The molecular mechanisms and adverse outcome pathways underlying carbon nanomaterials induced pro-inflammatory responses and related indirect effects

&nbsp;&nbsp;&nbsp;&nbsp; 随着纳米产业的发展，纳米材料在原材料加工、消费品生成及消费品使用的过程中，不可避免的会进入到环境中，并可能造成人群暴露。碳纳米材料，如一维的碳纳米管、二维的石墨烯、三维的富勒烯，以及其它衍生物，因优良的物理化学性质，在生产、生活中得到广泛应用，尤其是在生物医疗领域的应用潜能及优越性日益突出。因此，探究碳纳米材料生物安全性及潜在的环境及健康风险也越来越重要。尽管目前关于碳纳米管生物效应的研究很多，但是大部分工作集中在探究碳纳米材料暴露后对暴露部位细胞、组织、器官的直接毒性效应，很少有研究工作关注伴随着炎症反应状态改变产生的继发性或者代偿性效应。对于碳纳米材料暴露诱发的&ldquo;有害结局路径&rdquo;也称为&ldquo;毒性通路&rdquo;（Adverse outcome pathways，AOP）并没有明确的认识，实用的针对碳纳米材料风险评估的框架并没有建立。<br /> &nbsp;&nbsp;&nbsp; 机体的免疫系统是监控、清除入侵机体的外源物质的第一道防线，免疫状态的改变会影响机体其他代谢过程及内稳态，探究纳米材料的免疫毒性机制，识别机体以免疫状态改变为起始事件的毒性通路，及其相关的间接性及代偿性效应对于更全面、深入的理解纳米材料组织、细胞相容性，揭示其生物安全性具有重要意义。探究材料本身理化性质对于生物相容性及毒性效应的影响，对于进一步基于构效关系进行纳米材料的修饰、改性，拓展其在生物医药以及环境污染控制方面的应用也具有一定的指导意义。<br /> &nbsp;&nbsp;&nbsp; 本论文主要探讨碳纳米管暴露诱发周身炎症反应的间接毒性效应及氧化石墨烯活化炎症反应的理化性质决定机制。采用了原始态（P-MWCNTs），氨基化（MWCNTs -NH2）、聚乙二醇化（MWCNTs-PEG）、聚醚酰亚胺化（MWCNTs-PEI）和羧基化（MWCNTs-COOH）多壁碳纳米管模拟真实环境中的人群暴露方式及暴露剂量进行实验研究。我们发现，原始态及功能化修饰的碳纳米管（CNTs）均能诱发促炎症反应并不同程度的改变机体的铁代谢稳态。表现为：不管是什么修饰类型的&nbsp; CNTs并以何种方式暴露（腹腔暴露或者肺暴露），均能检测到实验小鼠发生明显的炎症反应及机体铁代谢紊乱。从整体上考虑表面修饰对这些生物效应的影响，我们发现原始态的碳纳米管对炎症反应状态及铁稳态的扰动作用最强，而 PEG和&nbsp;&nbsp; COOH的引入会部分减弱这种效应，而NH2则可以进一步加剧CNTs的促炎症反应。进一步的机制研究表明：碳纳米管能显著活化炎性细胞（如巨噬细胞）发生促炎症反应，并在暴露部位招募炎性细胞促进炎症反应的级联放大，导致以肝脏 hepcidin为核心的铁代谢紊乱，红细胞的合成发生障碍，脾脏内造血的代偿性增加，最终导致炎症性贫血的发生。与此同时，我们还发现碳纳米管暴露后对机体远端组织-关节存在潜在的健康风险。选择相较于其他修饰类型诱发炎症反应较为温和的羧基化碳纳米管（ MWCNTs-COOH）进行机制探索发现：经碳纳米管暴露后小鼠关节部位出现以滑膜增厚、炎性细胞浸润为特征的滑膜炎症反应。进一步的机制探究说明：MWCNTs-COOH能直接活化巨噬细胞表达促炎症因子，利用巨噬细胞炎性上清孵育滑膜/软骨细胞后，可明显检测到与软骨降解相关的基质金属蛋白酶（MMPs）以及与炎症反应相关的环氧合酶（COXs）的表达量及活性增加。将 MWCNTs-COOH暴露巨噬细胞得到的炎性上清进行抗体中和后再暴露细胞发现其 MMPs及COXs的表达量降低。说明碳纳米管暴露可能使得机体的免疫敏感性增加，患免疫相关疾病的风险增加。<br /> &nbsp;&nbsp;&nbsp; 氧化石墨烯（GO）因其独特的理化性质（表面积大，稳定性高）以及在水溶液中的亲水性及可分散性，在生物医药领域得到广泛应用。但是，由于 GO的理化性质差异较大，对于其生物相容性及生物安全性的认识充满争议。对材料自身的理化性质，如水平尺寸，在 GO的生物学效应及毒性效应中的影响尚不清楚。在这部分工作中，我们重点关注 GO水平尺寸对其生物效应的影响。我们利用相同的原始材料，制备得到以水平尺寸为单一变量的一组 GO样品，模拟真实环境中人群接触碳纳米材料的途径（呼吸暴露、经腹腔的内暴露及经尾静脉的血液暴露）进行体内实验，我们发现，不同尺寸 GO在激活巨噬细胞和诱发局部与系统性炎症反应方面存在很大差异：与小尺寸的 GO相比，大尺寸的&nbsp; GO更易活化巨噬细胞并促发炎症反应。一系列的细胞与分子生物学研究表明：相较于尺寸较小的 GO，大尺寸材料更倾向结合于巨噬细胞的细胞膜表面，与膜的相互作用更强。<br /> &nbsp;&nbsp;&nbsp; 进一步的机制研究揭示，不同大小的&nbsp; GO在不同程度上激活位于细胞膜表面的Toll样受体，并差异活化其介导的下游NF-&kappa;B炎症反应信号通路。同时发现大尺寸的 GO显著刺激巨噬细胞向M1亚型极化，放大炎性级联反应。这些发现对于揭示纳米材料的潜在毒性效应，深入理解纳米材料的健康风险及生物安全性具有重要价值。