大黄抗内毒素性休克大鼠炎性介质作用的实验研究

目的：研究大黄对内毒素性休克大鼠炎性介质作用的机制。方法：选用大鼠内毒素性休克模型。随机分为６组：单纯手术组、内毒素组、大黄预防用药组（１５０ｍｇ／ｋｇ组和７５０ｍｇ／ｋｇ组）和大黄治疗组（１５０ｍｇ／ｋｇ组和７５０ｍｇ／ｋｇ组）。检测磷脂酶Ａ２（ＰＬＡ２）和血小板活化因子（ＰＡＦ）的活性。结果：内毒素注射前６组大鼠平均动脉压（ＭＡＰ）无显著性差异；注射内毒素后４小时ＭＡＰ明显降低；大黄预防用药组和大黄治疗组ＭＡＰ则与注射内毒素前及单纯手术组比较均无明显变化，并均显著高于内毒素组注射内毒素４小时后。注射内毒素后４小时，血清和小肠组织中ＰＬＡ２活性及ＰＡＦ含量均明显增高；与内毒素组注射内毒素后４小时比较，大黄预防组和治疗组则血清和小肠组织中ＰＬＡ２活性和ＰＡＦ含量显著降低。结论：大黄对内毒素性休克所致炎症反应有明显的预防和治疗作用     

Kupffer cells and neutrophils as paracrine regulators of the heme oxygenase-1 gene in hepatocytes after hemorrhagic shock

Heme oxygenase (HO) plays a pivotal role for the maintenance of liver blood flow and hepatocellular integrity after hemorrhagic shock. We investigated the role of Kupffer cells and neutrophils as paracrine modulators of hepatocellular HO-1 gene expression in a rat model of hemorrhage and resuscitation. Male Sprague-Dawley rats (n = 6-10/group) were anesthetized (pentobarbital, 50 mg/kg intraperitonal) and subjected to hemorrhagic shock (mean arterial blood pressure: 35 mmHg for 60 min) or a sham protocol. Based on the time course of HO-1 gene expression, the effect of various antioxidants, Kupffer cell blockade [gadolinium chloride (GdCl3); 10 mg/kg; 24 h prior to hemorrhage or dichloromethylene diphosphonate (Cl2MDP); 1 mg/kg; 2 days prior to hemorrhage], or neutrophil depletion (vinblastine, 0.5 mg/kg, 5 days prior to hemorrhage) on induction of the HO-1 gene was assessed at 5 h of resuscitation, i.e., the time point of maximal induction. Kupffer cell blockade and antioxidants abolished HO-1 mRNA and protein induction after hemorrhage, while neutrophil depletion failed to affect hepatocellular HO-1 gene expression. In addition, Kupffer cell blockade aggravated hepatocellular injury. N-formyl-methionine-leucyl-phenylalanin (fMLP) induced a substantial influx of neutrophils into the liver but failed to induce hepatocellular HO-1 mRNA expression. These data suggest that Kupffer cells but not neutrophils induce an adaptive hepatocellular stress response after hemorrhage and resuscitation. Oxygen-free radicals released by Kupffer cells may serve as paracrine regulators of a hepatocellular stress gene which is necessary to maintain liver blood flow and integrity under stress conditions.     

Low-dose dexamethasone ameliorates circulatory failure and renal dysfunction in conscious rats with endotoxemia

Corticosteroids have long been proposed as a therapeutic adjuvant in septic renal dysfunction because of their anti-inflammatory properties and favorable results from animal experiments. However, some reports suggested the potential for harm associated with the administration of early high-dose corticosteroids in patients with severe sepsis and septic shock. Thus, we examined the effects of low-dose dexamethasone (0.01 and 0.1 mg/kg) on hemodynamics and renal function in conscious rats with endotoxemia. Intravenous injection of rats with endotoxin (E. coli lipopolysaccharide, LPS, 1 mg/kg) caused hypotension, vascular hyporeactivity, and tachycardia as well as renal dysfunction. Circulatory failure and renal dysfunction caused by LPS were significantly attenuated in the dexamethasone 0.1 mg/kg-treated group. The nitric oxide (NO) production in plasma and renal tissue and the iNOS protein expression in the kidney were suppressed by cotreatment of LPS rats with dexamethasone, 0.1 mg/kg. Light microscopy showed that 0.1 mg/kg dexamethasone reduced marked infiltration of neutrophils in renal tissues from LPS rats. Moreover, the survival rate at 18 h was significantly increased in the dexamethasone 0.1 mg/kg-treated group when compared with the LPS group. These results suggest that the beneficial effects of low-dose dexamethasone (0.1 mg/kg) in conscious rats with endotoxic shock are associated with amelioration of circulatory failure and renal dysfunction, and this is attributed to inhibition of NO production.     

