Effect of a neutrophil elastase inhibitor on ventilator-induced lung injury in rats

Objective

We hypothesized that pretreatment with sivelestat therapy could attenuate ventilator-induced lung injury (VILI) and lung inflammation in a rat model.

Methods

The neutrophil elastase inhibitor was administered intraperitoneally 30 min before and at the initiation of ventilation. The rats were categorized as (I) sham group; (II) VILI group; (III) sivelestat group; (IV) early sivelestat group. Wet-to-dry weight ratio, bronchoalveolar lavage fluid (BALF) neutrophil and protein, tissue malondialdehyde (MDA) and histologic VILI scores were investigated.

Results

The ratio of wet-to-dry weight, BALF neutrophil and protein, tissue MDA and VILI scores were significantly increased in the VILI group compared to the sham group [3.85±0.32 vs. 9.05±1.02, P<0.001; (0.89±0.93)×10⁴ vs. (7.67±1.41)×10⁴ cells/mL, P<0.001; 2.34±0.47 vs. 23.01±3.96 mg/mL, P<0.001; 14.43±1.01 vs. 36.56±5.45 nmol/mg protein, P<0.001; 3.78±0.67 vs. 7.00±1.41, P<0.001]. This increase was attenuated in the early sivelestat group compared with the sivelestat group [wet-to-dry ratio: 6.76±2.01 vs. 7.39±0.32, P=0.032; BALF neutrophil: (5.56±1.13)×10⁴ vs. (3.89±1.05)×10⁴ cells/mL, P=0.021; BALF protein: 15.57±2.32 vs. 18.38±2.00 mg/mL, P=0.024; tissue MDA: 29.16±3.01 vs. 26.31±2.58, P=0.049; VILI scores: 6.33±1.41 vs. 5.00±0.50, P=0.024].

Conclusions

Pretreatment with a neutrophil elastase inhibitor attenuates VILI in a rat model.     

呼吸机所致肺损伤的生物伤机制与易感性

呼吸机所致肺损伤（VILI）的生物伤机制颇受关注。机械牵拉能刺激肺泡上皮细胞和肺内皮细胞、活化多形核白细胞和肺泡巨噬细胞，激活系列信号转导通路，导致细胞因子的释放、黏附分子的表达、细胞外基质及其降解酶的改变、花生四烯酸通路的活化、氧化与抗氧化失衡、核苷酸谱改变、凝血与纤溶异常、凋亡与坏死的变化。VILI存在诸多易感因素，如局部缺血和再灌注、体温、年龄、血流速度、内毒素等。     

Heliox attenuates lung inflammation and structural alterations in acute lung injury

Low-density gas mixtures, such as heliox, were shown to reduce the work of breathing and facilitate the distribution of inspired gas. Since supplemental ventilatory and oxygen requirements may lead to pulmonary inflammation and structural alterations, we hypothesized that by reducing these requirements, heliox breathing may attenuate the acute inflammatory and structural changes associated with acute lung injury. Spontaneously breathing neonatal pigs were anesthetized, instrumented, supported with continuous positive airway pressure (CPAP), injured with oleic acid, and randomized to nitrox (n = 6) or heliox (n = 5).F(I)O(2) was titrated for pulse oximetry (SpO(2)) 95 +/- 2% for 4 hr. Gas exchange and pulmonary mechanics were measured. Lungs were analyzed for myeloperoxidase (MPO), interleukin-8 (IL-8), and histomorphometery. Relationships between physiologic indices and cumulative lung structure and inflammatory indices were evaluated. With heliox, compliance was significantly greater, while tidal volume, frequency, minute ventilation, F(I)O(2), arterial carbon dioxide tension (PaCO(2)), MPO, and IL-8 were significantly lower compared to nitrox. The expansion index and number of exchange units were significantly greater with heliox, while the exchange unit area (EUA) was smaller. MPO was significantly and positively correlated with F(I)O(2) (r = 0.76) and EUA (r = 0.63), and negatively correlated with number of open exchange units/field (r = -0.73). Compared to breathing nitrox, these data indicate that heliox improved the distribution of inspired gas, thereby recruiting more gas exchange units, improving gas exchange efficiency, reducing ventilatory and oxygen requirements, and attenuating lung inflammation. These data suggest that heliox breathing may have the combined therapeutic benefits of attenuating lung inflammation by reducing mechanical and oxidative stress in the clinical management of acute lung injury.     

