Calpains对机械通气肺损伤早期炎性反应的作用及机制研究
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摘要
研究背景与目的
对于呼吸功能严重受损的患者,机械通气是必不可少的治疗手段。然而机械通气是把双刃剑,在提供有效呼吸支持的同时,还能导致肺部严重损伤,即机械通气所致肺损伤(ventilator-induced lung injury VILI).这是机械通气最严重的并发症,易诱发炎症反应综合征(SIRS)和多器官功能衰竭(MSOF),死亡率近50%。
肺牵张是VILI的主要原因。机械牵张可使气道上皮屏障功能降低,肺泡毛细血管通透性增强,中性粒细胞迁移浸润。而机体如何将机械信号转为生物信号并释放各种致炎因子促进中性粒细胞(PMN)浸涧肺,最终导致SIRS及MSOF,其早期机制仍未知,因此阐明VILI的早期机制,了解起始免疫反应,有助于VILI的早期治疗,意义重大。
Calpains是存在于细胞浆的Ca2+依赖性半胱氨酸蛋白酶(蛋白水解酶),自从1964年由Guroff第一次将Calpain描述为钙激活的中性蛋白酶,关于其形态、结构、活性、位置、生理及病理作用已经被越来越多的阐述。到目前,哺乳动物中已被识别的Calpain蛋白酶家族成员有15个,基于其组织表现形式不同分为普遍存在型和组织特异型。其中Calpain1、Calpain2、Calpain10存在于所有哺乳动物细胞中Calpain3(肌肉P94)、Calpain8(胃nCL-2)是组织特异表达型。Calpain1和Calpain2是最主要的两种亚型,它们由两种不同的催化亚基(80KDa)和一种相同的调节亚基(28KDa)组成,两者的激活分别需要微摩尔和毫摩尔的Ca2+浓度来活化,所以又分别被称为u-Calpain和m-Calpain。当细胞内Ca2+浓度超过各亚型所需阈浓度时,其两种亚基产生催化、自溶反应,使其构象改变从而产生了活性。
目前Calpains的生理作用还未完全阐明,但研究表明Calpains参与了细胞运动、凋亡、炎症等过程。短暂的Calpains活性参与细胞信号和蛋白逆转录过程,而持续的Calpains活性与急性神经系统损伤和阿尔茨海默病有关。Calpainl基因缺失小鼠将导致血小板功能障碍,Calpainl和Calpain2基因缺失小鼠将导致胚胎死亡。Calpainl调节了脓毒血症中肺血管内皮细胞的凋亡,Calpain抑制剂降低了酵母多糖所致的多器官功能衰竭和出血性休克大鼠的多器官损伤和NF-kB活性。
目前Calpain在VILI的作用及调节机理,未见报道。
本研究显示大潮气量机械通气早期即诱发了迅速的Caiapain活性和以中性粒细胞浸润、肺毛细血管通透性增高、肺水肿形成为特征的炎性反应：分别以体内转染小干扰RNA基因沉默技术和药物途径干预Calpain表达后,降低了支气管灌洗液(BAL)内中性粒细胞数和蛋白含量、同时TNF-a、IL-6降低,肺毛细血管外肺水含量(ELW)减少、肺MPO活性降低,说明Calpain在VILI发病机理中起关键性作用,是机械通气肺损伤早期炎性反应发病机理的关键因素。进‘步的研究显示,大潮气量机械通气诱发Calpain活性后,介导了NOS-3磷酸化,使NOS-3磷酸化来源的N()产物聚集增多,后者刺激细胞粘附因子ICAM-1磷酸化,使P-ICAM-1蛋白表达增高,刺激PMN浸润入肺产生VILI。提示由大潮气量机械通气刺激Calpain活性增高触发的早期肺炎性反应是通过Akt_P-NOS3_NO_Src_P-ICAM1_PMN途径而产生的。
研究方法
动物实验。
本研究分四部分
第一部分小鼠机械通气肺损伤模型的建立及大潮气量机械通气肺炎性反应
1.小鼠机械通气肺损伤模型：育龄8-12周、体重25-30克重的雄性C57BL／6J小鼠,以氯胺酮腹腔注射麻醉(75mg/kg),后行气管切开,分别以大潮气量(28ml/kg、呼吸频率60次／分钟)和普通潮气量(7ml／kg、呼吸频率120次／分钟)行机械通气。1%异氟醚与空气混合维持麻醉。机械通气维持呼气末二氧化碳35-45cmH2O、保温灯维持体温37-38℃。(Fig1)
2.大潮气量机械通气肺炎性反应：机械通气2小时后分别测定支气管肺泡灌洗液(BAL)内的蛋白含量、中性粒细胞数目、TNF-a、IL-6、肺毛细血管外肺水(ELW)、肺MPO活性测定。每种检测指标需要小鼠6-8只。
第二部分Calpain活性分析及Calpain抑制剂对VILI的作用
1.Calpain活性测定：分别取大潮气量机械通气15分钟、30分钟、60分钟、120分钟肺组织,测定Calpain活性。每组3-4只,数据取其平均值。
2. Calpain抑制剂对其活性的影响：Calpain抑制剂Ⅰ分别以0、5mg/kg、10mg/kg、20mg/kg于机械通气前1小时行腹腔注射,机械通气2小时后测定Calpain活性。每组3-4只,数据取其平均值。
3.Calpain抑制剂对VILI的作用：Calpain抑制剂10mg/kg使用前后观察机械通气对支气管肺泡灌洗液(BAL)内的蛋白含量、中性粒细胞数目、TNF-a、 IL-6、肺毛细血管外肺水(ELW)、肺MPO活性的影响
第三部分脂质体转染Calpain1/2siRNA基因模型的建立及Calpain1/2基因knockdown对VILI的作用
1.采用新鲜制备的脂质体介导的基因转染法。新鲜制备脂质体,将脂质体与Calpain1或Calpain2siRNA以一定比例混匀,于机械通气前48小时行尾静脉注射,Calpain scsiRNA相同比例与脂质体混合作为对照。Calpain1或Calpain2siRNA是否成功转染由Western blotting分析验证。
2. Calpain1/2基因knockdown对VILI的作用：Calpain1/2基因knockdown后对ELW、BAL内(蛋白含量、PMN数目、TNF-a、IL-6)、肺MPO活性的作用。小鼠分普通潮气量机械通气、大潮气量机械通气Sc siRNA、Calpainl siRNA、Calpain2siRNA共四组,每组6只,取均值。于机械通气48小时前行尾静脉注射Sc siRNA或Calpainl/2siRNA,后行Western blotting分析,确定成功转染的标本保留其ELW、BAL内(蛋白含量、PMN数目、TNF-a、IL-6)、肺MPO活性数据,未转染者剔除。