Influence of heme-based solutions on stress protein expression and organ failure after hemorrhagic shock

OBJECTIVE: Hemoglobin-based oxygen carriers (e.g., diaspirin-cross-linked hemoglobin [DCLHb] and hemoglobin glutamer-200 [HbG]) may have potential in the treatment of hemorrhagic shock. The nitric oxide scavenging and direct vasoconstrictive side effects of free hemoglobin of currently available preparations may increase organ injury after shock in contrast to non-oxygen-carrying heme solutions (e.g., hemin arginate [HAR]). However, both classes of substances might induce the protective enzyme heme oxygenase (HO)-1, particularly in the liver. The aim of the study was to assess the role of pretreatment with DCLHb, HbG, or HAR on HO-1 expression and organ injury after hemorrhagic shock. DESIGN: Prospective controlled laboratory study. SETTING: Animal research laboratory at a university hospital. SUBJECTS: Male Sprague-Dawley rats (200-300 g body weight, n = 5-12/group). INTERVENTIONS: Twenty-four hours after different doses of DCLHb, HbG (each 1, 2, or 3 g/kg of body weight), or HAR (5, 25, or 75 mg/kg of body weight), the protein expression of HO-1 and heat shock protein-70 in liver, kidney, heart, lungs, and aorta was determined. Twenty-four hours after pretreatment with DCLHb, HbG, or HAR, rats were subjected to hemorrhage (mean arterial blood pressure, 35-40 mm Hg for 1 or 2 hrs)/resuscitation (5 or 4 hrs, respectively). Animals treated with Ringer's solution (30 mL/kg of body weight) served as controls. In additional experiments, HO activity was blocked with tin mesoporphyrin-IX. MEASUREMENTS AND MAIN RESULTS: DCLHb, HbG, and HAR dose-dependently induced HO-1 protein but not heat shock protein-70. Pretreatment with DCLHb or HbG shortened the onset of decompensation in shock (DCLHb, 40 +/- 11 mins; HbG, 36 +/- 4 mins) compared with vehicle (68 +/- 4 mins, p < .05) and HAR pretreatment (81 +/- 7 mins, p < .05). High doses of DCLHb pretreatment increased mortality (2 hrs of shock, 80%; p < .05 vs. vehicle or HAR). Pretreatment with HAR led to higher shed blood volumes (p < .05) and higher hepatocellular ATP levels (2 hrs of shock, p < .05 vs. DCLHb and HbG). Blockade of HO activity by tin mesoporphyrin-IX abolished the protection mediated by HAR. CONCLUSIONS: Although DCLHb, HbG, and HAR induce HO-1 in the absence of an unspecific stress response, only HAR pretreatment protects against shock-induced organ failure. Although the underlying mechanisms of positive HAR priming are not completely understood, the induction of HO-1 expression and the lack of nitric oxide scavenging through HAR may play an important role.     

