Prevention of ischemia reperfusion injury by positive pulmonary venous pressure in isolated rat lung

Pulmonary ischemia-reperfusion (I/R) without tissue hypoxia induces inflammatory cytokine mRNA expression in the lung under the condition of 0 mm Hg pulmonary venous pressure (0PVP), which might be a cause of I/R injury. Our aim is to determine whether the pulmonary vascular endothelium expresses cytokine mRNAs and their corresponding proteins or develops I/R injury when positive PVP is maintained during ischemia to provide a positive stretch to the endothelium throughout the ischemic period. In isolated, perfused, and ventilated rat lungs, the right and left pulmonary arteries were isolated, and the left lung was selectively occluded for 60 min and then reperfused for 30 min. During ischemia, the left atrial pressure was maintained at 5 mm Hg (5PVP) or 0PVP. TNF-alpha, IL-1beta, IL-6, and IL-10 mRNA expression in the lungs was evaluated by RT-PCR and in situ hybridization, and the production and localization of corresponding proteins were determined by staining with fluorescence-labeled antibodies against the cytokines and an antibody against CD34. Pulmonary vascular/epithelial permeability was evaluated by measuring albumin content in bronchoalveolar lavage (BAL) fluid and wet/dry ratio. At 5PVP, there were no increases in the left lung perfusion pressure, albumin content in BAL fluid, wet/dry ratio, or expression of cytokine mRNAs and their corresponding proteins on the vascular endothelium by I/R. In contrast, at 0PVP, the increased expression of cytokine mRNAs and their corresponding proteins on the vascular endothelium by I/R was verified. The finding that the application of 5PVP during ischemia abolished the expression of cytokine mRNAs and their corresponding proteins as well as the I/R injury gives us new insights in the study of lung preservation for transplantation.     

支气管肺泡灌洗对肠源性感染致早期肺损伤的诊断价值

目的　观察肠源性感染致肺损伤时炎症介质的变化 ,探讨支气管肺泡灌洗对早期肺损伤的诊断价值。方法　采用大鼠盲肠结扎并穿孔造成腹腔感染制备肠源性感染动物模型 ,并设假手术对照组。分别在术后 0、2 4、4 8、72、96和 12 0 h处死一组大鼠 ,取支气管肺泡灌洗液 (BAL F)进行细胞学分析 ,检测 BAL F、肺组织和血浆的内毒素、磷脂酶 A2 (PL A2 )和肿瘤坏死因子 α(TNFα)含量。结果　 BAL F的中性粒细胞百分率逐渐增加 ,时间越长越明显。BAL F、肺组织和血浆的内毒素、PL A2 逐渐增加 ,三者之间两两相关 (BAL F与肺 :r=0 .90 4 ,P<0 .0 5 ;BAL F与血浆 :r=0 .895 ,P<0 .0 5 ;肺与血浆 :r=0 .94 6 ,P<0 .0 1) ;TNFα也随时间的延长而逐渐增加 ,BAL F和肺组织的 TNFα显著相关 (r=0 .95 2 ,P<0 .0 1) ,两者与血浆的 TNFα无明显相关性 (r1 =0 .6 84 ,r2 =0 .6 0 8,P均 >0 .0 5 )。结论　支气管肺泡灌洗可发现早期的肠源性肺损伤。     

