Effects of thermal preconditioning on the ischemia-reperfusion-induced acute lung injury in minipigs

Lung ischemia-reperfusion (I/R) injury plays an important role in many clinical issues. A series of mechanisms after I/R has been uncovered after numerous related studies. Organ preconditioning (PC) is a process whereby a brief antecedent event, such as transient ischemia, oxidative stress, temperature change, or drug administration, bestows on an organ an early or delayed tolerance to further insults by the same or different stressors. In this study, we want to uncover the optimal thermal PC patterns that cause maximal early or delayed protective effect on the subsequent pulmonary I/R with the use of miniature pig model. Twenty-eight 15- to 20-kg weight Lanyu miniature pigs are used and divided into four groups (seven sham operation control [NC], seven PC only [PC], seven I/R [I/R], and seven PC followed by I/R [PC + I/R]). The PC was performed with the animals being anesthetized and, using an alternative hyperthermic (40 degrees C) and normothermic moist air to ventilate their lungs for 15 min, respectively, for 2 cycles, followed by I/R, which consists of 90 min of blocking the perfusion and ventilation of the left lung followed by 240 min of reperfusion. Control animals had a thoracotomy with hilar dissection only. Indicators of lung injury included hemodynamic parameters, blood gas analysis, histopathological (lung pathology, wet/dry weight ratio, myeloperoxidase assay), and molecular biological profiles (interleukin-1beta [IL-1beta], IL-6, tumor necrosis factor-alpha by enzyme-linked immunosorbent assay analysis). Lung tissue heat shock protein 70 (HSP-70) expression was also detected by Western blotting. This model of lung I/R induced significant lung injury with pulmonary hypertension, increased pulmonary vascular resistance, and pulmonary venous hypoxemia at the ischemia side, increased pulmonary tissue injury score and neutrophil infiltration, increased wet/dry ratio, myeloperoxidase assay, tumor necrosis factor-alpha, IL-1beta, and IL-6 assay. This type of thermal PC would not injure the lung parenchyma or tracheal epithelium. Moreover, it could attenuate the I/R-related lung injury, with some of these parameters improved significantly. Increased expression of HSP-70 was also found in the group of PC plus I/R than the I/R only. Less prominent and transient increase in expression of HSP-70 was found in the PC group. We concluded that the intratracheal thermal PC can effectively attenuate I/R-induced lung injury through various mechanisms, including the decrease of various proinflammatory cytokines. The mechanism of its protective effect might be related to the increased expression of HSP-70.     

炎症因子在小鼠肾缺血再灌注损伤相关的肺损伤中的表达

目的观察炎症因子在肾缺血再灌注损伤小鼠全身和肺组织局部表达的变化,探讨炎症反应在肾缺血再灌注损伤相关的急性肺损伤中的作用。方法选取8～10周龄雄性C57BL/6小鼠30只,随机分为假手术组（Sham组n=10）、缺血再灌注组（I/R组n=10）和双肾切除组（BNx组n=10）。分别于术后6、24h留取小鼠肾和肺组织及血浆标本,观察肾和肺组织学改变,计算肺组织中中性粒细胞浸润数,称肺湿干重,计算肺湿干重比（W/D）;ELISA法检测小鼠血清和肺冲洗液中的IL-6、IL-1β、TNF-α的浓度,实时定量PCR检测小鼠肺组织IL-6、IL-1β、TNF-α基因表达,免疫组化检测肺组织IL-6、IL-1β和TNF-α蛋白的表达。结果 I/R组和BNx组小鼠术后24h时的BUN和Scr均显著高于Sham组（P均〈0.05）,I/R组和BNx组小鼠术后24h出现肺组织炎症细胞浸润、肺泡周围毛细血管出血和肺间质水肿,两组肺组织中性粒细胞计数均高于Sham组（P〈0.05）。术后6h时I/R组和BNx组小鼠血清IL-6、IL-1β和TNF-α浓度与Sham组相比显著升高（分别为606.32±59.07和300.22±169.73vs121.52±9.12pg/ml;443.93±91.98和959.47±184.46vs21.71±2.47pg/ml,119.67±21.66和132.33±62.64vs30.21±2.46pg/ml,P均〈0.05）;I/R组和BNx组小鼠肺组织冲洗液中IL-6、IL-1β和TNF-α的水平与Sham组比较显著升高（109.74±15.91和70.00±2.42vs37.69±7.96pg/mg,117.02±27.46和215.35±18.49vs42.10±5.20pg/mg,512.31±71.95和988.25±133.55vs52.76±12.82pg/mg,P〈0.05）;术后6h肺组织IL-6、IL-1β、TNF-α的mRNA表达较Sham组显著升高（P均〈0.05）;免疫组化显示了相似的结果。结论肾缺血再灌注损伤可以介导急性肺损伤,全身和肺组织局部的炎症因子表达明显升高可能参与了肾缺血再灌注损伤相关的肺损伤。     

