Burn-induced acute lung injury requires a functional Toll-like receptor 4

The role of the Toll-like receptor 4 (TLR4), a component of the innate immune system, in the development of burn-induced acute lung injury (ALI) has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation. We hypothesized that burn-induced ALI is a TLR4-dependent process. Male C57BL/6J (TLR4 wild-type [WT]) and C57BL/10ScN (TLR4 knockout [KO]) mice were subjected to a 30% total body surface area steam burn. Animals were killed at 6 and 24 h after the insult. Lung specimens were harvested for histological examination after hematoxylin-eosin staining. In addition, lung myeloperoxidase (MPO) and intercellular adhesion molecule 1 immunostaining was performed. Lung MPO was measured by an enzymatic assay. Total lung keratinocyte-derived chemoattractant (IL-8) content was measured by enzyme-linked immunosorbent assay. Western blot was performed to quantify phosphorylated IκBα, phosphorylated nuclear factor κB p65 (NF-κBp65), and high mobility group box 1 expression. Acute lung injury, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intraalveolar hemorrhage, and neutrophil infiltration, was seen in WT but not KO animals at 24 h. Myeloperoxidase and intercellular adhesion molecule 1 immunostaining of KO animals was also similar to sham but elevated in WT animals. In addition, a reduction in MPO enzymatic activity was observed in KO mice as well as a reduction in IL-8 levels compared with their WT counterparts. Burn-induced ALI develops within 24 h after the initial thermal insult in our model. Toll-like receptor 4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR4-dependent process.     

Toll-like receptor 4-dependent responses to lung injury in a murine model of pulmonary contusion

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We previously demonstrated that toll-like receptor 2 (TLR-2) participates in the inflammatory response to lung injury. We hypothesized that the TLR-4, in an MyD88-dependent manner, may also participate in the response to lung injury. To investigate this, we used a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans and evaluated postinjury lung function, pulmonary neutrophil recruitment, and the systemic innate immune response. Comparisons were made between wild-type mice and mice deficient in TLR-4 or MyD88. We found TLR-4-dependent responses to pulmonary contusion that include hypoxemia, edema, and neutrophil infiltration. Increased expression of IL-6 and chemokine (C-X-C motif) ligand 1 in the bronchoalveolar lavage and serum was also dependent on TLR-4 activation. We further demonstrated that these responses to pulmonary contusion were dependent on MyD88, an adapter protein in the signal transduction pathway mediated by TLRs. These results show that TLRs have a primary role in the response to acute lung injury. Lung inflammation and systemic innate immune responses are dependent on TLR activation by pulmonary contusion.     

Toll样受体4在急性肺损伤中的作用

目的观察Toll样受体4(toll-like receptor 4,TLR4)基因突变型小鼠与野生型小鼠内毒素攻击致急性肺损伤(acute lung injury,ALI)的差异,分析TLR4在ALI中的作用。方法采用TLR4基因突变小鼠(C3H/HeJ品系,TLR4~(-/-)及野生型小鼠(CBA品系,TLR4~( / )),用内毒素攻击复制小鼠ALI模型,用显微病理及肺干湿重比(W/D)分析肺损伤程度,检测肺组织髓过氧化物酶(MPO)水平,探讨损伤差异的可能机制。结果用1,5 mg·kg~(-1)LPS溶液经尾静脉注射复制ALI模型,结果示TLR4突变小鼠肺组织大体及显微病理损伤较野生型小鼠减轻,同时肺组织水肿(W/D)亦较野生小鼠减轻,尤其是在大剂量(5mg·kg~(-1))时,差异有显著性[(4．08±0．1)vs．(4．55±0．2),n=10,t=12．71,P＜0．01]。突变小鼠肺组织PMN浸润水平较野生型低[MPO：1 mg·kg~(-1)组,(20．73±4．58)vs．(39．97±3．66),n=10,t=13．43,P＜0．01];5 mg·kg~(-1)组,[(24．0±3．94)vs．(48．5±4．07),n=10,t=15．33,P＜0．01],且两者均较假手术组高。结论TLR4可能通过介导中性粒细胞肺内聚集而导致ALI的发生发展。     

