静脉麻醉药物对脂多糖诱导的急性肺损伤大鼠肺组织CD14和TLR4受体的影响
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摘要
目的
急性肺损伤的发病率为78.9/10万人年,医院内死亡率较高。随着手术和麻醉技术的提高,越来越多的急性肺损伤患者在麻醉状态下接受手术,而且在重症监护室中急性肺损伤的患者经常使用到各种麻醉药物。麻醉药物的使用一方面可以抑制手术和疼痛应激反应,另一方面由于各种麻醉药物作用机制的不同,对免疫系统功能直接或间接地产生不同的影响。而在需要麻醉和重症监护的病人中有免疫抑制的患者比例在不断增加;免疫功能失常会引起术后和重症监护病人长时间的感染和败血症,重者可发生感染性休克和多器官功能衰竭,甚至死亡。因此,麻醉药物对机体免疫功能的影响已倍受关注。
引起急性肺损伤和急性呼吸窘迫综合征(ARDS)的肺脏炎症是导致人类死亡的主要疾病之一。急性肺损伤主要表现为中性粒细胞大量内流入肺脏,促炎性介质的生成及肺上皮损伤。宿主受体识别脂多糖(LPS)是激发肺内多种细胞信号传导瀑布的最重要的第一步。LPS能刺激多种细胞使其激活,与靶细胞表面的CD14受体相结合。Toll受体4(TLR4)是LPS/CD14复合体的近端跨膜受体,作用于CD14下游,传导LPS信号。病原微生物抗原和内源性抗原等被细胞表面的TLR4识别后,通过依赖MyD88或非依赖MyD88信号转导途径激活NF-κB,进而诱导TNF-α、IL-1β、环氧合酶2、ICAM-1等多种细胞因子和化学因子的生成与释放,引发以中性粒细胞浸润、微血管内皮细胞损伤与蛋白液体渗漏为特征的炎症反应,TLR4和CD14被证实是脂多糖激活内在免疫信号转导途径进而激活NF-κB所必需的,而后者的活化是炎症反应的共同途径。CD14和TLR4在激活内在免疫系统、细菌吞噬作用和清除凋亡细胞等各种免疫反应中起重要作用。有研究显示LPS介导的宿主反应完全依赖于TLR4,仅部分依赖于CD14。另有研究显示吸入内毒素引起的支气管收缩和急性肺炎症完全依赖于TLR4/CD14/MD2复合体的表达。迄今,对于LPS介导的肺损伤机制尚未完全清楚。
临床常用的静脉麻醉药包括非阿片类静脉麻醉药(丙泊酚、咪达唑仑等)和阿片类静脉麻醉药(雷米芬太尼、芬太尼等)。丙泊酚作为常用的非阿片类静脉麻醉药物,通过强化GABA-A受体发挥麻醉作用。咪达唑仑是苯二氮卓类镇静剂,作用迅速,半衰期短,对心脏呼吸影响小,也广泛应用于麻醉和重症监护室病人。雷米芬太尼是超短效阿片类受体激动剂,起效快,消除迅速,越来越多地用于临床麻醉。既往关于静脉麻醉药对急性肺损伤免疫机制的研究都局限于NF-κB下游细胞因子的研究。唯一关于丙泊酚对TLR4和CD14的研究仅在大鼠肝细胞系有报道:100μM丙泊酚预处理24小时能明显抑制TLR4,CD14,TNF-α等基因的表达。提示其可能减轻败血症相关性肝脏炎性反应。而关于丙泊酚对于肺组织NF-κB上游基因CD14和TLR-4的基因水平及蛋白表达的影响尚未有报道。其他麻醉药如咪达唑仑,雷米芬太尼等对急性肺损伤的作用研究很少。
合理使用麻醉药物,减少手术和麻醉应激,尽量维持机体免疫系统的正常功能是非常重要的。本研究旨在进一步在在体水平及细胞水平探讨LPS诱导的急性肺损伤炎性反应通路以及不同种静脉麻醉药物对炎性反应通路的影响机制,找出能更好的保护肺脏的麻醉药物,降低机体的免疫反应,并为开发更好的抑制炎症反应的麻醉药物提供理论上的支持。
实验材料
1.实验动物:
雄性Wistar大鼠,体重250-350克(在体实验);SPF级雄性Wistar大鼠,体重180-250克(离体实验)。由中国医科大学实验动物部提供。
2.主要实验试剂:
LPS,丙泊酚,咪达唑仑,雷米芬太尼,Takara反转录试剂盒,兔抗大鼠CD-14一抗,羊抗大鼠TLR4一抗,小鼠抗大鼠β-actin一抗,羊抗兔二抗,兔抗羊二抗,TNF-αELISA试剂盒,SYBR PrimeScriptTM实时定量PCR试剂盒。
3.主要实验仪器:
呼吸机,微量输液泵,电泳凝胶成像分析系统,电泳仪,半干转印仪,酶标仪,分光光度计,荧光显微镜带自动照像和数字成像装置,ABI 7500型Real-TimePCR仪。
实验方法
1.脂多糖气管内注入制造急性肺损伤模型戊巴比妥钠50 mg/kg腹腔注射麻醉。戊巴比妥钠和哌库溴铵通过左颈内静脉泵入,右颈动脉插管连接监护仪行持续动脉压监测。正中气管切开,插入气管插管(14号套管针)。连接呼吸机行压力控制通气。气管内注入LPS 5 mg/kg(1ml/kg)。
2.在体实验动物分组
Wistar大鼠40只,随机分为5组,每组8例。
空白对照组:气管内注入生理盐水,静脉泵入生理盐水
LPS组:气管内注入LPS,静脉泵入生理盐水;
丙泊酚组:气管内注入LPS,静脉泵入丙泊酚
咪达唑仑组:气管内注入LPS,静脉泵入咪达唑仑
雷米芬太尼组:气管内注入LPS,静脉泵入雷米芬太尼
分别于气管内给药前、给药1h、3h和5h记录MAP和HR、采动脉血行血气分析。实时定量PCR测定肺组织CD14和TLR4mRNA表达。Western-blot法测定肺组织CD14和TLR4蛋白表达。ELISA法测定BALF中TNF-α水平。
3.大鼠肺泡Ⅱ型上皮细胞原代培养
戊巴比妥和肝素腹腔内麻醉,无菌开胸,气管插管。用生理盐水行肺循环冲洗,经气管导管用生理盐水灌洗肺泡腔。蛋白酶消化30min,将肺去气道后放入其中剪成1-2mm~3的小块。100目和500目滤网依次过滤。Percoll梯度离心,取离心管内轻重比重Percoll溶液中间的细胞层,再次离心后DMEM液重悬细胞培养皿中放于培养箱中孵育。
4.细胞实验分组,
每只大鼠分离的ATII细胞随机分于五组:
C组:对照组,不添加任何药物,培养3小时;
LPS组:1μg.ml~(-1)LPS培养3小时;
P1组:1μg.ml~(-1)LPS和25μM丙泊酚培养:3小时;
P2组:1μg.ml~(-1)LPS和50μM丙泊酚培养3小时;
P3组:1μg.ml~(-1)LPS和100μM丙泊酚培养3小时。
实时定量PCR测定CD14和TLR4mRNA表达。Western-blot肺组织CD14和TLR4蛋白表达。免疫荧光双标检测CD14和TLR4在ATII细胞上的表达。ELISA法测定上清液中TNF-α水平。
5.统计学处理:
采用SPSS 13.0统计软件行统计分析。计量资料以均数±标准差表示,组间比较采用单因素方差分析,post hoc Tukey检验比较各组间差异。P＜0.05为差异有统计学意义。
实验结果
1.急性肺损伤模型制作成功
本研究采用气管内注入LPS 5mg/kg建立ALI模型,以PaO_2/FiO_2≤300mmHg作为ALI模型制备成功标准。LPS、丙泊酚、咪达唑仑和雷米芬太尼组3h和5hMAP与对照组比较均有降低。LPS、丙泊酚、咪达唑仑和雷米芬太尼组3h和5hMAP与0h比较有降低。LPS、丙泊酚、咪达唑仑和雷米芬太尼组1、3、5小时PaO_2低于0小时的PaO_2。LPS、丙泊酚、咪达唑仑和雷米芬太尼组1、3、5小时PaCO_2,高于0小时的PaCO_2。HE染色可见明显肺泡壁水肿、出血和增厚以及炎性细胞浸润,动脉血气分析结果及病理学结果证明模型制备成功。
2.在体动物实验
LPS、丙泊酚、咪达唑仑和雷米芬太尼组CD14 mRNA表达与对照组比较均有增加。丙泊酚组CD14 mRNA表达明显低于LPS组和雷米芬太尼组。LPS组和雷米芬太尼组TLR4 mRNA表达明显高于对照组。丙泊酚组TLR4 mRNA表达明显低于LPS组。丙泊酚组和咪达唑仑组TLR4 mRNA表达明显低于雷米芬太尼组。
LPS、丙泊酚、咪达唑仑、雷米芬太尼组CD14蛋白表达明显高于对照组。LPS、丙泊酚、咪达唑仑、雷米芬太尼组各组间CD14表达无显著差异。LPS、咪达唑仑、雷米芬太尼组TLR4蛋白表达明显高于对照组,丙泊酚组TLR4表达明显低于LPS组。丙泊酚、咪达唑仑组TLR4表达明显低于雷米芬太尼组。
光镜下肺组织HE切片丙泊酚组及咪达唑仑组损伤较轻,雷米芬太尼组损伤较重。
与对照组比较,LPS、咪达唑仑和雷米芬太尼组BALF中TNF-α明显增加,与LPS组比较,丙泊酚组BALF中TNF-α明显降低,雷米芬太尼组BALF中TNF-α明显增高。丙泊酚组和咪达唑仑组BALF中TNF-α明显低于雷米芬太尼组。
CD14蛋白、CD14 mRNA、TLR4蛋白和TLR4 mRNA表达与TNF-α水平Pearson相关系数分别为0.515(P＜0.05),0.672(P＜0.05),0.543(P＜0.05)和0.787(P＜0.05)。
3.原代培养ATII细胞
AKP染色蓝染细胞为ATII细胞。透射电镜显示了ATII细胞的标准板层小体。ATII细胞纯度为85±5%。经Trypan蓝染色证实各组细胞活性为90-95%。
4.细胞实验结果
1μg/ml的LPS可以明显增加ATII细胞CD14mRNA和TLR4 mRNA表达。丙泊酚可以剂量依赖性的抑制LPS引起的ATII细胞的CD14mRNA和TLR4 mRNA表达。
1μg/ml的LPS明显增加CD14蛋白和TLR4蛋白表达。丙泊酚剂量依赖性降低CD14蛋白和TLR4蛋白表达,并减少TNF-α产生。
CD14和TLR4免疫双标检测证实二者在ATII细胞表面表达部位一致。
CD14蛋白表达、CD14mRNA表达、TLR4蛋白表达和TLR4mRNA表达与TNF-α水平Pearson相关系数分别为0.639(P＜0.05),0.643(P＜0.05),0.774(P＜0.05)和0.634(P＜0.05)。
结论
1.LPS导致急性肺损伤大鼠在体肺组织及离体ATII细胞CD14和TLR4基因和蛋白水平的表达,增加TNF-α产生。
2.丙泊酚下调在体大鼠肺组织及离体ATII细胞由LPS诱导的CD14和TLR4基因和蛋白的表达,并减少其TNF-α产生。
3.丙泊酚和咪达唑仑与雷米芬太尼相比,能改善肺损伤。
4.CD14和TLR4在ATII细胞表达部位一致,
Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are major causes of acute respiratory failure,which increases the risk of morbidity and mortality in critically ill patients.Intratracheal instillation of lipopolysaccharides(LPS) in the rat is a well characterized model of acute lung injury,and closely resembles in many important aspects the clinical presentation of ALI/ARDS