依布硒啉对急性肺损伤大鼠氧化应激的影响

目的 观察依布硒啉对内毒素性急性肺损伤的保护作用.方法 健康雄性SD大鼠,随机分成6组:正常对照组、模型组、依布硒啉高剂量组、中剂量组、低剂量组和地塞米松对照组.通过尾静脉注射LPS(5 mg/kg)建立模型,治疗组于造模前30 min,大鼠腹腔注射依布硒啉(7.5 mg/kg、15 mg/kg、30 mg/kg),对照组和模型组分别注入等量溶剂.造模后6h,麻醉放血处死动物,并取肺组织,测定肺湿质量/干质量.采用硫代巴比妥酸和黄嘌呤氧化法分别检测肺组织中超氧化物歧化酶SOD活性和丙二醛MDA及髓过氧化物酶(MPO)含量.光镜下观察肺组织病理学改变.结果 依布硒啉治疗组与模型组相比,肺湿质量/干质量降低,肺组织SOD的活性明显增高,MDA和MPO含量显著降低(P＜0.05),病理改善明显.结论 依布硒啉对内毒素性急性肺损伤有一定保护作用,其机制可能与提高抗氧化能力有关.     

内皮素-1在呼吸机所致肺损伤犬肺中的表达变化研究

目的 检测内皮素-1(ET-1)在呼吸机所致肺损伤(VILI)模型肺组织的表达分布及变化.方法 普通级犬随机分为正常对照组(N组,n=6)、急性肺损伤(ALI)组(n=14).用油酸静脉内注射制备ALI模型,制备成功后随机取2只作为ALI组,其他随机分为小潮气量机械通气组(LV组,n=6)、大潮气量机械通气组(即VIL...     

骨髓间充质干细胞对急性肺损伤大鼠氧化应激的影响

目的观察骨髓间充质干细胞对内毒素性急性肺损伤的保护作用及机制。方法健康4月龄SPF级雄性C57BL/6J小鼠8只,用于骨髓间充质干细胞分离培养;SPF级雄性SD大鼠90只,随机分成:正常对照组(A组);内毒素组(B组)、骨髓间充质干细胞高剂量组(C组)、骨髓间充质干细胞低剂量组(D组)、骨髓间充质干细胞对照组(E组)。于造模后6、24、72小时,处死动物,每个时间点6只。分别取肺组织,测定肺湿质量/干质量。检测肺组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)及髓过氧化物酶(MPO)含量。观察肺组织病理学改变。结果 LPS的24小时组死亡1只,两个骨髓间充质干细胞治疗组与模型组相比,肺组织SOD的活力明显增高,MDA和MPO含量显著降低(P<0.05);病理明显的改善,肺湿质量/干质量降低。正常对照组和骨髓间充质干细胞组相比,上述指标差异无统计学意义。结论骨髓间充质干细胞对内毒素性急性肺损伤有一定保护作用,其机制与抗氧化能力有关。不同种属骨髓间充质干细胞移植后,未见明显损伤。     

Neutrophil elastase activity in acute lung injury and respiratory distress syndrome

Background and objective:   Neutrophil elastase (NE) may play a key role in the development of acute lung injury (ALI) or ARDS. NE activity (NEA) was measured in patients with ALI treated with a selective NE inhibitor.
Methods:   NEA and NE-α1-antitrypsin (NE-AT) complex were measured in plasma before, during and after the administration of the selective NE inhibitor, sivelestat, in 32 patients with a diagnosis of ALI or ARDS. NEA index (NEAI) was calculated as NEA/NE-AT. The sequential organ failure assessment (SOFA) score and the ratio PaO₂/fraction of inspired oxygen (FiO₂) were measured.
Results:   NEA and NE-AT was raised in all patients. Sivelestat reduced NEAI and NEA ( P  < 0.01 for both) but not NE-AT and NEA, and NEAI returned to pretreatment levels. NEA correlated closely with NE-AT before, but not after treatment. No relationship was observed between these indices and SOFA score or PaO₂/FiO₂ ratio.
Conclusions:   Sivelestat reduced NEA and NEAI in patients with ALI or ARDS suggesting NE inhibition. A larger study is needed to determine the relationship of NEA, NE-AT and NEAI with the outcome of ALI/ARDS.     