异丙酚后处理对急性肺损伤大鼠肺组织Toll样受体4表达的影响

目的 探讨异丙酚后处理对脂多糖(LPS)诱导急性肺损伤(ALI)大鼠肺组织中Toll样受体4(TLR4)表达的影响.方法 将30只SD大鼠按随机数字表法分为对照组、ALI组及异丙酚后处理组3组,每组10只.经股静脉泵入8 mg/kg LPS 30 min诱导ALI模型,对照组给予等量生理盐水.异丙酚后处理组于制模后股静脉推注20 mg/kg异丙酚,再以40 mg· kg-1·h-1微量泵匀速泵入维持1h.于给药结束后6h处死大鼠,取肺脏测定湿/干重(W/D)比值,计算肺通透性指数(LPI),用酶联免疫吸附试验(ELISA)测定支气管肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)水平,用逆转录-聚合酶链反应(RT-PCR)测定肺组织TLR4 mRNA表达.结果 与对照组比较,ALI组肺W/D比值、LPI、TLR4 mRNA表达及BALF中TNF-α水平(ng/L)均明显升高[肺W/D比值:5.30±0.28比4.21 ±0.14,LPI(×10-3):8.7±2.2比3.3±2.0,TLR4 mRNA:2.451±0.028比0.998±0.021,TNF-α:643.46±62.31比120.43±12.65,均P＜0.05];而异丙酚后处理组肺W/D比值、LPI、TLR4 mRNA表达及BALF中TNF-α水平均较ALI组明显降低[肺W/D比值:4.68±0.19比5.30±0.28,LPI(×10-3):5.8±2.0比8.7±2.2,TLR4 mRNA:1.126±0.025比2.451±0.028,TNF-α:290.53±32.01比643.46±62.31,均P＜ 0.05],但仍显著高于对照组(均P＜0.05).结论 异丙酚后处理能明显改善LPS诱导的ALI,其作用机制可能是通过下调TLR4 mRNA表达,从而抑制“瀑布样”炎症反应.     

TLR4在脓毒症大鼠急性肺损伤中作用

目的:研究Toll样受体(Toll like receptors,TLR)4在脓毒症急性肺损伤(acute lung injury,ALI)大鼠肺内的动态表达情况.方法:32只Wistar大鼠随机分成对照组12只与ALI组20只.实验6,12,18,24 h后分别处死大鼠.采用Real-time PCR测定肺组织TLR4 mRNA表达,免疫组织化学观察肺组织TLR4蛋白的表达情况.结果:ALI组与对照组比较,肺组织TLR4蛋白和TLR4 mRNA表达增加(P<0.01).ALI组TLR4蛋白和TLR4 mRNA在盲肠结扎穿孔术术后逐渐增加至18 h达高峰,后逐渐下降.结论:TLR4升高与脓毒症ALI的发生、发展密切相关.     

高氧致大鼠急性肺损伤Toll样受体2和4的变化及意义

目的 探讨Toll样受体(TLRs)在高氧致急性肺损伤(ALI)发病过程中的作用.方法 将32只SD大鼠按随机数字表法分为空气对照组和高氧暴露24、48、72 h组,每组8只.分别于相应时间点活杀8只大鼠,取肺组织标本,测定肺湿/干重(W/D)比值并行肺组织病理评分,用实时荧光定量逆转录-聚合酶链反应(RT-PCR)测定肺组织TLR2和TLR4的mRNA表达,用蛋白质免疫印迹法(Western blotting)测定肺组织TLR2和TLR4的蛋白表达,用酶联免疫吸附法(ELISA)测定肺组织匀浆中白细胞介素-6(IL-6)含量.结果 各高氧暴露组肺W/D比值均较空气对照组明显增高(P均<0.01).高氧暴露48 h、72 h肺组织病理评分[(2.69±0.53)分,(3.94±0.62)分]均明显高于空气对照组[(0.41±0.38)分,P均<0.01].RT-PCR结果显示,高氧暴露组肺组织TLR2和TLR4 mRNA表达均明显高于空气对照组(0.67±0.15和0.63±0.19),并均于24 h达高峰(1.82±0.33,1.35±0.26,P均<0.05).Western blotting结果显示,高氧暴露组TLR2和TLR4蛋白表达均较空气对照组[(7.20±0.51)%和(14.26±0.19)%]明显增高,分别于48 h、72 h达峰值[(28.12±0.24)%,(81.35±0.82)%,P均<0.05].ELISA结果显示,高氧暴露组肺组织匀浆IL-6含量较空气对照组[(639.38±95.24)pg/L]明显增高,于72 h达高峰[(1 300.58±442.24)pg/L,P<0.05].结论 在高氧引起的ALI中,TLR2和TLR4在肺组织炎症启动和维持中起重要作用.     