脓毒症大鼠肺组织中尿激酶型纤溶酶原激活剂受体的表达及其与肺损伤程度的相关性研究

目的探讨脓毒症大鼠肺组织中尿激酶型纤溶酶原激活剂受体（uPAR）表达变化及其与肺损伤的关系。 方法将48只雄性成年大鼠分为假手术组及脓毒症组,每组各24只大鼠。采用盲肠结扎穿孔术建立脓毒症损伤模型,假手术组大鼠只行开腹手术、不行结扎及穿孔,各组分别在术后6、12、24 h各取8只大鼠分批处死后收集肺泡灌洗液并留取肺脏。观察所有大鼠各时间点肺组织病理变化,测定肺组织干湿重比及肺泡灌洗液中蛋白变化,并采用免疫组织化学染色测定uPAR表达情况。 结果光镜下可见,假手术组大鼠肺组织肺泡结构完整,术后24 h只有少量炎症细胞浸润;脓毒症组大鼠肺泡腔内大量炎症细胞浸润,肺泡结构破坏,并以术后24 h最明显。两组大鼠各时间点肺组织干湿重比、肺泡灌洗液蛋白水平及uPAR表达比较,差异均有统计学意义（F = 32.000、97.770、72.780,P均< 0.001）。与假手术组相比,脓毒症组大鼠6、12、24 h肺组织干湿重比[（0.75 ± 0.03）vs.（0.78 ± 0.03）;（0.77 ± 0.04）vs.（0.83 ± 0.05）;（0.78 ± 0.04）vs.（0.89 ± 0.05）]、肺泡灌洗液蛋白水平[（79 ± 22）mg/L vs.（110 ± 40）mg/L;（79 ± 23）mg/L vs.（168 ± 36）mg/L;（94 ± 24）mg/L vs.（199 ± 56）mg/L]及uPAR表达[（2.2 ± 1.3）分vs.（4.6 ± 1.9）分;（2.3 ± 0.9）分vs.（6.4 ± 1.8）分;（2.4 ± 0.9）分vs.（7.0 ± 1.8）分]均明显升高（P均< 0.05）;且脓毒症组大鼠12、24 h亚组肺组织干湿重比、肺泡灌洗液蛋白水平均显著高于6 h亚组,而uPAR表达仅24 h亚组显著高于6 h亚组（P均< 0.05）。同时,肺组织中uPAR表达与肺组织干湿重比、肺泡灌洗液蛋白浓度均呈正相关（r = 0.618,P< 0.001;r = 0.652,P< 0.001）。 结论与假手术组相比,uPAR在脓毒症大鼠肺组织中表达明显增加,并且与肺损伤程度呈正相关。     

Role of p38 mitogen-activated protein kinase in lung injury after burn trauma

This study was undertaken to evaluate the effect of SB203580, a specific p38 mitogen-activated protein (MAP) kinase inhibitor, on burn-induced lung injury as well as the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in rats to characterize the role of p38 MAP kinase in lung injury after burn trauma. Sprague-Dawley rats were divided into three groups: 1) sham group, or rats who underwent sham burn; 2) control group, or rats given third-degree burns over 30% total body surface area (TBSA) and lactated Ringer solution for resuscitation; and 3) SB203580 group, or rats given burn injury and lactated Ringers solution with SB203580 inside for resuscitation. Pulmonary injury was assessed at 24 h by pulmonary capillary permeability determined with fluorescein isothiocyanate-labeled albumin and lung histologic analysis. TNF-alpha and IL-1beta protein in bronchoalveolar lavage fluid and serum were measured by enzyme-linked immunosorbent assay and p38 MAP kinase was activity determined in lung by Western blot analysis. These studies showed that significant activation of p38 MAP kinase at 24 h postburn compared with control. Burn trauma resulted in increased pulmonary capillary leakage permeability, elevated levels of TNF-alpha and IL-1beta in bronchoalveolar lavage fluid and serum, and worsened histologic condition. SB203580 inhibited the activation of p38 MAP kinase, reduced the levels of TNF-alpha and IL-1beta, and prevented burn-mediated lung injury. These data suggest that p38 MAP kinase activation is one important aspect of the signaling event that may mediate the release of TNF-alpha and IL-1beta and contributes to burn-induced lung injury.     