&nbsp;&nbsp;&nbsp; With&nbsp; the&nbsp;&nbsp; development&nbsp; of&nbsp;&nbsp; Nano-technology&nbsp; Industrialization,&nbsp;&nbsp; the&nbsp; expanding releasing of nanomaterial from the process of raw materials, the production of consumer goods&nbsp; and usage&nbsp; of commercial&nbsp; products&nbsp; will certainly&nbsp; increase&nbsp; the opportunity&nbsp; of human exposure,&nbsp; and this&nbsp; has raised&nbsp; general concerns&nbsp; on their&nbsp; safety. Owing&nbsp; to the novel&nbsp;&nbsp; physicochemical&nbsp;&nbsp;&nbsp; properties,&nbsp;&nbsp; carbon-based&nbsp;&nbsp;&nbsp; nanomaterials&nbsp;&nbsp; such&nbsp;&nbsp;&nbsp; as&nbsp;&nbsp; one-dimensional carbon nanotubes, two-dimensional graphene, three-dimensional fullerene and their derivatives have continuously attracted great attention in a wide range fields,especially in&nbsp; biomedical and pharmaceutical&nbsp; applications. Meanwhile, the&nbsp; increasing use of carbon nanomaterial may cause adverse health effect through ingestion, injection and inhalation. Despite a wide spectrum of reported toxicities for carbon nanomaterial,most of them studied the toxic effects to the directly targeted tissues/organs that provide little insight&nbsp; into their indirect&nbsp; effects beyond their&nbsp; primary targets. And there&nbsp; is few research about adverse&nbsp; outcome (AOP) for nanomaterials with&nbsp; high exposure risk for human&nbsp; beings&nbsp; and a&nbsp; validated&nbsp; framework&nbsp; for&nbsp; human&nbsp; health&nbsp; risk assessment&nbsp; upon nanomaterials has not been developed yet. The inflammatory system is the first line of surveillance,&nbsp;&nbsp; clearance,&nbsp;&nbsp; defense&nbsp;&nbsp; at&nbsp;&nbsp; the&nbsp;&nbsp; portal-of-entry&nbsp;&nbsp; against&nbsp;&nbsp; foreign&nbsp;&nbsp;&nbsp; agents.<br /> &nbsp;&nbsp;&nbsp; Inflammation disrupts various pathways&nbsp; responsible for diverse homeostasis and may cause diverse detrimental health problems, such as anemia of inflammation and arthritis. Therefore, to investigate the mechaims about immunotoxicity and to identify AOP with the&nbsp; activation&nbsp;&nbsp; of&nbsp; inflammatory&nbsp; pathways&nbsp;&nbsp; as&nbsp; initiating&nbsp; events&nbsp;&nbsp; and&nbsp; inflammation-associated metabolic disorders and secondary effects are of great importance for deeply understanding&nbsp;&nbsp; the&nbsp; biosafety&nbsp;&nbsp; of&nbsp; nanomaterials.