Toll样受体4单克隆抗体预处理对脂多糖诱发小鼠急性肺损伤的影响

目的 探讨预先使用Toll样受体4单克隆抗体(TLR4mAb)对LPS诱发的小鼠脓毒症肺损伤的保护作用.方法 45只健康清洁级雄性BALB/c小鼠以随机数字法分为对照组(C 组)、脓毒症组(S组)、预处理组(P组),每组15只.P组小鼠在造模前1h预先腹腔注射TLR4mAb 5 μg/g.S组、P组均腹腔注射LPS 10 mg/kg以制备脓毒症急性肺损伤模型,C组经腹腔注射等量生理盐水.各组分别于注射后6,12,24h,采用酶联免疫吸附法测定血清TNF-a和IL-6浓度;取肺脏组织,分别检测TLR4 mRNA的表达、肺组织含水量、病理学改变.组内比较采用双因素方差分析,组间比较采用单因素方差分析,以P ＜0.05为差异有统计学意义.结果 与C组比较,S组、P组肺组织含水量在12,24h时均显著性升高;与S组比较,P组肺组织含水量在12,24h时均显著性降低.与C组比较,S组、P组血清IL-6,TNF-α质量浓度在各时点均显著性升高;与S组比较,P组IL-6,TNF-α质量浓度在各时点均显著性降低.与C组比较,S组、P组TLR4 mRNA在各时点时均显著性升高;与S组比较,P组TLR4mRNA在各时点均显著性降低.与S组比较,P组病理改变程度均明显减弱.结论 预先使用TLR4mAb可以减轻LPS所诱发的急性肺损伤程度,可抑制炎性因子的过度表达;调控TLR4通路可能是治疗脓毒症及其肺损伤的有效靶点.     

Toll-like receptor 4 contributes to microvascular inflammation and barrier dysfunction in thermal injury

Systemic and microvascular inflammation plays a key role in the development of multiple organ failure after infection, sepsis, and traumatic injury. Toll-like receptors (TLRs) regulate host responses to pathogens and sterile, injury-associated inflammatory responses. We investigated whether TLR-4 contributes to microvascular dysfunction during thermal injury in vivo in anesthetized wild-type or TLR-4 (-/-) mice receiving either a 25% total body surface area full-thickness scald burn or sham treatment on the dorsal skin. Using intravital microscopy, we assessed the hemodynamics and leukocyte dynamics in the mesenteric microvasculature as representative of the splanchnic microcirculation at a site remote from the burn wound. The transvascular flux of fluorescein isothiocyanate-albumin across mesenteric venules was measured as an indicator of microvascular permeability. Furthermore, cultured microvascular endothelial cell models were used to evaluate the endothelial-specific mechanisms involved in TLR-4-mediated barrier dysfunction. The results showed significantly elevated microvascular permeability in wild-type mice after burn, whereas this response was markedly attenuated in TLR-4 (-/-) mice. Burn injury also increased leukocyte adhesion in mesenteric venules of wild-type mice, and a blunted leukocyte response was seen in the TLR-4 mice. Treatment of endothelial cell monolayers with burn plasma induced a rapid reduction in the transendothelial electrical resistance measured by electric cell-substrate impedance sensing, indicative of endothelial cell-cell adhesive barrier dysfunction. Reducing expression of TLR-4 with siRNA treatment attenuated this response. Taken together, these data indicate that TLR-4 plays an important role in microvascular leakage and leukocyte adhesion under the inflammatory condition associated with nonseptic thermal injury.     