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。     

Hypercapnic acidosis modulates inflammation, lung mechanics, and edema in the isolated perfused lung

OBJECTIVE: Low tidal volume (V(T)) ventilation strategies may be associated with permissive hypercapnia, which has been shown by ex vivo and in vivo studies to have protective effects. We hypothesized that hypercapnic acidosis may be synergistic with low V(T) ventilation; therefore, we studied the effects of hypercapnia and V(T) on unstimulated and lipopolysaccharide-stimulated isolated perfused lungs. MATERIALS AND METHODS: Isolated perfused rat lungs were ventilated for 2 hours with low (7 mL/kg) or moderately high (20 mL/kg) V(T) and 5% or 20% CO(2), with lipopolysaccharide or saline added to the perfusate. RESULTS: Hypercapnia resulted in reduced pulmonary edema, lung stiffness, tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in the lavage and perfusate. The moderately high V(T) did not cause lung injury but increased lavage IL-6 and perfusate IL-6 as well as TNF-alpha. Pulmonary edema and respiratory mechanics improved, possibly as a result of a stretch-induced increase in surfactant turnover. Lipopolysaccharide did not induce significant lung injury. CONCLUSIONS: We conclude that hypercapnia exerts a protective effect by modulating inflammation, lung mechanics, and edema. The moderately high V(T) used in this study stimulated inflammation but paradoxically improved edema and lung mechanics with an associated increase in surfactant release.     

γ-干扰素对博莱霉素诱导大鼠肺损伤的影响

目的 观察γ-干扰素（IFN-γ）对博莱霉素（BLM）诱导大鼠肺损伤的影响及其作用机制。方法66只大鼠随机分为正常对照组（24只）、BLM组（24只）和IFN-γ组（18只）。分别观察气管内给药后14、17、21和28d肺组织损伤程度，以及血清和支气管肺泡灌洗液（BALF）中肿瘤坏死因子-α（TNF-α）、IFN-γ含量。结果 与正常对照组比较，BLM和IFN-γ组在给予BLM后17、21和28d肺组织损伤加重（P均〈0．05），IFN-γ组在给予BLM后21和28d肺损伤程度虽重于BLM组，但差异无显著性，IFN-γ组于17、21和28dBALF中TNF-α含量和21和28d血清中TNF-α含量均显著高于正常对照组和BLM组，21和28dBALF和血清中IFN-γ含量均显著高于其余两组（P均〈0．05）。结论 IFN-γ可能会加重BLM诱导大鼠肺损伤，其作用可能与TNF-α相互协作有关。     

橙皮苷对过敏性哮喘小鼠肺组织炎症及肺泡灌洗液中细胞因子水平影响

目的研究橙皮苷对过敏性哮喘小鼠肺组织炎症及肺泡灌洗液中细胞因子水平影响。方法实验分为:对照组、模型组、实验1组、实验2组和实验3组。模型组、实验1组、实验2组和实验3组构建哮喘模型,实验1组、实验2组和实验3组在实验第25天、26天、27天灌胃橙皮苷(浓度分别为:12 mg/kg、24 mg/kg、36 mg/kg),对照组给予等量的磷酸盐缓冲液(PBS)代替。对小鼠进行哮喘发作情况行为学评分和肺组织炎症评分,酶联免疫吸附(ELISA)检测肺泡灌洗液中白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-13(IL-13)、干扰素γ(IFN-γ)、白细胞介素-10(IL-10)水平,同时对肺泡灌洗液中炎性细胞数目进行分类计数,Western blot检测肺组织中NF-κBp65蛋白水平。结果与对照组比较,模型组小鼠哮喘发作情况行为学评分和肺组织炎症评分升高,肺泡灌洗液中IL-4、IL-5、IL-13水平也升高,肺泡灌洗液中炎性细胞总数、中性粒细胞数目、嗜酸性粒细胞数目、淋巴细胞数目升高,肺组织中NF-κBp65蛋白水平升高。橙皮苷灌胃处理后过敏性哮喘小鼠哮喘发作情况行为学评分、肺组织炎症评分降低,肺泡灌洗液中炎性细胞总数、中性粒细胞数目、嗜酸性粒细胞数目、淋巴细胞数目减少,IL-4、IL-5、IL-13水平降低,IFN-γ水平升高,IL-10水平也升高,与未经橙皮苷处理的哮喘小鼠比较,差异有统计学意义(P<0.05),呈剂量依赖性。结论橙皮苷能够呈剂量依赖性减轻过敏性哮喘小鼠炎症,调节肺泡灌洗液中细胞因子表达。     