第四部分Calpain对VILI的作用机制
1. Calpain活性对P-ICAM-1、CAM-1蛋白表达的影响
2. Calpain活性对NO产物的影响
3.NOS抑制剂L-NAME对P-ICAM-1蛋白表达的影响
4.ICAM-1在Tyr518位点磷酸化抑制剂Genistein对P-ICAM-1蛋白表达的影响和对BAL内PMN数目的影响
5.Calpain活性对P-NOS-3、NOS3、NOS2蛋白表达的影响
6.Calpan活性对P-Akt、Akt蛋白表达的影响
结果
1与普通潮气量机械通气相比,大潮气量机械通气2小时产生了以肺泡-毛细血管通透性增强、肺水肿形成、中性粒细胞浸润入肺为特征的VILI
使ELW增加三倍、BAL的中性粒细胞数目由正常的几乎为零上升到8.5*105、BAL的总蛋白含量增加5倍、肺组织MPO活性增加4倍、BAL内IL-6、 TNF-a由正常的几乎为零分别上升到110pg/ml和360pg/ml。(Fig2)
2大潮气量机械通气产生了迅速的Calpain活性,Calpain抑制剂显著降低VILI(Fig3.4)
大潮气量机械通气15分钟即引起Calpain活性加倍并维持至2小时,而普通潮气量机械通气2小时Calpain活性不变。Calpain抑制剂降低其活性呈剂量依赖性,10mg/kg浓度的抑制剂即可完全阻滞其活性。
Calpain抑制剂减轻了机械通气肺炎性反应：提示药物抑制Calpain活性对减轻VILI有显著作用。
3Calpainl或Calpain2基因knockdown降低了VILI(Fig5)
提示以基因沉默技术干预Calpainl或Calpain2蛋白表达对减轻VILI有显著作用。两种亚型对VILI的作用无显著差异,提示两种亚型在VILI中发挥相同作用
4.1Calpain活性在VILI中通过调节ICAM-1磷酸化发挥作用(Fig6)
最近的研究显示ICAM-1位于络氨酸的磷酸化介导了早期炎性反应的白细胞的迁移。本实验我们假定大潮气量机械通气通过Calpain活性诱发ICAM-1磷酸化。实验数据显示大潮气量机械通气15分钟ICAM-1磷酸化开始发生于Tyr518位点并持续至机械通气2小时。Calpain抑制剂显著降低了机械通气诱发的ICAM-1tyrosine的磷酸化：与此相似,Calpainl或Calpain2knockdown也明显降低了VILI的ICAM-1磷酸化。Calpain抑制剂或Calpainl、Calpain2siRNA本身对ICAM-1磷酸化没有影响。数据还显示：2小时的机械通气没有改变ICAM-1的蛋白表达。
以上结果显示Calpain介导了ICAM一1的tyrosine磷酸化。
4.2Calpain活性介导了NO产物的形成刺激ICAM-1磷酸化从而引起PMN浸润入肺(Fig7)
体内研究发现肺炎性反应中NO来源于NOS-2和NOS-3两种途径,NO在VILI的发病机制中起重要作用。本研究数据显示：与自主呼吸相比,机械通气15分钟NO即升高3倍并在2小时的机械通气中继续缓慢升高至4倍,Calpain抑制剂阻滞了这种表现,而Calpain抑制剂本身没有改变基础NO浓度；与此相同,Calpainl或Calpain2基因knockdown也显著抑制了机械通气NO浓度的升高。
4.3NO刺激了一系列蛋白的磷酸化(Fig8)
本实验我们重点判定在小鼠肺内,大潮气量机械通气诱发的ICAM-1磷酸化和相应的PMN浸润是否为NO依赖机制。数据显示：非特异性NOS抑制剂L-NAME消除了ICAM-1在Tyr518位点的磷酸化并使BAL内PMN显著降低。
4.4ICAM-1tyrosine磷酸化在肺PMN浸润的作用
我们采用tyrosine特异性抑制剂genistein预先于机械通气1小时前腹腔注射,显著消除了ICAM-1在Tyr518位点的磷酸化并显著减轻了肺内PMN浸润(Fig8)。
4.5Calpain活性上调了NOS-3磷酸化水平,使NOS-3来源而非NOS-2来源的NO产物增加
实验数据显示：大潮气量机械通气诱导产生的NO产物来自于NOS-3磷酸化和NOS-2两种途径。如图7所示：机械通气15分钟,NOS-3磷酸化提高2.5倍并持续维持高水平至2小时,NOS-2蛋白表达增高,NOS-3蛋白表达不变；Calpain抑制剂显著降低NOS-3磷酸化而对NOS-2蛋白表达没有影响；与此相似,Calpainl或Calpain2基因knockdown后也显著降低NOS-3磷酸化而对NOS-2蛋白表达没有影响。这些结果说明大潮气量机械通气时,Calpain通过NOS-3磷酸化途径而非NOS-2途径介导NO产物增加(Fig9)。
4.6为了明确Calpain对NOS-3活化(磷酸化)的作用,我们观察了Calpain对NOS-3上游信号AKt磷酸化的作用
结果显示：Akt磷酸化程度在大潮气量机械通气的增强是时间依赖性的,机械通气1小时,达到最高,2小时较1小时下降。Calpain抑制剂或Calpain knockdown后,AKt磷酸化活性也降低。说明Calpain在VILI使NOS-3磷酸化是通过活化其上游信号Akt而产生的(Fig9)。
结论
1大潮气量机械通气产生了迅速的Calpain(?)舌性
2Calpain活性诱发了以中性粒细胞浸润、肺泡-毛细血管通透性增高、肺水肿形成为特征的炎性反应；运用脂质体介导小干扰RNA体内转染基因沉默技术和药物干预Calpain表达后,炎性反应降低,产生了一系列肺保护作用。
3机械通气早期Calpain1/2都被激活,两种亚型在VILI中具有相同作用
4Calpain活性增高介导的早期肺部炎性反应是通过Akt-P-NOS3-NO-Src-P-ICAM1-PMN途径而产：生的。(Fig10)
意义
首次探讨Calpain在机械通气肺损伤早期炎性反应的发病机理中起决定性作用,是治疗机械通气肺损伤的关键靶点。基因沉默技术或药物干预Calpain活性、对VILI的早期治疗有重要意义,为VILI的早期治疗提供了理论依据和实验基础。
