【作者】 吴剑波；

【导师】 于金贵； 胡国昌；

【作者基本信息】 山东大学 ， 麻醉学， 2014， 博士

【摘要】 研究背景机械通气是重要的生命支持手段也会导致并发症急性肺损伤,即机械通气所致肺损伤(centilator-induced lung,VILI),加重免疫系统功能的紊乱,影响呼吸窘迫综合征等重症患者预后。但机械通气致肺损伤作用机制有待探讨。因此,开展VILI机制研究,对探寻防治VILI的措施、改善患者预后具有重要意义。研究表明固有免疫在机械通气所致肺损伤过程中发挥重要作用,明确机械通气引发的免疫效应及其分子机制对于VILI的预防和治疗至为关键。机械牵张诱发细胞因子和炎性介质的释放,最终导致弥漫性肺泡的损害,病理特征包括渗透性的肺水肿、透明膜的形成以及炎性细胞的浸润、促炎因子释放等。肺泡巨噬细胞(AMs)是重要的靶细胞,肺泡巨噬细胞消减可以明显改善机械通气所致肺损伤。肺泡巨噬细胞大约占肺泡腔外周细胞数的5%左右,机械通气可以快速激活AMs并可能在VILI发病机制中发挥重要作用,其中包含促炎细胞因子白介素-1β(interleukin-1β, IL-1β)和IL-18释放,IL-1p受体拮抗剂和抗IL-1p抗体都可以减轻患者急性肺损伤炎性反应。促炎细胞因子IL-1p和IL-18是机械通气肺损伤肺部炎症的明确标记。IL-1β和IL-18产生与炎性体的激活有密切关联。炎性体(Inflammasome)是由多种蛋白质组成的复合体,可以调节固有免疫系统应激反应。主要的炎性体有：NLRP1、NLRP3、NLRC4、IPAF、AIM2炎性体等。其中NLRP3炎性体可感知伤害性刺激信号而被激活,伤害性刺激包括组织损伤、代谢、应激、感染等,现在己知其参与了多种无菌性炎性疾病进程。NLRP3炎性体复合体被激活后进一步激活半胱氨酸蛋白酶Caspase-1,后者的成熟体通过蛋白水解作用切割加工促炎细胞因子pro-IL-1β(?)(?)pro-1L-18成熟并促进它们分泌。然而,机械力被细胞感受以及被转化为生物化学信号并传导入细胞的机制、机械通气导致IL-1β和IL-18释放机制尚不明确,本文首次探讨了NLRP3炎性体机械牵张肺损伤的调节作用。我们的预实验显示,机械牵张可以促使线粒体ROS产生。研究表明抑制或清除ROS可明显抑制NLRP3的激活。最初的假设是ROS来源于吞噬相关NADPH氧化酶ROS,但是随后的研究表明NADPH基因敲除小鼠实验中,NLRP3炎性体的激活并未受明显影响。说明ROS可能来源于另一重要途径：线粒体ROS。其他研究证据表明：线粒体ROS是NLRP3炎性体激活所需的主要因素,线粒体功能障碍诱发NLRP3炎性体激活。另外我们发现VILI伴随NLRP3炎性体激活。机械牵张是否通过调节ROS的产生影响NLRP3炎性体的激活呢?相关研究未见报道,据此,我们提出假说：机械牵张可能激活ROS依赖性NLRP3炎性体/炎性因子通路,最终导致肺损伤。研究目的明确机械通气对NLRP3炎性体活性的影响,并探讨其作用机制；探讨VILI中ROS对NLRP3炎性体活性的影响；明确ROS/NLRP3/炎性因子信号通路对VILI的调节作用。研究方法本研究通过肺泡巨噬细胞、基因敲除小鼠、小鼠VILI模型等研究对象,采用Western Blot、siRNA转染基因下调、流式细胞术、ELISA等手段,多层面探讨机械通气肺损伤中ROS/NLRP3炎性体/细胞因子IL-1p通路的调节作用,为探寻防治机械通气肺损伤对策提供新的理论依据。第一部分机械牵张激活肺泡巨噬细胞NLRP3炎性体/IL-1β信号通路1.1肺泡巨噬细胞分离提取和机械牵张从C57BL/6J SD小鼠支气管肺泡灌洗液分离收集肺泡巨噬细胞,将收集的AMs种植在6孔弹性底培养皿内(BioFlex baseplate),培养基为含10%胎牛血清的MCDB-131培养基(不含抗生素)；细胞种植密度为1.0×105/cm2,培养皿内预铺0.1%胶原蛋白Ⅳ,置于5%CO2-95%空气的孵育箱内培养(37℃,饱和湿度)。BioFlex培养皿固定在FX-4000T Flexercell田胞环形牵张仪(Flexcell International,McKeesport, PA)25-mm BioFlex承载台,进行牵张模拟机械通气,内环境5%CO2-95%空气(37℃,饱和湿度)。计算机设定牵张参数,牵张频率30cycles/min(0.5Hz),牵张/松弛比率1：1。培养皿底部牵张度分别设定为8、15、和20%,分别对应机械通气肺通气量为50、64、80%的肺活量,牵张时间设为4h。不同牵张时间设定为0(对照)、1、2、4小时,牵张度设为20%。1.2siRNA转染技术选择合适siRNA下调相关基因表达：NLRP3/AIM2/IPAF。用无血清培养基稀释siRNA和DharmaFECT转染试剂,室温孵育5分钟,混合后室温孵育20分钟,加入无抗生素的完全培养基。换成转染培养基,在37℃孵育24-48小时。设立转染试剂、阴性、阳性对照。1.3ELIS试剂盒检测各组细胞上清pro-IL-1β、pro-1L-18、IL-1β和IL-18含量变化。1.4Western Blo和IP检测各组NLRP3/ASC/Caspase-1表达及相互作用、IL-1β和IL-18含量。采用RIPA缓冲液裂解细胞,提纯后用BCA法测定蛋白浓度,然后用WB测定NLRP3、ASC、Caspase-1、IL-1β和IL-18含量等,最后用ImageJ软件分析。第二部分机械牵张激活NLRP3炎性体信号通路需要线粒体ROS2.1分离C57BL/6J SD小鼠,gp91phox-/-小鼠AMs,不同组予以ROS抑制剂、激活剂,ROS荧光标记试剂预处理后进行机械牵张实验。2.2流式细胞术检测各组细胞线粒体ROS变化避光10μM MitoSOX Red (Invitrogen-Molecular Probes)标记实验细胞30min,进入各组牵张实验流程,然后胰蛋白酶-EDTA收集细胞,将各试验组细胞转入流式细胞技术专用试管,流式细胞术检测线粒体ROS表达,验证机械牵张是否通过线粒体ROS激活NLRP3炎性体。