By triggering a physical association between CD14 and TLR4,LPS is a principal initiator of host antimicrobial innate immune responses.Inflammatory response to endotoxins is largely mediated through CD14 and TLR4.The activation of CD14 and TLR4 leads to proinflammatory cascade through NF-κB,which induces expression of inflammatory mediators {tumor necrosis factor(TNF)-α,etc}.Elevated CD 14 is reported to be associated with increased infection and greater mortality in critically ill patients.The level of expression of TLR4 is closely associated with the extent of acute pulmonary response to inhaled endotoxin.
Anaesthetics are administered to sedate intubated,mechanically ventilated ALI and ARDS patients.These anaesthetics may modulate the immune system and influence the clinical course and outcome of patients.Previous reports have shown that anaesthetics affect inflammatory mediation in ALI.However,the exact mechanism of anaesthetic-induced effects on ALI has yet to be clarified.
The lung represents a site for the invasion of various bacteria or bacterial products. Along with alveolar macrophages,pulmonary epithelial cells are the first cells to be challenged by pathogenic microorganisms.Rat ATII cells synthesize and secrete surfactant,control the volume and composition of fluid in the alveolar space,and proliferate and differentiate into alveolar typeⅠepithelial cells after lung injury to maintain the integrity of the alveolar lining.Recently,there have been publications reporting the involvement of ATⅡin modulating the development of inflammatory reactions within the alveolus.ATⅡsecrete chemokines in response to inflammatory stimuli in vitro,suggesting a potential physiologic role in acute inflammatory lung injury.Little is known about the interaction of generally used intravenous anesthetic propofol with ATⅡ.
As a means of investigating how anaesthetics affect the inflammatory system,we designed a study to examine the effects of propofol,midazolam,and remifentanil on the mRNA and protein expression of both CD14 and TLR4 in rats with LPS-induced ALI and to investigate whether LPS induced inflammation in ATⅡis through CD14 and TLR4 and the effect of different dosage of propofol on the inflammation.
Materials
1.Animals:
Adult male Wistar rats weighting 250-350g for in vivo experiment and weighting 180-250 g for in vitro experiment,were provided by the Experimental Animal Center of China Medical University
2.Major reagents:
LPS,propofol,midazolam,remifentanil,Takara reverse transcript kit,rabbit anti-rat CD-14 primary antibody,goat anti-rat TLR4 primary antibody,mouse anti-ratβ-actin primary antibody,goat anti-rabbit secondary antibody,rabbit anti-goat secondary antibody,TNF-αELISA kit,SYBR PrimeScriptTM real-timePCR kit.
3.Major instruments:
Respiratory machine,micro-infusion pump,photo analysis system electrophoresis apparatus,semi-dry blotter,spectrophotometer,bechtop,CO_2 Incubators,Olympus BX61 photo analysis system,ABI 7500 Real-Time PCR system.
Methods
1.Animal model of the acute lung injury
Anaesthesia was induced with 50 mg kg~(-1) pentobarbital sodium administered intraperitoneally and maintained with pentobarbital sodium and pipecuronium bromide via left jugular vein.Tracheal cannulation(14 gauge) was performed after tracheostomy.The right carotid artery was catheterized for continuous arterial blood pressure measurements and blood sampling.The animals were ventilated using Servo 900C ventilator.LPS 5mg/kg was instilled intratracheally to induce acute lung injury.
2.In vivo experiment
According to the intravenous anaesthetics they received,the rats were randomly assigned to 5 groups with 8 in each group:
Control group:saline IT and salineⅳ
LPS group:LPS IT and salineⅳ
Propofol group:LPS IT and propofolⅳ
Midazolam group:LPS IT and midazolamⅳ
Remifentanil group:LPS IT and remifentanilⅳ
This intravenous infusion continued to the end of five-hour protocol.At 0 h,and at 1,3 and 5 h,MAP and HR were recorded and arterial blood was sampled for blood gas analysis.After the protocol,CD14 and TLR4 gene expression and protein expression in lung tissues were evaluated using real time PCR and western blot,respectively.TNF-αin bronchoalveolar lavage fluid(BALF) was detected using ELISA.