Backgroung and objective Mechanical ventilation is currently a therapeutic mainstay in the critically ill patients with acute lung injury and acute respiratory distress syndrome. It is now well recognized from animal experiments and increasing evidence in humans that mechanical ventilation per se can cause further lung injury and perhaps lead to the development of multiple organ failure
Ventilator-induced lung injury (VILI) as a result of mechanical stretch is characterized by massive inflammatory responses in the lung as evidenced by alveolar-capillary hyperpermeability, protein-rich edema formation and polymorphonuclear neutrophil (PMN) infiltration into lung interstitium and alveoli Calpains are Ca2+-dependent. non-lysosomal cysteine proteases (proteolytic enzymes). Several calpain isoforms, including calpain1(or μ-calpain), calpain2(or m-calpain), and calpain10, are ubiquitously expressed in mammals, whereas calpain3(muscle p94) and calpain8(stomach nCl-2) reveal tissue-specific expression patterns (25). Calpain1and calpain2, the two major isoforms of calpain. are heterodimers comprised of a unique large (80kDa) catalytic subunit and a common small (28kDa) regulatory subunit. Calpain1and calpain2require micromolar (1to20μM) and millimolar (0.25to0.75mM) concentration of intracellular Ca2+for activation. respectively. Elevations in intracellular Ca2+above isoform-specific thresholds induce Ca2+binding and subsequent activation via a conformational change that creates an active catalytic triad, as well as autolysis of both subunits. Calpain1-deficient mice have a platelet dysfunction (5), whereas knocking down both calpain1and2simultaneously leads to an embryonic-lethal phenotype. The physiological functions of calpains are not yet fully elucidated. Transient calpain activation has been shown to be involved in essential functions such as cell signaling and protein turnover, while sustained activation of calpains contributes to acute neurological injuries and Alzheimer's disease. Recently, calpains have been implicated in apoptotic cell death and tissue injury, and appear to be an essential regulator of inflammation. During sepsis, calpain1has been shown to induce apoptosis in pulmonary microvascular endothelial cells. In addition, inhibition of calpains attenuated zymosan-induced multiple organ failure, the activation of NF-κB and organ injury in hemorrhagic shock in the rat. There is not any about Calpain in VILI and metabolism. However,whether and how Calpain participates in the regulation of Ventilator-induced lung injury induced by ventilator remains important and unknown.
