【作者】 温宗梅；

【导师】 石学银； 范杰；

【作者基本信息】 第二军医大学 ， 麻醉学， 2013， 博士

【摘要】 研究背景失血性休克是临床上常见的急重症，复苏后常发生全身炎症反应综合征，甚至发展为多器官功能衰竭，其中急性肺损伤（acute lung injury, ALI）是最常见的复苏后并发症，是患者死亡的主要原因之一。对失血性休克/复苏（hemorrhagic shock/resuscitation, HS/R）后ALI的机制研究，目前公认的是“二次打击”学说，该学说把HS/R视为一种全身性缺血再灌注损伤，为第一次打击，使机体的免疫系统敏感性增强，当遇到第“二次打击”时，如轻微的细菌感染或炎性刺激，也可能导致机体爆发严重的全身炎症反应，甚至发展为多器官功能障碍。因为肺是一个开放器官，极易受到细菌或病原体的侵袭，在“二次打击”中往往成为最易受损伤的器官，所以ALI是HS/R后最常见的并发症。因此探索HS/R后ALI的发生机制，对防治ALI，降低HS/R患者的病死率具有重要意义。目前的研究集中在中性粒细胞与肺泡巨噬细胞之间，肺泡巨噬细胞与肺血管内皮细胞之间的相互作用，以及受体分子的相互作用，如TLR4和TLR2的相互作用。这些细胞或受体分子的相互作用，都是促进肺炎症反应的单向调控作用。有没有一种机制是可以具有促进炎症或抑制炎症的双向调控作用？又是什么分子信号通路介导这种双向调控作用？肺泡巨噬细胞（alveolar macrophages, AM）在介导肺炎症反应和ALI中具有重要作用，细胞表面的模式识别受体分子（Pattern recognition receptor molecules，PRRs），可以感受来自病原体的病原相关分子模式(pathogen associated molecularpatterns, PAMPs)以及由受损或死亡细胞释放的损伤相关分子模式(damageassociated molecular patterns, DAMPs)的刺激,启动感染性和非感染性炎症反应过程。前期研究发现HS/R后，高迁移率族蛋白1(HMGB1)释放增加，通过作用于AM的TLR4可以上调TLR2的表达，在HS/R诱导ALI发病机制中起到重要的作用。NOD2为一类存在于细胞内的PRR分子，只在巨噬细胞、树突状细胞和上皮细胞表达，其特异性配体为MDP，MDP是一种细菌细胞壁成分，为革兰氏阴性菌和革兰氏阳性菌都表达的细菌成分，因此NOD2识别细菌谱很广，NOD2与NF-κB炎症信号通路联系密切，介导炎症反应。有研究发现NOD2与自噬的关系非常密切，NOD2缺陷的树突状细胞不能合成自噬，并且细胞对细菌的易感性增强。NOD2与炎症、自噬和细胞凋亡等关系密切，而自噬和细胞凋亡对炎症反应的调控作用，目前尚无统一的定论，因此研究NOD2信号通路在HS/R后ALI中的调控作用，可能为该研究领域提供新的发现，为临床上有效干预HS/R后ALI提供理论依据和线索。第一部分失血性休克/复苏对肺泡巨噬细胞NOD2表达的影响目的：研究HS/R对AM的NOD2表达的影响及其机制。方法：体内实验：使用野生型（WT）和TLR4-/-小鼠建立HS/R模型，分别在复苏后2小时和4小时，收集和分离AM，用Western Blot的方法检测AM的NOD2蛋白表达；WT小鼠用抗HMGB1中和抗体预处理后，再进行HS/R，检测AM的NOD2表达；使用WT和NOD2-/-小鼠建立HS/R模型，检测AM的NOD1表达。体外实验：分离WT小鼠AM，体外培养，用HMGB1刺激，检测AM的NOD2表达。结果： HS/R处理后4小时，WT小鼠AM的NOD2表达明显增高，而TLR4-/-小鼠的NOD2表达无变化；提前给予HMGB1中和抗体预处理的小鼠HS/R处理后，AM的NOD2表达上调受到抑制； HS/R处理不能上调WT和NOD2-/-小鼠AM的NOD1的表达；HMGB1体外刺激WT小鼠AM，可诱导NOD2表达，呈时间和剂量依赖性。结论： HS/R通过HMGB1-TLR4信号通路特异性上调AM的NOD2表达。第二部分NOD2对“二次打击”后早期炎症反应的影响目的：研究NOD2对“二次打击”后AM早期炎症介质如MIF、MIP2、TNF-α表达的影响，以及NOD2信号通路对“二次打击”后小鼠肺泡灌洗液中IL-1β含量的影响。研究NOD2对AM的IL-1β成熟和释放的作用机制。方法：体内实验：使用WT和NOD2-/-小鼠建立HS/R+MDP的“二次打击”模型，部分小鼠在“二次打击”处理前，注射抗HMGB1中和抗体，“二次打击”处理后6小时，收集和分离AM，用Western Blot方法检测AM的MIP2、MIF和TNF-α的表达；使用WT、TLR4-/-、NOD2-/-、NF-κB-/-小鼠建立HS/R+MDP的“二次打击”模型，处理6小时后进行肺泡灌洗，用EILSA方法检测小鼠肺泡灌洗液中IL-1β的含量。体外实验：分离WT小鼠AM，在体外用HMGB1和MDP刺激6小时，用Western Blot方法检测AM的caspase-1和Pro-IL-1β表达。结果：“二次打击”处理后，小鼠AM的MIP2、MIF和TNF-α这些早期炎症介质表达上调， NOD2-/-和抗HMGB1中和抗体可以降低这些炎症介质的表达；“二次打击”处理后，WT小鼠肺泡灌洗液中IL-1β的含量显著升高，而TLR4-/-、NOD2-/-、NF-κB-/-小鼠肺泡灌洗液中IL-1β的含量无明显增加；HMGB1+MDP体外刺激AM，使AM的caspase-1活化的剪切体P10蛋白和成熟的IL-1β蛋白表达量上调。结论：HS/R上调AM的NOD2表达，使AM对MDP的反应性增强，“二次打击”处理后，小鼠AM的MIP2、MIF和TNF-α等早期炎症介质表达上调，可进一步活化巨噬细胞和诱导中性粒细胞聚集。