Apocynin attenuates lipopolysaccharide-induced lung injury in an isolated and perfused rat lung model

Apocynin (Apo) suppresses the generation of reactive oxygen species that are implicated in lipopolysaccharide (LPS)-induced lung injury (LPSLI). We thus hypothesized that Apo may attenuate LPSLI. In addition, we explored the cellular and molecular mechanisms of Apo treatment in LPSLI. Lipopolysaccharide-induced lung injury was induced by intratracheal instillation of 10 mg/kg LPS in isolated and perfused rat lung model. Apocynin was administered in the perfusate at 15 min before LPS was administered. Hemodynamics, lung injury indices, inflammatory responses, and activation of apoptotic pathways were assessed. There was an increase in lung vascular permeability associated with lung weight gain after LPS exposure. The levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), macrophage inflammatory protein 2, H2O2, and albumin increased in the bronchoalveolar lavage fluid. Adhesion molecule of neutrophil (CD31) was upregulated. The expression of TNF-α, IL-1β, glutathione, myeloperoxidase, JNK, P38, caspase 3, p-AKT, and plasminogen activator inhibitor 1 in lung tissue was greater in the LPS groups when compared with the control group. Upregulation and activation of nuclear factor κB occurred along with increased histopathologic lung injury score in LPSLI. The Apo attenuated these inflammatory responses including the levels of CD31, H2O2, TNF-α, IL-1β, myeloperoxidase, P38, and nuclear factor κB along with downregulation of apoptosis as reflected by caspase 3 and p-AKT. In addition, Apo attenuated the increase in lung weight, bronchoalveolar lavage fluid albumin content, and the histopathologic lung injury score. In conclusion, LPSLI is associated with increased inflammatory responses, apoptosis, and coagulation. The administration of Apo attenuates LPSLI through downregulation of the inflammatory responses and apoptosis.     

白细胞介素-10对急性肺损伤炎症/抗炎介质表达的影响

目的探讨白细胞介素-10(IL—10)对急性肺损伤(ALI)大鼠炎症介质／抗炎介质表达的影响。方法向气道内滴注内毒素(LPS，10mg／kg)建立大鼠ALI模型。54只雄性SD大鼠随机分为对照组、LPS损伤组、LPS加IL-10组，每组18只，各组又分为2、6和24h3个亚组，每个亚组各6只。按各时间点观察大鼠动脉血氧分压(PaO2)、支气管肺泡灌洗液(BALF)中细胞总数及分类计数、肺系数、BALF总蛋白水平及肺病理，同时用逆转录-聚合酶链反应(RT—PCR)方法检测肺组织中炎症介质／抗炎介质的表达。结果①LPS损伤组大鼠PaO2呈进行性降低；肺系数、BALF总蛋白水平及BALF中细胞总数均明显增加，分类以中性粒细胞为主；肺病理示肺内中性粒细胞大量浸润，伴出血、透明膜形成。LPS加IL-10组的各项指标均较LPS损伤组减轻。②LPS损伤组肺组织肿瘤坏死因子-α(TNF—α)mRNA表达于2h达高峰，随后迅速下降；白细胞介素-1β(IL—1β)mRNA表达于2h显著升高，6h达高峰，随后迅速下降；IL-1受体拮抗剂(IL—1ra)mRNA表达6h开始升高，且为峰值。24h仍高于对照组。LPS加IL-10组肺组织TNF—αmRNA、IL-1βmRNA表达受抑，而IL—1ra mRNA表达不受影响。结论①ALI早期TNF—αmRNA、IL-1βmRNA表达明显增加，而IL—1ra mRNA表达滞后，提示在无外来干预情况下，ALI早期存在炎症介质／抗炎介质的失衡。②IL-10可明显抑制炎症介质表达，不影响抗炎介质表达，有利于重建炎症介质／抗炎介质平衡，减轻LPS所致ALI。     