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice

Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen- induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.     

Protective effects of exogenous interleukin 18-binding protein in a rat model of acute renal ischemia-reperfusion injury

Ischemia-reperfusion (I/R) renal injury is considered the most common cause of acute kidney injury (AKI). The pathophysiology of I/R AKI involves a complex interplay among tubular epithelial cell injury, microcirculation dysfunction, and inflammation. Interleukin 18-binding protein (IL-18BP) is a natural inhibitor of IL-18 a cytokine that plays an important role in the pathogenesis of AKI. Therefore, we hypothesized that exogenous IL-18BP could protect against renal injuries after kidney I/R. Male Sprague-Dawley rats were divided into three groups: a sham operation group, I/R with vehicle injection, and I/R with IL-18BP injection. Rats underwent bilateral renal pedicle clamping, and IL-18BP or vehicle was administered just before reperfusion. Rats were killed 6, 24, and 72 h after reperfusion. After IL-18BP treatment, renal tubule epithelium showed reduced apoptosis and enhanced proliferation. For peritubular capillary (PTC) endothelium, apoptosis was inhibited, and there was an increase in PTC endothelium density. Macrophage infiltration was inhibited, and inflammatory cytokines were downregulated. Increased expression of vascular endothelial growth factor and decreased expression of thrombospondin 1 were also observed. Exogenous IL-18BP attenuated renal injury caused by I/R via inhibiting inflammation in the renal tissue and protecting tubular epithelium and PTC endothelium.     

Fingolimod attenuates renal ischemia/reperfusion-induced acute lung injury by inhibiting inflammation and apoptosis and modulating S1P metabolism

ObjectiveThis study examined whether the immunomodulator fingolimod (FTY720) could alleviate renal ischemia/reperfusion (I/R)-induced lung injury and explored the potential mechanisms.MethodsRenal I/R was established in a rat model, and FTY720 (0.5, 1, or 2 mg/kg) was injected intraperitoneally after 15 minutes of ischemia. Pro-inflammatory cytokine levels, oxidative stress, apoptosis, and the mRNA expression of the sphingosine-1-phosphate (S1P)-related signaling pathway genes sphingosine kinase-1 (SphK1) and sphingosine kinase-2 were analyzed in lung tissue.ResultsIncreased pro-inflammatory cytokine levels; decreased total superoxide dismutase, catalase, and glutathione peroxidase levels; increased apoptosis; and increased S1P lyase and SphK1 expression were observed following renal I/R. FTY720 reversed renal I/R-induced changes and effectively attenuated lung injury.ConclusionFTY720 protected against acute lung injury in rats subjected to renal I/R by decreasing pulmonary inflammation and apoptosis, increasing oxidative stress, and modulating S1P metabolism.     

Inhibition of nitric oxide synthesis by L-name exacerbates acute lung injury induced by hepatic ischemia-reperfusion.

