Preventive effects of edaravone, a free radical scavenger, on lipopolysaccharide-induced lung injury in mice

Background and objective:   Reactive oxygen species (ROS) play an important role in the pathogenesis of acute lung injury (ALI) and pulmonary fibrosis. It was hypothesized that edaravone, a free radical scavenger, would be able to attenuate LPS-induced lung injury in mice by decreasing oxidative stress.
Methods:   For the in vivo experiments, lung injury was induced in female BALB/c mice by the intranasal instillation of LPS. Edaravone was given by intraperitoneal administration 1 h before the LPS challenge. For the in vitro experiments, MH-S cells (murine alveolar macrophage cell line) were exposed to edaravone, followed by stimulation with LPS.
Results:   In the LPS-induced ALI mouse model, the administration of edaravone attenuated cellular infiltration into and the concentrations of albumin, IL-6, tumour necrosis factor-α, keratinocyte-derived chemokine and macrophage inflammatory protein-2 in BAL fluid. In addition, the in vitro studies showed that the elevated IL-6 secretion from MH-S cells in response to LPS was significantly attenuated by co-incubation with edaravone.
Conclusions:   In an experimental murine model, a free radical scavenger may prevent ALI via repression of pro-inflammatory cytokine production by lung macrophages.     

地塞米松对急性肺损伤大鼠信号转导细胞外信号调节激酶的影响

目的观察地塞米松对急性肺损伤(ALI)大鼠细胞信号转导中细胞外信号调节激酶(ERK)表达的影响,从而为临床治疗提供理论依据。方法健康雄性SD大鼠随机分为脂多糖(LPS)模型组(静脉注射5.0mg/kgLPS)、LPS+地塞米松低、中、高(0.2mg/kg、1.0mg/kg、5.0mg/kg)3个剂量组和对照组,每组5只,4h时测定动脉血气,肺湿/干(W/D)比值,Bradford法测定支气管肺泡灌洗液(BALF)和血浆中蛋白浓度并计算肺通透指数(LPI=BALF蛋白含量/血浆蛋白含量),Westernblot法检测不同剂量地塞米松对p-ERK蛋白表达的影响;另取大鼠,在注射LPS后1h、4h、8h、12h和0h(对照组:注射生理盐水)5个时相点(每个时相点5只)分别用Westernblot法检测地塞米松对p-ERK蛋白表达的影响。结果 LPS模型组大鼠动脉血气各项指标、肺W/D比值、LPI及肺组织p-ERK表达显著高于对照组(P0.05或P0.01)。地塞米松中、高剂量组能显著降低LPS诱导的ALI大鼠动脉血气分析、肺W/D比值、LPI(P0.05或P0.01),同时还能显著抑制LPS诱导的ALI大鼠肺组织p-ERK蛋白表达(P0.05);地塞米松在注射LPS后4h时能显著抑制p-ERK的蛋白表达。结论地塞米松抑制LPS诱导的ALI大鼠肺组织通透性增加和ERK的表达,可能这种通过抑制ERK活化的作用,对ALI的预后有益。     

Resolvin D1 protects mice from LPS-induced acute lung injury

Resolvin D1 (RvD1), an endogenous lipid molecule derived from docosahexaenoic acid (DHA), has been described to promote inflammatory resolution. The present study aimed to determine the protective effects and the underlying mechanisms of RvD1 on lipopolysaccharide (LPS)-induced acute lung injury (ALI). Pretreatment RvD1 to mice 30 min before inducing ALI by LPS decreased the mortality and improved lung pathological changes, inhibited LPS-induced increases in polymorphonulear and mononuclear leukocytes recruitment, total proteins content, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) production in the bronchoalveolar lavage fluids (BALFs). In addition, RvD1 markedly reduced LPS-induced the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and adhesion molecules, as well as myeloperoxidase (MPO) activity. Moreover, RvD1 markedly inhibited LPS-induced the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). Furthermore, pretreatment with Boc, a lipoxin A4 receptor (ALX) antagonist, significantly reversed these beneficial effects of RvD1 on LPS-induced acute lung injury in mice. Taken together, our study showed that RvD1 improved survival rate and attenuated ALI in mice induced by LPS, and the protective mechanisms might be related to selective reaction with ALX, which inhibits MAPKs and NF-κB pathway.     