In the present study, using genetic and pharmacological approaches we identified a crucial role for calpains in the pathogenesis of ventilator-induced lung inflammation and injury. Rapid calpain activation following mechanical stretch triggers PMN infiltration into the lung via endothelial nitric oxide synthase or eNOS (NOS-3)-nitric oxide (NO)-mediated ICAM-1phosphorylation. Our data suggest that calpains may be a key mechanical sensor of early lung inflammation and injury.
Methods
Experimental protocols
Mice model of VILI
Mice were anesthetized with ketamine (75mg/kg). underwent tracheotomy and ventilated for2h with1%isoflurane in room air to maintain anesthesia, using either high or normal tidal volume. For high tidal volume ventilation, mice were connected to a Harvard Apparatus ventilator (MiniVent. Harvard Biosciences:Holliston. MA), with a tidal volume of28ml/kg, a respiratory rate of60breath/min. and0cm H2O end-expiratory pressure. For normal volume ventilation, mice received7ml/kg of tidal volume, a respiratory rate of120breath/min and0cm H2O end expiratory pressure. In order to maintain PaCO2between35and45torr (4.7-6.0kPa),～5ml of dead space was added to the ventilator circuit. Body temperature was maintained between37℃and38℃, using a heating lamp (15). In some experiments, animals were pretreated with inhibitor of both calpain1and calpain2. calpain inhibitor I (ALLN,5-20mg/kg, intraperitoneally. i.p.). NOS inhibitor L-NAME (15mg/kg, i.p.) or tyrosine phosphorylation inhibitor genistein (50mg/kg, i.p.)1h before exposure to mechanical ventilation. After2h of mechanical ventilation, various measurements were obtained. Depletion of calpain1and calpain2in mouse lungs
Calpain1and2small interfering RNAs (siRNAs) are a pool of3target-specific20-25nt siRNAs (Santa Cruz). Calpain1/2was depleted in mouse pulmonary vasculature by retro-orbital injection of cationic liposome-siRNA complexes as described previously. The liposome-siRNA complexes were prepared by addition of0.5mg/kg siRNA into150μl of liposome suspensions. A scramble siRNA was employed as control. Successful transfection of calpain1and calpain2siRNAs was confirmed by Western blotting analysis of lung homogenates. All animal experiments were performed48h after transfection.
Calpain activity assays
Analysis of calpain activity in the lung was performed using a calpain activity assay kit (Abeam, Cambridge, MA) according to the manufacturer's instructions
Western blot analysis
At the end of experiments, lungs were homogenized and lysed in lysis buffer (50mM Tris-HCl, pH7.5,150mM NaCl,1mM EDTA,0.25%sodium deoxycholate,1.0%NP-40,0.1%SDS,1mM Na3VO4,1mM NaF,1mM PMSF, and protease inhibitor mixture). Protein concentrations were determined by the Bicinchoninic Acid (BCA) assay. Equal amounts of protein from each lysate were separated by SDS/PAGE and transferred to nitrocellulose membranes, blocked, and then incubated with relevant blotting antibodies.Protein bands were visualized using Pierce ECL reagent. Densotometric measurement was preformed from scanned films using Imagej software (NIH).
NO production measurement
NO production in the lung was determined using a commercial nitrate/nitrite colorimetric assay kit (Cayman Chemical Company. Ann Arbor. MI) according to the manufacturer's instructions.
Lung MPO assay
The azurophilic granules of PMNs contain MPO, a potent oxidizing and chlorinating agent which can be used as a marker for PMN infiltration into the lung MPO activity was determined using a MPO assay kit (Invitrogen) according to manufacturer's protocol.
Determination of PMN counts in bronchoalveolar lavage (BAL) fluid
Assessment of pulmonary vascular permeability and edema formation
Cytokine assay
Statistical analysis
RESULTS
1Rapid calpain activation by high tidal volume mechanical ventilation in the mouse lung
2Calpain inhibitor attenuates mechanical ventilation-induced lung inflammation
3Depletion of calpain1or calpain2in pulmonary vasculature reduced mechanical ventilation-induced lung inflammation
4Calpain activation regulates ICAM-1phosphorylation during VILI
5Calpain-mediated NO production stimulates ICAM-1phosphorylation and subsequent PMN infiltration into the lung
6A critical role of calpain in NOS-3-derived NO production after mechanical ventilation
Conclusion
1Mechanical ventilation with high tidal volume induced rapid (within15minutes) and persistent calpain activation
2Calpain triggered lung inflammation evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition and Depletion of calpain1or calpain2significantly attenuated these inflammatory responses caused by lung hyperinflation and had a protective effect against ventilator-induced lung inflammatory responses.
3Both calpain land calpain2were activated in the early stage of the mechanical ventilator. They have the same enfluence inVILI.
4.calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1phosphorylation and neutrophil recruitment.
In summary. our study identifies calpain activation as an early event of lung inflammatory injury induced by hyperinflation.These findings suggest that calpain may represent a novel therapeutic target for the prevention and treatment of VILI.