盐酸戊乙奎醚对急性肺损伤患者Toll样受体4表达的影响

目的 观察急性肺损伤(ALI)患者早期应用盐酸戊乙奎醚治疗效果;早期应用盐酸戊乙奎醚治疗ALI患者外周血单核细胞Toll样受体4(TLR4)表达及其下游炎症因子和血清中抗炎因子的表达变化;探讨TLR4在ALI过程中的参与机制.方法 2007/年9月至2008年8月,南京军区南京总医院急救医学科重症监护病房收治的45例ALI患者.全部患者符合国内外普遍应用的ALI诊断标准.45例ALI患者随机分成盐酸戊乙奎醚治疗组(实验组,n=21)和常规治疗组(对照组,n=24).两组均给予常规综合治疗,实验组在常规综合治疗基础上加用盐酸戊乙奎醚治疗,1 mg,肌注,1次/12h.观察两组48 h临床疗效、动脉血气、细胞因子等的变化,流式细胞仪即时检测血液中TLR4的表达变化.两组间均数比较用成组t检验,组内各时间点与0 h的比较采用配对t检验.两组率的比较采用Fisher精确概率法.结果 治疗后心率、呼吸频率48 h两组均较0 h有改善,组间无统计学差异(P>0.05),两组PaO_2和PaO_2/FiO_2均逐渐升高,6 h,24 h,48 h实验组改善优于对照组(P<0.05);加用盐酸戊乙奎醚治疗可明显缩短患者在ICU监护治疗的时间(P<0.01);流式细胞仪TLR4检测显示,0 h两组患者TLR4表达水平明显高于正常健康体检者(P<0.01);治疗后24 h、48 h两组患者TLR4均较0 h下降(P<0.05),实验组下降幅度大于对照组24 h(P<0.05),48 h(P<0.01),且两组TLR4水平0 h就明显升高的患者预后不良,大部分发展为ARDS;24 h ARDS发生率实验组为23.8%,对照组为29.17%,至48 h对照组又有2例进展为ARDS,实验组无再发患者;细胞因子检测:0～48 h IL-1、IL-广8和TNF-α浓度两组均进行性下降,其中24 h IL-8和TNF-α实验组较对照组下降明显(P<0.05),48 h三者实验组均较对照组下降明显(P<0.05);IL-13在0～24 h进行性升高,48 h较24 h略有下降,与0 h相比组内有统计学差异(P<0.05),组间无统计学差异.结论 盐酸戊乙奎醚能够早期改善氧合、抑制TLE4的表达,使其传导通路下游炎症因子表达下降,这种对炎症因子的抑制作用并非是通过早期上调抗炎介质而是通过下调TLR4来实现的,盐酸戊乙奎醚可能成为急性肺损伤治疗的重要药物.TLR4在急性肺损伤发生、发展的病理过程中有重要作用,并可以考虑作为判断ALI预后的参考标志.     

甘草酸二铵对百草枯中毒大鼠肺组织TLR -4 mRNA 表达的影响

目的探讨甘草酸二铵（ DG）对百草枯中毒（ PQ）致急性肺损伤（ ALI）大鼠肺Toll样受体4（TLR-4） mRNA、NF-κB mRNA表达及对血清白细胞介素-6（IL-6）、白细胞介素-17（IL-17）浓度的影响。方法将50只SD大鼠采用随机数字表法分为PQ组（n＝20）、DG治疗组（n＝20）和正常对照组（S组,n＝10）,PQ组和DG治疗组予百草枯100 mg/kg灌胃1次,DG组于灌胃后立即腹腔注射DG 50 mg/kg（1次/d）,S组和PQ组注射等剂量的生理盐水,观察至48 h。取肺组织检测肺湿干质量比（ W/D）;采用RT-PCR法检测肺组织TLR-4 mRNA、NF-κB mRNA的表达情况;ELISA法测定血清IL-6、IL-17浓度。另选择健康SD大鼠50只,分组及各组处置方法同上,观察其7 d内死亡率。结果与S组比较,PQ组W/D、TLR-4 mRNA、NF-κB mRNA表达均明显升高（P＜0．01）,血清IL-6、IL-17亦升高（P＜0．01）;DG组干预后,W/D降低, TLR-4 mRNA、NF-kB mRN、AIL-6及IL-17有所降低,与PQ组比较差异有统计学意义（ P＜0.01或P＜0．05）。 PQ组大鼠7 d死亡18只（死亡率90％）,DG组死亡8只（死亡率40％）,NS组无死亡（死亡率0）。结论甘草酸二铵可抑制PQ致ALI大鼠肺组织TLR-4 mRNA、NF-κB mRNA及血清IL-6、IL-17浓度的过度表达,从而减轻肺损伤程度。     