3.Primary culture of typeⅡalveolar epithelial cell
ATⅡwere isolated from male SPF degree Wistar rats(180-250g),following the protocol according to the method of Richard et al~(12).Briefly,after intraperitoneal sodium pentobarbital and heparin(400U.kg~(-1)),the thoracic cavity was opened and the inferior vena cava was cut.The lungs were perfused with sterile saline.After removal from the thoracic cavity,they were washed 6 times with normal saline and digested enzymatically with typsin.The lungs were then minced,and the resulting suspension was filtered through 150μm and 30μm mesh once.The cell mixture was purified by centrifugation on a discontinuous Percoll gradient.The interface was collected and after rewashing the cells were seeded onto 6-well culture dishes in DMEM at 37℃in the humidified atmosphere of 95%air/5%CO2 and cultured for 18-20 hours.
4.In vivo experiment
The cultured ATⅡcells from the same rat were randomly assigned to one of the following five groups:
Group C:ATⅡcells was cultured in the absence of propofol and LPS;
Group LPS:treated with 1μg.~(-1) ml LPS;
Group P1:treated with 1μg~(-1) LPS and 25μM propofol;
Group P2:treated with 1μg~(-1) LPS and 50μM propofol;
Group P3:treated with 1μg~(-1) LPS and 100μM propofol.
ATⅡcells in untreated control group and propofol groups were cultured for 3 h. CD14 and TLR4 mRNA were detected using real-time PCR.Western blot were used to detect CD14 and TLR4 protein expression.CD14 and TLR4 expression on the ATⅡcells were imaged using immunofluorescence.TNF-α.production were determined using ELISA kit.
5.Data analysis and statistics:
The data were normally distributed as determined by the Levene's test.ANOVA followed by a post hoc Tukey's test was used for multiple-group comparisons.Values are expressed as means±SD.The significance level was set at P<0.05.SPSS 13.0 software was used for all statistical work.
Results
1.Rat model of acute lung injury
This study regards PaO_2/FiO_2≤300mmHg as the successful setup of ALI rat model. After LPS 5mg/kg IT,LPS group,C,D and E all satisfied this standard.PaO_2 values at 3 h and 5 h were lower than at 0 h.HE stain showed edema,hemorrhage and thickness of alveolar wall as well as obvious inflammatory ceils infiltration into the septum.
2.Results of in vivo experiment
CD 14 mRNA expression in LPS group,propofol group,midazolam group and remifentanil group were significantly increased compared with control group.TLR4 mRNA expression in LPS group and remifentanil group were significantly increased compared with control group.TLR4 mRNA expression in propofol group and midazolam group were lower than in midazolam group.TLR4 mRNA in propofol group was lower than in LPS group.
CD14 protein expression in LPS,propofol,midazolam and remifentanil groups were significantly higher than in control group and there were no statistical difference among LPS,propofol,midazolam and remifentanil groups.TLR4 protein expression in LPS,midazolam and remifentanil groups were higher than in control group.TLR4 protein expression in propofol and midazolam groups were lower than in remifentanil group and TLR4 protein expression in propofol group was lower than in LPS group.
Propofol and midazolam group showed milder lung injury compared with remifentanil group in HE stain.
Compared with LPS group,TNF-αin BALF was decreased in propofol group and elevated in remifentanil group.
3.Primary culture of ATⅡcells
The cell mix,obtained by the discontinuous Percoll gradient centrifugation method,contained 85±5%of ATⅡwhich were stained blue with AKP histochemical staining.Transmission electron microscopy showed typical lamella bodies in ATⅡ.The cell viability in each group,determined by trypan blue dye exclusion test ranged from 90 to 95%.
4.Results of in vitro experiment
LPS stimulation resulted in an increased CD 14 and TLR4 expression and increased TNF-αproduction in ATⅡcells.Propofol,at concentrations≥50μM, significantly and dose-dependently decreased CD14 and TLR4 mRNA expression as well as their protein expression in ATⅡcells.This was accompanied by decreases in TNF-αproduction.
Conclusion
1.Both the mRNA and protein expressions of CD14 and TLR4 can be increased by LPS stimulation in the lung tissue in vivo and the ATⅡcells in vitro,so as with TNF-αproduction.
2.Propofol can downregulate both the mRNA and protein expressions of CD14 and TLR4 in the lung tissue in vivo and the ATⅡcells in vitro,and can decrease TNF-αproduction.
3.Compared with remifentanil,propofol and midazolam can relieve acute lung injury.
4.The study showed the collocation of CD14 and TLR4 expression.
急性肺损伤的发病率为78.9/10万人年,医院内死亡率较高。随着手术和麻醉技术的提高,越来越多的急性肺损伤患者在麻醉状态下接受手术,而且在重症监护室中急性肺损伤的患者经常使用到各种麻醉药物。麻醉药物的使用一方面可以抑制手术和疼痛应激反应,另一方面由于各种麻醉药物作用机制的不同,对免疫系统功能直接或间接地产生不同的影响。而在需要麻醉和重症监护的病人中有免疫抑制的患者比例在不断增加;免疫功能失常会引起术后和重症监护病人长时间的感染和败血症,重者可发生感染性休克和多器官功能衰竭,甚至死亡。因此,麻醉药物对机体免疫功能的影响已倍受关注。
