环境化学与生态毒理学国家重点实验室知识库

       因人类活动产生的大气细颗粒物污染及由此引起的人体健康风险，已成为全球关注的重要议题。大气 细 颗粒物 成分复杂 、理化性质独特 ，现有毒理学数据大多局限于这类污染物的靶器官毒性效应， 不能充分解释 其 暴露与健康危害间的因果关系， 其 毒性模式与作用机制仍需进一步 研究 。 考虑到污染物在进入机体到达靶器官前大多会进入血液循环系统，而血浆体系中富含多种重要酶系，这些酶系相互作用共同调节血液稳态及心血管系统中诸多生物学过程，因此大气细颗粒物与血浆酶系可能存在的相互作用值得探索。此外，血小板是由巨核细胞胞浆脱落下来的小块胞质，在血液中广泛存在，在生理止血过程中发挥着重要的功能。肺脏是大气细颗粒物暴露的重要靶器官，同时也被报道是体内血小板生成的重要器官之一。大气细颗粒物暴露是否可能影响机体血小板的生成，这对客观评价这类污染物引发心血管疾病风险具有重要意义。本研究分别选用无机石英滤膜（QFF）和有机聚丙烯滤膜（PPF），采集了北京市冬季大气细颗粒物 PM1和 PM2.5样本 。
      在对颗粒物样本进行物理化学性质表征的基础上， 研究了 大气细颗粒物对血浆关键蛋白酶系级联活化的调控作用，并进而探讨了这类污染物暴露对血小板生成的影响。 首先，采用 超声与冷冻复合提取法，获得北京市 2016年12月至2017年2月间四种 类 型 大气细颗粒物 样品 ，即 QFF PM1、 PPF PM1、 QFF PM2.5、 PPF PM2.5发现 QFF膜颗粒物回收率大于 PPF。电镜 结果显示， 四种 类 型 大气细颗粒物样品形貌不规则，水合粒径在纳米级别范围，表面带负电荷。 分析两种采样膜来源的颗粒物样品中非金属元素、金属元素、离子组分以及内毒素，发现两种膜采集的颗粒物 中 碳 元素含量相差较大，可能与 颗粒物 提取时采样膜对有机组分吸附力的不同 相关 ，其它组分相差不大。
    其次，通过设计一系列体外、离体或活体实验发现，有机滤膜和无机滤膜采集的大气细颗粒物均可以有效促进血管舒缓素 激肽系统（ KKS 关键酶原蛋白活化，并且呈现剂量依赖和时间依赖效应；通过大气细颗粒物成分分析以及模拟组分的KKS激活实验显示，带负电荷的颗粒物以及附着的内毒素在大气细颗粒物引起的KKS系统激活中发挥重要作用。在大气细颗粒物调控KKS活化过程中，起始酶原FXII可在大气细颗粒物作用下发生自激活剪切，活化的FXIIa进而触发下游PPK与HK级联激活。此外，大气细颗粒物暴露引起补体系统关键因子—补体3和补体5的活化，以及凝血酶原的激活。抑制剂实验表明，大气细颗粒物诱导的KKS激活介导了补体和凝血系统的级联活化。
      最后，研究发现不同粒径大气细颗粒物PM1和PM2.5均可以有效促进人巨核细胞向血小板分化，同时伴随巨核细胞分化过程增强，包括明显的细胞巨核化现象、 DNA倍体化、 髓系特异性分子 CD33的表达减少以及巨核细胞成熟分子CD41a表达增加。差异蛋白质组学 分析 显示， 大气细颗粒物对巨核细胞分化影响的主要细胞和分子事件集中在代谢通路、线粒体氧化磷酸化过程 OxPHOS 、细胞骨架、剪切体、核糖体、血小板激活等通路。 进一步机制研究 发现，大气细颗粒物诱导 的线粒体 OxPHOS和线粒体 活性氧 的产生共同调控 巨核细胞分化和血小板 的 生成 。
      综上，本研究探讨了大气细颗粒污染物与血浆蛋白酶的相互作用，揭示了颗粒物在血液稳态中可能存在的干扰效应；阐明了大气细颗粒物对巨核细胞分化为血小板的影响及作用机制，为雾霾颗粒物引发的心血管疾病风险评价与机制阐释提供了重要科学依据。

      Particulate air pollution from human activities has drawn global attention due to its potential health risks. Due to various physical and chemical properties of airborne fine particulate matter (PM), the currently available toxicological data on PM is still not sufficient to explain their cause-and-effect. Considering the inevitable contact of inhaled airborne PM with the plasma, the hematological effects of PM are worthy of being studied. The complement, coagulation and kallikrein-kinin system (KKS), key zymogens in plasma, are working in concert with each other to provide both positive and negative feedback for hematological hemostasis. In addition, platelet, derived from the differentiation of megakaryocyte and widely found in blood, can regulate various physiological and pathological processes. Lung tissue belongs to the important target of PM and a primary site of platelet production. Hence, the effects of airborne fine particles on key zymogens and platelet formation may be an important prerequisite for determining their health risks. Herein, airborne PM samples were collected by inorganic quartz fiber filters (QFFs) and organic polypropylene filters (PPFs) in Beijing during winter. Based on the physicochemical characterization of these particle samples, their potential effectss on plasma zymogens and platelet generation were investigated.