红景天苷对脂多糖所致急性肺损伤治疗作用的研究

目的:观察红景天苷（SDS）对脂多糖（LPS）引起的大鼠急性肺损伤（ALI）的治疗作用,并初步探讨其作用的机制。方法: 将36只SD大鼠随机分为生理盐水（NS）对照组、LPS组和LPS+SDS组(每组n=12),通过向气管内滴注LPS建立大鼠ALP模型。采用微量注射泵向大鼠右颈外静脉注射液体并留置的给药方法,NS组和LPS组注射NS并留置4 h;LPS+SDS组先于气管内滴注LPS,0.5 h后再注射SDS并留置4 h。4.5 h后处死实验动物,取肺组织观察其形态学改变,测定肺湿/干质量的比值（W/D）、肺泡灌洗液（BALF）中蛋白的含量及肺组织匀浆中TNF-α、IL-6、IL-10、乳酸脱氢酶(LDH)和髓过氧化物酶(MPO)的含量。结果: 形态学观察表明,LPS组肺组织水肿,有点、片状出血及大量的炎性细胞浸润,肺泡间隔显著增厚,肺泡腔变窄、结构消失。LPS+SDS组的肺组织损伤明显减轻,肺组织结构趋于正常,肺泡腔及支气管腔炎细胞及渗出物与LPS组相比明显减少。LPS组肺W/D与NS组相比明显增加（P<0.05）,BALF中蛋白的含量显著增加（P<0.05）,肺组织匀浆中TNF-α、IL-6、IL-10、LDH和MPO的含量显著增加（P<0.05）;而给予SDS治疗后,肺W/D、BALF中蛋白含量、肺组织匀浆中TNF-α、IL-6、LDH和MPO的含量与LPS组相比均明显减少（P<0.05）,IL-10含量显著增加（P<0.05）。结论: SDS对LPS所致ALI具有治疗作用,这种治疗作用可能与其通过抑制肺组织中炎症介质的作用有关。     

Protective effect of bicyclol on lipopolysaccharide-induced acute lung injury in mice

Bicyclol is synthesized based on schisandrin, which is one of the main active components of Chinese herb Fructus Schisandrae. The purpose of this study is to investigate whether bicyclol has a beneficial effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Bicyclol was given to mice by gavage for three times. ALI was induced by vena caudalis injection of LPS. The last dose of bicyclol was administrated 1 h before LPS given. Mice in each group were sacrificed at different time point after LPS administration. As revealed by survival study, pretreatment with high doses of bicyclol reduced the mortality of mice from ALI. Bicyclol pretreatment significantly improved LPS-induced lung pathological changes, inhibited myeloperoxidase (MPO) activity, and reduced lung/body and lung wet/dry weight ratios. Bicyclol also inhibited the release of TNF-α, IL-1β and HMGB1, whereas simultaneously increased the expression of IL-10. Furthermore, the phosphorylation level of NF-κB p65 was markedly decreased by bicyclol. Taken together, our study showed that bicyclol improves survival rate and attenuates LPS-induced ALI. The protective mechanism may be due to the inhibition of NF-κB activation and regulation of cytokine secretion.     

