炎症因子TNF-α的活性,升高HSP70的表达。     

Neutrophil heat shock protein expression and activation correlate with increased apoptosis following transmigration through the endothelial barrier.

Polymorphonuclear leukocytes (PMN) undergo endothelial transmigration upon activation or in response to a chemoattractant. Such cells are stressed and have an increased capacity to incite tissue injury. Little is known about the effect of transmigration on PMN stress gene responses, PMN activation, and ultimately programmed cell death (apoptosis). Human endothelial cells (ECV-304) were plated onto transwell membranes to form an endothelial monolayer and PMN transendothelial migration through this endothelial barrier was examined. Chemotaxis was induced by formyl-methionyl-leucyl-phenylalanine (fMLP). Flow cytometry was used to determine PMN receptor expression (CD11b, CD14, CD16, CD18, CD54), phagocytosis, and apoptosis. Heat shock protein (Hsp) expression was evaluated by Western blotting. fMLP-induced PMN transendothelial migration resulted in increased adhesion receptor expression and phagocytosis. Migrated PMN also had an increased rate of apoptosis as evaluated by uptake of propidium iodide and decreased FcgammaR III (CD16) expression. Increased PMN apoptosis coincided with induction of Hsp72 following transmigration. Thus, naive PMN that migrate through endothelium in response to a chemoattractant undergo activation as represented by increased phagocytosis and expression of adhesion receptors.     

高温联合辐射对胚胎神经发育及脑热应激蛋白的影响

目的探讨高温联合辐射致胚胎神经发育与早期胚胎脑蛋白质和HSPs表达的关系。方法将130只孕鼠按随机抽签分为13组,每组10只。在鼠妊娠第8d,第9d分别接受60Co及γ-射线全身照射各1次,照射剂量1.0Gy;第10d各置温箱中,分别使其肛温保持在37±0.5℃,41±0.5℃,42±0.5℃,并持续3,4,5min。移出温箱2h后,每组每时段随机取5只脱颈法处死,采用Westerndotblot和lowry方法定量分析胚胎脑HSP70和蛋白质含量与胚胎神经系统畸形。结果高温联合辐射对胚胎神经毒性明显增加,并与高温强度及作用时间呈正相关;高温联合辐射作用于胚胎早期对胚胎脑蛋白质降低有复合作用(P<0.01),并可诱导脑HSP70合成。结论高温与辐射联合致神经发育与胚胎脑蛋白质合成及热应激蛋白的诱导有一定的相关性。     

骨骼肌小热休克蛋白在离心运动后的表达

背景：研究证明离心方式的训练可使骨骼肌产生保护作用,避免离心运动引起的损伤,但是机械负荷引起的小热休克蛋白的保护作用至今却少有报道。目的：观察骨骼肌小热休克蛋白家族中αB-晶体蛋白在离心运动后的表达,以此探讨机械负荷诱导的小热休克蛋白对骨骼肌细胞的保护作用机制。方法：将Wistar大鼠随机分为安静对照组和运动训练组。运动训练组使用动物跑台进行6周间歇性离心运动训练,每周训练5d,安静对照组正常喂养。训练6周休息48h后,安静对照组与运动训练组随机选出6只大鼠做1次性大负荷离心运动。观察两组血清肌酸激酶变化;蛋白免疫印迹法检测两组腓肠肌αB-晶体蛋白含量变化,用免疫荧光组织化学法分析两组腓肠肌αB-晶体蛋白亚细胞表达特征。结果与结论：大负荷离心运动后,安静对照组血清肌酸激酶与运动前相比显著增高（P〈0.05）,说明骨骼肌细胞出现严重的损伤,而运动训练组这种损伤不明显。蛋白免疫印迹结果表明,运动训练组做一次性大负荷离心运动后αB-晶体蛋白表达水平比安静对照组增加（P〈0.05）。从免疫荧光组化切片可见,αB-晶体蛋白在细胞内发生了移位变化,从胞浆移位于Z盘和细胞膜。提示αB-晶体蛋白在离心运动训练后表达增多,并通过移位于肌细胞Z-盘和细胞膜发挥对骨骼肌细胞的保护作用。     