慢性乙醇摄取对急性肺损伤大鼠气道上皮细胞间连接蛋白occludin和E-cadherin及屏障功能的影响

目的 观察慢性乙醇摄取及内毒素处理对大鼠气道上皮屏障功能及紧密连接(TJ)特征性蛋白occludin和黏附连接(AJ)蛋白E-cadherin的影响.方法 将40只SD大鼠随机均分为对照组、慢性乙醇摄取组(乙醇组)、内毒素处理组(LPS组)、慢性乙醇摄取合并内毒素处理组(乙醇+LPS组).利用荧光示踪剂异硫氰酸荧光素标记的右旋糖酐(FD4)测定支气管肺泡上皮的通透性;免疫荧光共聚焦显微镜下观察大鼠气道上皮occludin和E-cadherin蛋白分布及表达;蛋白质免疫印迹法(Western blotting)和逆转录-聚合酶链反应(RT-PCR)测定肺组织中occludin和E-cadherin的蛋白及mRNA表达;并观察肺组织病理学改变.结果 乙醇组及LPS组支气管肺泡上皮通透性均较对照组明显增高(P均＜0.05);乙醇+LPS组支气管肺泡上皮的通透性进一步增高(P＜0.01).occludin和E-cadherin蛋白在对照组大鼠气道上皮呈连续、均匀的胞膜及胞质中表达;在乙醇组、LPS组胞膜呈部分断裂、不连续的表达,且胞膜和胞质的表达下降;在乙醇+LPS组的表达显著下降,且胞膜表达呈明显的断裂甚至消失.Western blotting和RT-PCR显示,乙醇组和LPS组肺组织中occludin和E-cadherin的蛋白及mRNA表达均较对照组明显下降(P均＜0.05);乙醇+LPS组中蛋白及mRNA表达下降最为明显,与其余各组比较差异均有统计学意义(P均＜0.01).结论 慢性乙醇摄取通过降低TJ蛋白occludin和AJ蛋白E-cadherin的蛋白及mRNA表达水平,并干扰各蛋白在胞膜上的定位,最终导致气道上皮屏障功能受损,加重内毒素诱导的急性肺损伤.     

Inhaled aerosolized insulin ameliorates hyperglycemia-induced inflammatory responses in the lungs in an experimental model of acute lung injury

Introduction

Previous studies have shown that patients with diabetes mellitus appear to have a lower prevalence of acute lung injury. We assumed that insulin prescribed to patients with diabetes has an anti-inflammatory property and pulmonary administration of insulin might exert beneficial effects much more than intravenous administration.

Methods

Twenty-eight mechanically ventilated rabbits underwent lung injury by saline lavage, and then the animals were allocated into a normoglycemia group (NG), a hyperglycemia group (HG), an HG treated with intravenous insulin (HG-VI) group or an HG treated with aerosolized insulin (HG-AI) group with continuous infusion of different fluid solutions and treatments: normal saline, 50% glucose, 50% glucose with intravenous insulin, or 50% glucose with inhaled aerosolized insulin, respectively. After four hours of treatment, the lungs and heart were excised en bloc, and then high-mobility group B1 concentration in bronchoalveolar lavage fluid, interleukin-8 and toll-like receptor 4 mRNA expression in bronchoalveolar lavage fluid cells, and lung myeloperoxidase activity were measured.

Results

Treatment with both aerosolized insulin and intravenous insulin attenuated toll-like receptor 4 mRNA expressions in the bronchoalveolar lavage fluid cells. Interleukin-8 and toll-like receptor 4 mRNA expression was significantly lower in the HG-AI group than in the HG-IV group. The lung myeloperoxidase activity in the normal healthy group showed significantly lower levels compared to the NG group but not different compared to those of the HG, HG-VI and HG-AI groups.

Conclusions

The results suggest that insulin attenuates inflammatory responses in the lungs augmented by hyperglycemia in acute lung injury and the insulin''s efficacy may be better when administered by aerosol.     