胰岛素对内毒素所致的急性肺损伤的防治作用

目的 观察胰岛素（insulin,ISL）对内毒素（LPS）引起的兔急性肺损伤（ALI）的预防与治疗作用,并初步探讨其作用机制。方法 将新西兰兔随机分为生理盐水（NS）对照组、LPS组、ISL+LPS组及LPS+ISL组,气管内滴注LPS建立兔ALI模型。采用微量注射泵经兔耳缘静脉滴注不同的液体,NS组和LPS组:滴注生理盐水;ISL+LPS组:滴注ISL混合液（ISL+葡萄糖+KCl）0.5 h后,再于气管内滴注LPS;LPS+ISL组:先经气管内滴注LPS 0.5 h后,再滴注ISL混合液。滴注时间持续4 h,期间监测兔平均颈总动脉压（mCAP）、血糖及血钾的变化。实验结束后处死实验动物,取肺组织观察形态学改变、测定肺湿/干质量的比值（W/D）,并分别用亚硝酸盐显色法和硫代巴比妥法测定肺组织匀浆中超氧化物歧化酶（SOD）和丙二醛（MDA）的含量及活性。结果 LPS组兔随时间 mCAP逐渐下降,实验结束时下降至(8.09±1.12)kPa,与NS组相比差异显著(P<0.05)。应用ISL预防和治疗后, mCAP下降的趋势缓慢,基本保持平稳,与LPS组相比有显著性差异（P<0.05）。LPS组兔的血糖随时间延长逐渐增高,实验结束时达峰值至(12.8±5.6)mmol/L,与NS组相比差异显著（P<0.05）。应用ISL预防和治疗后,兔的血糖随时间变化不明显,与LPS组相比差异明显（P<0.05）。实验期间各组兔的血钾基本保持平稳。形态学观察表明,LPS组肺组织水肿有点、片状出血,大量炎性细胞浸润,肺泡间隔显著增厚,肺泡腔变窄,结构消失。ISL+LPS组及LPS+ISL组的肺损伤明显减轻,肺组织结构均趋于正常;肺泡腔及支气管腔炎细胞及渗出物明显减少。与NS组相比,LPS组兔的肺W/D明显增加（P<0.05）,肺组织匀浆中SOD的活性显著降低（P<0.05）,MDA的含量显著升高（P<0.05）;而ISL+LPS组及LPS+ISL组兔的肺W/D明显减少（P<0.05）,肺组织匀浆中SOD的活性显著增加（P<0.05）,MDA的含量显著降低（P<0.05）。结论 ISL对LPS所致兔ALI具有预防与治疗的作用,其作用可能与抑制肺组织中的氧化应激作用有关。     

Inhibition of cyclooxygenase-2 by natriuretic peptides

The atrial natriuretic peptide (ANP) has been suggested to possess immunomodulatory potential because of its property to alter macrophage functions via its guanylate-cylcase- coupled A-receptor (NPR-A), such as inhibiting the expression of inducible nitric oxide synthase or TNF-alpha. The aim of this study was to investigate whether ANP influences COX-2. COX-2 expression in murine macrophages and in mice was induced by lipopolysaccharide. Release of PGE(2) and thromboxane B(2) was significantly reduced in the presence of ANP. C-type natriuretic peptide (CNP) also significantly reduced PGE(2)-accumulation in macrophages. Northern and Western blots showed that ANP attenuates COX-2 mRNA and protein. Reduction of neither COX-2 nor of PGE(2) production was significantly abrogated by an NPR-A antagonist, suggesting a pathway independent of cGMP. Furthermore, dibutyryl-cGMP did not affect PGE(2)-accumulation. cANF, the specific ligand for the natriuretic peptide (NP) clearance-receptor (NPR-C), significantly inhibited PGE(2)-production. Because some biological activities of ANP have been reported to be mediated via an NPR-C-mediated inhibition of adenylate-cyclase, we determined cAMP levels. ANP, CNP, and cANF significantly attenuated intracellular cAMP. In summary, ANP was shown to attenuate PGE(2)-production of lipopolysaccharide-activated macrophages predominantly via the NP clearance-receptor. ANP reduces COX-2-protein and -mRNA levels. The inhibition seems to be mediated via NPR-C and related to an attenuation of cAMP production.     

β-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) occur in approximately 200,000 patients per year. Studies indicate that lung endothelium plays a significant role in ALI. The authors' recent in vitro studies demonstrate a novel mechanism of β-nicotinamide adenine dinucleotide (β-NAD)-induced protection against gram-positive (pneumolysin, PLY) and gram-negative (lipopolysaccharide, LPS) toxin-induced lung endothelial cell (EC) barrier dysfunction. The objective of the current study was to evaluate the protective effect of β-NAD against LPS-induced ALI in mice. C57BL/6J mice were randomly divided into 4 groups: vehicle, β-NAD, LPS, and LPS/β-NAD. After surgery, mice were allowed to recover for 24 hours. Evans blue dye-albumin (EBA) was given through the internal jugular vein 2 hours prior to the termination of the experiments. Upon sacrificing the animals, bronchoalveolar lavage fluid (BALF) was collected and the lungs were harvested. β-NAD treatment significantly attenuated the inflammatory response by means of reducing the accumulation of cells and protein in BALF, blunting the parenchymal neutrophil infiltration, and preventing capillary leak. In addition, the histological examination demonstrated decreased interstitial edema in the LPS/β-NAD specimens, as compared to the LPS-only specimens. The mRNA levels of the anti-inflammatory cytokines were up-regulated in the LPS group treated with β-NAD compared to the LPS-only-treated group. β-NAD treatment down-regulated the mRNA levels of the proinflammatory cytokines. These findings suggest that β-NAD could be investigated as a therapeutic option against bacterial toxin-induced lung inflammation and ALI in mice.     