Hepatic Kupffer cells and pulmonary alveolar macrophages together constitute a macrophage-axis involved in the regulation of regional and systemic inflammatory responses. Systemic inflammatory response syndrome induced by overproduced pro-inflammatory mediators is the major cause of adult respiratory distress syndrome. In the present study, we examined the anti-inflammatory role of nitric oxide (NO) in a rat model of acute lung injury induced by hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia by clamping the relevant branches of hepatic artery and portal vein, followed by a 4-h reperfusion achieved by removal of the vascular clamp. Four groups of animals were studied: sham control + saline; sham control + N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v., 10 min before reperfusion); HI/R + saline; HI/R + L-NAME. Results show that (1) administration of L-NAME to rats subjected to HI/R decreased plasma NO levels; however, the attenuation of NO increased plasma alanine aminotransferase (ALT) activity and superoxide generation in the ischemic lobes of liver, compared to HI/R alone. (2) Inhibition of NO synthesis with L-NAME in rats subjected to HI/R also enhanced systemic inflammatory response as assessed by the increase in the number of circulating leukocytes and levels of plasma tumor necrosis factor-alpha (TNFalpha) and interleukin 1-beta (IL-1beta). (3) The overwhelming systemic inflammatory response induced by administration of L-NAME in rats subjected to HI/R also augmented pulmonary vascular permeability and superoxide generation in the lung tissue. (4) Pulmonary alveolar macrophages isolated from rats subjected to HI/R + L-NAME produced higher levels of TNFalpha and IL-1beta in the supernatant of culture medium than that of rats subjected to HI/R alone. (5) There were no differences between the groups of sham + saline and sham + L-NAME in terms of plasma NO levels and ALT activity, circulating leukocytes, superoxide generation in the liver and lung, lavage protein levels, and TNFalpha and IL-1beta levels in plasma and bronchoalveolar lavage fluid. Our results suggest that inhibition of NO synthesis by L-NAME in rats subjected to HI/R not only augments ischemic liver injury, but also enhances the systemic inflammatory response and exacerbates remote lung injury. The increase in TNFalpha and IL-1beta production by alveolar macrophages may, in part, account for L-NAME-induced enhancement of acute lung injury.     

线粒体ATP敏感钾通道开放剂对大鼠肺缺血-再灌注损伤的保护作用

目的 探讨线粒体ATP敏感钾通道开放剂二氮嗪(DE)对肺缺血-再灌注损伤(I/R)的保护作用.方法 建立大鼠肺I/R模型,随机设立假手术(sham)组、I/R组、DE组、线粒体ATP敏感钾通道阻断剂5-羟基葵酸(5-HD)组,每组10只,观察各组肺组织病理形态学变化,测定肺湿/干质量比,检测肺组织髓过氧化物酶(MPO)活性、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性.结果 与sham组比较,I/R组肺组织出现明显损伤性病理形态学变化,肺湿/干质量比明显增加(P<0.05),肺组织MPO活性显著增高、MDA含量显著增加和SOD活性显著降低(P<0.05).与I/R组比较,DE组肺组织损伤明显减轻,肺湿/干质量比降低(P<0.05),肺组织MPO活性降低、MDA含量减少、SOD活性增高(P< 0.05).5-HD组各观察指标与I/R组差异无统计学意义(P>0.05).结论 线粒体ATP敏感钾通道开放剂DE可通过抑制中性粒细胞聚集、减少自由基产生、增强抗氧化能力对大鼠肺缺血-再灌注损伤产生明显保护作用,该保护作用可被线粒体ATP敏感钾通道阻断剂5-HD所拮抗.     