引起急性肺损伤和急性呼吸窘迫综合征(ARDS)的肺脏炎症是导致人类死亡的主要疾病之一。急性肺损伤主要表现为中性粒细胞大量内流入肺脏,促炎性介质的生成及肺上皮损伤。宿主受体识别脂多糖(LPS)是激发肺内多种细胞信号传导瀑布的最重要的第一步。LPS能刺激多种细胞使其激活,与靶细胞表面的CD14受体相结合。Toll受体4(TLR4)是LPS/CD14复合体的近端跨膜受体,作用于CD14下游,传导LPS信号。病原微生物抗原和内源性抗原等被细胞表面的TLR4识别后,通过依赖MyD88或非依赖MyD88信号转导途径激活NF-κB,进而诱导TNF-α、IL-1β、环氧合酶2、ICAM-1等多种细胞因子和化学因子的生成与释放,引发以中性粒细胞浸润、微血管内皮细胞损伤与蛋白液体渗漏为特征的炎症反应,TLR4和CD14被证实是脂多糖激活内在免疫信号转导途径进而激活NF-κB所必需的,而后者的活化是炎症反应的共同途径。CD14和TLR4在激活内在免疫系统、细菌吞噬作用和清除凋亡细胞等各种免疫反应中起重要作用。有研究显示LPS介导的宿主反应完全依赖于TLR4,仅部分依赖于CD14。另有研究显示吸入内毒素引起的支气管收缩和急性肺炎症完全依赖于TLR4/CD14/MD2复合体的表达。迄今,对于LPS介导的肺损伤机制尚未完全清楚。
临床常用的静脉麻醉药包括非阿片类静脉麻醉药(丙泊酚、咪达唑仑等)和阿片类静脉麻醉药(雷米芬太尼、芬太尼等)。丙泊酚作为常用的非阿片类静脉麻醉药物,通过强化GABA-A受体发挥麻醉作用。咪达唑仑是苯二氮卓类镇静剂,作用迅速,半衰期短,对心脏呼吸影响小,也广泛应用于麻醉和重症监护室病人。雷米芬太尼是超短效阿片类受体激动剂,起效快,消除迅速,越来越多地用于临床麻醉。既往关于静脉麻醉药对急性肺损伤免疫机制的研究都局限于NF-κB下游细胞因子的研究。唯一关于丙泊酚对TLR4和CD14的研究仅在大鼠肝细胞系有报道:100μM丙泊酚预处理24小时能明显抑制TLR4,CD14,TNF-α等基因的表达。提示其可能减轻败血症相关性肝脏炎性反应。而关于丙泊酚对于肺组织NF-κB上游基因CD14和TLR-4的基因水平及蛋白表达的影响尚未有报道。其他麻醉药如咪达唑仑,雷米芬太尼等对急性肺损伤的作用研究很少。
合理使用麻醉药物,减少手术和麻醉应激,尽量维持机体免疫系统的正常功能是非常重要的。本研究旨在进一步在在体水平及细胞水平探讨LPS诱导的急性肺损伤炎性反应通路以及不同种静脉麻醉药物对炎性反应通路的影响机制,找出能更好的保护肺脏的麻醉药物,降低机体的免疫反应,并为开发更好的抑制炎症反应的麻醉药物提供理论上的支持。
实验材料
1.实验动物:
雄性Wistar大鼠,体重250-350克(在体实验);SPF级雄性Wistar大鼠,体重180-250克(离体实验)。由中国医科大学实验动物部提供。
2.主要实验试剂:
LPS,丙泊酚,咪达唑仑,雷米芬太尼,Takara反转录试剂盒,兔抗大鼠CD-14一抗,羊抗大鼠TLR4一抗,小鼠抗大鼠β-actin一抗,羊抗兔二抗,兔抗羊二抗,TNF-αELISA试剂盒,SYBR PrimeScriptTM实时定量PCR试剂盒。
3.主要实验仪器:
呼吸机,微量输液泵,电泳凝胶成像分析系统,电泳仪,半干转印仪,酶标仪,分光光度计,荧光显微镜带自动照像和数字成像装置,ABI 7500型Real-TimePCR仪。
实验方法
1.脂多糖气管内注入制造急性肺损伤模型戊巴比妥钠50 mg/kg腹腔注射麻醉。戊巴比妥钠和哌库溴铵通过左颈内静脉泵入,右颈动脉插管连接监护仪行持续动脉压监测。正中气管切开,插入气管插管(14号套管针)。连接呼吸机行压力控制通气。气管内注入LPS 5 mg/kg(1ml/kg)。
2.在体实验动物分组
Wistar大鼠40只,随机分为5组,每组8例。
空白对照组:气管内注入生理盐水,静脉泵入生理盐水
LPS组:气管内注入LPS,静脉泵入生理盐水;
丙泊酚组:气管内注入LPS,静脉泵入丙泊酚
咪达唑仑组:气管内注入LPS,静脉泵入咪达唑仑
雷米芬太尼组:气管内注入LPS,静脉泵入雷米芬太尼
分别于气管内给药前、给药1h、3h和5h记录MAP和HR、采动脉血行血气分析。实时定量PCR测定肺组织CD14和TLR4mRNA表达。Western-blot法测定肺组织CD14和TLR4蛋白表达。ELISA法测定BALF中TNF-α水平。
3.大鼠肺泡Ⅱ型上皮细胞原代培养
戊巴比妥和肝素腹腔内麻醉,无菌开胸,气管插管。用生理盐水行肺循环冲洗,经气管导管用生理盐水灌洗肺泡腔。蛋白酶消化30min,将肺去气道后放入其中剪成1-2mm~3的小块。100目和500目滤网依次过滤。Percoll梯度离心,取离心管内轻重比重Percoll溶液中间的细胞层,再次离心后DMEM液重悬细胞培养皿中放于培养箱中孵育。
4.细胞实验分组,
每只大鼠分离的ATII细胞随机分于五组:
C组:对照组,不添加任何药物,培养3小时;
LPS组:1μg.ml~(-1)LPS培养3小时;
P1组:1μg.ml~(-1)LPS和25μM丙泊酚培养:3小时;
P2组:1μg.ml~(-1)LPS和50μM丙泊酚培养3小时;
P3组:1μg.ml~(-1)LPS和100μM丙泊酚培养3小时。
实时定量PCR测定CD14和TLR4mRNA表达。Western-blot肺组织CD14和TLR4蛋白表达。免疫荧光双标检测CD14和TLR4在ATII细胞上的表达。ELISA法测定上清液中TNF-α水平。
5.统计学处理:
采用SPSS 13.0统计软件行统计分析。计量资料以均数±标准差表示,组间比较采用单因素方差分析,post hoc Tukey检验比较各组间差异。P＜0.05为差异有统计学意义。
实验结果
1.急性肺损伤模型制作成功
本研究采用气管内注入LPS 5mg/kg建立ALI模型,以PaO_2/FiO_2≤300mmHg作为ALI模型制备成功标准。LPS、丙泊酚、咪达唑仑和雷米芬太尼组3h和5hMAP与对照组比较均有降低。LPS、丙泊酚、咪达唑仑和雷米芬太尼组3h和5hMAP与0h比较有降低。LPS、丙泊酚、咪达唑仑和雷米芬太尼组1、3、5小时PaO_2低于0小时的PaO_2。LPS、丙泊酚、咪达唑仑和雷米芬太尼组1、3、5小时PaCO_2,高于0小时的PaCO_2。HE染色可见明显肺泡壁水肿、出血和增厚以及炎性细胞浸润,动脉血气分析结果及病理学结果证明模型制备成功。
2.在体动物实验
LPS、丙泊酚、咪达唑仑和雷米芬太尼组CD14 mRNA表达与对照组比较均有增加。丙泊酚组CD14 mRNA表达明显低于LPS组和雷米芬太尼组。LPS组和雷米芬太尼组TLR4 mRNA表达明显高于对照组。丙泊酚组TLR4 mRNA表达明显低于LPS组。丙泊酚组和咪达唑仑组TLR4 mRNA表达明显低于雷米芬太尼组。
LPS、丙泊酚、咪达唑仑、雷米芬太尼组CD14蛋白表达明显高于对照组。LPS、丙泊酚、咪达唑仑、雷米芬太尼组各组间CD14表达无显著差异。LPS、咪达唑仑、雷米芬太尼组TLR4蛋白表达明显高于对照组,丙泊酚组TLR4表达明显低于LPS组。丙泊酚、咪达唑仑组TLR4表达明显低于雷米芬太尼组。
光镜下肺组织HE切片丙泊酚组及咪达唑仑组损伤较轻,雷米芬太尼组损伤较重。
与对照组比较,LPS、咪达唑仑和雷米芬太尼组BALF中TNF-α明显增加,与LPS组比较,丙泊酚组BALF中TNF-α明显降低,雷米芬太尼组BALF中TNF-α明显增高。丙泊酚组和咪达唑仑组BALF中TNF-α明显低于雷米芬太尼组。
CD14蛋白、CD14 mRNA、TLR4蛋白和TLR4 mRNA表达与TNF-α水平Pearson相关系数分别为0.515(P＜0.05),0.672(P＜0.05),0.543(P＜0.05)和0.787(P＜0.05)。
3.原代培养ATII细胞
AKP染色蓝染细胞为ATII细胞。透射电镜显示了ATII细胞的标准板层小体。ATII细胞纯度为85±5%。经Trypan蓝染色证实各组细胞活性为90-95%。
4.细胞实验结果
1μg/ml的LPS可以明显增加ATII细胞CD14mRNA和TLR4 mRNA表达。丙泊酚可以剂量依赖性的抑制LPS引起的ATII细胞的CD14mRNA和TLR4 mRNA表达。
1μg/ml的LPS明显增加CD14蛋白和TLR4蛋白表达。丙泊酚剂量依赖性降低CD14蛋白和TLR4蛋白表达,并减少TNF-α产生。
CD14和TLR4免疫双标检测证实二者在ATII细胞表面表达部位一致。
CD14蛋白表达、CD14mRNA表达、TLR4蛋白表达和TLR4mRNA表达与TNF-α水平Pearson相关系数分别为0.639(P＜0.05),0.643(P＜0.05),0.774(P＜0.05)和0.634(P＜0.05)。
结论
1.LPS导致急性肺损伤大鼠在体肺组织及离体ATII细胞CD14和TLR4基因和蛋白水平的表达,增加TNF-α产生。
2.丙泊酚下调在体大鼠肺组织及离体ATII细胞由LPS诱导的CD14和TLR4基因和蛋白的表达,并减少其TNF-α产生。
3.丙泊酚和咪达唑仑与雷米芬太尼相比,能改善肺损伤。
4.CD14和TLR4在ATII细胞表达部位一致,
Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are major causes of acute respiratory failure,which increases the risk of morbidity and mortality in critically ill patients.Intratracheal instillation of lipopolysaccharides(LPS) in the rat is a well characterized model of acute lung injury,and closely resembles in many important aspects the clinical presentation of ALI/ARDS
By triggering a physical association between CD14 and TLR4,LPS is a principal initiator of host antimicrobial innate immune responses.Inflammatory response to endotoxins is largely mediated through CD14 and TLR4.The activation of CD14 and TLR4 leads to proinflammatory cascade through NF-κB,which induces expression of inflammatory mediators {tumor necrosis factor(TNF)-α,etc}.Elevated CD 14 is reported to be associated with increased infection and greater mortality in critically ill patients.The level of expression of TLR4 is closely associated with the extent of acute pulmonary response to inhaled endotoxin.