HDAC3、IL-17RA、miR-19a-3p在类风湿性关节炎相关间质性肺病小鼠中的表达及意义

目的研究HDAC3、IL-17RA、miR-19a-3p在酵母多糖诱导的类风湿性关节炎相关间质性肺病(RA-ILD)小鼠中的表达情况。方法采用注射酵母多糖的方式构建RA-ILD小鼠模型,通过关节肿胀评分及肺脏组织病理评估造模是否成功;测定肺脏羟脯氨酸(HYP)的含量验证模型的可靠性;测定肺组织和肺泡灌洗液中HDAC3、IL-17RA和miR-19a-3p的表达情况。结果 12周后,RA-ILD组小鼠均患有严重的关节炎且部分小鼠肺组织出现明显炎症,HYP含量高于Normal组(P<0.01)。RA-ILD组小鼠肺组织和肺泡灌洗液中的HDAC3、IL-17RA的mRNA表达量均明显高于Normal组,miR-19a-3p表达量低于Normal组(P<0.05)。RA-ILD组小鼠肺组织和肺泡灌洗液中的HDAC3、IL-17RA蛋白表达量均明显高于Normal组(P<0.05)。结论 HDAC3和IL-17RA在RA-ILD纤维化小鼠模型肺组织中高表达,而miR-19a-3p低表达,推测三者可能参与了RA-ILD的发病。     

重度烟雾吸入致大鼠急性肺损伤的免疫应答及其机制探讨

目的分析重度烟雾吸入致吸人性急性肺损伤(ALI)对大鼠肺自然免疫及特异性免疫反应。方法分别复制一氧化碳(CO)浓度为2×10~(-3)(低浓度)和4×10~(-3)(高浓度)重度烟雾吸入致大鼠吸人性ALI模型。观察染毒后0～24 h大鼠肺组织病理学变化;检测支气管肺泡灌洗液(BALF)中致炎及抗炎细胞因子的浓度;用流式细胞仪检测外周血及BALF中淋巴细胞亚群数,BALF中CD45~+淋巴细胞和非淋巴细胞数量以及CD4~+/CD8~+变化。结果肺组织病理学检查证实染毒后可致明显肺损伤。染毒2 h BALF中肿瘤坏死因子-α(TNF-α)呈一过性升高,高浓度组较低浓度组更明显,之后下降;4 h白细胞介素-6(IL-6)、γ-干扰素(IFN-γ)开始升高,其中IL-6低浓度组较高浓度组明显,IFN-γ高浓度组较低浓度组明显,至12 h达高峰,24 h开始下降,但仍高于正常对照组水平(P＜0．05或P＜0．01);6～24 h IL-10与正常对照组比较均显著升高,尤以24 h明显(P＜0．05或P＜0．01)。外周血及BALF中CD4~+、CD8~+、自然杀伤细胞、B细胞及总T细胞均较正常对照组明显下降(P＜0．05或P＜0．01)。BALF中CD45~+淋巴细胞数和CD4~+/CD8~+均较正常对照组明显减少,非淋巴细胞数较正常对照组明显增多,且高浓度组较低浓度组变化趋势明显(P＜0．05或P＜0．01)。结论重度烟雾吸入致吸入性ALI的过程中伴有持续且过度的肺自然免疫反应,这种自然免疫反应部分由活化的中性粒细胞及巨噬细胞所介导;而肺特异性免疫反应受到明显的抑制。     

Response to exogenous surfactant is different during open lung and conventional ventilation