Pulmonary endothelium in acute lung injury: from basic science to the critically ill

Background Pulmonary endothelium is an active organ possessing numerous physiological, immunological, and metabolic functions. These functions may be altered early in acute lung injury (ALI) and further contribute to the development of acute respiratory distress syndrome (ARDS). Pulmonary endothelium is strategically located to filter the entire blood before it enters the systemic circulation; consequently its integrity is essential for the maintenance of adequate homeostasis in both the pulmonary and systemic circulations. Noxious agents that affect pulmonary endothelium induce alterations in hemodynamics and hemofluidity, promote interactions with circulating blood cells, and lead to increased vascular permeability and pulmonary edema formation.Objective We highlight pathogenic mechanisms of pulmonary endothelial injury and their clinical implications in ALI/ARDS patients.     

Effects of interleukin-10 and modulators of cyclic AMP formation on endotoxin-induced inflammation in rat lung

Abstract— The aim of this study was to investigate the effects of IL-10, a cell permeable analogue of cyclic AMP, dibutyryl-cAMP (db-cAMP), modulators of intracellular cyclic AMP such as phosphodiesterase (PDE) inhibitors and a β₂-adrenoceptor agonist, salmeterol, on pulmonary inflammation following acute lung injury induced by endotoxin exposure in rats. Pulmonary inflammation was induced in adult Wistar rats by a 60-min exposure to endotoxin (lipopolysaccharide, LPS, 100 μg/mL). 4 h later bronchoalveolar lavage (BAL) was performed. The PDE inhibitors, rolipram (3 and 5 mg/kg) and theophylline (30 and 100 mg/kg) inhibited neutrophil recruitment, TNF-α release and cellular activation in BAL. Salmeterol (0.5 mg/mL) and IL-10 (0.1 μg) only inhibit TNF-α increase in the BAL fluid and db-AMPc (2.5 μg/rat) was ineffective. The present data show that the selective PDE4 inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline, markedly reduced the pulmonary inflammation associated with acute lung injury in the rat. These effects may be mediated in part by IL-10 rather than by cyclic AMP, as demonstrated by the potent inhibitory activity of exogenous IL-10 on the increase in TNF-α release in BAL fluid of rats exposed to LPS.     

Nitric oxide mediates lung vascular permeability and lymph-borne IL-6 after an intestinal ischemic insult

Acute lung injury following intestinal I/R depends on neutrophil-endothelial cell interactions and on cytokines drained from the gut through the lymph. Among the mediators generated during I/R, increased serum levels of IL-6 and NO are also found and might be involved in acute lung injury. Once intestinal ischemia itself may be a factor of tissue injury, in this study, we investigated the presence of IL-6 in lymph after intestinal ischemia and its effects on human umbilical vein endothelial cells (HUVECs) detachment. The involvement of NO on the increase of lung and intestinal microvascular permeability and the lymph effects on HUVEC detachment were also studied. Upon anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2-h intestinal reperfusion. Rats were treated with the nonselective NO synthase (NOS) inhibitor L-NAME (N(omega)-nitro-L-arginine methyl ester) or with the selective inhibitor of iNOS aminoguanidine 1 h before superior mesenteric artery occlusion. Whereas treatment with L-NAME during ischemia increased both IL-6 levels in lymph and lung microvascular permeability, aminoguanidine restored the augmented intestinal plasma extravasation due to ischemia and did not induce IL-6 in lymph. On the other hand, IL-6 and lymph of intestinal I/R detached the HUVECs, whereas lymph of ischemic rats upon L-NAME treatment when incubated with anti-IL-6 prevented HUVEC detachment. It is shown that the intestinal ischemia itself is sufficient to increase intestinal microvascular permeability with involvement of iNOS activation. Intestinal ischemia and absence of constitutive NOS activity leading to additional intestinal stress both cause release of IL-6 and increase of lung microvascular permeability. Because anti-IL-6 prevented the endothelial cell injury caused by lymph at the ischemia period, the lymph-borne IL-6 might be involved with endothelial cell activation. At the reperfusion period, this cytokine does not seem to be modulated by NO.     