对于呼吸功能严重受损的患者,机械通气是必不可少的治疗手段。然而机械通气是把双刃剑,在提供有效呼吸支持的同时,还能导致肺部严重损伤,即机械通气所致肺损伤(ventilator-induced lung injury VILI).这是机械通气最严重的并发症,易诱发炎症反应综合征(SIRS)和多器官功能衰竭(MSOF),死亡率近50%。
肺牵张是VILI的主要原因。机械牵张可使气道上皮屏障功能降低,肺泡毛细血管通透性增强,中性粒细胞迁移浸润。而机体如何将机械信号转为生物信号并释放各种致炎因子促进中性粒细胞(PMN)浸涧肺,最终导致SIRS及MSOF,其早期机制仍未知,因此阐明VILI的早期机制,了解起始免疫反应,有助于VILI的早期治疗,意义重大。
Calpains是存在于细胞浆的Ca2+依赖性半胱氨酸蛋白酶(蛋白水解酶),自从1964年由Guroff第一次将Calpain描述为钙激活的中性蛋白酶,关于其形态、结构、活性、位置、生理及病理作用已经被越来越多的阐述。到目前,哺乳动物中已被识别的Calpain蛋白酶家族成员有15个,基于其组织表现形式不同分为普遍存在型和组织特异型。其中Calpain1、Calpain2、Calpain10存在于所有哺乳动物细胞中Calpain3(肌肉P94)、Calpain8(胃nCL-2)是组织特异表达型。Calpain1和Calpain2是最主要的两种亚型,它们由两种不同的催化亚基(80KDa)和一种相同的调节亚基(28KDa)组成,两者的激活分别需要微摩尔和毫摩尔的Ca2+浓度来活化,所以又分别被称为u-Calpain和m-Calpain。当细胞内Ca2+浓度超过各亚型所需阈浓度时,其两种亚基产生催化、自溶反应,使其构象改变从而产生了活性。
目前Calpains的生理作用还未完全阐明,但研究表明Calpains参与了细胞运动、凋亡、炎症等过程。短暂的Calpains活性参与细胞信号和蛋白逆转录过程,而持续的Calpains活性与急性神经系统损伤和阿尔茨海默病有关。Calpainl基因缺失小鼠将导致血小板功能障碍,Calpainl和Calpain2基因缺失小鼠将导致胚胎死亡。Calpainl调节了脓毒血症中肺血管内皮细胞的凋亡,Calpain抑制剂降低了酵母多糖所致的多器官功能衰竭和出血性休克大鼠的多器官损伤和NF-kB活性。
目前Calpain在VILI的作用及调节机理,未见报道。
本研究显示大潮气量机械通气早期即诱发了迅速的Caiapain活性和以中性粒细胞浸润、肺毛细血管通透性增高、肺水肿形成为特征的炎性反应：分别以体内转染小干扰RNA基因沉默技术和药物途径干预Calpain表达后,降低了支气管灌洗液(BAL)内中性粒细胞数和蛋白含量、同时TNF-a、IL-6降低,肺毛细血管外肺水含量(ELW)减少、肺MPO活性降低,说明Calpain在VILI发病机理中起关键性作用,是机械通气肺损伤早期炎性反应发病机理的关键因素。进‘步的研究显示,大潮气量机械通气诱发Calpain活性后,介导了NOS-3磷酸化,使NOS-3磷酸化来源的N()产物聚集增多,后者刺激细胞粘附因子ICAM-1磷酸化,使P-ICAM-1蛋白表达增高,刺激PMN浸润入肺产生VILI。提示由大潮气量机械通气刺激Calpain活性增高触发的早期肺炎性反应是通过Akt_P-NOS3_NO_Src_P-ICAM1_PMN途径而产生的。
研究方法
动物实验。
本研究分四部分
第一部分小鼠机械通气肺损伤模型的建立及大潮气量机械通气肺炎性反应
1.小鼠机械通气肺损伤模型：育龄8-12周、体重25-30克重的雄性C57BL／6J小鼠,以氯胺酮腹腔注射麻醉(75mg/kg),后行气管切开,分别以大潮气量(28ml/kg、呼吸频率60次／分钟)和普通潮气量(7ml／kg、呼吸频率120次／分钟)行机械通气。1%异氟醚与空气混合维持麻醉。机械通气维持呼气末二氧化碳35-45cmH2O、保温灯维持体温37-38℃。(Fig1)
2.大潮气量机械通气肺炎性反应：机械通气2小时后分别测定支气管肺泡灌洗液(BAL)内的蛋白含量、中性粒细胞数目、TNF-a、IL-6、肺毛细血管外肺水(ELW)、肺MPO活性测定。每种检测指标需要小鼠6-8只。
第二部分Calpain活性分析及Calpain抑制剂对VILI的作用
1.Calpain活性测定：分别取大潮气量机械通气15分钟、30分钟、60分钟、120分钟肺组织,测定Calpain活性。每组3-4只,数据取其平均值。
2. Calpain抑制剂对其活性的影响：Calpain抑制剂Ⅰ分别以0、5mg/kg、10mg/kg、20mg/kg于机械通气前1小时行腹腔注射,机械通气2小时后测定Calpain活性。每组3-4只,数据取其平均值。
3.Calpain抑制剂对VILI的作用：Calpain抑制剂10mg/kg使用前后观察机械通气对支气管肺泡灌洗液(BAL)内的蛋白含量、中性粒细胞数目、TNF-a、 IL-6、肺毛细血管外肺水(ELW)、肺MPO活性的影响
第三部分脂质体转染Calpain1/2siRNA基因模型的建立及Calpain1/2基因knockdown对VILI的作用
1.采用新鲜制备的脂质体介导的基因转染法。新鲜制备脂质体,将脂质体与Calpain1或Calpain2siRNA以一定比例混匀,于机械通气前48小时行尾静脉注射,Calpain scsiRNA相同比例与脂质体混合作为对照。Calpain1或Calpain2siRNA是否成功转染由Western blotting分析验证。
2. Calpain1/2基因knockdown对VILI的作用：Calpain1/2基因knockdown后对ELW、BAL内(蛋白含量、PMN数目、TNF-a、IL-6)、肺MPO活性的作用。小鼠分普通潮气量机械通气、大潮气量机械通气Sc siRNA、Calpainl siRNA、Calpain2siRNA共四组,每组6只,取均值。于机械通气48小时前行尾静脉注射Sc siRNA或Calpainl/2siRNA,后行Western blotting分析,确定成功转染的标本保留其ELW、BAL内(蛋白含量、PMN数目、TNF-a、IL-6)、肺MPO活性数据,未转染者剔除。