OBJECTIVE: Previous studies have shown that the efficacy of exogenous surfactant is dose-dependent during conventional positive pressure ventilation (PPVCON). The present study aimed to determine whether this dose-dependent relationship is also present during open lung (OLC) ventilation. We also explored the effect of exogenous surfactant on the ventilation pressures applied during ventilation. DESIGN: Animal study. SETTING: University-affiliated research laboratory. SUBJECTS: Seventy-two newborn piglets. INTERVENTIONS: After repeated whole lung lavage, animals were randomly allocated to two surfactant groups receiving either 100 mg/kg surfactant (S100) or 25 mg/kg surfactant (S25) or to a control group receiving a bolus of air. Within each group, animals were randomly assigned to either PPVCON, open lung PPV (PPVOLC), or open lung high-frequency oscillatory ventilation (HFOVOLC) and ventilated for 5 hrs. MEASUREMENTS AND MAIN RESULTS: The ventilation pressures decreased in a dose-dependent way, showing the largest reduction in the S100 group. In both OLC groups, oxygenation, lung mechanics, and polymorphonuclear neutrophils analyzed in bronchoalveolar lavage were independent of the surfactant dose. In the PPVCON group, however, there was a clear dose-dependency, resulting in a deterioration of oxygenation and lung mechanics and an increase in polymorphonuclear neutrophils as the surfactant dose decreased. Although comparable between the three ventilation groups, bronchoalveolar lavage interleukin-8 concentrations significantly increased in all ventilation groups as the surfactant dose increased. Alveolar protein influx and conversion of large to small aggregate surfactant were higher during PPVCON compared with both OLC groups. There were no differences in the surfactant treatment response between PPVOLC and HFOVOLC. CONCLUSION: Exogenous surfactant enables a reduction in ventilation pressures. Compared with PPVCON, the efficacy of surfactant treatment is less dose-dependent during open lung ventilation. Surfactant conversion during open lung ventilation is reduced compared with PPVCON. Exogenous surfactant seems to up-regulate bronchoalveolar lavage interleukin-8 concentrations, independent of the ventilation strategy.     

Protective effects of N-acetylcysteine and erdosteine on hemorrhagic shock-induced acute lung injury.

BACKGROUND: The drugs N-acetylcysteine and erdosteine were used to evaluate their protective effects in hemorrhagic shock-induced acute lung injury in an animal model. METHODS: Forty rats were used and randomly allocated into four groups (n=10). Animals in group III were fed with water containing 1 mg/dl erdosteine, and those in group IV were given 0.5 mg/dl N-acetylcysteine 3 days before the experiment. Group I was taken as the control and group II was taken as the hemorrhagic shock group. Hemorrhagic shock was initiated by blood withdrawal and reduction of the mean arterial pressure to 40 mmHg within 10 min via the right carotid artery. After a hypotensive period of 2 h, animals were resuscitated by transfusion of the shed blood and Ringer lactate in a volume equal to the shed blood. After a period of 1 h, blood samples were taken via the carotid artery. Bronchoalveolar lavage was performed to recover cells from the alveolar space with 40 ml of bronchoalveolar lavage fluid. Lung tissues were also resected to measure tissue malondialdehyde and L-gamma-glutamyl-L-cysteinyl-glycine levels with high performance liquid chromatography. The numbers of neutrophils and alveolar macrophages in bronchoalveolar lavage fluid were counted. RESULTS: Serum malondialdehyde levels were significantly higher in the shock groups (P<0.05), but there was no significant difference (P>0.05). Lung malondialdehyde levels were also significantly increased in the shock groups (P<0.05). In the erdosteine-applied group, tissue malondialdehyde levels were lower than in group II and the NAC-applied group (P<0.05). In the analyses of serum and lung tissue L-gamma-glutamyl-L-cysteinyl-glycine, the values of groups I, II and IV were found to be below the calibration graphics. The alveolar macrophage count was found to be the highest and the neutrophil count the lowest in group III when compared with the other groups in bronchoalveolar lavage fluid analyses (P<0.05). CONCLUSION: We may say that in the model of hemorrhagic shock-induced acute lung injury, it was found that erdosteine has a protective effect on lung tissue.     