脓毒症大鼠急性肺损伤肺血管通透性、炎症因子、诱导型一氧化氮合酶的变化及意义

目的:观察脓毒症大鼠急性肺损伤肺血管通透性、炎症因子、诱导型一氧化氮合酶(iNOS)的变化特点及其意义.方法:24只雄性SD大鼠随机分为脓毒症组和对照组,脓毒症组按5 mg/kg体重腹腔注射脂多糖(LPS),对照组给予等量生理盐水,于注射后2h、6h经腹主动脉采血、处死,比较动脉血气分析、ELISA法检测的肺组织iNOS、白介素-6(IL-6)含量、右下肺肺组织病理切片计算的病理学肺损伤积分.结果:与对照组比较,脓毒症组大鼠氧合指数显著下降、肺组织iNOS、IL-6含量、病理学肺损伤积分均显著增加,P＜0.01.结论:脓毒症大鼠肺iNOS的过度表达增加了炎症因子的释放、增加肺血管通透性,加重肺损伤.     

烟碱对心肌缺血/再灌注损伤大鼠炎症细胞因子的影响

目的 探讨烟碱对心肌缺血/再灌注(I/R)损伤大鼠炎症细胞因子的影响.方法 50只健康雄性SD大鼠按随机数字表法分为假手术组、I/R组、烟碱高剂量(400μg/kg)组、烟碱低剂量(40μg/kg)组及α-银环蛇毒素(α-BGT,1μg/kg)组5组,每组10只.采用结扎心脏左冠状动脉前降支30 min、再灌注90 min制作大鼠心肌I/R损伤模型;假手术组仅穿线不结扎.制模前30 min各药物组颈静脉注射相应剂量药物干预,假手术组和I/R组给予等量生理盐水.于再灌注末取右颈动脉血,测定肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)、IL-10浓度和肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)活性;然后处死动物,取缺血区心肌组织测定髓过氧化物酶(MPO)活性;采用免疫组化和逆转录-聚合酶链反应检测心肌组织细胞间黏附分子-1(ICAM-1)蛋白及mRNA表达,并观察心肌超微结构.结果 与假手术组比较,I/R组血浆TNF-α、IL-8、IL-10、CK-MB、cTnI、心肌MPO活性及ICAM-1蛋白和mRNA表达均显著升高[TNF-α(ng/L):158.7±32.7比31.5±5.8,IL-8(ng/L):0.71±0.06比0.30±0.04,IL-10(ng/L):69.0±7.8比41.4±4.3,CK-MB(U/L):2 540±169比1 120±102,cTnI(μg/L):26.2±4.6比0.9±0.2,MPO(U/g):4.2±0.6比1.6±0.4,ICAM-1蛋白:0.210±0.025比0.100±0.018,ICAM-1 mRNA:1.82±0.23比1.18±0.20,P＜0.05或P＜0.01],病理学显示心肌组织损伤较重.与I/R组比较,烟碱高剂量组血浆TNF-α、IL-8降低[TNF-α(67.3±9.8)ng/L,IL-8(0.47±0.04)ng/L],IL-10升高[(147.5±12.5)ng/L],CK-MB、cTnI及心肌MPO活性、ICAM-1蛋白和mRNA均降低[CK-MB(1 282±145)U/L,cTnI(4.7±1.4)μg/L,MPO(2.5±0.4)U/g,ICAM-1蛋白0.140±0.026,ICAM-1 mRNA 1.31±0.25,P＜0.05或P＜0.01],心肌组织损伤减轻;而烟碱低剂量组和α-BGT组上述指标与I/R组比较差异无统计学意义.结论 烟碱可阻断内皮细胞表达黏附分子,阻断中性粒细胞黏附、游出,改善抗炎/促炎反应平衡,从而拮抗大鼠心肌I/R损伤时的过度炎症反应.     