Calcitonin gene-related peptide mediates acid-induced lung injury in mice

BACKGROUND AND OBJECTIVE: Acid-induced lung injury from aspiration is one of the most important causes of ARDS. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has various biological actions. The current study investigated whether CGRP might have pathophysiological roles in acid-induced lung injury. METHODS: The investigations employed CGRP gene-disrupted mice--mutant mice (CGRP(-/-)) and their littermate controls (CGRP(+/+)). Anaesthetized and mechanically ventilated mice received 2 mL/kg HCl (pH = 1.5) intratracheally. Lung wet-to-dry weight ratios were calculated to assess pulmonary oedema, total and differential cell counts of the BALF were determined, and measurements of myeloperoxidase activity were performed. RESULTS: Acid-induced lung injury was characterized by an increase in lung permeability and respiratory failure. Disruption of the CGRP gene significantly attenuated acid-induced injury, oedema and respiratory failure. CONCLUSIONS: This study suggests that CGRP is involved in the pathogenesis of acute lung injury caused by acid aspiration and CGRP mutant mice may provide an appropriate model to study molecular mechanisms in this context.     

Patient-specific transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-associated mortality. The inadvertent transfusion of neutrophil antibodies can cause pulmonary transfusion reactions and TRALI. However, not all patients transfused with neutrophil antibodies experience transfusion reactions. A 22-year-old man with severe aplastic anaemia (SAA) experienced TRALI after a platelet transfusion. The donor was found to be alloimmunized to human neutrophil antigen (HNA)-3a, an antigen expressed by neutrophils from approximately 90% of Caucasians. Eleven other platelet components from this donor were transfused prior to this event and two caused reactions: one chills and one TRALI. Both episodes of TRALI occurred in the same male patient with SAA. The fact that one patient experienced TRALI following both exposures to anti-HNA-3a from the same donor whereas nine other recipients did not adds evidence to the observation that patient factors make a significant contribution to neutrophil antibody-mediated transfusion reactions.     

N-乙酰半胱氨酸对急性肺损伤的防治作用研究进展

N-乙酰半胱氨酸是L-半胱氨酸的乙酰化物,作为巯基的供体,不仅具有强大的抗氧化作用,还能有效补充机体内降低的还原型谷胱苷肽,保护细胞免受损伤,其对急性肺损伤的防治作用已获得动物实验和临床研究结果的支持。     

氨溴索对大鼠胃内容物吸入性肺损伤细胞凋亡的影响

目的研究细胞凋亡在胃内容物吸入性肺损伤中的作用机制,并初步探讨氨溴索对肺损伤发生后细胞凋亡的影响。方法 30只健康Sprague-Dawley大鼠随机分为3组:对照组、损伤组、氨溴索组。采用蛋白免疫印迹法(Western blot)检测Bc12(B cell lymphoma/lewkmia-2)蛋白、Bax(Bc12 associated x protein)蛋白及Caspase-3蛋白表达;DNA原位末端标记(TUNEL)法测定肺组织细胞凋亡指数。结果与对照组比较,损伤组中Bax蛋白、Caspase-3蛋白表达增多,细胞凋亡指数增高;Bc12蛋白表达减少;差异均显著(P<0.05)。与损伤组比较,氨溴索组Bax蛋白、Caspase-3蛋白表达减少,细胞凋亡指数降低;Bc12蛋白表达增高;差异均显著(P<0.05)。结论细胞凋亡参与了胃内容物吸入后肺损伤发病过程,氨溴索在吸入性肺损伤组织中发挥抗细胞凋亡作用,可能机制在于调控死亡信号通路中Bax/Bc12蛋白表达相关。     