“二次打击”处理后，小鼠肺泡中IL-1β的含量明显上升，TLR4、NOD2、NF-κB分子参与IL-1β成熟和释放，并且AM中NOD2分子介导对caspase-1活化，启动IL-1β成熟和释放。第三部分NOD2在“二次打击”中对肺泡巨噬细胞自噬体形成的影响目的：研究NOD2对“二次打击”后小鼠AM自噬体形成的影响，以及HMGB1+MDP联合刺激对小鼠AM自噬体形成的影响。方法：体内/体外实验：使用WT小鼠建立HS/R模型，处理后4小时，分离AM，体外培养，用MDP刺激，分别在不同时间点收集细胞，用免疫荧光和Western Blot方法检测AM的LC3蛋白表达。体内实验：用WT和NOD2-/-小鼠“二次打击”处理后8小时，收集和分离AM，用免疫荧光和Western Blot方法检测AM的LC3蛋白表达，部分小鼠在“二次打击”处理前，3-MA预先给药2小时。体外实验：分离WT和NOD2-/-小鼠AM，在体外用HMGB1和MDP刺激8小时，收集细胞，用免疫荧光和Western Blot方法检测AM的LC3表达，部分组别处理前，用3-MA预处理2小时。结果：体内/体外实验结果显示HS/R处理后的小鼠AM，对MDP敏感性增强，上调AM中LC3荧光颗粒的形成和LC3-II蛋白的表达。体内实验结果显示，HS/R+MDP“二次打击”可上调小鼠AM自噬体的形成，而该作用可被3-MA阻断，NOD2-/-小鼠对“二次打击”的作用无反应。体外实验结果显示HMGB1+MDP刺激可以诱导AM自噬体形成，NOD2分子的缺失和3-MA可阻断自噬形成。结论：NOD2在“二次打击”中，上调AM自噬体形成，并且自噬体出现较晚，在刺激8小时开始出现，3-MA可以阻断“二次打击”对自噬的诱导作用，NOD2-/-小鼠对“二次打击”诱导自噬体形成无反应，证明NOD2是介导细胞自噬体重要分子。第四部分NOD2-自噬对“二次打击”后急性肺损伤的影响目的：研究NOD2-自噬对“二次打击”后小鼠的生存率和肺损伤的影响，以及AM在“二次打击”后急性肺损伤中的作用。方法：使用WT和NOD2-/-小鼠分别建立HS/R、“二次打击”、3-MA+“二次打击”模型，观察处理后36小时小鼠的生存状况，计算生存率；建立WT和NOD2-/-小鼠HS/R、“二次打击”、3-MA+“二次打击”模型，分别在处理后8小时和24小时，取小鼠肺组织做病理切片染色，观察肺组织损伤情况；建立WT和NOD2-/-小鼠HS/R、“二次打击”、3-MA+“二次打击”模型，分别在处理后8小时和24小时，取小鼠肺组织，用ELISA的方法检测小鼠肺组织MPO的表达水平；建立WT和NOD2-/-小鼠HS/R、“二次打击”、3-MA+“二次打击”模型，分别在处理后8小时和24小时，收集肺泡灌洗液，用ELISA方法检测肺泡灌洗液中IL-6和IL-12炎症因子的表达；建立WT和NOD2-/-小鼠HS/R、“二次打击”、3-MA+“二次打击”模型，分别在处理后8小时和24小时，收集和分离AM，用Real-TimePCR的方法，检测AM中TNF-α、IL-6和IL-12mRNA的表达。结果： WT小鼠HS/R组36小时生存率100%，“二次打击”组生存率为75%，3-MA+“二次打击”组生存率为12.5%，而NOD2-/-小鼠三组的生存率均为100%；肺组织病理切片染色结果显示，在8小时处理组中，WT小鼠“二次打击”组肺损伤明显严重于HS/R组，与3-MA+“二次打击”组肺损伤程度相似，但在24小时处理组，“二次打击”组肺损伤明显减轻，而3-MA+“二次打击”组肺损伤却加重，NOD2-/-小鼠两组中肺组织均未见明显肺损伤表现；在8小时处理组中，WT小鼠“二次打击”组的肺泡灌洗液中IL-6和IL-12与HS/R组相比，明显上升，与与3-MA+“二次打击”组差异性较小，在24小时处理组，“二次打击”组IL-6和IL-12显著降低，而3-MA+“二次打击”组却继续升高，NOD2-/-小鼠两组中肺泡灌洗液中IL-6和IL-12均未见明显升高；检测小鼠AM中TNF-α、IL-6和IL-12mRNA的表达结果显示，在8小时组中，WT小鼠“二次打击”组的AM中TNF-α、IL-6和IL-12mRNA表达明显上调，但24小时处理组，“二次打击”组的AM中TNF-α、IL-6和IL-12mRNA表达没有进一步上调，而3-MA+“二次打击”组AM的TNF-α、IL-6和IL-12mRNA表达显著升高，NOD2-/-小鼠两组中AM的TNF-α、IL-6和IL-12mRNA表达水平均未见明显改变。结论：NOD2在“二次打击”后早期时相，促进肺炎症反应，在“二次打击”后晚期时相诱导自噬形成，抑制肺炎症反应，如果阻断自噬可加重肺损伤，降低小鼠的生存率，NOD2-/-可明显降低“二次打击”带来的肺损伤，因此NOD2信号通路是介导“二次打击”后ALI的重要调控机制。第五部分NOD2-自噬调控“二次打击”后急性肺损伤的机制目的：研究NOD2-自噬调控“二次打击”后小鼠AM凋亡的影响，探讨NOD2-自噬诱导AM凋亡对“二次打击”后ALI的影响。方法：体外实验：分离WT和NOD2-/-小鼠AM，体外培养，用MDP刺激，分别在不同时间点收集细胞，用TUNNEL免疫荧光法检测AM凋亡表达，部分细胞用3-MA预处理2小时。体内实验：用WT和NOD2-/-小鼠“二次打击”处理后24小时，收集和分离AM，用流式细胞术法检测AM的Annexin V和7-AAD染色，分析凋亡细胞的比例，部分组别小鼠“二次打击”前注射3-MA。结果：体外实验：结果显示MDP处理24小时后WT小鼠AM出现凋亡，呈时间依赖性；HMGB1+MDP刺激AM24小时，出现大量细胞凋亡，而3-MA预处理组，细胞凋亡反而减少，NOD2-/-小鼠AM在各组中未见明显凋亡。体内实验：检测“二次打击”后24小时小鼠体内AM的凋亡情况，流式细胞学结果显示，WT小鼠“二次打击”处理24小时后，AM有较高比例死亡，达18.8±1.1%，而3-MA+“二次打击”组中的AM凋亡比例显著降低，只有3.3±0.4%，而NOD2-/-小鼠两组AM均无明显细胞凋亡出现，分别为2.0±0.1%和0.5±0.2%。结论：NOD2在“二次打击”晚期时相，促进肺泡巨噬细胞自噬形成，诱导细胞凋亡，3-MA阻断自噬减少细胞凋亡或坏死细胞的清除，使炎症反应放大，加重肺损伤程度。总结：综上所述，本研究证实在HS/R+MDP“二次打击”过程中，NOD2信号通路在早期时相，促进炎症细胞活化、炎症因子的合成与分泌，从而促进炎症反应；在晚期时相通过促进自噬和诱导细胞凋亡抑制炎症反应。因此，本课题揭示了“二次打击”过程中NOD2介导的重要始动和调控机制，并可以作为防治失血性休克/复苏后急性肺损伤的重要靶点，具有很高的科学意义和临床价值。更多还原