Anaesthetics are administered to sedate intubated,mechanically ventilated ALI and ARDS patients.These anaesthetics may modulate the immune system and influence the clinical course and outcome of patients.Previous reports have shown that anaesthetics affect inflammatory mediation in ALI.However,the exact mechanism of anaesthetic-induced effects on ALI has yet to be clarified.
The lung represents a site for the invasion of various bacteria or bacterial products. Along with alveolar macrophages,pulmonary epithelial cells are the first cells to be challenged by pathogenic microorganisms.Rat ATII cells synthesize and secrete surfactant,control the volume and composition of fluid in the alveolar space,and proliferate and differentiate into alveolar typeⅠepithelial cells after lung injury to maintain the integrity of the alveolar lining.Recently,there have been publications reporting the involvement of ATⅡin modulating the development of inflammatory reactions within the alveolus.ATⅡsecrete chemokines in response to inflammatory stimuli in vitro,suggesting a potential physiologic role in acute inflammatory lung injury.Little is known about the interaction of generally used intravenous anesthetic propofol with ATⅡ.
As a means of investigating how anaesthetics affect the inflammatory system,we designed a study to examine the effects of propofol,midazolam,and remifentanil on the mRNA and protein expression of both CD14 and TLR4 in rats with LPS-induced ALI and to investigate whether LPS induced inflammation in ATⅡis through CD14 and TLR4 and the effect of different dosage of propofol on the inflammation.
Materials
1.Animals:
Adult male Wistar rats weighting 250-350g for in vivo experiment and weighting 180-250 g for in vitro experiment,were provided by the Experimental Animal Center of China Medical University
2.Major reagents:
LPS,propofol,midazolam,remifentanil,Takara reverse transcript kit,rabbit anti-rat CD-14 primary antibody,goat anti-rat TLR4 primary antibody,mouse anti-ratβ-actin primary antibody,goat anti-rabbit secondary antibody,rabbit anti-goat secondary antibody,TNF-αELISA kit,SYBR PrimeScriptTM real-timePCR kit.
3.Major instruments:
Respiratory machine,micro-infusion pump,photo analysis system electrophoresis apparatus,semi-dry blotter,spectrophotometer,bechtop,CO_2 Incubators,Olympus BX61 photo analysis system,ABI 7500 Real-Time PCR system.
Methods
1.Animal model of the acute lung injury
Anaesthesia was induced with 50 mg kg~(-1) pentobarbital sodium administered intraperitoneally and maintained with pentobarbital sodium and pipecuronium bromide via left jugular vein.Tracheal cannulation(14 gauge) was performed after tracheostomy.The right carotid artery was catheterized for continuous arterial blood pressure measurements and blood sampling.The animals were ventilated using Servo 900C ventilator.LPS 5mg/kg was instilled intratracheally to induce acute lung injury.
2.In vivo experiment
According to the intravenous anaesthetics they received,the rats were randomly assigned to 5 groups with 8 in each group:
Control group:saline IT and salineⅳ
LPS group:LPS IT and salineⅳ
Propofol group:LPS IT and propofolⅳ
Midazolam group:LPS IT and midazolamⅳ
Remifentanil group:LPS IT and remifentanilⅳ
This intravenous infusion continued to the end of five-hour protocol.At 0 h,and at 1,3 and 5 h,MAP and HR were recorded and arterial blood was sampled for blood gas analysis.After the protocol,CD14 and TLR4 gene expression and protein expression in lung tissues were evaluated using real time PCR and western blot,respectively.TNF-αin bronchoalveolar lavage fluid(BALF) was detected using ELISA.
3.Primary culture of typeⅡalveolar epithelial cell
ATⅡwere isolated from male SPF degree Wistar rats(180-250g),following the protocol according to the method of Richard et al~(12).Briefly,after intraperitoneal sodium pentobarbital and heparin(400U.kg~(-1)),the thoracic cavity was opened and the inferior vena cava was cut.The lungs were perfused with sterile saline.After removal from the thoracic cavity,they were washed 6 times with normal saline and digested enzymatically with typsin.The lungs were then minced,and the resulting suspension was filtered through 150μm and 30μm mesh once.The cell mixture was purified by centrifugation on a discontinuous Percoll gradient.The interface was collected and after rewashing the cells were seeded onto 6-well culture dishes in DMEM at 37℃in the humidified atmosphere of 95%air/5%CO2 and cultured for 18-20 hours.
4.In vivo experiment
The cultured ATⅡcells from the same rat were randomly assigned to one of the following five groups:
Group C:ATⅡcells was cultured in the absence of propofol and LPS;
Group LPS:treated with 1μg.~(-1) ml LPS;
Group P1:treated with 1μg~(-1) LPS and 25μM propofol;
Group P2:treated with 1μg~(-1) LPS and 50μM propofol;
Group P3:treated with 1μg~(-1) LPS and 100μM propofol.
ATⅡcells in untreated control group and propofol groups were cultured for 3 h. CD14 and TLR4 mRNA were detected using real-time PCR.Western blot were used to detect CD14 and TLR4 protein expression.CD14 and TLR4 expression on the ATⅡcells were imaged using immunofluorescence.TNF-α.production were determined using ELISA kit.
5.Data analysis and statistics:
The data were normally distributed as determined by the Levene's test.ANOVA followed by a post hoc Tukey's test was used for multiple-group comparisons.Values are expressed as means±SD.The significance level was set at P<0.05.SPSS 13.0 software was used for all statistical work.
Results
1.Rat model of acute lung injury
This study regards PaO_2/FiO_2≤300mmHg as the successful setup of ALI rat model. After LPS 5mg/kg IT,LPS group,C,D and E all satisfied this standard.PaO_2 values at 3 h and 5 h were lower than at 0 h.HE stain showed edema,hemorrhage and thickness of alveolar wall as well as obvious inflammatory ceils infiltration into the septum.
2.Results of in vivo experiment
CD 14 mRNA expression in LPS group,propofol group,midazolam group and remifentanil group were significantly increased compared with control group.TLR4 mRNA expression in LPS group and remifentanil group were significantly increased compared with control group.TLR4 mRNA expression in propofol group and midazolam group were lower than in midazolam group.TLR4 mRNA in propofol group was lower than in LPS group.
CD14 protein expression in LPS,propofol,midazolam and remifentanil groups were significantly higher than in control group and there were no statistical difference among LPS,propofol,midazolam and remifentanil groups.TLR4 protein expression in LPS,midazolam and remifentanil groups were higher than in control group.TLR4 protein expression in propofol and midazolam groups were lower than in remifentanil group and TLR4 protein expression in propofol group was lower than in LPS group.
Propofol and midazolam group showed milder lung injury compared with remifentanil group in HE stain.
Compared with LPS group,TNF-αin BALF was decreased in propofol group and elevated in remifentanil group.
3.Primary culture of ATⅡcells
The cell mix,obtained by the discontinuous Percoll gradient centrifugation method,contained 85±5%of ATⅡwhich were stained blue with AKP histochemical staining.Transmission electron microscopy showed typical lamella bodies in ATⅡ.The cell viability in each group,determined by trypan blue dye exclusion test ranged from 90 to 95%.
4.Results of in vitro experiment
LPS stimulation resulted in an increased CD 14 and TLR4 expression and increased TNF-αproduction in ATⅡcells.Propofol,at concentrations≥50μM, significantly and dose-dependently decreased CD14 and TLR4 mRNA expression as well as their protein expression in ATⅡcells.This was accompanied by decreases in TNF-αproduction.
Conclusion
1.Both the mRNA and protein expressions of CD14 and TLR4 can be increased by LPS stimulation in the lung tissue in vivo and the ATⅡcells in vitro,so as with TNF-αproduction.
2.Propofol can downregulate both the mRNA and protein expressions of CD14 and TLR4 in the lung tissue in vivo and the ATⅡcells in vitro,and can decrease TNF-αproduction.