大鼠棉花烟雾吸入性肺损伤中的氧化应激

目的 探讨大鼠棉花烟雾吸入性肺损伤中的氧化应激反应机制.方法 18只雄性SD大鼠随机分成对照组、6h组、24 h组,每组6只.复制大鼠棉花烟雾吸入性肺损伤模型,6h组、24 h组大鼠分别在烟雾吸入后6h或24 h行安乐死,ELISA法检测大鼠肺组织丙二醛(MDA)、谷胱甘肽(GSH)、一氧化氮(NO)、诱导型一氧化氮合酶(iNOS)、γ-谷氨酰半胱氨酸合成酶(γ-GCS)浓度,荧光定量PCR法行肺组织iNOS mRNA、γ-GCS mRNA定量.结果 6h组、24 h组大鼠肺组织匀浆中的MDA、NO、iNOS、γ-GCS浓度、iNOS mRNA和γ-GCS mRNA的相对表达量均高于对照组,且NO浓度、iNOSmRNA和γ-GCS mRNA的相对表达量24 h组高于6h组(P值均＜0.05);而6h组和24 h组GSH浓度均低于对照组,且24 h组低于6h组(P值均＜0.05).结论 在大鼠棉花烟雾吸入性肺损伤中,氧化应激反应加剧,同时抗氧化体系的激活不足以对抗氧化应激损伤,氧化/抗氧化体系失衡,导致肺损伤逐渐加重.     

Heat shock response decreases endotoxin-induced acute lung injury in rats

OBJECTIVE: Transient whole-body hyperthermia was reported to reduce lung damage in a rat with intra-abdominal sepsis produced by caecal perforation. METHODOLOGY: In order to determine the effect of heat shock response on acute lung injury induced by endotoxin, which plays a central role in the pathogenesis of sepsis, we instilled either saline or lipopolysaccharide (LPS) intravenously with and without heat pretreatment in rats. The heated rats had their rectal temperature raised to more than 40 degrees C for 13 min 18 h before intravenous administration of saline or LPS. RESULTS: We found that the lung leak was significantly increased among the rats given LPS intravenously with (median, 0.17; range, 0.15-0.22; n = 10) and without heat pretreatment (0.23; 0.17-0.30; n = 10) compared with those of saline-treated rats (0.13; 0.10-0.14; n = 10) (P < 0.05 in each). However, rats given LPS after heat pretreatment had significantly decreased lung leak index compared with those of LPS-treated rats without heat pretreatment (P < 0.05). Rats administered LPS intravenously showed increased myeloperoxidase activity without heat pretreatment (19.01; 9.34-28.00 U/g; n = 10) compared with that of saline-treated rats (7.09; 4.49-10.56 U/g; n = 5) (P < 0.05) (Fig. 2). Myeloperoxidase activity of the rats treated with LPS with heat pretreatment (5.57; 2.87-8.96 U/g; n = 10) was significantly decreased to the level of normal control compared with that of LPS-treated rats without heat pretreatment (P < 0.05). The levels of heat shock proteins (HSP72) in lung tissue, which were examined by western blot analysis, were increased over baseline levels at 23 h after hyperthermic stress. CONCLUSIONS: These observations show that brief heat shock response is associated with the induction of HSP72 protein synthesis and attenuated neutrophil recruitment and acute lung leak is induced by endotoxin in rats.     

Antibody-mediated (immune) transfusion-related acute lung injury

Antibody-mediated transfusion-related acute lung injury (immune TRALI) is now recognized as the most common cause of transfusion-associated major morbidity and death in the Western world. Among the implicated leucocyte antibodies, these ones directed against the human leucocyte antigens (HLA) class II, human neutrophil alloantigens (HNA)-3a and HLA-A2 antigens are frequently associated with severe (artificial ventilation required) and fatal cases. There is accumulating evidence that preventive measures such as transfusion of plasma-rich blood components from male donors or from donors tested negative for leucocyte antibodies are effective in the reduction of severe and fatal immune TRALI.     