高迁移率族蛋白B1 / Toll样受体4信号通路在脓毒症大鼠致急性肺损伤中的作用研究

目的探讨高迁移率族蛋白B1（HMGB1） / Toll样受体4（TLR4）信号通路对脓毒症导致的急性肺损伤大鼠的影响。 方法将60只清洁级雄性Sprague Dawley大鼠分为假手术组、脓毒症组和实验组,每组各20只。假手术组大鼠麻醉后开腹翻动肠道,随即关腹;脓毒症组和实验组行盲肠结扎穿孔（CLP）术后0.5 h于尾静脉分别注射等渗NaCl溶液（4 mL / kg）及抗HMGB1单克隆抗体（2 mg / kg）。各组分别取10只用于观察大鼠CLP建模后7 d存活情况,其余大鼠于造模后24 h处死并留取肺组织标本。计算各组大鼠的肺损伤Smith评分,并比较各组大鼠HMGB1和TLR4阳性蛋白在肺组织中的表达水平。采用酶联免疫吸附测定检测各组大鼠肺泡灌洗液（BALF）中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、HMGB1和TLR4水平,计算每个巨噬细胞内微球蛋白数以比较各组大鼠肺泡巨噬细胞（AM）的吞噬功能,并采用Western-blotting法测定AM内HMGB1、TLR4蛋白表达水平。 结果假手术组大鼠建模后7 d全部存活,脓毒症组大鼠仅3只存活,实验组大鼠5只存活。各组大鼠间存活情况的比较,差异有统计学意义（ χ² = 10.833,P = 0.004）;且假手术组大鼠的存活情况明显优于脓毒症组与实验组大鼠（P均< 0.017）,而脓毒症组与实验组大鼠间存活情况比较,差异无统计学意义（P = 0.120）。假手术组、脓毒症组及实验组大鼠间肺组织损伤评分[（2.20 ± 0.27）、（8.20 ± 1.27）、（4.25 ± 2.21）分,F = 56.432,P < 0.001],肺组织HMGB1阳性蛋白[（10.4 ± 1.5）、（34.4 ± 5.0）、（26.6 ± 6.9）,F = 35.203,P = 0.003]、TLR4阳性蛋白[（10.6 ± 2.1）、（48.0 ± 5.8）、（38.2 ± 5.3）,F = 103.414,P = 0.002],BALF中TNF-α[（19 ± 4）、（45 ± 4）、（35 ± 4）μg / L,F = 2.749,P < 0.001]、IL-6[（56 ± 19）、（86 ± 15）、（70 ± 19）μg / L,F = 4.648,P = 0.001]、HMGB1[（41 ± 18）、（70 ± 15）、（56 ± 12）μg / L,F = 7.254,P = 0.002]、TLR4[（20.9 ± 1.8）、（51.2 ± 1.6）、（49.8 ± 2.6）μg / L,F = 3.978,P = 0.035],AM的吞噬功能[（21.8 ± 2.7）、（3.1 ± 1.9）、（12.6 ± 2.2）个,F = 32.821,P = 0.001]及AM中HMGB1蛋白[（11 ± 3）、（40 ± 15）、（24 ± 13）,F = 7.253,P < 0.001]、TLR4蛋白[（0.9 ± 0.4）、（1.2 ± 0.6）、（1.1 ± 0.4）,F = 3.984,P = 0.028]的比较,差异均有统计学意义。进一步两两比较发现,脓毒症组与实验组大鼠肺组织损伤评分,肺组织HMGB1阳性蛋白、TLR阳性蛋白表达水平,BALF中TNF-α、IL-6、HMGB1水平及AM中HMGB1蛋白表达水平均明显高于假手术组大鼠,且脓毒症组大鼠更高（P均< 0.05）;脓毒症组及实验组大鼠AM的吞噬功能均显著低于假手术组,且脓毒症组更低（P均< 0.05）;脓毒症组及实验组大鼠BALF中TLR4水平及AM中TLR4蛋白表达水平均显著高于假手术组（P均< 0.05）,但脓毒症组与实验组间上述指标的比较,差异均无统计学意义（P均> 0.05）。 结论通过抑制HMGB1 / TLR4信号通路可以缓解脓毒症致肺损伤大鼠肺组织的炎症损伤,抑制炎症介质过度释放,并增强AM的细胞吞噬功能。     