骨骼肌小热休克蛋白在离心运动后的表达

背景:研究证明离心方式的训练可使骨骼肌产生保护作用,避免离心运动引起的损伤,但是机械负荷引起的小热休克蛋白的保护作用至今却少有报道。目的:观察骨骼肌小热休克蛋白家族中αB-晶体蛋白在离心运动后的表达,以此探讨机械负荷诱导的小热休克蛋白对骨骼肌细胞的保护作用机制。方法:将Wistar大鼠随机分为安静对照组和运动训练组。运动训练组使用动物跑台进行6周间歇性离心运动训练,每周训练5d,安静对照组正常喂养。训练6周休息48h后,安静对照组与运动训练组随机选出6只大鼠做1次性大负荷离心运动。观察两组血清肌酸激酶变化;蛋白免疫印迹法检测两组腓肠肌αB-晶体蛋白含量变化,用免疫荧光组织化学法分析两组腓肠肌αB-晶体蛋白亚细胞表达特征。结果与结论:大负荷离心运动后,安静对照组血清肌酸激酶与运动前相比显著增高(P<0.05),说明骨骼肌细胞出现严重的损伤,而运动训练组这种损伤不明显。蛋白免疫印迹结果表明,运动训练组做一次性大负荷离心运动后αB-晶体蛋白表达水平比安静对照组增加(P<0.05)。从免疫荧光组化切片可见,αB-晶体蛋白在细胞内发生了移位变化,从胞浆移位于Z盘和细胞膜。提示αB-晶体蛋白在离心运动训练后表达增多,并通过移位于肌细胞Z-盘和细胞膜发挥对骨骼肌细胞的保护作用。     

人骨保护素-分枝杆菌热休克蛋白70融合蛋白的克隆与表达及其活性鉴定

目的:为解决类风湿性关节炎中骨破坏及炎症损伤两大难题,拟克隆入骨保护素功能区与分枝杆菌热休克蛋白70肽段融合基因,进一步观察其在大肠杆菌中的表达并鉴定活性.方法:实验于2006-05/2007-09在首都医科大学免疫学系实验室完成.采用反转录-聚合酶链反应技术从人骨肉瘤细胞系MG63总RNA中扩增骨保护素成熟肽段编码区基因,构建pGEM-TEasy-骨保护素重组质粒.以此为模板,聚合酶链反应扩增骨保护索-热休克蛋白70功能区DNA,构建重组表达载体pET-28a-骨保护素-热休克蛋白70,将其转化E.coli.BL21(DE3),经异丙基硫代半乳糖苷诱导后收集菌体蛋白,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及蛋白免疫印迹鉴定该融合蛋白的表达,以破骨细胞生长抑制实验及抑炎实验检测该融合蛋白的生物学活性.结果:①实验最终获得人骨保护素全长基因片段,人骨保护素-热休克蛋白70功能区DNA片段已被正确插入到pET-28a载体中,转化菌株可表达人骨保护素-热休克蛋白70融合蛋白.②十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示约在Mr22000处有蛋白的超表达,而未诱导菌株未发现有此条带.③蛋白免疫印迹检测表明,融合蛋白能与抗人骨保护素单克隆抗体特异性结合.④破骨细胞生长抑制实验表明,该融合蛋白能够减少破骨细胞的生成数量,具有体外抑制骨破坏的生物活性.⑤抑炎实验表明,融合蛋白具有显著减轻迟发型超敏反应小鼠模型炎症反应程度的作用,说明融合蛋白中热休克蛋白70具有抑制炎症的生物学活性.结论:在E.coli.BL21(DE3)中表达了骨保护素-热休克蛋白70融合蛋白,体外功能实验证实该融合蛋白具有一定的生物学活性.     

Death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways

Induction of heat shock proteins (Hsps) following cellular damage can prevent apoptosis induced by both the intrinsic and the extrinsic pathways. The intrinsic pathway is characterized by mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, apoptosome assembly, and caspase activation. Hsps promote cell survival by preventing MOMP or apoptosome formation as well as via regulation of Akt and JNK activities. Engagement of the TNF death receptors induces the extrinsic pathway that is characterized by Fas-associated death domain-dependent (FADD-dependent) caspase-8 activation or induction of NF-kappaB to promote cellular survival. Hsps can directly suppress proapoptotic signaling events or stabilizing elements of the NF-kappaB pathway to promote cellular survival.     