肺缺血再灌注损伤模型大鼠肺组织细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶3表达的动态变化

背景:肺缺血再灌注损伤过程中肺组织细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶3表达的动态变化及其可能的作用机制有待观察。目的:观察大鼠肺缺血再灌注损伤中肺组织细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶3表达的动态变化,并分析细胞凋亡在肺缺血再灌注损伤中作用及可能机制。设计:随机对照动物实验。单位:南京医科大学附属南京第一医院急诊中心。材料:实验于2006-04/2006-09在南京医科大学附属南京第一医院动物实验室及南京市放射免疫检测中心完成。选用28只雄性健康清洁级SD大鼠,体质量250￣350g,鼠龄49￣76d,由南京医科大学实验动物中心提供。随机数字表法将大鼠分为实验组及对照组,每组14只。方法:①实验干预:实验组大鼠参照Eppinger等的方法进行建立肺缺血再灌注模型,大鼠给予阻断肺门45min(观察肺无舒缩为阻断标准),再开放3,6h(再灌注以肺恢复舒缩为标准)后取材,每次取7只,对照组仪行开胸术,于相同时间点同样方法分离肺组织。②实验评估:采用Annexin-V-PI双染法通过流式细胞仪检测肺组织凋亡细胞,计算凋亡率;采用免疫组织化学法及图像分析法观察肺脏天冬氨酸特异性半胱氨酸蛋白酶3的表达;计算2组大鼠左肺湿干质量比;计算肺泡损伤数比值进行肺组织损伤定量评价;光镜下苏木精-伊红染色观察肺脏的病理形态学变化。主要观察指标:①肺组织细胞凋亡率及天冬氨酸特异性半胱氨酸蛋白酶3的表达。②肺湿干质量比及肺组织损伤定量评价结果。③肺组织病理形态学变化。结果:纳入大鼠28只均进入结果分析,无脱落。①实验组肺缺血再灌注3,6h肺组织细胞凋亡率较对照组有明显增加,差异有统计学意义(P<0.01),实验组缺血再灌注6h凋亡率较3h有所降低(P<0.05)。②实验组大鼠肺组织天冬氨酸特异性半胱氨酸蛋白酶3表达3h达到最高,6h稍有下降,较对照组各时间点表达增加(P<0.01)。③实验组肺缺血再灌注3,6h肺泡损伤数比值及肺脏湿干质量比值较对照组均明显增加(P<0.01),实验组缺血再灌注6h两比值较3h有所增加(P<0.05)。④实验组肺泡结构破坏、塌陷、消失,肺泡间隔有大量的炎性细胞浸润,肺缺血再灌注6h较3h更严重。对照组无明显改变。结论:肺缺血再灌注早期可能通过天冬氨酸特异性半胱氨酸蛋白酶3表达的改变,激活细胞的凋亡途径,诱导肺组织细胞凋亡的发生,从而导致肺损伤的发生。     

Attenuation of reperfusion lung injury and apoptosis by A2A adenosine receptor activation is associated with modulation of Bcl-2 and Bax expression and activation of extracellular signal-regulated kinases

Adenosine receptors (AR) and extracellular signal-regulated kinases (ERK) have been implicated in tissue protection and apoptosis regulation during ischemia/reperfusion (I/R) injury. This study tests the hypothesis that reduction of reperfusion lung injury after A2A AR activation is associated with attenuation of apoptosis, modulation of ERK activation, and alterations in antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). Experiments were performed in intact-chest, spontaneously breathing cats in which the arterial branch of the left lower lung lobe was occluded for 2 h and reperfused for 3 h (I/R group). Animals were treated with the selective A2A AR agonist ATL313 given 5 min before reperfusion alone or in combination with the selective A2A AR antagonist ZM241385. Western blot analysis showed significant reduction in expression of Bcl-2 and increase in expression of Bax after reperfusion, compared with control lungs. Phosphorylated ERK1/2 levels were also increased after reperfusion. Compared with the I/R group, ATL313 markedly (P < 0.01) attenuated indices of injury and apoptosis including the percentage of injured alveoli, wet-dry weight ratio, myeloperoxidase activity, in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells, and caspase 3 activity and expression. Furthermore, compared with reperfused lungs, in ATL313-pretreated lungs, Western blot analysis demonstrated substantial ERK1/2 activation, increased expression of Bcl-2, and attenuated expression of Bax. The protective effects of ATL313 were blocked by pretreatment with ZM241385. In summary, the present study shows that in vivo activation of A2A AR confers protection against reperfusion lung injury. This protection is associated with decreased apoptosis and involves ERK1/2 activation and alterations in antiapoptotic Bcl-2 and proapoptotic Bax proteins.     

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。