3.Compared with remifentanil,propofol and midazolam can relieve acute lung injury.
4.The study showed the collocation of CD14 and TLR4 expression.
引文
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12 Jeyaseelan S, Manzer R, Young SK, et al. Induction of CXCL5 during inflammation in the rodent lung involves activation of alveolar epithelium. Am J Respir Cell Mol Biol.2005;32:531-9.
13 Chu CH, David Liu D, Hsu YH, et al. Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats. Pulm Pharmacol Ther 2007; 20:503-12.
14 Kim SN, Son SC, Lee SM, et al. Midazolam inhibits proinflammatory mediators in the lipopolysaccharide-activated macrophage. Anesthesiology 2006; 105:105-10.
15 Sacerdote P, Gaspani L, Rossoni G, et al. Effect of the opioid remifentanil on cellular immune response in the rat. Int Immunopharmacol 2001;1:713-9
16 Beilin B, Shavit Y, Hart J, et al. Effects of anesthesia based on large versus small doses of fentanyl on natural killer cell cytotoxicity in the perioperative period.. Anesth Analg 1996;82:492-7.
17 Jawan B, Kao YH, Goto S, et al. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-kappaB translocation. Toxicol Appl Pharmacol.2008;229:362-73.
18 van Helden HP, Kuijipers WC, Steenvooorden D, et al. Intratracheal aerosolization of endotoxin (LPS) in the rat: a comprehensive animal model to study adult (acute) respiratory distress syndrome. Exp Lung Res, 1997, 23(4):297-316.
19 Kawai T, Takeuchi O, Fujita T, et al. Lipopoiysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol, 2001, 167 (10):5887.
20 Jiang Q, Akashi S, Miyake K, et al. Lipopoiysaccharide induces physical proximity between CD14 and Toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-κB. J Immunol,2000, 165 (7):3541-4.
21 Demiralay R, Gursan N, Ozbilim G, Erdogan G, Demirci E. Comparison of the effects of erdosteine and N-acetylcysteinne on apoptosis regulation in endotoxin-induced acute lung injury. J Appl Toxicol. 2006;26(4):301-8.
22 Fragen RJ. Pharmacokinetics and pharmacodynamics of midazolam given via continuous intravenous infusion in intensive care units. Clin Ther. 1997; 19(3):405-19;
23 Barr J, Donner A. Optimal intravenous dosing strategies for sedatives and analgesics in the intensive care unit. Crit Care Clin. 1995;11(4):827-47.
24 Muellejans B, Lopez A, Cross MH, Bonome C, Morrison L, Kirkham AJ. Remifentanil versus fentanyl for analgesia based sedation to provide patient comfort in the intensive care unit: a randomized, double-blind controlled trial. Crit Care. 2004 Feb;8(1):R1-R11.
25 Muellejans B, Matthey T, Scholpp J, Schill M. Sedation in the intensive care unit with remifentanil/propofol versus midazolam/fentanyl: a randomised, open-label,pharmacoeconomic trial Crit Care. 2006;10(3):R91.
26 Huettemann E, Jung A, Vogelsang H, Hout N, Sakka SG: Effects of propofol vs methohexital on neutrophil function and immune status in critically ill patients. J Anesth.2006; 20:86-91.
27 Evans MJ, Cabral LJ, Stephens RJ, Freeman G. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol, 1975, 22:142-50.
28 Crandall ED, Matthay MA. Alveolar epithelial transport. Basic science to clinical medicine. Am J Respir Crit Care Med, 2001,163:1021 -9.
29 Chen RM, Wu GJ, Tai YT, et al. Propofol reduces nitric oxide biosynthesis in lipopolysaccharideactivated macrophages by downregulating the expression of inducible nitric oxide synthase. Arch Toxicol. 2003, 77:418-423.
30 Richards RJ, Davies N, Atkins J, Oreffo VI. Isolation, biochemical characterization, and culture of lung type II cells of the rat. Lung 1987;165:143-158.
31 Kwak SH,Choi Jl,Park JT.Effects of Propofol on endotoxin-induced acute lung injury in Rabbit.J Korean Med Sci,2004,19:55-61.
32 胡晓敏,吕阳,杨艳等.丙泊酚对大鼠肠缺血再灌注后肺损伤的影响.中华麻醉学杂志,2007.27:256-259.
33 Gepts E,Camu F,Cockshott ID,Douglas EJ.Disposition of propofol administered as constant rate intravenous infusions in humans.Anesth Analg,1987,66:1256-63.
34 Servin F,Desmonts JM,Haberer JP,et al.Pharmacokinetics and protein binding of propofol in patients with cirrhosis.Anesthesiology,1988,69:887-91.
1 Luhr OR, Antonsen.K., Karlsson M, et al. Incidence and mortality after acute respiratory failure and acute respiratory distress syndrome in Sweden, Denmark, and Iceland. The ARF Study Group. Am J Respir Crit Care Med, 1999, 159(6): 1849-61.
2 Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury. N Engl J Med, 2006; 354:416-417.
3 Vernooy JH, Dentener MA, van Suylen RJ,et al. Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells. Am J Respir Cell Mol Biol, 2001,24(5):569-576
4 Becker MN, Diamond G, Verghese MW, et al. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem, 2000,275(38):29731-6.
5 Frevert CW, Matute-Bello G, Skerrett SJ, et al. Effect of CD14 blockade in rabbits with Escherichia coli pneumonia and sepsis. J Immunol, 2000. 164(10): 5439-5445.
6 Shimazu R, Akashi S, Ogata H, et al. MD-2, a Molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med, 1999,189(11): 1777-1782.
7 Barsness KA, Arcaroli J, Harken AH, et al. Hemorrhage-induced acute lung injury is TLR-4 dependent. Am J Physiol Regul Integr Comp Physiol, 2004 , 287(3):R592-9.
8 Lorenz E, Jones M, Wohlford-Lenane C, et al. Genes other than TLR4 are involved in the response to inhaled LPS. Am J Physiol Lung Cell Mol Physiol, 2001,281(5):L1106-14.
9 Jeyaseelan S, Manzer R, Young SK, et al. Induction of CXCL5 during inflammation in the rodent lung involves activation of alveolar epithelium. Am J Respir Cell Mol Biol.2005;32:531-9.
10 Lin SM, Frevert CW, Kajikawa O, et al. Differential regulation of membrane CD14 expression and endotoxin-tolerance in alveolar aacrophages. Am J Respir Cell Mol Biol, 2004,31(2): 162-70.
11 Edelman DA, Jiang Y, Tyburski J, et al. Toll-like receptor-4 message is up-regulated in lipopolysaccharide-exposed rat lung pericytes. J Surg Res, 2006,134(1): 22-27.
12 Togbe D, Schnyder-Candrian S, Schnyder B, et al. TLR4 gene dosage contributes to endotoxin-induced acute respiratory inflammation. J Leukoc Biol, 2006, 80(3): 451-7.
13 Muzio M, Bosisio D, Polentarutti N, Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol, 2000, 164(11): 5998-6004.
14 Blackwell TS, Christman JW.The Role of Nuclear Factor-kappa B in Cytokine Gene Regulation. Am J Respir Cell Mol Biol, 1997, 17(1): p. 3-9
15 Blackwell TS, Blackwell TR, Holden EP, et a 1. In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation.. J Immunol,1996,157(4):1630-1637.
16 Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med,2000,342:1334-49.
17 Janeway CN, Travers P, Walport M, et al. Immunobiology.. 6th Edition ed.
18 Beck-Schimmer B, Madjdpour C, Kneller S,Role of alveolar epithelial ICAM-1 in lipopolysaccharide-induced lung inflammation. Eur Respir J, 2002,19(6): 1142-1150.
19 Lamy M, Deby-Dupont G, Deby C, et al. Measurements of mediator cascade during adult respiratory distress syndrome. In: Artigs A, Lemaire F, Suter PM, Zapol WM, Adult Respiratory Distress Syndrome. New York: Churchill Livingstone, 1992: 71-88.
20 Samuelsson B, Dahlen SE, Lindgren JA, et al. Leukotrienes and lipoxins: structures,biosynthesis, and biological effects. Science, 1987, 237(4819):1171-6.