Effects of intratracheal tumor necrosis factor-alpha plasmid vector on lipopolysaccharide lethality and lung injury in mice

Bacterial lipopolysaccharide (LPS) causes acute lung injury (ALI) and contributes to inflammation in the acute respiratory distress syndrome (ARDS) and sepsis, making mechanisms of resistance to LPS critically important in clinical settings. The authors postulated that intratracheal administration of a plasmid (pcDNA3. 0-rTNFalpha) encoding rat tumor necrosis factor-alpha (TNF-alpha) would increase resistance of mice to LPS-induced ALI or mortality. They investigated the time course and dose-response for development of LPS-induced ALI in C57/BL6 mice and sought possible protective effects of 100 microg pcDNA3.0-rTNFalpha intratracheally 1, 2, or 3 weeks before LPS challenge. Lung myeloperoxidase (MPO) activity and alveolar lavage fluid (BALF) cell counts increased significantly 48 hours after intraperitoneal (IP) LPS challenges. After pcDNA3.0-rTNFalpha pretreatment, mice challenged with LPS had lower lung/body weight ratios than mice treated with pcDNA3.0; however, other indices of lung injury did not differ. Survival of mice challenged with lethal IP LPS 2 weeks after intratracheal pcDNA3.0-rTNFalpha vector improved significantly, compared to mice pretreated with the control vector, pcDNA3.0. However, pcDNA3.0-pretreated mice tolerated LPS challenge less well than saline-pretreated controls. LPS causes neutrophilic lung injury and mortality, but pcDNA3.0-TNFalpha does not prevent ALI due to LPS. Intratracheal pcDNA3.0-rTNFalpha pretreatment significantly improves survival of mice after LPS challenge, compared to those pretreated with pcDNA3.0.     

Inhibition of HA Synthase 3 mRNA Expression, with a Phosphodiesterase 3 Inhibitor, Blocks Lung Injury in a Septic Ventilated Rat Model

Low-molecular-weight hyaluronan produced by hyaluronan synthase 3 (HAS3) has been shown to play a role in acute lung injury secondary to high-tidal-volume ventilation. Phosphodiesterase 3 inhibitors have been shown to decrease HAS3 expression. We hypothesized that low-molecular-weight hyaluronan (LMW HA) produced by HAS3 mediates LPS-induced lung injury in the mechanically ventilated rat and that milrinone (MIL), by blocking HAS3 mRNA expression, would prevent the injury. Rats were randomized to four groups: controls with mechanical ventilation at 7 cc/kg MV, MV+LPS, MV+MIL, and MV+LPS+MIL. Rats were intubated and ventilated without PEEP for 4 h. Lipopolysaccharide (LPS) (1 mg/kg) was infused into the arterial line 1 h prior to MV. MIL 10 μg/kg/min (or an equivalent volume of saline) was infused through the venous line at the beginning of MV. Bronchoalveolar lavage fluid (BAL) was collected after 4 h of ventilation and lungs were saved for histopathology. LPS significantly increased neutrophil infiltration and protein concentration in the BAL and augmented lung injury score on histology. MIL significantly lowered alveolar protein and neutrophil infiltration as well as lung injury in response to LPS. Furthermore, MIL decreased the mRNA expression for HAS3 and MIP2 in lung tissue and decreased the protein content in BAL. MIL, a commonly used inotropic agent, inhibited LPS-induced lung inflammation and lung injury in mechanically ventilated rats. The anti-inflammatory properties of MIL may be mediated by inhibition of HAS3 and/or MIP2 and could be beneficial in the treatment of sepsis.     