第四部分Calpain对VILI的作用机制
1. Calpain活性对P-ICAM-1、CAM-1蛋白表达的影响
2. Calpain活性对NO产物的影响
3.NOS抑制剂L-NAME对P-ICAM-1蛋白表达的影响
4.ICAM-1在Tyr518位点磷酸化抑制剂Genistein对P-ICAM-1蛋白表达的影响和对BAL内PMN数目的影响
5.Calpain活性对P-NOS-3、NOS3、NOS2蛋白表达的影响
6.Calpan活性对P-Akt、Akt蛋白表达的影响
结果
1与普通潮气量机械通气相比,大潮气量机械通气2小时产生了以肺泡-毛细血管通透性增强、肺水肿形成、中性粒细胞浸润入肺为特征的VILI
使ELW增加三倍、BAL的中性粒细胞数目由正常的几乎为零上升到8.5*105、BAL的总蛋白含量增加5倍、肺组织MPO活性增加4倍、BAL内IL-6、 TNF-a由正常的几乎为零分别上升到110pg/ml和360pg/ml。(Fig2)
2大潮气量机械通气产生了迅速的Calpain活性,Calpain抑制剂显著降低VILI(Fig3.4)
大潮气量机械通气15分钟即引起Calpain活性加倍并维持至2小时,而普通潮气量机械通气2小时Calpain活性不变。Calpain抑制剂降低其活性呈剂量依赖性,10mg/kg浓度的抑制剂即可完全阻滞其活性。
Calpain抑制剂减轻了机械通气肺炎性反应：提示药物抑制Calpain活性对减轻VILI有显著作用。
3Calpainl或Calpain2基因knockdown降低了VILI(Fig5)
提示以基因沉默技术干预Calpainl或Calpain2蛋白表达对减轻VILI有显著作用。两种亚型对VILI的作用无显著差异,提示两种亚型在VILI中发挥相同作用
4.1Calpain活性在VILI中通过调节ICAM-1磷酸化发挥作用(Fig6)
最近的研究显示ICAM-1位于络氨酸的磷酸化介导了早期炎性反应的白细胞的迁移。本实验我们假定大潮气量机械通气通过Calpain活性诱发ICAM-1磷酸化。实验数据显示大潮气量机械通气15分钟ICAM-1磷酸化开始发生于Tyr518位点并持续至机械通气2小时。Calpain抑制剂显著降低了机械通气诱发的ICAM-1tyrosine的磷酸化：与此相似,Calpainl或Calpain2knockdown也明显降低了VILI的ICAM-1磷酸化。Calpain抑制剂或Calpainl、Calpain2siRNA本身对ICAM-1磷酸化没有影响。数据还显示：2小时的机械通气没有改变ICAM-1的蛋白表达。
以上结果显示Calpain介导了ICAM一1的tyrosine磷酸化。
4.2Calpain活性介导了NO产物的形成刺激ICAM-1磷酸化从而引起PMN浸润入肺(Fig7)
体内研究发现肺炎性反应中NO来源于NOS-2和NOS-3两种途径,NO在VILI的发病机制中起重要作用。本研究数据显示：与自主呼吸相比,机械通气15分钟NO即升高3倍并在2小时的机械通气中继续缓慢升高至4倍,Calpain抑制剂阻滞了这种表现,而Calpain抑制剂本身没有改变基础NO浓度；与此相同,Calpainl或Calpain2基因knockdown也显著抑制了机械通气NO浓度的升高。
4.3NO刺激了一系列蛋白的磷酸化(Fig8)
本实验我们重点判定在小鼠肺内,大潮气量机械通气诱发的ICAM-1磷酸化和相应的PMN浸润是否为NO依赖机制。数据显示：非特异性NOS抑制剂L-NAME消除了ICAM-1在Tyr518位点的磷酸化并使BAL内PMN显著降低。
4.4ICAM-1tyrosine磷酸化在肺PMN浸润的作用
我们采用tyrosine特异性抑制剂genistein预先于机械通气1小时前腹腔注射,显著消除了ICAM-1在Tyr518位点的磷酸化并显著减轻了肺内PMN浸润(Fig8)。
4.5Calpain活性上调了NOS-3磷酸化水平,使NOS-3来源而非NOS-2来源的NO产物增加
实验数据显示：大潮气量机械通气诱导产生的NO产物来自于NOS-3磷酸化和NOS-2两种途径。如图7所示：机械通气15分钟,NOS-3磷酸化提高2.5倍并持续维持高水平至2小时,NOS-2蛋白表达增高,NOS-3蛋白表达不变；Calpain抑制剂显著降低NOS-3磷酸化而对NOS-2蛋白表达没有影响；与此相似,Calpainl或Calpain2基因knockdown后也显著降低NOS-3磷酸化而对NOS-2蛋白表达没有影响。这些结果说明大潮气量机械通气时,Calpain通过NOS-3磷酸化途径而非NOS-2途径介导NO产物增加(Fig9)。
4.6为了明确Calpain对NOS-3活化(磷酸化)的作用,我们观察了Calpain对NOS-3上游信号AKt磷酸化的作用
结果显示：Akt磷酸化程度在大潮气量机械通气的增强是时间依赖性的,机械通气1小时,达到最高,2小时较1小时下降。Calpain抑制剂或Calpain knockdown后,AKt磷酸化活性也降低。说明Calpain在VILI使NOS-3磷酸化是通过活化其上游信号Akt而产生的(Fig9)。
结论
1大潮气量机械通气产生了迅速的Calpain(?)舌性
2Calpain活性诱发了以中性粒细胞浸润、肺泡-毛细血管通透性增高、肺水肿形成为特征的炎性反应；运用脂质体介导小干扰RNA体内转染基因沉默技术和药物干预Calpain表达后,炎性反应降低,产生了一系列肺保护作用。
3机械通气早期Calpain1/2都被激活,两种亚型在VILI中具有相同作用
4Calpain活性增高介导的早期肺部炎性反应是通过Akt-P-NOS3-NO-Src-P-ICAM1-PMN途径而产：生的。(Fig10)
意义
首次探讨Calpain在机械通气肺损伤早期炎性反应的发病机理中起决定性作用,是治疗机械通气肺损伤的关键靶点。基因沉默技术或药物干预Calpain活性、对VILI的早期治疗有重要意义,为VILI的早期治疗提供了理论依据和实验基础。