甲泼尼龙和纳洛酮对大鼠肺缺血/再灌注损伤的保护作用研究

目的 研究甲泼尼龙、纳洛酮在大鼠肺缺血/再灌注(I/R)损伤中的作用,并探讨其可能机制.方法 雄性SD大鼠70只,随机均分为假手术组(sham组)、I/R组、甲泼尼龙组(MP组)、纳洛酮组(Na组)、甲泼尼龙联合纳洛酮组(MP+Na组).术后3 h和6 h取标本,用钙结合蛋白Ⅴ-碘化丙啶(AnnexinⅤ-PI)双染法、流式细胞仪检测肺组织凋亡细胞并计算凋亡率;用免疫组化及图像分析法观察肺脏核转录因子-κB抑制因子-α(IκB-α)、天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)的表达;计算肺湿/干重(W/D)比值;苏木素-伊红(HE)染色,光镜下观察肺组织病理学变化,电镜下观察肺脏超微结构的改变.结果 ①6 h MP组较I/R组肺脏IκB-α的表达有所增加,差异有统计学意义(P＜0.01);3 h和6 h Na组较I/R组肺组织caspase-3表达明显减少,差异有统计学意义(P均＜0.05);MP组和Na组3 h和6 h的细胞凋亡率较I/R组均有所减少(P均＜0.01),肺组织的病理形态及超微结构损害均有所减轻.②MP+Na组较MP组、Na组、I/R组肺组织凋亡率、caspase-3表达均有不同程度的减少(P＜0.05或P＜0.01),肺脏IκB-α的表达较6 h Na组显著增多(P＜0.05),肺组织的病理形态及超微结构损害明显减轻,6 h较3 h减轻更明显.结论 甲泼尼龙、纳洛酮分别通过抗炎、减少caspase-3的激活两个不同途径抑制凋亡的发生,早期联合应用有协同效应,更能有效抑制肺组织I/R损伤凋亡的发生,对肺组织有明显的保护作用.     

Regulatory effects of endogenous interleukin-1 receptor antagonist protein in immunoglobulin G immune complex-induced lung injury.

IL-1 receptor antagonist (IL-1Ra) has regulatory effects on IL-1 activity both in vitro and in vivo. In the IgG immune complex model of lung injury in rats, exogenously administered human IL-1Ra suppressed neutrophil recruitment and ensuing lung injury. In this study, we sought to determine if endogenous rat IL-1Ra might regulate this lung-inflammatory response. By Northern blot analysis of lung mRNA and Western analysis of bronchoalveolar lavage (BAL) fluids, rat IL-1Ra expression was found to increase during development of inflammation in IgG immune complex-mediated alveolitis. By immunostaining, alveolar macrophages and recruited neutrophils were the apparent sources of IL-1Ra. In vivo blocking of endogenous IL-1Ra resulted in a 53% increase in lung vascular permeability and a 180% increase in BAL fluid neutrophils. In companion studies, a significant increase in IL-1beta was found, whereas no significant change in TNF-alpha activity was observed. Whereas the in vivo regulatory effects of IL-1R appear to be limited to IL-1beta, IL-10 regulates both IL-1beta and TNF-alpha in this model, reflected by a 48% increase in BAL IL-1beta in rats treated with anti-IL-10. These findings suggest that IL-1Ra is an intrinsic regulator of inflammatory injury after deposition of IgG immune complexes and that it regulates production of IL-1beta.     