基因组学技术在机械通气相关性肺损伤基因活化中的应用

机械通气是临床广泛应用的通气方式,它大大增加了重危患者抢救的成功率。然而,不合理的应用也可能产生和加重原有肺损伤,引起机械通气相关性肺损伤（VILI）。目前,VILI的基因表达及基因产物研究取得了很大进展,如早期诱导基因活化、临床敏感基因定位、基因信号传导通路等。而探讨基因组学技术在VILI中病理生理机制的应用,丰富和完善VILI理论,并为VILI提供有效的药物治疗方法。通过识别VILI中的相关因素,我们可利用其对VILI的危险人群进行识别。随着VILI的学科机制的完善与发展,关于VILI发病机制的完整性描述将会成为可能。     

TGF-β1/Smad2信号通路参与海水淹溺肺损伤引起的细胞凋亡

目的探讨TGF-β1/Smad2信号通路是否参与海水淹溺肺损伤引发的细胞凋亡过程。 方法体外培养A549人肺癌细胞,用浓度为20%、40%、60%的海水进行处理,根据MTT实验结果,筛选出最适浓度的海水用于后续研究;用抑制剂SB431542预处理细胞后,再用最适浓度的海水进行处理,然后采用Real-time PCR与Western blot检测凋亡相关指标Cleaved Caspase-3的表达水平、流式细胞术与Hoechst染色观察细胞凋亡情况,同时应用Real-time PCR与Western blot对TGF-β1、Smad2和p-Smad2的表达水平进行检测。 结果随着海水浓度的增加,A549细胞的活力逐渐降低,最终选用浓度为40%的海水进行后续研究;海水处理可使细胞发生明显的凋亡现象,而抑制剂SB431542可明显恢复由海水引发的凋亡水平;同时,发现海水使TGF-β1的表达水平显著上调,抑制剂SB431542处理后这种异常上调可显著被缓解,而Smad2的表达水平无显著性变化,但海水可使p-Smad2表达水平显著上调,且抑制剂SB431542处理后p-Smad2的表达水平可明显被抑制。 结论海水淹溺肺损伤可能通过激活TGF-β1/Smad2信号通路,调节凋亡执行蛋白Caspase-3的活性,从而促使肺组织细胞发生凋亡。     

The effects of neutrophil depletion on a completely noninvasive model of acute pancreatitis-associated lung injury

Summary Conclusion A completely noninvasive animal model of acute pancreatitis-associated lung injury was used to show that neutrophils, activated by pancreatitis, play a key role in mediating pancreatitis-associated lung injury. Background Significant pulmonary complications have been known to occur in over 50% of patients with severe acute pancreatitis. Recent studies using a variety of animal models of pancreatitis have suggested that neutrophil activation may play an important role in mediating lung injury. However, in these models, the interpretation of the results is complicated because surgical manipulations alone could have resulted in the activation of neutrophils. Methods Young female mice were fed a choline deficient ethionine (CDE) supplemented diet. The severity of pancreatitis was evaluated by measuring hyperamylasemia, acinar cell necrosis, and pancreatic myeloperoxidase activity. Lung injury was quantified by measuring lung microvascular permeability and lung myeloperoxidase activity. To evaluate the role of neutrophils in CDE diet-induced pancreatitis-associated lung injury, animals were pretreated with antineutrophil serum. Results Mice fed the CDE diet develop pancreatitis-associated lung injury. Pretreatment of mice with antineutrophil serum results in marked depletion of circulating neutrophils. Under these conditions, the severity of pancreatitis is reduced and lung injury is completely prevented.     