Mechanisms of the beneficial effect of NHE1 inhibitor in traumatic hemorrhage: inhibition of inflammatory pathways

This study evaluated the effects of sodium-hydrogen exchanger (NHE1) inhibition on enhancing fluid resuscitation outcomes in traumatic hemorrhagic shock, and examined the mechanisms related to NHE1 inhibitor-induced protection and recovery from hemorrhagic shock. Traumatic hemorrhage was modeled in anesthetized pigs by producing tibia fractures followed by hemorrhage of 25 ml/kg for 20 min, and then a 4mm hepatic arterial tear with surgical repair after 20 min. Animals then underwent low volume fluid resuscitation with either hextend (n=6) or 3mg/kg BIIB513 (NHE1 inhibitor)+hextend (n=6). The experiment was terminated 6h after the beginning of resuscitation. In association with traumatic hemorrhagic shock, there was a decrease in cardiac index, stimulation of the inflammatory response, myocardial, liver and kidney injury. The administration of the NHE1 inhibitor at the time of resuscitation attenuated shock-resuscitation-induced myocardial hypercontracture and resulted in a significant increase in stroke volume index, compared to vehicle-treated controls. NHE1 inhibition also reduced the inflammatory response, and lessened myocardial, liver and kidney injury. In addition, NHE1 inhibition reduced NF-κB activation and iNOS expression, and attenuated of ERK1/2 phosphorylation. Results from the present study indicate that NHE1 inhibition prevents multiple organ injury by attenuating shock-resuscitation-induced myocardial hypercontracture and by inhibiting NF-κB activation and neutrophil infiltration, reducing iNOS expression and ERK1/2 phosphorylation, thereby, reducing systemic inflammation and thus multi-organ injury.     

Dihydralazine treatment limits liver injury after hemorrhagic shock in rats

OBJECTIVE: Impaired hepatic perfusion after hemorrhagic shock frequently results in hepatocellular dysfunction associated with increased mortality. This study characterizes the effect of the vasodilators dihydralazine and urapidil on hepatocellular perfusion and integrity after hemorrhagic shock and resuscitation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University experimental laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: To register systemic and regional hepatic hemodynamics, rats (n=6 per group) were instrumented and randomly assigned to the following groups: shock+vehicle; shock+dihydralazine (1.5 mg/kg); or shock+urapidil (3 mg/kg). After 1 hr of hemorrhagic shock, animals were resuscitated for 5 hrs and mean arterial pressure was maintained at 70+/-5 mm Hg by administration of dihydralazine or urapidil. To evaluate hepatic heme oxygenase-1 expression and liver injury (determination of levels of alanine and aspartate aminotransferase [ALT, AST] and histology), an additional series of experiments with six animals per group was performed. At the end of each experiment, animals were killed and blood and liver tissue was obtained for subsequent analyses. MEASUREMENTS AND MAIN RESULTS: Dihydralazine increased cardiac output and portal and hepatic microvascular flow (p<.05) and reduced liver injury after shock (lower ALT and AST levels [p<.05]; improvement of histopathological changes). In contrast, urapidil had no effect on portal flow or liver injury. Hepatic heme oxygenase-1 mRNA expression was upregulated in animals subjected to hemorrhagic shock but did not differ among experimental groups. CONCLUSIONS: Dihydralazine increases nutritive portal and hepatic microvascular flow and limits liver injury after hemorrhagic shock. This protective effect appears to be the result of increased cardiac output and increased portal flow. These findings may offer a new strategy for hepatic protection after hemorrhagic shock.     

regulatory effects of hypoxia-inducible factor 1alpha on vascular reactivity and its mechanisms following hemorrhagic shock in rats

The purpose of the present study is to investigate the regulatory effect of hypoxia-inducible factor 1alpha (HIF-1alpha) on vascular reactivity and its mechanism after hemorrhagic shock (HS). Gene expression of HIF-1alpha and its downstream molecules, including eNOS, iNOS, cyclooxygenase 2 (COX-2), and heme oxygenase 1 (HO-1), and plasma nitric monoxide (NO), prostaglandin (PGI), and whole blood carbon monoxide (CO) were determined after HS in rats with or without oligomycin, the specific antagonist of HIF-1alpha. The vascular reactivity was determined via observing the constriction initiated by norepinephrine in isolated organ perfusion system. The results indicated that HIF-1alpha, eNOS, iNOS, HO-1, and COX-2 messenger RNA expression exhibited a time-dependent increase after HS, although the expression of these genes and their products, NO, CO, and PGI were suppressed by oligomycin to some extent. The vascular reactivity revealed a biphasic change, which was increased compensatorily at the early stage of HS (immediate to 1 h after shock) and decreased progressively at the decompensatory period after 4 h of shock. Oligomycin treatment partly inhibited the vascular reactivity at early stage (immediate to 1 h after shock) and improved it at decompensatory period at 4 to 6 h after shock (P < 0.01). The results suggested that HIF-1alpha plays an important regulatory role in the change of vascular reactivity after HS in rats. The possible mechanism of HIF-1alpha regulating vascular reactivity is closely related to its regulation on the expression of eNOS, iNOS, HO-1, COX-2 and the production of NO, CO and PGI.     