Local expression of Toll-like receptor 4 at the site of ruptured plaques in patients with acute myocardial infarction

Several reports suggest that a chronic inflammatory process plays a key role in coronary artery plaque instability and subsequent occlusive thrombosis. In a previous study, we found that TLR4 (Toll-like receptor 4) mediates the synthesis of cytokines in circulating monocytes of patients with AMI (acute myocardial infarction); however, it remains unclear whether TLRs are expressed at the site of the ruptured plaque in these patients. The aim of the present study was to determine whether TLR2 and TLR4 are expressed at the site of ruptured plaques in patients with AMI and to compare this with systemic levels. The study included 62 patients with AMI, 20 patients with SA (stable angina) and 32 subjects with a normal coronary angiogram (control). Local samples from the site of the ruptured plaque were taken from patients with AMI using aspiration catheterization. Systemic blood samples from the aorta were taken from patients with AMI and SA and controls. Systemic levels of TLR2 and TLR4 were higher in patients with AMI than in patients with SA and controls. In patients with AMI, local TLR4 levels were higher than systemic levels. There was no significant difference in TLR2 levels between local and systemic samples. TLR4 immunostaining was positive in infiltrating macrophages in ruptured plaque material. Cardiac events were observed in 16 patients with AMI at the time of the 6-month follow-up study. Local and systemic levels of TLR4 were higher in patients with AMI with cardiac events than in those without. These results indicate an increase in monocytic TLR4 expression not only in the systemic circulation, but also at the site of plaque rupture. In conclusion, expression of both systemic and local plaque TLR4 may be one of the mechanisms responsible for the pathogenesis of AMI.     

失血性休克鼠肺组织Toll样受体基因的表达

目的探讨单纯性失血性休克（无复苏）对肺组织中Toll样受体（TLR）mRNA表达的影响及意义。方法C57BL／6小鼠45只。随机分为失血组、脂多糖（LPS）组（阳性对照组。尾静脉注射LPS5mg／kg）、假手术组（阴性对照组）。每组15只。心脏穿刺复制失血性休克模型，在不同时间点取出肺组织。提取总RNA，通过逆转录-聚合酶链反应（RT—PCR）半定量方法检测肺组织TLR2 mRNA和TLR4 mRNA的表达水平。结果在失血性休克和LPS刺激后肺出现明显的中性粒细胞浸润、红细胞渗出。正常肺组织中有TLR2 mRNA、TLR4 mRNA表达；住失血性休克和LPS刺激后0、1、2、4和6h肺组织TLR2 mRNA、TLR4 mRNA表达均逐渐增加；而假手术组未发生明显改变。结论失血性休克后肺组织TLR2 mRNA、TLR4 mRNA表达增加与急性肺损伤（ALI）的发生有密切关系。除增强了机体非特异性免疫能力外，同时增加了宿主对随后各种刺激的易感性。过度表达的TLR2、TLR4可能造成组织、器官结构和功能损害。     