2.3Western Blot检测NLRP3炎性体表达采用RIPA缓冲液裂解细胞,提纯后用BCA法测定蛋白浓度,然后用WB测定NLRP3、ASC、Caspase-1、IL-1β和IL-18含量等,最后用ImageJ软件分析。2.4ELISA试剂盒检测细胞培养上清pro-IL-1β、pro-IL-18、IL-1β和IL-18含量变化。第三部分机械通气对小鼠肺NLRP3炎性体的激活作用3.1VILI模型建立按照文献报道和预实验方案,建立机械通气肺损伤肺损伤模型。3.2NLIP3炎性体表达机械通气后分别取各组支气管肺泡灌洗液和肺组织,Western Blot检测各组细胞裂解液中TLR4、NLRP3、ASC、Caspase-1以及pro-1L-1β、pro-IL-18、IL-1p和IL-18含量变化。ELISA法检测各组支气管肺泡灌洗液中IL-1β和IL-18含量变化。3.3线粒体ROS拮抗剂SS-31预处理实验小鼠SS-31雾化吸入预处理24h,机械通气后分别取各组支气管肺泡灌洗液和肺组织,Western Blot检测各组细胞裂解液中TLR4、NLRP3、ASC、Caspase-1以及IL-1β和IL-18含量变化。Elisa法检测各组支气管肺泡灌洗液中IL-1β和IL-18含量变化。结果第一部分：机械牵张激活肺泡巨噬细胞NLRP3炎性体/IL-1β信号通路机械牵张引起肺泡巨噬细胞促炎因子IL-1β释放,并随牵张时间延长、牵张程度增大而增强。CS诱发小鼠肺泡巨噬细胞IL-1β释放具有caspase-1依赖性。Caspase-1抑制剂明显减少IL-1β含量。机械牵张激活小鼠肺泡巨噬细胞NLRP3炎性体/IL-1β信号通路,而非其他炎性体通路。第二部分：机械牵张激活NLRP3炎性体信号通路需要线粒体ROS机械牵张诱导肺泡巨噬细胞线粒体ROS产生。机械牵张激活AMs NLRP3炎性体需要线粒体ROS。NADPH氧化酶ROS未参与机械牵张所致NLRP3炎性体激活。机械牵张可能通过AMs尿酸释放致线粒体ROS产生并激活NLRP3炎性体。第三部分：机械通气激活小鼠肺ROS/NLRP3炎性体/IL-1β通路高潮气量机械通气激活小鼠肺NLRP3炎性体。线粒体ROS抑制剂SS-31抑制机械通气所致NLRP3炎性体激活,并降低IL-1β和IL-18产生量。通过肺泡巨噬细胞消减技术证明VILI中肺泡巨噬细胞起关键作用。IL-1β受体拮抗剂减轻机械通气所致肺损伤炎性反应。结论机械牵张激活肺泡巨噬细胞线粒体ROS依赖性NLRP3炎性体信号通路并导致炎性因子IL-1β和IL-18释放,是VILI早期的重要机制。创新点及意义本研究首次发现机械牵张通过激活NLRP3炎性体诱导Caspase-1的活化及IL-1β和IL-18产生,并发现机械牵张通过线粒体ROS通路激活NLRP3炎性体。本研究结果提示机械牵张可能通过激活NLRP3炎性体诱导免疫性肺损伤,RNA转染技术或药物干预NLRP3活性、对VILI的早期治疗有重要意义。本研究为VILI发病机制研究提供了新的思路,并为其临床防治提供理论基础。更多还原

【Abstract】 Background and ObjectiveMechanical ventilation is necessary to support patients with acute lung injury (ALI) or its most severe form, acute respiratory distress syndrome (ARDS); however, it has also been shown to exacerbate lung injury, the so-called ventilator-induced lung injury (VILI). VILI is characterized by inflammation associated with robust release of proinflammatory cytokines and activation of inflammatory signaling pathways. A variety of inflammatory mediators are released into the distal air spaces during ALI, and among these is IL-1β, a potent proinflammatory cytokine initiating and amplifying lung inflammation in patients. IL-1β can stimulate the production of a variety of chemokines (e.g., IL-8, MCP-1, and MIP-1a). A recent study indicates that IL-1β is critical for the pathogenesis of VILI. Activation of the inflammatory response, including increased IL-1signaling, is a major mechanism of alveolar barrier dysfunction in VILI. Studies in patients have demonstrated that IL-1β is among the best markers of ventilator-induced lung inflammation. Findings also suggest that IL-1is a key regulator of inflammation. IL-1β receptor