21 Murphy PG, Bennett JR, Myers DS, et al. The effect of propofol anaesthesia on free radical-induced lipid peroxidation in rat liver microsomes. Eur J Anaesthesiol, 1993, 10 (4):261-6.
22 Mathy-Hartert M, Deby-Dupont G, Hans P, et al. Protective activity of propofol, Diprivan and intralipid against active oxygen species. Mediators Inflamm. 1998, 7(5):327-33.
23 Murphy PG, Myers DS, Davies MJ, et al. The antioxidant potential of propofol(2,6-diisopropylphenol). Br JAnaesth, 1992, 68(6): 613-8.
24 Galley HF, Dubbels AM, Webster NR.The effect of midazolam and propofol on interleukin-8 from human polymorphonuclear leukocytes. Anesth Analg, 1998, 86(6): 1289-93.
25 Taniguchi T, Kanakura H, Takemoto Y, et al. Effects of ketamine and propofol on the ratio of interleukin-6 to interleukin-10 during endotoxemia in rats. Tohoku J Exp Med, 2003, 200(2):85-92.
26 Taniguchi T, Yamamoto K, Ohmoto N, et al Effects of propofol on hemodynamic and inflammatory responses to endotoxemia in rats. Crit Care Med, 2000, 28(4): 1101-6.
27 Taniguchi T, Kanakura H, Yamamoto K.Effects of posttreatment with propofol on mortality and cytokine responses to endotoxin-induced shock in rats. Crit Care Med, 2002, 30(4):904-7.
28 Mikawa K, Akamatsu H, Nishina K, et al. Propofol inhibits human neutrophil functions. Anesth Analg, 1998, 87(3):695-700.
29 Hofbauer R, Frass M, Salfinger H, et al. Propofol reduces the migration of human leukocytes through endothelial cell monolayers. Crit Care Med, 1999, 27(9): 1843-7.
30 Takao Y, Mikawa K, Nishina K, et al. Attenuation of Acute Lung Injury with Propofol in Endotoxemia. Anesth Analg, 2005, 100(3):810-6
31 Gao J, Zhao WX, Zhou LJ, et al.Protective effects of propofol on lipopolysaccharide-activated endothelial cell barrier dysfunction. Inflamm Res, 2006, 55(9):385-392.
32 Gao J, Zeng BX, Zhou LJ, et al. Protective effects of early treatment with propofol on endotoxininduced acute lung injury in rats. Br J Anaesth, 2004, 92(2):277-9.
33 Kwak SH, Choi JI, Park JT. Effects of Propofol on Endotoxin-Induced Acute Lung Injury in Rabbit. J Korean Med Sci, 2004, 19(1):55-61.
34 Inada T, Taniuchi S, Shingu K, et al. Propofol depressed neutrophil hydrogen peroxide production more than midazolam, whereas adhesion molecule expression was minimally affected by both anesthetics in rats with abdominal sepsis. Anesth Analg, 2001,92(2): 437-41.
35 Chu CH, David Liu D, Hsu YH, et al. Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats. Pulm Pharmacol Ther, 2007,20:503-12.
36 Kim SN, Son SC, Lee SM, et al. Midazolam inhibits proinflammatory mediators in the lipopolysaccharide-activated macrophage. Anesthesiology, 2006, 105:105-10.
2 Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol 2004; 4:499-511.
3 Dianhua Jiang, Jiurong Liang, Juan Fan, et al. Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nature medicine. 2005; 11: 1173-1179.
4 Phan HH, Cho K, Sainz-Lyon KS, et al.CD14-dependent modulation of NF-kappaB alternative splicing in the lung after burn injury. Gene 2006; 371:121 -9.
5 Wright SD, Ramos RA, Tobias PS, et al.CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990;249:1431-1433.
6 Smith LS, Kajikawa O, Elson G, et al. Effect of Toll-like receptor 4 blockade on pulmonary inflammation caused by mechanical ventilation and bacterial endotoxin. Exp Lung Res.2008;34:225-43.
7 Jeyaseelan S, Chu HW, Young SK, et al. Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury Infect Immun. 2005 Mar;73(3):1754-63.
8 Togbe D, Schnyder-Candrian S, Schnyder B, et al. Toll-like receptor and tumor necrosis factor dependent endotoxin-induced acute lung injury. Int J Exp Pathol. 2007;88:387-91.
9 Crandall ED, Matthay MA. Alveolar epithelial transport. Basic science to clinical medicine. Am J Respir Crit Care Med. 2001; 163:1021-9.
10 Evans MJ, Stephens RJ, Freeman G. Renewal of pulmonary epithelium following oxidant injury.Lung cells in disease. Amsterdam, The Netherlands: North-Holland; 1976.p165-178.
11 Rose F, Guthmann B, Tenenbaum T, et al. Apical, but not basolateral, endotoxin preincubation protects alveolar epithelial cells against hydrogen peroxide-induced loss of barrier function: the role of nitric oxide synthesis. J Immunol. 2002; 169:1474-81.
12 Jeyaseelan S, Manzer R, Young SK, et al. Induction of CXCL5 during inflammation in the rodent lung involves activation of alveolar epithelium. Am J Respir Cell Mol Biol.2005;32:531-9.
13 Chu CH, David Liu D, Hsu YH, et al. Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats. Pulm Pharmacol Ther 2007; 20:503-12.
14 Kim SN, Son SC, Lee SM, et al. Midazolam inhibits proinflammatory mediators in the lipopolysaccharide-activated macrophage. Anesthesiology 2006; 105:105-10.
15 Sacerdote P, Gaspani L, Rossoni G, et al. Effect of the opioid remifentanil on cellular immune response in the rat. Int Immunopharmacol 2001;1:713-9
16 Beilin B, Shavit Y, Hart J, et al. Effects of anesthesia based on large versus small doses of fentanyl on natural killer cell cytotoxicity in the perioperative period.. Anesth Analg 1996;82:492-7.
17 Jawan B, Kao YH, Goto S, et al. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-kappaB translocation. Toxicol Appl Pharmacol.2008;229:362-73.
18 van Helden HP, Kuijipers WC, Steenvooorden D, et al. Intratracheal aerosolization of endotoxin (LPS) in the rat: a comprehensive animal model to study adult (acute) respiratory distress syndrome. Exp Lung Res, 1997, 23(4):297-316.
19 Kawai T, Takeuchi O, Fujita T, et al. Lipopoiysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol, 2001, 167 (10):5887.
20 Jiang Q, Akashi S, Miyake K, et al. Lipopoiysaccharide induces physical proximity between CD14 and Toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-κB. J Immunol,2000, 165 (7):3541-4.
21 Demiralay R, Gursan N, Ozbilim G, Erdogan G, Demirci E. Comparison of the effects of erdosteine and N-acetylcysteinne on apoptosis regulation in endotoxin-induced acute lung injury. J Appl Toxicol. 2006;26(4):301-8.
22 Fragen RJ. Pharmacokinetics and pharmacodynamics of midazolam given via continuous intravenous infusion in intensive care units. Clin Ther. 1997; 19(3):405-19;
23 Barr J, Donner A. Optimal intravenous dosing strategies for sedatives and analgesics in the intensive care unit. Crit Care Clin. 1995;11(4):827-47.
24 Muellejans B, Lopez A, Cross MH, Bonome C, Morrison L, Kirkham AJ. Remifentanil versus fentanyl for analgesia based sedation to provide patient comfort in the intensive care unit: a randomized, double-blind controlled trial. Crit Care. 2004 Feb;8(1):R1-R11.
25 Muellejans B, Matthey T, Scholpp J, Schill M. Sedation in the intensive care unit with remifentanil/propofol versus midazolam/fentanyl: a randomised, open-label,pharmacoeconomic trial Crit Care. 2006;10(3):R91.
26 Huettemann E, Jung A, Vogelsang H, Hout N, Sakka SG: Effects of propofol vs methohexital on neutrophil function and immune status in critically ill patients. J Anesth.2006; 20:86-91.