余氏止痛消炎膏与芙黄膏合用治疗急性软组织损伤

急性软组织损伤早期不宜温热外敷。伤科广泛使用的活血消肿止痛药膏多由温热药组成,对以皮下出血、浆液渗出为主的早期损伤不宜单独使用。笔者采用余氏止痛消炎膏与芙黄膏(余芙膏)外敷治疗急性软组织损伤760例,收效显著。现报告如下。1　临床资料　　760例均为伤后2d～3d且无皮     

内皮源性toll样受体4在急性肺损伤中的作用

目的 探索内皮源性或髓源性细胞表面toll样受体4(TLR4)介入急性肺损伤(ALI)的作用. 方法 采用TLR4基因突变(C3H/HeJ品系,TLR4mut/mut)及野生型(C3H/HeN,TLB4+/+)小鼠,通过骨髓移植建立"内皮细胞TLR4+/+髓系细胞TLR4mut/mut"(WT/Mutant:受体/供体)及Mutant/WT嵌合体小鼠,尾静脉注射LPS(5 mg·kg-1)复制ALI模型,5 h后测肺组织湿干重比(W/D),肺通透指数(LPI),肺组织髓过氧化物酶(MPO)水平、炎症因子及黏附分子水平. 结果 TLR4mut/mut小鼠肺损伤较TLR4+/+小鼠较轻,WT/Mutant组小鼠肺组织损伤较Mutant/WT组重,且WT/Mutant组与WT/WT组差异无统计学意义.WT/Mutant组小鼠肺组织MPO水平、ICAM-1表达水平较Mutant/WT组高,且ICAM-1表达水平WT/Mutant组小鼠与WT/WT小鼠比较差异无统计学意义.炎症因子TNF-α、IL-1β的水平Mutant/WT组较WT/Mutant组高. 结论 内皮源性TLR4通过调控黏附分子表达而促进PMN肺组织招募,在介导LPS诱导的ALI中的发挥核心作用.     

Pulmonary lactate release in patients with acute lung injury is not attributable to lung tissue hypoxia

OBJECTIVE: To determine whether pulmonary lactate production in patients with acute lung injury is attributable to lung tissue hypoxia. DESIGN: Prospective, controlled, clinical study. SETTING: A multidisciplinary university intensive care unit in a general hospital. PATIENTS: Seventy consecutive critically ill patients requiring mechanical ventilation and invasive hemodynamic monitoring. Of these patients, 18 had no acute lung injury (no ALI); 33 had acute lung injury (ALI) (Lung Injury Score [LIS] < or =2.5); and 19 had acute respiratory distress syndrome (ARDS) (LIS >2.5). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: After hemodynamic measurements, lactate and pyruvate concentrations were assessed in simultaneously drawn arterial (a) and mixed venous (v) blood samples. Pulmonary lactate release was calculated as the product of transpulmonary a-v lactate difference (L[a-v]) times the cardiac index. Two indices of anaerobic metabolism of the lung, i.e., the transpulmonary a-v difference of lactate pyruvate ratio (L/P[a-v]) and excess lactate formation across the lungs (XL), were calculated. L(a-v) and pulmonary lactate release were higher in patients with ARDS than in the other groups (p<.001), and they were also higher in patients with ALI compared with patients with no ALI (p<.001). In patients with ALI and ARDS (n = 52), pulmonary lactate release correlated significantly with LIS (r2 = .14, p<.01) and venous admixture (r2 = .13, p<.01). When all patients were lumped together (n = 70), pulmonary lactate release directly correlated with LIS (r2 = .30, p<.001), venous admixture (r2 = .26, p<.001), and P(A-a)O2 (r2 = .14, p<.01). Neither L/P(a-v) nor XL was significantly different among the three groups. CONCLUSION: The lungs of patients with ALI produce lactate that is proportional to the severity of lung injury. This lactate production does not seem to be attributable to lung tissue hypoxia.     