Backgroung and objective Mechanical ventilation is currently a therapeutic mainstay in the critically ill patients with acute lung injury and acute respiratory distress syndrome. It is now well recognized from animal experiments and increasing evidence in humans that mechanical ventilation per se can cause further lung injury and perhaps lead to the development of multiple organ failure
Ventilator-induced lung injury (VILI) as a result of mechanical stretch is characterized by massive inflammatory responses in the lung as evidenced by alveolar-capillary hyperpermeability, protein-rich edema formation and polymorphonuclear neutrophil (PMN) infiltration into lung interstitium and alveoli Calpains are Ca2+-dependent. non-lysosomal cysteine proteases (proteolytic enzymes). Several calpain isoforms, including calpain1(or μ-calpain), calpain2(or m-calpain), and calpain10, are ubiquitously expressed in mammals, whereas calpain3(muscle p94) and calpain8(stomach nCl-2) reveal tissue-specific expression patterns (25). Calpain1and calpain2, the two major isoforms of calpain. are heterodimers comprised of a unique large (80kDa) catalytic subunit and a common small (28kDa) regulatory subunit. Calpain1and calpain2require micromolar (1to20μM) and millimolar (0.25to0.75mM) concentration of intracellular Ca2+for activation. respectively. Elevations in intracellular Ca2+above isoform-specific thresholds induce Ca2+binding and subsequent activation via a conformational change that creates an active catalytic triad, as well as autolysis of both subunits. Calpain1-deficient mice have a platelet dysfunction (5), whereas knocking down both calpain1and2simultaneously leads to an embryonic-lethal phenotype. The physiological functions of calpains are not yet fully elucidated. Transient calpain activation has been shown to be involved in essential functions such as cell signaling and protein turnover, while sustained activation of calpains contributes to acute neurological injuries and Alzheimer's disease. Recently, calpains have been implicated in apoptotic cell death and tissue injury, and appear to be an essential regulator of inflammation. During sepsis, calpain1has been shown to induce apoptosis in pulmonary microvascular endothelial cells. In addition, inhibition of calpains attenuated zymosan-induced multiple organ failure, the activation of NF-κB and organ injury in hemorrhagic shock in the rat. There is not any about Calpain in VILI and metabolism. However,whether and how Calpain participates in the regulation of Ventilator-induced lung injury induced by ventilator remains important and unknown.