Caspase inhibitor zVAD-fmk protects against acute pancreatitis-associated lung injury via inhibiting inflammation and apoptosis

Background/objectivesPulmonary apoptosis is an important pathogenic mechanism of acute lung injury induced by many factors. This study aims to investigate whether the caspase inhibitor zVAD-fmk has a protective effect against lung injury in the severe acute pancreatitis model (SAP) in rats.MethodsSeventy-two Sprague-Dawley rats were randomly divided into Sham, SAP, and SAP + zVAD-fmk groups. The SAP model was established by injection of 5% sodium taurocholate into the pancreatic duct. Animals were sacrificed at 3 h, 6 h, 12 h, and 24 h after operation and then HE staining analysis was performed to assess the lung injury. ELISA was used to detect the activity of myeloperoxidase (MPO) and the concentrations of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Western blotting was used to detect the expression of cleaved caspase-3 in the lung tissues.ResultsRats in SAP group showed obvious lung injury through pathologic examination. Pretreatment with zVAD-fmk significantly inhibited a post-SAP increase in the activation of MPO, TNF-α, IL-1β, and caspase-3, and decreased lung injury induced by SAP as determined by the pathologic score.ConclusionOur results suggest that apoptosis plays an important role in acute pancreatitis-associated lung injury (APALI), and inhibition of caspase activity may represent a new therapeutic approach for the treatment of APALI.     

地塞米松通过加强自噬和抑制凋亡减轻海水吸入型肺损伤

目的 探讨海水吸入型肺损伤中自噬过程,以及地塞米松对其干预作用.方法 40只大鼠完全随机分为正常对照组、1、3、6、12 h海水处理组,每组8只.采用气管内滴注海水（3ml/kg）的方法制作海水吸入型急性肺损伤大鼠模型,观察病理变化,检测肺组织湿干比,western blot检测beclin-1的表达变化.体外实验A549细胞分为正常对照组、12 h海水组、地塞米松＋12 h海水组、3-MA＋ 12 h海水组,采用western blot检测beclin-1和bax的表达变化.结果 海水吸入后,大鼠肺部严重损伤,水肿明显,beclin-1随损伤时间延长表达量增加,在海水处理后12 h达到高峰.体外实验中海水刺激增加了beclin-1和bax的表达,然而与海水组相比,地塞米松预处理增加了beclin-1的表达,同时使bax的表达下降.3-MA预处理则效果相反.结论 自噬参与了海水吸入型肺损伤的修复过程,地塞米松能够通过加强自噬并且抑制凋亡减轻肺损伤.     

人IL-10对呼吸机相关性肺损伤大鼠炎性细胞因子的影响

目的：探讨人白介素10(hIL-10)预先给药对呼吸机相关性肺损伤(VILI)大鼠炎性因子的影响。方法健康雄性 SD大鼠36只,体质量220~300 g,随机分为3组(n=12)：对照组(C组)、大潮气量机械通气组(H 组)和 hIL-10干预组(HI组)。其中C组经口插管后维持自主呼吸4 h;H 组采用大潮气量机械通气建立大鼠 VILI 模型,阿曲库铵静脉输注抑制自主呼吸、维持肌松,接小动物呼吸机机械通气,呼吸参数设定：呼吸频率40次/min,通气时间4 h,吸呼比为1∶3,呼气末正压通气0 cmH2 O,吸入氧浓度为21%,潮气量30 ml/kg,根据体质量调节潮气量;HI组在大潮气量机械通气开始前30 min从尾静脉注射 hIL-10(剂量为8000 U/kg),余同 H 组,C组、H 组给予等量的生理盐水。各组于通气4 h后放血处死大鼠,收集大鼠血清和 BALF,采用酶联免疫吸附测定法检测肿瘤坏死因子α(TNF-α)、IL-8和细胞间黏附分子1(ICAM-1)的浓度;取右上肺组织做病理切片,HE染色镜下观察形态学变化。结果光镜下观察显示 C组肺泡结构无明显损伤;H 组肺泡结构破坏严重,肺泡内渗出物、出血和间质水肿明显,可见明显肺泡腔融合,肺间隔增宽,大量炎性细胞浸润;HI组肺组织结构有一定程度损伤,肺间隔有少量增厚,但较 H 组减轻。与C组相比,H 组、HI组血清和BALF中TNF-α、IL-8和 ICAM-1水平均升高(P <0.05或 P <0.01);与 H 组相比, HI组血清和BALF中TNF-α、IL-8和 ICAM-1水平明显降低(P值均<0.01)。结论 hIL-10作为机体重要的抗炎因子,可通过调节肺组织炎症反应,降低炎性细胞因子水平,在一定程度上减轻大鼠VILI。