Role of Toll-like receptor 4 on pancreatic and pulmonary injury in a mice model of acute pancreatitis associated with endotoxemia

OBJECTIVE: Infection of pancreatic necrosis is a severe complication of acute pancreatitis. Because Toll-like receptor 4 (TLR4) has been identified as a receptor necessary to transduct the signal of bacteria-derived lipopolysaccharide into cells, we investigated the role of TLR4 on pancreatic and pulmonary injury in acute pancreatitis and acute pancreatitis associated with endotoxemia in wild-type and TLR4-deficient mice. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Heterozygous TLR4 mice. INTERVENTIONS: Mice were injected intraperitoneally with a supramaximal dose of cerulein each hour for 10 hrs. To mimic infection, additional groups of mice were injected with lipopolysaccharide in the presence or absence of cerulein injections. MEASUREMENTS AND MAIN RESULTS: The severity of acute pancreatitis was assessed by serum amylase activity, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity. Lung injury was quantitated by lung microvascular permeability and lung myeloperoxidase activity. Injections of cerulein induced an edematous pancreatitis that was of similar severity in wild-type and TLR4-deficient mice. Lipopolysaccharide alone had no toxic effect on pancreas and lungs and did not worsen the pancreatic injury induced by cerulein in wild-type and TRL4-deficient mice. In contrast, lipopolysaccharide worsened pancreatitis-associated lung injury, and the deficiency in TLR4 fully prevented this aggravation. CONCLUSIONS: TLR4 may not play a role in the pancreatitis-associated lung injury but participates in the pulmonary injury mediated by endotoxemia.     

Toll-like receptor 2 participates in the response to lung injury in a murine model of pulmonary contusion

Blunt chest trauma resulting in pulmonary contusion with an accompanying acute inflammatory response is a common but poorly understood injury. We report that Toll-like receptor (TLR) 2 participates in the inflammatory response to lung injury. To show this, we use a model of pulmonary contusion in the mouse that is similar to that observed clinically in humans based on histologic, morphologic, and biochemical criteria of acute lung injury. The inflammatory response to pulmonary contusion in our mouse model is characterized by pulmonary edema, neutrophil transepithelial migration, and increased expression of the innate immunity proinflammatory cytokines IL 1beta and IL 6, the adhesion intracellular adhesion molecule 1, and chemokine (CXC motif) ligand 1. Compared with wild-type animals, contused Tlr2(-/-) mice have significantly reduced pulmonary edema and neutrophilia. These findings are associated with decreased levels of circulating chemokine (CXC motif) ligand 1. In contrast, systemic IL 6 levels remain elevated in the TLR2-deficient phenotype. These results show that TLR2 has a primary role in the neutrophil response to acute lung injury. We suggest that an unidentified noninfectious ligand generated by pulmonary contusion acts via TLR2 to generate inflammatory responses.     

Toll-like receptor 4 regulates heme oxygenase-1 expression after hemorrhagic shock induced acute lung injury in mice: requirement of p38 mitogen-activated protein kinase activation

Acute lung injury (ALI) leading to respiratory distress is a common sequela of shock or trauma. The toll-like receptors (TLRs) stand at the interface of innate immune activation in the settings of both infection and sterile injury by responding to a variety of microbial and endogenous ligands alike. This work explored the effects of TLR-4 on hemorrhage-induced ALI and characterizes the signaling pathways and the mechanisms involved in noninfectious ALI. Mice underwent hemorrhagic shock and resuscitation (HSR). Arterial blood gases; expressions of TLR-4, heme oxygenase 1 (HO-1), and p38 mitogen-activated protein kinase (p38MAPK); myeloperoxidase activity; lung wet/dry ratios; and IL-10 levels in lung tissues were obtained at 6, 24, and 48 h after HSR. Hemorrhagic shock and resuscitation induced significant expressions of TLR-4, HO-1, and p38MAPK in C3H/HeN mice. IL-10 and myeloperoxidase were markedly increased at 24 h after HSR, and C3H/HeN mice had ALI with PaO2/fraction of inspired oxygen less than 300 mmHg. The induced amount of each cytokine level and the expressions of TLR-4, HO-1, and p38MAPK of C3H/HeN mice were significantly higher compared with C3H/HeJ mice. This study demonstrated that lung p38MAPK is activated after HSR, and p38MAPK inhibitor FR167653 suppresses HO-1 induction after ALI. We concluded that TLR-4 might induce HO-1 messenger RNA expression, which is probably involved in p38MAPK activation in the development of the lung dysfunction after HSR.