      Firstly, four types of PM samples, including QFF PM1, PPF PM1, QFF PM2.5 and PPF PM2.5, were collected from Beijing during December, 2016 Febuary, 2017. PM recoveries of QFFs were much higher than those of PPFs, due to the stronger adsorbtion of PM on organic filters. Morphological observation showed that both PM1 and PM2.5 samples collected by two kinds of filters were irregularly particulate aggregates. The hydrodynamic diameters of airborne fine particles were at nano scale, and zeta potential analysis revealed that they were negatively charged in both water and plasma.The analysis of main components, such as C, N, S, some non metal and metal elements, and anions was performed in different types of PM samples, and the element C levels in QFF PM2.5 and PPF PM2.5 samples were relatively higher than those of the other tested elements, while little difference was observed for the other physicochemical properties in PM samples collected by different filters.
      Secondly, the studies based on ex vivo, in vitro and in vivo KKS activation assays indicated that PM samples could efficiently cause the cascade activation of key zymogens in the KKS, wherein, the particles and the attached lipopolysaccharide (LPS) components provided substantial contribution. The binding of Hageman factor XII (FXII) with PM samples and its subsequent auto-activation initiated the activation of the KKS. The crucial elements in the complement cascade, including complement 3 (C3) and complement 5 (C5), and coagulation system (prothrombin) were also found to be actively induced by PM exposure. More specifically, these reactions were mediated by triggering KKS system activation, which was demonstrated by the inhibition effect induced by the inhibitors of KKS system (aprotinin) and FXII (CTI).
      Finally, both PM1 and PM2.5 sample exposure promoted the process of megakaryocyte differentiation and platelet formation. The differentiation of megakaryocyte, including the growth of cell nucleus and size, occurrence of DNA ploidy, and alteration of platelet differentiation factor levels, were apparently induced by PM exposure. The data from proteomics demonstrated that the main pathways involved in the differentiation of megakaryocytes were related with cellular metabolism, mitochondrial oxidative phosphorylation (OXPHOS) process, cytoskeleton, etc. More specially, mitochondrial OXPHOS accompanied with mitochondrial ROS played pivotal roles in the differentiation of megakaryocytes into platelets upon airborne PM treatments.
      This research unveiled the potential perturbation of haze particles in hematological homeostasis by disturbing the cascade activation of the pivotal plasma protease systems, and platelet generation. The findings provided important scientific evidences for the evaluation of the potential health hazards due to airborne PM exposure.