Impairment of the natriuretic peptide system in follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause

Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [(125)I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.     

地塞米松对急性损伤大鼠肺组织细胞凋亡及Fas基因与Fas基 …

目的 探讨细胞凋亡与急性肺损伤（ＡＬＩ）发生、发展的关系以及地塞米松在全身炎症反应及急性肺损伤中的作用机制及意义。方法 通过复制内毒素性大鼠ＡＬＩ模型,采用ＴＵＮＥＬ法、原位杂交、ＳｑＲＴ－ＰＣＲ以及免疫组化等技术观察ＡＬＩ肺组织细胞凋亡变化、Ｆａｓ基因与Ｆａｓ基因配体系统ｍＲＮＡ和蛋白质表达的改变以及地塞米松对细胞凋亡及Ｆａｓ、ＦａｓＬ表达的影响。结果 ＡＬＩ早期肺组织细胞凋亡明显增加;Ｆdispla     

Exogenous porcine surfactants increase the infiltration of leukocytes in the lung of rats

BackgroundSeveral studies have investigated the influence of exogenous surfactants on inflammatory response in the lung, however results reported about effects of surfactants on the lung infiltration of leukocytes are controversial. Our previous study noticed that treatment of porcine surfactant (PS) significantly increased the lung infiltration of leukocytes in rats with acute lung injury (ALI). The objective of this study was to verify the effect of exogenous PS on the lung infiltration of leukocytes in vivo and investigate the possible mechanisms involved in vitro.MethodsThe number of leukocytes in bronchoalveolar lavage fluid (BALF) of rats with or without lipopolysaccharide (LPS)-induced ALI was determined after treatment with different concentrations of PS, dexamethasone (Dex) or PS + Dex. The effect of PS and Curosurf, a commercially available porcine surfactant, on human peripheral neutrophil migration was determined by the Boyden Chamber Assay.ResultsInstillation of PS significantly increased the number of leukocytes in BALF of normal rats and rats with LPS-induced ALI. Most of the increased leukocytes were neutrophils. Dex significantly decreased the number of leukocytes and TNF-α concentration in BALF caused by LPS, but did not significantly reduce the number of leukocytes increased by PS. In vitro experiments further demonstrated that both PS and Curosurf had direct chemotactic effects on neutrophils.ConclusionsThese results suggest that PS contain chemoattractant(s) which induce the infiltration of leukocytes, especially neutrophils, into lung.     

Phosphorylated ERM Mediates Lipopolysaccharide Induced Pulmonary Microvascular Endothelial Cells Permeability Through Negatively Regulating Rac1 Activity

IntroductionThe endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood.MethodsRat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1 mg/L, 1 mg/L, and 10 mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway.ResultsLPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other.ConclusionPhosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.     

Attenuation of lipopolysaccharide-induced acute lung injury by treatment with IL-10

Background and objective:   The aim of this study was to characterize the changes in neutrophils and cytokines in BAL fluid following acute lung injury (ALI), and to determine the protective effect of post-injury treatment with IL-10.
Methods:   A rat model of ALI was established by evenly spraying LPS (16 mg/kg) into the lungs followed by observation for 48 h. Histological changes and the kinetics of neutrophil infiltration were evaluated in the injured lungs. The cytokines (TNF-α, IL-6, IL-10 and interferon-γ) and macrophage-inflammatory protein (MIP-2) were measured in BAL fluid by ELISA. The activation of BAL fluid neutrophils was investigated after treatment with IL-10 in vitro . The protective effect on histology and MIP-2 levels of intra-tracheal instillation of IL-10 12 and 16 h after LPS treatment was studied in vivo.
Results:   Intra-tracheal instillation of LPS caused significant lung injury and the activation of neutrophils. The levels of TNF-α and IL-6 in BAL fluid peaked at 8 and 16 h after LPS instillation respectively. IL-10 levels reached a maximum at 16–24 h, at the beginning of resolution of tissue injury. IL-10 inhibited the activation of neutrophils in vitro and MIP-2 induction in vivo . IL-10 had a protective effect if it was administered 12 but not 16 h after LPS.
Conclusions:   Neutrophils appeared to play an important role in ALI. Time-dependent treatment with IL-10 after intra-tracheal instillation of LPS was effective in protecting rats from ALI, probably by suppressing pulmonary infiltration with activated neutrophils.     