27 Evans MJ, Cabral LJ, Stephens RJ, Freeman G. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol, 1975, 22:142-50.
28 Crandall ED, Matthay MA. Alveolar epithelial transport. Basic science to clinical medicine. Am J Respir Crit Care Med, 2001,163:1021 -9.
29 Chen RM, Wu GJ, Tai YT, et al. Propofol reduces nitric oxide biosynthesis in lipopolysaccharideactivated macrophages by downregulating the expression of inducible nitric oxide synthase. Arch Toxicol. 2003, 77:418-423.
30 Richards RJ, Davies N, Atkins J, Oreffo VI. Isolation, biochemical characterization, and culture of lung type II cells of the rat. Lung 1987;165:143-158.
31 Kwak SH,Choi Jl,Park JT.Effects of Propofol on endotoxin-induced acute lung injury in Rabbit.J Korean Med Sci,2004,19:55-61.
32 胡晓敏,吕阳,杨艳等.丙泊酚对大鼠肠缺血再灌注后肺损伤的影响.中华麻醉学杂志,2007.27:256-259.
33 Gepts E,Camu F,Cockshott ID,Douglas EJ.Disposition of propofol administered as constant rate intravenous infusions in humans.Anesth Analg,1987,66:1256-63.
34 Servin F,Desmonts JM,Haberer JP,et al.Pharmacokinetics and protein binding of propofol in patients with cirrhosis.Anesthesiology,1988,69:887-91.
1 Luhr OR, Antonsen.K., Karlsson M, et al. Incidence and mortality after acute respiratory failure and acute respiratory distress syndrome in Sweden, Denmark, and Iceland. The ARF Study Group. Am J Respir Crit Care Med, 1999, 159(6): 1849-61.
2 Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury. N Engl J Med, 2006; 354:416-417.
3 Vernooy JH, Dentener MA, van Suylen RJ,et al. Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells. Am J Respir Cell Mol Biol, 2001,24(5):569-576
4 Becker MN, Diamond G, Verghese MW, et al. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem, 2000,275(38):29731-6.
5 Frevert CW, Matute-Bello G, Skerrett SJ, et al. Effect of CD14 blockade in rabbits with Escherichia coli pneumonia and sepsis. J Immunol, 2000. 164(10): 5439-5445.
6 Shimazu R, Akashi S, Ogata H, et al. MD-2, a Molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med, 1999,189(11): 1777-1782.
7 Barsness KA, Arcaroli J, Harken AH, et al. Hemorrhage-induced acute lung injury is TLR-4 dependent. Am J Physiol Regul Integr Comp Physiol, 2004 , 287(3):R592-9.
8 Lorenz E, Jones M, Wohlford-Lenane C, et al. Genes other than TLR4 are involved in the response to inhaled LPS. Am J Physiol Lung Cell Mol Physiol, 2001,281(5):L1106-14.
9 Jeyaseelan S, Manzer R, Young SK, et al. Induction of CXCL5 during inflammation in the rodent lung involves activation of alveolar epithelium. Am J Respir Cell Mol Biol.2005;32:531-9.
10 Lin SM, Frevert CW, Kajikawa O, et al. Differential regulation of membrane CD14 expression and endotoxin-tolerance in alveolar aacrophages. Am J Respir Cell Mol Biol, 2004,31(2): 162-70.
11 Edelman DA, Jiang Y, Tyburski J, et al. Toll-like receptor-4 message is up-regulated in lipopolysaccharide-exposed rat lung pericytes. J Surg Res, 2006,134(1): 22-27.
12 Togbe D, Schnyder-Candrian S, Schnyder B, et al. TLR4 gene dosage contributes to endotoxin-induced acute respiratory inflammation. J Leukoc Biol, 2006, 80(3): 451-7.
13 Muzio M, Bosisio D, Polentarutti N, Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol, 2000, 164(11): 5998-6004.
14 Blackwell TS, Christman JW.The Role of Nuclear Factor-kappa B in Cytokine Gene Regulation. Am J Respir Cell Mol Biol, 1997, 17(1): p. 3-9
15 Blackwell TS, Blackwell TR, Holden EP, et a 1. In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation.. J Immunol,1996,157(4):1630-1637.
16 Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med,2000,342:1334-49.
17 Janeway CN, Travers P, Walport M, et al. Immunobiology.. 6th Edition ed.
18 Beck-Schimmer B, Madjdpour C, Kneller S,Role of alveolar epithelial ICAM-1 in lipopolysaccharide-induced lung inflammation. Eur Respir J, 2002,19(6): 1142-1150.
19 Lamy M, Deby-Dupont G, Deby C, et al. Measurements of mediator cascade during adult respiratory distress syndrome. In: Artigs A, Lemaire F, Suter PM, Zapol WM, Adult Respiratory Distress Syndrome. New York: Churchill Livingstone, 1992: 71-88.
20 Samuelsson B, Dahlen SE, Lindgren JA, et al. Leukotrienes and lipoxins: structures,biosynthesis, and biological effects. Science, 1987, 237(4819):1171-6.
21 Murphy PG, Bennett JR, Myers DS, et al. The effect of propofol anaesthesia on free radical-induced lipid peroxidation in rat liver microsomes. Eur J Anaesthesiol, 1993, 10 (4):261-6.
22 Mathy-Hartert M, Deby-Dupont G, Hans P, et al. Protective activity of propofol, Diprivan and intralipid against active oxygen species. Mediators Inflamm. 1998, 7(5):327-33.
23 Murphy PG, Myers DS, Davies MJ, et al. The antioxidant potential of propofol(2,6-diisopropylphenol). Br JAnaesth, 1992, 68(6): 613-8.
24 Galley HF, Dubbels AM, Webster NR.The effect of midazolam and propofol on interleukin-8 from human polymorphonuclear leukocytes. Anesth Analg, 1998, 86(6): 1289-93.
25 Taniguchi T, Kanakura H, Takemoto Y, et al. Effects of ketamine and propofol on the ratio of interleukin-6 to interleukin-10 during endotoxemia in rats. Tohoku J Exp Med, 2003, 200(2):85-92.
26 Taniguchi T, Yamamoto K, Ohmoto N, et al Effects of propofol on hemodynamic and inflammatory responses to endotoxemia in rats. Crit Care Med, 2000, 28(4): 1101-6.
27 Taniguchi T, Kanakura H, Yamamoto K.Effects of posttreatment with propofol on mortality and cytokine responses to endotoxin-induced shock in rats. Crit Care Med, 2002, 30(4):904-7.
28 Mikawa K, Akamatsu H, Nishina K, et al. Propofol inhibits human neutrophil functions. Anesth Analg, 1998, 87(3):695-700.
29 Hofbauer R, Frass M, Salfinger H, et al. Propofol reduces the migration of human leukocytes through endothelial cell monolayers. Crit Care Med, 1999, 27(9): 1843-7.
30 Takao Y, Mikawa K, Nishina K, et al. Attenuation of Acute Lung Injury with Propofol in Endotoxemia. Anesth Analg, 2005, 100(3):810-6
31 Gao J, Zhao WX, Zhou LJ, et al.Protective effects of propofol on lipopolysaccharide-activated endothelial cell barrier dysfunction. Inflamm Res, 2006, 55(9):385-392.
32 Gao J, Zeng BX, Zhou LJ, et al. Protective effects of early treatment with propofol on endotoxininduced acute lung injury in rats. Br J Anaesth, 2004, 92(2):277-9.
33 Kwak SH, Choi JI, Park JT. Effects of Propofol on Endotoxin-Induced Acute Lung Injury in Rabbit. J Korean Med Sci, 2004, 19(1):55-61.
34 Inada T, Taniuchi S, Shingu K, et al. Propofol depressed neutrophil hydrogen peroxide production more than midazolam, whereas adhesion molecule expression was minimally affected by both anesthetics in rats with abdominal sepsis. Anesth Analg, 2001,92(2): 437-41.
35 Chu CH, David Liu D, Hsu YH, et al. Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats. Pulm Pharmacol Ther, 2007,20:503-12.
36 Kim SN, Son SC, Lee SM, et al. Midazolam inhibits proinflammatory mediators in the lipopolysaccharide-activated macrophage. Anesthesiology, 2006, 105:105-10.