In the present study, using genetic and pharmacological approaches we identified a crucial role for calpains in the pathogenesis of ventilator-induced lung inflammation and injury. Rapid calpain activation following mechanical stretch triggers PMN infiltration into the lung via endothelial nitric oxide synthase or eNOS (NOS-3)-nitric oxide (NO)-mediated ICAM-1phosphorylation. Our data suggest that calpains may be a key mechanical sensor of early lung inflammation and injury.
Methods
Experimental protocols
Mice model of VILI
Mice were anesthetized with ketamine (75mg/kg). underwent tracheotomy and ventilated for2h with1%isoflurane in room air to maintain anesthesia, using either high or normal tidal volume. For high tidal volume ventilation, mice were connected to a Harvard Apparatus ventilator (MiniVent. Harvard Biosciences:Holliston. MA), with a tidal volume of28ml/kg, a respiratory rate of60breath/min. and0cm H2O end-expiratory pressure. For normal volume ventilation, mice received7ml/kg of tidal volume, a respiratory rate of120breath/min and0cm H2O end expiratory pressure. In order to maintain PaCO2between35and45torr (4.7-6.0kPa),～5ml of dead space was added to the ventilator circuit. Body temperature was maintained between37℃and38℃, using a heating lamp (15). In some experiments, animals were pretreated with inhibitor of both calpain1and calpain2. calpain inhibitor I (ALLN,5-20mg/kg, intraperitoneally. i.p.). NOS inhibitor L-NAME (15mg/kg, i.p.) or tyrosine phosphorylation inhibitor genistein (50mg/kg, i.p.)1h before exposure to mechanical ventilation. After2h of mechanical ventilation, various measurements were obtained. Depletion of calpain1and calpain2in mouse lungs
Calpain1and2small interfering RNAs (siRNAs) are a pool of3target-specific20-25nt siRNAs (Santa Cruz). Calpain1/2was depleted in mouse pulmonary vasculature by retro-orbital injection of cationic liposome-siRNA complexes as described previously. The liposome-siRNA complexes were prepared by addition of0.5mg/kg siRNA into150μl of liposome suspensions. A scramble siRNA was employed as control. Successful transfection of calpain1and calpain2siRNAs was confirmed by Western blotting analysis of lung homogenates. All animal experiments were performed48h after transfection.
Calpain activity assays
Analysis of calpain activity in the lung was performed using a calpain activity assay kit (Abeam, Cambridge, MA) according to the manufacturer's instructions
Western blot analysis
At the end of experiments, lungs were homogenized and lysed in lysis buffer (50mM Tris-HCl, pH7.5,150mM NaCl,1mM EDTA,0.25%sodium deoxycholate,1.0%NP-40,0.1%SDS,1mM Na3VO4,1mM NaF,1mM PMSF, and protease inhibitor mixture). Protein concentrations were determined by the Bicinchoninic Acid (BCA) assay. Equal amounts of protein from each lysate were separated by SDS/PAGE and transferred to nitrocellulose membranes, blocked, and then incubated with relevant blotting antibodies.Protein bands were visualized using Pierce ECL reagent. Densotometric measurement was preformed from scanned films using Imagej software (NIH).
NO production measurement
NO production in the lung was determined using a commercial nitrate/nitrite colorimetric assay kit (Cayman Chemical Company. Ann Arbor. MI) according to the manufacturer's instructions.
Lung MPO assay
The azurophilic granules of PMNs contain MPO, a potent oxidizing and chlorinating agent which can be used as a marker for PMN infiltration into the lung MPO activity was determined using a MPO assay kit (Invitrogen) according to manufacturer's protocol.
Determination of PMN counts in bronchoalveolar lavage (BAL) fluid
Assessment of pulmonary vascular permeability and edema formation
Cytokine assay
Statistical analysis
RESULTS
1Rapid calpain activation by high tidal volume mechanical ventilation in the mouse lung
2Calpain inhibitor attenuates mechanical ventilation-induced lung inflammation
3Depletion of calpain1or calpain2in pulmonary vasculature reduced mechanical ventilation-induced lung inflammation
4Calpain activation regulates ICAM-1phosphorylation during VILI
5Calpain-mediated NO production stimulates ICAM-1phosphorylation and subsequent PMN infiltration into the lung
6A critical role of calpain in NOS-3-derived NO production after mechanical ventilation
Conclusion
1Mechanical ventilation with high tidal volume induced rapid (within15minutes) and persistent calpain activation
2Calpain triggered lung inflammation evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition and Depletion of calpain1or calpain2significantly attenuated these inflammatory responses caused by lung hyperinflation and had a protective effect against ventilator-induced lung inflammatory responses.
3Both calpain land calpain2were activated in the early stage of the mechanical ventilator. They have the same enfluence inVILI.
4.calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1phosphorylation and neutrophil recruitment.
In summary. our study identifies calpain activation as an early event of lung inflammatory injury induced by hyperinflation.These findings suggest that calpain may represent a novel therapeutic target for the prevention and treatment of VILI.
引文
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