热休克蛋白与肿瘤的关系

热休克蛋白 (HSP)是生物进化过程中高度保守的一个蛋白家族 ,参与了多种生理过程 ,具有重要的生理作用。近年来又发现它与肿瘤的发生、发展及预后等方面有着密切的关系。现概述HSP与肿瘤之间的关系。     

Adrenomedullin reduces vascular hyperpermeability and improves survival in rat septic shock

Objective Current therapies of sepsis and septic shock require administration of a large volume of fluid to maintain hemodynamic stability. The vasoregulatory peptide adrenomedullin has been shown to prevent the transition to the fatal hypocirculatory septic state by poorly understood mechanisms. We tested the hypothesis that therapeutic administration of adrenomedullin would reduce vascular hyperpermeability, thereby contributing to improved hemodynamics and survival. Design Prospective randomized controlled animal study. Subjects Male Sprague–Dawley rats (270 g). Interventions We used 4.8 × 10³ U/kg of Staphylococcus aureus α-toxin, a pore-forming exotoxin, to induce vascular leakage and circulatory shock in rats. The infusion rate was 24 μg/kg per hour. Adrenomedullin was started 1 h after α-toxin administration. Measurement and results Infusion of α-toxin in rats induced cardiocirculatory failure resulting in a 6-h mortality of 53%. α-Toxin provoked massive vascular hyperpermeability, which was indicated by an enrichment of Evans blue dye albumin in the tissues of lung, liver, ileum and kidney. Plasma fluid loss led to a significant hemoconcentration. Hemodynamic impairment observed after α-toxin infusion was closely correlated to vascular hyperpermeability. Therapeutic administration of 24 μg/kg per hour adrenomedullin reduced 6-h mortality from 53% to 7%. Stabilization of the endothelial barrier by adrenomedullin was indicated by reduced extravasation of albumin and plasma fluid and may have contributed to hemodynamic improvement. Conclusions These data suggest that adrenomedullin-related reduction of vascular hyperpermeability might represent a novel and important mechanism contributing to the beneficial effects of this endogenous vasoregulatory peptide in sepsis and septic shock. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users. Bettina Temmesfeld-Wollbrück and Bernhard Brell contributed equally to this study     

Hypertonic saline-dextran improves intestinal perfusion and survival in porcine endotoxin shock

OBJECTIVE: To examine the effects of hypertonic (7.5%) saline-6% dextran 70 (HSD) and isotonic (0.9%) saline-6% dextran 70 (ISD) on cardiovascular function and intestinal perfusion in experimental endotoxin shock. DESIGN: Experimental, randomized, unblinded, interventional study. SETTING: University experimental animal laboratory. SUBJECTS: Anesthetized and mechanically ventilated landrace pigs (n = 24). INTERVENTIONS: Induction of endotoxin (ET) shock by infusion of Escherichia coil lipopolysaccharide endotoxin (serotype 0111: B4) followed by no fluid treatment (control; C) or small-volume (4 mL/kg) treatment with HSD or ISD. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure, central venous pressure, pulmonary artery pressure, pulmonary artery occlusion pressure, cardiac output, portal vein blood flow, intestinal microcirculation, intramucosal (regional) P(CO2), intestinal-arterial gap of CO2, and intramucosal pH were monitored, and blood gases were analyzed. Infusion of ET resulted in hypokinetic shock, which in untreated animals led to cardiovascular deterioration and a survival rate of only 33% at 300 mins after start of ET infusion. ISD treatment transiently improved hemodynamic variables and mucosal blood flow but did not affect the survival rate vs. C. Significant beneficial, long-lasting effects of HSD infusion on hemodynamics, especially on mucosal blood flow and intramucosal pH, were demonstrable, resulting in a survival rate of 86%. The relative risk of death at 300 mins was 1.20 for ISD vs. C and 0.17 for HSD vs. C. CONCLUSION: Small-volume HSD resuscitation is much more effective than ISD resuscitation. Variables that were improved include cardiac output, portal blood flow, and intestinal mucosal blood flow in ET shock, all of which improve survival. Such beneficial effects of HSD on splanchnic perfusion may be of value in treating critically ill septic patients in the intensive care unit.