一氧化氮合酶对早期内毒素休克猕猴肾脏的影响

目的 探讨早期内毒素休克肾脏损伤的发病机制。方法 猕猴11只，随机分成两组：对照组5只静脉注射生理盐水；实验组6只，静脉注射脂多糖（LPS）2．8mg／kg。LPS攻击后120min，处死动物，取肾脏标本，用免疫组化方法[卵白素一生物素一过氧化物酶法（ABc法）]进行内皮型一氧化氮合酶（eNOs）和诱导型一氧化氮合酶（iNOS）染色。观察eNOS和iN0s在早期猕猴内毒素休克肾脏中的表达，同时观察肾脏超微结构的改变、酸性磷酸酶（ACP）活性变化。结果LPS攻击后120min，可见肾脏组织超微结构明显破坏和细胞内溶酶体数量增加。实验组iN0s在肾小球血管内皮、肾小管上皮、肾小球血管内和肾小管周围均有表达，对照组无表达。eNOS在实验组和对照组的肾小球血管内皮及周围血管内皮细胞均有表达。结论 一氧化氮在猕猴内毒素休克早期肾脏损伤中发挥了重要作用。     

失血性休克大鼠肺细胞HO-1mRNA表达和凋亡的相关性研究

目的检测和分析失血性休克大鼠肺细胞HO-1 mRNA表达及凋亡的发生情况,从而探讨其临床意义。方法本研究将14只SD大鼠制成重度失血性休克模型,使用SYBR Green I实时荧光定量逆转录-聚合酶链反应技术和流式细胞仪FITC-Annexin V/PI荧光染色法,定量检测各组大鼠肺细胞HO-1 mRNA表达及细胞凋亡的发生情况,并加以比较和分析。结果经过失血性休克和急救后,大鼠的肺细胞均存在较高的细胞凋亡率,达（4.0±1.4）%,也存在一定程度的HO-1 mRNA表达,具体表达强度为：（中位数：3.7 869,4分位数间距：0.1 379-5.7 169）表达指数。大鼠肺细胞的HO-1 mRNA表达和凋亡之间存在着显著的负相关性（r=-0.653,P=0.011）。结论在该重度失血性休克大鼠模型,肺细胞HO-1 mRNA高表达可能有助于减少失血性休克/急救后的肺细胞凋亡的发生率,从而减少肺损伤。     

Lung, spleen, and kidney are the major places for inducible nitric oxide synthase expression in endotoxic shock: role of p38 mitogen-activated protein kinase in signal transduction of inducible nitric oxide synthase expression

Bacterial lipopolysaccharide (LPS) is known to induce endotoxic shock with inducible nitric oxide (NO) synthase (iNOS) expression and NO production. However, the major place for NO production in shock remains unclear. Although there is some literature about p38 mitogen-activated protein kinase (MAPK) in regulating LPS-induced iNOS expression, the results are contradictory. To interpret the precise cell mechanism and the role of p38 MAPK in the expression of iNOS during endotoxic shock, we carried out the following investigations. A severe endotoxic shock model was reproduced in mice 6 h after LPS injection. The plasma NO level was increased in a dose- and time-dependent manner after LPS stimulation and was suppressed by administration of SB203580 [4-(4-fluorophenyl)-2-4-methylsulfonylphenyl-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK. The iNOS expression was increased in many organs, including heart, liver, spleen, lung, gut, and kidney in endotoxic shock. Among them, the highest expression of iNOS mRNA and protein was in the lung, moderate expression was in the spleen and kidney, and the lowest expression was in the heart, gut, and liver. The level of expression in lung was 5.5 times that of iNOS mRNA and was 3.1 times that of iNOS protein than in heart, and 1.6 and 1.8 times that of iNOS mRNA and 1.7 and 1.4 times that of iNOS protein than in spleen and kidney, respectively. The p38 MAPK activity increased after LPS injection, and SB203580 markedly reduced LPS-induced expressions of iNOS protein and mRNA in the lung. The results indicates that lung, spleen, and kidney are the major places for iNOS expression in endotoxic shock and are important therapeutic target organs for attenuating NO production in shock treatment.     