【Abstract】 ‘Second hit’ is used to describe the phenomenon that body reaction to infections isenhanced after hemorrhagic shock/resuscitation (HS/R), with slight infection beingable to cause systemic inflammation and multiple organ dysfunctions. As an openorgan, lung is susceptible to acute lung injury (ALI) during this HS/R process. Ourpreliminary data show that high-mobility group box1(HMGB1) increases theexpression of TLR2in alveolar macrophages (AM) by binding to TLR4after HS/R,and thereafter plays an important role in mediating ALI. Based on these findings andthe knowledge that NOD2, an intracellular pattern recognition receptor, is closelyassociated with inflammation, autophagy and apoptosis, we come up with ourhypothesis: HS/R may activate NOD2and MDP-NOD2signal, a ‘second hit’, mayaffect ALI after HS/R.Part1. The effect of HS/R on NOD2expression in AMTo study the effect of HS/R on NOD2expression in AM, we employed an HS/Rmodel in mice. We found that NOD2expression increased significantly in WT, but notTLR4-/-, mice, indicating that HS/R activates NOD2secondarily by binding to TLR4.Administration of neutralizing anti-HMGB1prior to HS/R prevented the upregulationof NOD2. These data demonstrated HS/R upregulates NOD2expression throughHMGB1-TLR4signaling pathway in AM. In the in vitro experiment, cultured WTAM were stimulated with HMGB1. We found that NOD2expression upregulationwas induced by HMGB1stimulation. Part2. The effect of NOD2on inflammatory response early after the‘second hit’To determine the effect of NOD2on lung inflammation after HS/R, we usedHS/R+MDP injection, a ‘second hit’ model mimicking clinical situations. Datashowed that the expression of MIP2, MIF and TNF-α was significantly increased,indicating that the upregulation of NOD2expression in AM after ‘second hit’mediated ALI. In addition, after performing the ‘second hit’ model in WT, TLR4-/-,NOD2-/-and NF-κB-/-mice, we examined the content of IL-1β in bronchoalveolarlavage (BAL) fluid. We found that TLR4, NOD2and NF-κB mediated the release ofIL-1β and also mediated lung inflammation both directly and indirectly. We alsostimulate AM sequentially with HMGB1and MDP, mimicking in vivo ‘second hit’model, to measure the activation of caspase-1and maturation of IL-1β. We foundthat HS/R mediated NOD2upregulation and thus increase the sensitivity of NOD2toMDP through HMGB1-TLR4signaling pathway. NOD2was also shown tomodulate IL-1β maturation and release and thus promote the release ofproinflammatory factors by regulating the activity of caspase-1.Part3. The role of NOD2in autophagosome formation during the‘second hit’To demonstrate whether MDP induces autophagy in macrophages, WT and NOD2-/-mice were treated with HS/R operation, then harvest the AM and stimulate them withMDP. T he cells were examined with immunofluorescence and Westren Blot for LC3protein. We found that HS/R could promote autophagy, which was caused by MDPadministration and could be blocked by3-MA. We also found that upregulation ofNOD2by HS/R promoted autophagy in AM in a HS/R+MDP ‘second hit’ model withWT and NOD2-/-mice. To examine the effect of HMGB1and MDP on autophagy inAM, we cultured mouse AM and stimulate them with HMGB1and MDP. LC3inAM was measured. We found that HMGB1upregulated NOD2expression in AMand then induced autophagy together with MDP. Part4. The role of NOD2-autophagy in ALI after the ‘second hit’We looked into the overall effect of NOD2-autophagy on animal survival after ‘secondhit’ with MDP. All HS/R WT mice survived for36h.75%or12.5%WT micesurvived when treated with an ‘second hit’ or3-MA+‘second hit’, respectively.However, all NOD2-/-mice survived regardless of MDP or3-MA administration. Toinvestigate the effect of NOD2-autophagy signaling pathway in ALI after the ‘secondhit’, we examined the lung tissue histologically. Histological examinations showedremarkable lung injuries in the tissues harvested8h after MDP injection, while theinjuries stayed similar at24h. In contrast, when mice were pretreated with3-MA toblock autophagy, WT lung injuries aggravated with time during24h after the ‘secondhit’ while the NOD2-/-lung injuries stayed similar. These data indicated that NOD2played a key role in mediating lung injury and autophagy possessed ananti-inflammatory function. To study the effect of NOD2-autophagy signalingpathway on the myeloperoxidas (MPO) level in lung tissue from mice undergoing‘second hit’, we used the HS/R+MDP model and examined the activity of MPO in thelung tissue. The results showed that lung injury caused by neutrophils wasexacerbated when autophagy was blocked. Defect in NOD2expression could blockthe downstream inflammatory signals and