Endothelial FAK as a therapeutic target in disease

Focal adhesions (FA) are important mediators of endothelial cytoskeletal interactions with the extracellular matrix (ECM) via transmembrane receptors, integrins and integrin-associated intracellular proteins. This communication is essential for a variety of cell processes including EC barrier regulation and is mediated by the non-receptor protein tyrosine kinase, focal adhesion kinase (FAK). As FA mediate the basic response of EC to a variety of stimuli and FAK is essential to these responses, the idea of targeting EC FAK as a therapeutic strategy for an assortment of diseases is highly promising. In particular, inhibition of FAK could prove beneficial in a variety of cancers via effects on EC proliferation and angiogenesis, in acute lung injury (ALI) via the attenuation of lung vascular permeability, and in rheumatoid arthritis via reductions in synovial angiogenesis. In addition, there are potential therapeutic benefits of FAK inhibition in cardiovascular disease and diabetic nephropathy as well. Several drugs that target EC FAK are now in existence and include agents currently under investigation in preclinical models as well as drugs that are readily available such as the sphingolipid analog FTY720 and statins. As the role of EC FAK in the pathogenesis of a variety of diseases continues to be explored and new insights are revealed, drug targeting of FAK will continue to be an important area of investigation and may ultimately lead to highly novel and effective strategies to treat these diseases.     

内毒素致大鼠急性肺损伤模型的建立

目的建立经尾静脉注射脂多糖致大鼠急性肺损伤动物模型。方法大鼠尾静脉注射5 mg/kg脂多糖,造成急性肺损伤,于注射脂多糖2 h后,分别测定动脉血气分析、血清蛋白含量、肺泡灌洗液中蛋白、细胞间粘附分子-1的含量、肿瘤坏死因子-α、白介素-8,计算肺湿/干重比、肺通透指数,观察肺组织病理学改变。实验设置生理盐水对照组。结果肺组织病理切片提示脂多糖组肺间质大量炎性细胞浸润,出血、水肿,而生理盐水组肺组织损伤轻微。对照组肺湿/干重比、肺泡通透指数、肺泡灌洗液中中性粒细胞比均显著低于脂多糖组(P<0.05),而动脉血氧分压则高于脂多糖组(P<0.05)。脂多糖组肺泡灌洗液中肿瘤坏死因子-α、细胞间粘附分子-1、白介素-8浓度高于对照组(P<0.05)。结论此模型基本符合急性肺损伤诊断标准及动物模型的考察指标符合临床,说明本模型是成功可靠的。     

HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption

Acute lung injury (ALI) results from loss of alveolar-capillary barrier integrity and the evolution of high-permeability pulmonary edema resulting in alveolar flooding and significant morbidity and mortality. HMGB1 is a late mediator of sepsis which uniquely participates in the evolution of sepsis and sepsis-induced ALI. The molecular events by which HMGB1 contributes to ALI remain poorly characterized. We characterized the role of HMGB1 in endothelial cell (EC) cytoskeletal rearrangement and vascular permeability, events essential to paracellular gap formation and barrier dysfunction characteristic of ALI. Initial experiments demonstrated HMGB1-mediated dose-dependent (5-20 μg/ml) decreases in transendothelial cell electrical resistance (TER) in the human pulmonary artery EC, a reflection of loss of barrier integrity. Furthermore, HMGB1 produced dose-dependent increases in paracellular gap formation in concert with loss of peripheral organized actin fibers, dissociation of cell-cell junctional cadherins, and the development of central stress fibers, a phenotypic change associated with increased contractile activity and increased EC permeability. Using siRNA strategies directed against known HMGB1 receptors (RAGE, TLR2, TLR4), we systematically determined that the receptor for advanced glycation end products (RAGE) is the primary receptor signaling HMGB1-induced TER decreases and paracellular gap formation via p38 MAP kinase activation and phosphorylation of the actin-binding protein, Hsp27. These studies add to the understanding of HMGB1-induced inflammatory events and vascular barrier disruption and offer the potential for clinical intervention in sepsis-induced ALI.