antagonist and anti-IL-1β Ab have been demonstrated to prevent ALI.Alveolar macrophages (AMs) residing in the alveolar space account for5%of peripheral lung cells. Under e remainder being mainly dendritic cells and T cells. The lung parenchyma also contains macrophages. AMs have a central role in the maintenance of immunological homeostasis and in host defense. In response to inflammatory stimuli, AMs are the primary source of cytokines in lungs. AMs can be rapidly activated by mechanical ventilation and thus may play an important role in the pathogenesis of VILI. Depletion of AMs improved alveolar barrier dysfunction and lung inflammatory injury caused by high tidal volume ventilation.The inflammasome mainly consists of nucleotide-binding oligomerization domain-like receptor family members containing pyrin domain (NLRP), the adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. To date, four distinct inflammasome complexes including NLRP1(NALP1), NLRP3(NALP3), IPAF (NLRC4, IL-1-converting enzyme) protease-activating factor,and absent in melanoma2(AIM2), have been characterized. Among these inflammasome prototypes, NLRP3is involved in sensing endogenous danger signals, including uric acid crystals and amyloid-b protein. In response to danger signals, NLRP3interacts with procaspase-1through ASC, which leads to activation of caspase-1. Active caspase-1promotes cleavage and, therefore, maturation of proinflammatory cytokines (Pro-IL-1β, Pro-IL-18, and IL-33). The production of mature IL-1β is however tightly regulated.In this study, We will further address the role of ROS/NLRP3Inflammasomes signal pathway in Ventilator-induced lung injury. To test the hypothesis, we will use AMs, siRNA gene transfection, tlr4-/-gp91phox-/-mice and ROS inhibitors with immunology and biochemistry methods to explore the molecular mechanism of ROS/NLRP3inflammasomes signaling pathways mediating Ventilator-Induced Lung Injury.MehtodsIn vitro experimental protocolsIsolation of AMsAMs were isolated by bronchoalveolar lavage (BAL). Briefly, mice were anesthetized by i.p. injection of3mg/kg xylazine and75mg/kg ketamine and then sacrificed by cardiac exsanguination. The lungs and trachea were then excised en bloc, washed in HBSS, and lavaged.10times with light massaging by slowly instilling and withdrawing1ml warm (37℃) Ca2+/Mg2+-free HBSS (pH7.4) containing EDTA (0.6 mM). BAL fluid was collected and then centrifuged at400g for10min at4℃. The cells were then incubated in100-mm sterilized polystyrene Petri dishes for2h