thus relieve the lung inflammation causedby ‘second hit’. To investigate how the NOD2-autophaty signaling pathwaymodulates the inflammatory cytokines in lungs from ‘second hit’ model, we measuredIL-6and IL-12in the BAL fluid. Results showed that lung inflammation aggravatedwhen autophagy was blocked. To confirm effect of NOD2-autophagy of AM on theexpression and release of inflammatory cytokines, we performed the ‘second hit’model and measured the mRNA level of TNF-α, IL-6and IL-12in AM.Part5. Mechanisms of NOD2-autophagy modulation of ALI after‘second hit’To study NOD2-autophagy modulation on AM apoptosis after ‘second hit’ with MDPand its role in ALI after ‘second hit’, we cultured WT and NOD2-/-AM and treatedwith HMGB1and MDP with/without3-MA. We found with TUNNEL staining that 24h after treatment, WT cells underwent apoptosis when treated with MDP alone orHMGB1+MDP, with HMGB1+MDP resulting in profound apoptosis. When treatedwith3-MA, apoptosis decreased significantly. NOD2-/-AM showed little signs ofapoptosis. To test these findings in vivo, we used HS/R+MDP ‘second hit’ modelwith/without3-MA administration, in WT or NOD2-/-mice. We found24h after‘second hit’ WT AM underwent a significantly high level of apoptosis (18.8±1.1%)comparing with cells from mice treated with3-MA (3.3±0.4%). AM from NOD2-/-mice underwent relatively low levels of apoptosis,2.0±0.1%or0.5±0.2%when3-MA was injected or not, respectively.SummaryBased on the findings above, we conclude:1, HS/R upregulate NOD2expression inAM through HMGB1-TLR4signaling pathway;2, HS/R upregulated NOD2expression in AM and thus promote its sensitivity to MDP and therefore aggravate thelung inflammation and lung injury in the HS/R+MDP ‘second hit’ model;3, HS/Rinduces lung inflammation and lung injury in an IL-1β dependent manner, which ismodulated by NOD2;4, HS/R-mediated NOD2upregulation induces autophagy inlung inflammation and lung injury;5, NOD2-autophagy signaling pathway mediateslung inflammation and lung injury in the HS/R+MDP ‘second hit’ model;6,NOD2-autophagy signaling pathway plays Janus role in the HS/R+MDP ‘second hit’model, being both proinflammatory and anti-inflammatory;7, NOD2-autophagy maysuppress ‘second hit’-induced lung inflammation by promoting AM apoptosis.In brief, this study provides a novel mechanism by which ‘second hit’ induces ALI:NOD2-autophagy signaling pathway initiates and modulates ‘second hit’-induced ALI,which may provide a novel target for clinical intervention.更多还原