丙泊酚对大鼠肠缺血-再灌注后肺细胞间黏附分子-1蛋白表达的影响

目的 研究丙泊酚对大鼠肠缺血-再灌注(I／R)后肺细胞间黏附分子-1(ICAM-1)蛋白表达的 影响。方法 SD大鼠随机分为4组(n=8):①I／R组:暴露腹腔后夹闭肠系膜上动脉(SMA)1 h,开放再灌注 2 h;②丙泊酚预处理组(P1组):肠缺血前10 min给予丙泊酚;③丙泊酚治疗组(P2组):肠再灌注前10 min给 予丙泊酚;④假手术组:仅暴露SMA,不行肠I／R及丙泊酚输注。丙泊酚剂量为首剂10 mg／kg,然后以 10 mg·kg-1·h-1持续输注。所有动物于再灌注2 h处死,检测血浆和肺组织肿瘤坏死因子-α(TNF-α) 及肺组织MPO含量,免疫组化染色检测肺组织ICAM-1蛋白的表达。结果 肠I／R后动物血浆和肺组织 TNF-α含量及肺组织MPO含量、ICAM-1蛋白表达均增加。丙泊酚可以抑制上述改变,以P1组效果最明 显,其中血浆TNF-α及肺组织ICAM-1表达在I／R组和P1组间存在显著性差异(P均<0.05),而P2组上 述各指标显著高于假手术组。结论 ICAM-1在肠I／R后肺损伤的发生中发挥重要作用。肠I／R早期应用丙 泊酚可减少肺组织ICAM-1表达,在一定程度上减轻肺损伤。     

Nifedipine and diltiazem reduce pulmonary edema formation during postischemic reperfusion of the rabbit lung

A protective effect of calcium antagonists in pulmonary preservation for transplantation has been observed recently. This report focuses on the potential use of diltiazem and nifedipine in the early phase of reperfusion after normothermic pulmonary ischemia. Rabbits weighing 4–5 kg were tracheotomized and ventilated with 50% oxygen. In a control group (group I,n=7), the hilus of the right lung was clamped for 210 min without ischemia of the left lung. Lung ischemia was created in a second group (n=7) by clamping the left hilum for 2 h. Subsequently, reperfusion of the left lung was maintained for 210 min, while the right hilus was kept occluded. In group III (n=6) and group IV (n=8) the conditions were the same as in group II, but either diltiazem (62.5 μg/kg i.v., group III) or nifedipine (3 μg/kg i.v., group IV) was administered during the first 20 min of reperfusion. After 210 min of reperfusion, the pulmunary vascular resistance was elevated in group II (×: 5120 dyn·sec·cm⁻⁵), group III (5518 dyn·sec·cm⁻⁵), and group IV (4324 dyn·sec·cm⁻⁵), compared with group I (3390; n.s.). Arterial oxygenation showed no significant differences among group I (×: 257 mmHg), group II (261 mmHg), group III (208 mmHg), and group IV (247 mmHg). Pulmonary ischemia resulted in an increased extravascular lung water content in group II as compared to group I (73 and 64 g/g wet weight;P<0.0125 vs group I). No such increase was seen in groups III and IV (53 and 54 g/g wet weight respectively;P<0.001 vs group II). In this model of normothermic left lung ischemia, the application of diltiazem or nifedipine during the early phase of reperfusion limits formation of pulmonary edema. In constrast to previous findings using verapamil, both substances do not show significant influence on pulmonary vascular resistance. They may be of use in clinical lung transplantation by reducing fluid accumulation in the lung parenchyma.     

Cyclic arginine-glycine-aspartate attenuates acute lung injury in mice after intestinal ischemia/reperfusion

Introduction

Intestinal ischemia is a critical problem resulting in multiple organ failure and high mortality of 60 to 80%. Acute lung injury (ALI) is a common complication after intestinal ischemia/reperfusion (I/R) injuries and contributes to the high mortality rate. Moreover, activated neutrophil infiltration into the lungs is known to play a significant role in the progression of ALI. Integrin-mediated interaction is involved in neutrophil transmigration. Synthetic peptides containing an arginine-glycine-aspartate sequence compete with adhesive proteins and inhibit integrin-mediated interaction and signaling. Thus, we hypothesized that the administration of a cyclic arginine-glycine-aspartate peptide (cRGD) inhibited neutrophil infiltration and provided protection against ALI induced by intestinal I/R.

Methods

Ischemia in adult male C57BL/6 mice was induced by fastening the superior mesenteric artery with 4-0 suture. Forty-five minutes later, the vascular suture was released to allow reperfusion. cRGD (5 mg/kg body weight) or normal saline (vehicle) was administered by intraperitoneal injection 1 hour prior to ischemia. Blood, gut, and lung tissues were collected 4 hours after reperfusion for various measurements.