at37℃. The cells adhering to the bottom of dish were collected and replated for further experimental use. The purity of isolated AM was.95%as determined using fluorescently labeled Abs (mAbs) that specifically recognize proteins expressed by mice macrophages (surface Ags F4/80and CD11b). The viability was98%as evaluated by trypan blue exclusion.Cyclic stretching studies, Transfection of small interfering RNAs, Measurement of IL-1β and IL-18concentration via ELISA KIT, Western blotting and immunoprecipitation,Flow cytometric analysis were used. Mitochondria-associated ROS levels were measured in AMs by staining cells with MitoSOX (Invitrogen-Molecular Probes).In vivo experimental protocolsA well-established in vivo mouse model of VILI was used. In some experiments, animals were pretreated with a mitochondrial ROS inhibitor SS-31for24h. After mechanical ventilation, various measurements were obtained. In the IL-1β neutralization experiment, at10min before ventilation, mice were intratracheally in-stilled with100mg anti-IL-1β or IgG isotype control and then ventilated at28ml/kg for2h.Part I Cyclic stretch activates NLRP3inflammasome/IL-1β pathway signalling.Part II Mitochondria ROS are required for NLRP3inflammasome activation induced by cyclic stretch.Part III High tidal volume mechanical ventilation activates NLRP3inflammasome.ResultsCyclic stretch induces the release of IL-1β and IL-18by mouse AMsCyclic stretch-induced release of IL-1β is caspase-1dependentCyclic stretch activates NLRP3inflammasome pathwayMitochondria ROS are required for inflammasome activation induced by cyclic stretch Cyclic stretch-induced release of IL-1β is dependent of TLR4signalingHigh tidal volume mechanical ventilation activates NLRP3inflammasomeConclusionIn summary, this study for the first time, demonstrates the essential role of NLRP3inflammasome in AMs in the pathogenesis of VILI. Mechanical stretch stimulates mitochondrial ROS production in AMs, which in turn signals assembly of ASC, NLRP3, and caspase-1to activate NLRP3inflammasome, leading to the processing and maturation of Pro-IL-1β into the active IL-1β variant. TLR4signaling also plays an important role in Pro-IL-1β expression and subsequent IL-1β secretion. id production has an additional effect, which may be to enhance mitochondrial ROS generation and thereby amplify NLRP3inflammasome activation in response to cyclic stretch of AMs. The results point to potential therapeutic approaches to targeting NLRP3inflammasome in AMs for treatment of VILI.更多还原