α黑素细胞刺激素及其类似物对内毒素休克小鼠的保护作用

目的 研究α黑素细胞刺激素（α－NSH）及其类似物（NDP－MSH）对内毒素休克小鼠的保护作用。方法 腹腔注射LPS50ug/kg和D－半乳糖（D－Gal)900mg/kg复制小鼠内毒素休克模型,研究不同剂量α-MSH及NDP－MSH在不同时间或经不同途径给药给药对内毒素休克小鼠生存率的影响。用常规病理切片观察α-MSH对内毒素休克小鼠重要脏器病理变化的影响。结果 未给予α－MSH的内毒素休克小鼠在9h内全部死亡,经尾静脉、腹腔和皮下注射α-MSH2.5mg/kg或5.0mg/kg均可以起到一定的保护作用;给予LPS前后不同时间注射2.5mg/kgα-MSH都可以明显延长内毒素休克小鼠起始死亡的时间,提高存活率,其中以给予LPS后1h经腹腔给药疗效最好,72h的生存率达33.3%,但增加给药次 未见疗效提高。经腹腔注射2.5mg/kgNDP－MSH,小鼠平均起死亡时间较α-MSH组延长4h,72h的生存率升至44.4%。病理学检查结果表明α-MSH可以减轻LPS引起的肝脏、脾脏及肺脏瘀血及细胞坏死。结论 α-MSH及NDP－MSH可以减轻LPS引起的小鼠重要脏器的病理损害,对内毒素休克小鼠有明显的保护作用。     

Variable effects of inhibiting iNOS and closing the vascular ATP-sensitive potassium channel (via its pore-forming and sulfonylurea receptor subunits) in endotoxic shock

Excess production of NO and activation of vascular ATP-sensitive potassium (K(ATP)) channels are implicated in the hypotension and vascular hyporeactivity associated with endotoxic shock. Using a fluid-resuscitated endotoxic rat model, we compared the cardiovascular effects of an iNOS inhibitor and two distinct inhibitors of the K(ATP) channel. Endotoxin (LPS) was administered to anesthetized, spontaneously breathing, fluid-resuscitated adult male Wistar rats, in which MAP, aortic and renal blood flow, and hepatic microvascular oxygenation were monitored continuously. At 120 min, the iNOS inhibitor, GW273629, and the K(ATP)-channel inhibitors, PNU-37883A and glyburide, were administered separately, and their effects on hemodynamics and oxygenation were examined. We found that GW273629 increased MAP over and above the pressor effect achieved in sham animals. Inhibiting K(ATP) channels via the pore-forming subunit (PNU-37883A and high-dose glyburide) produced significant pressor effects, whereas inhibiting the sulfonylurea receptor with low-dose glyburide was ineffective. No agent reversed the fall in aortic or renal blood flow, the fall in hepatic microvascular oxygenation, or the metabolic acidosis that occurred in LPS-treated animals. We conclude that inhibition of the K(ATP) channel via the pore-forming, but not the sulfonylurea receptor subunit, increases blood pressure in a short-term endotoxic model. However, this was not accompanied by any improvement in macrocirculatory or microcirculatory organ blood flow nor reversal of metabolic acidosis. It therefore remains uncertain whether the iNOS pathway or the K(ATP) channel represents a potential target for drug development in the treatment of endotoxic shock.