Results

Intestinal I/R caused severe widespread injury to the gut and lungs. Treatment with cRGD improved the integrity of microscopic structures in the gut and lungs, as judged by histological examination. Intestinal I/R induced the expression of β₁, β₂ and β₃ integrins, intercellular adhesion molecule-1, and fibronectin. cRGD significantly inhibited myeloperoxidase activity in the gut and lungs, as well as neutrophils and macrophages infiltrating the lungs. cRGD reduced the levels of TNF-α and IL-6 in serum, in addition to IL-6 and macrophage inflammatory protein-2 in the gut and lungs. Furthermore, the number of TUNEL-staining cells and levels of cleaved caspase-3 in the lungs were significantly lowered in the cRGD-treated mice in comparison with the vehicle mice.

Conclusions

Treatment with cRGD effectively protected ALI and gut injury, lowered neutrophil infiltration, suppressed inflammation, and inhibited lung apoptosis after intestinal I/R. Thus, there is potential for developing cRGD as a treatment for patients suffering from ALI caused by intestinal I/R.     

缺血预适应对肺缺血-再灌注损伤中细胞凋亡与热休克蛋白70表达的影响

目的 观察缺血预适应(IP)对在体大鼠肺缺血-再灌注(I/R)损伤细胞凋亡及热休克蛋白(HSP70)表达的影响,探讨其作用的可能机制.方法 雄性SD大鼠36只,随机分为3组:假手术(SO)组,缺血.再灌注(I/R)组,缺血预适应(IP)组,每组12只.I/R组开胸后用无创微血管钳钳夹肺门远心端,阻断肺门(观察肺无舒缩为阻断标准),建立在体肺脏L/R损伤模型.IP组于缺血开始前,应用3个循环的5min缺血+5 min灌注进行预处理.假手术组仅予开胸术.各组均于2h、5 h时结扎肺门取出左肺.用原位末端标记法(TUNEL)检测细胞凋亡指数,免疫组化法测定HSPT0表达.计算肺湿干比(W/D),肺泡损伤数定最评价指标(IQA),同时在光镜与电镜下观察肺脏的病理形态学和超微结构的改变.为应用单因素方差分析,组间两两比较采用scheffe检验.结果 与SO组比较,I/R组凋亡指数2 h点为21.37±4.35、5h点为19.67±3.64,均增加(P=0.000),HSP 70表达2 h点为0.187±0.019、5 h点为0.207±0.021均增加(P=0.000),W/D 2 h点为6.65±0.85、5 h点为7.10±0.94,均增加(P=0.000),IQA 2 h点为45.95±2.82、5 h点为55.77±3.24均显著升高(P:0.000).与I/R组比较,IP组凋亡指数2 h点为14.02±3.15(P=0.005)、5 h点为12.18±2.29(P=0.001),均明显下降,HSP70表达2 h点为0.240±0.017(P=0.000)、5 h点为0.260±0.022(P=0.002),均增强,W/D 2 h点为5.39±0.36(P=0.074)、5 h点为5.47±0.44(P=0.003),有不同程度降低、IQA 2 h点为25.77±3.77、5 h点为30.35±3.69,差异具有统计学意义(P=0.000).肺脏超微结构损害和肺水肿程度明显减轻.结论 缺血预适应对肺缺血.再灌注损伤有保护作用,其机制可能是通过上调HSP70的表达而抑制细胞凋亡来实现的.     

Role of poly-(ADP-ribose) synthetase in lipopolysaccharide-induced vascular failure and acute lung injury in pigs

PURPOSE: To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs. MATERIALS AND METHODS: Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs. Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2. Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion. Only 3-aminobenzamide and not LPS was injected in group 4. All animals were examined for 180 minutes. Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period. Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals. RESULTS: LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure. Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion. Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals. No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion. Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance. CONCLUSIONS: We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.