&nbsp;&nbsp; To&nbsp;&nbsp; identify&nbsp; the&nbsp;&nbsp; physicochemical properties responsible for&nbsp; the biocompability and toxicology&nbsp; of specific nanomaterial is&nbsp;&nbsp; meanful&nbsp;&nbsp; to&nbsp;&nbsp; further&nbsp;&nbsp; modification&nbsp;&nbsp; according&nbsp;&nbsp; to&nbsp;&nbsp; developed&nbsp;&nbsp; structure-function relationship and&nbsp; to extend the&nbsp; applications in biomedicine and&nbsp; contamination control field.<br /> &nbsp;&nbsp;&nbsp; In&nbsp; this study,&nbsp; we&nbsp; endeavor&nbsp; to verify&nbsp; the&nbsp; indirect effects&nbsp; and&nbsp; adverse&nbsp; outcome pathways related to carbon nanomaterials induced alternation of systemic inflammatory status,&nbsp; and&nbsp; to&nbsp;&nbsp; illuminate&nbsp; the&nbsp; molecular&nbsp;&nbsp; mechanisms&nbsp; and&nbsp; size&nbsp; effect&nbsp;&nbsp; relationship underlying graphene oxide-induced pro-inflammatory effects.Apanel of CNTs were prepared including pristine multi-wall CNTs (P-MWCNTs),aminated (MWCNTs-NH2), polyethylene glycol (MWCNTs-PEG),&nbsp; polyethyleneimine (MWCNTs-PEI) and&nbsp; carboxylated&nbsp; MWCNTs (MWCNTs-COOH).&nbsp; We demonstrated that regardless of&nbsp; the type of CNTs&nbsp; and the exposure way (either&nbsp; peritoneal cavity or lung&nbsp; exposure),&nbsp; significant&nbsp;&nbsp; inflammation&nbsp; and&nbsp; alteration&nbsp; of&nbsp;&nbsp; iron&nbsp; parameters&nbsp; were observed&nbsp; in mice.&nbsp; The mechanisms&nbsp; of&nbsp; toxicity involved&nbsp; enhanced&nbsp; hepatic hepcidin mRNAexpression likely as a result of increased inflammatory cytokine IL-6 production,which&nbsp;&nbsp; resulted&nbsp;&nbsp; in&nbsp; disordered&nbsp;&nbsp; iron&nbsp;&nbsp; metabolism&nbsp;&nbsp; impacting&nbsp;&nbsp; RBC&nbsp;&nbsp; formation&nbsp; and consequently the occurrence&nbsp; of anemia of inflammation&nbsp; in mice. Further mechanistic investigations&nbsp; showed that&nbsp; CNTs&nbsp; activate&nbsp; macrophages and&nbsp; macrophage-associated pro-inflammatory responses&nbsp; (including secretion&nbsp; of&nbsp; pro-inflammatory cytokines&nbsp; and recruitment of inflammatory leukocytes), which&nbsp; at least partially account for hepcidin induction,&nbsp; iron&nbsp; sequestration for&nbsp; erythropoiesis,&nbsp; anemia&nbsp; and&nbsp; finally&nbsp; extramedullary erythropoiesis.<br /> &nbsp;&nbsp;&nbsp; Compared to&nbsp; other modified&nbsp; CNTs, carboxylated&nbsp; multi-wall CNTs&nbsp; (MWCNTs-COOH) induced milder&nbsp; systemic inflammatory response in&nbsp; some extent. We selected MWCNTs-COOH&nbsp; in&nbsp; the&nbsp; following&nbsp;&nbsp; experiment&nbsp; and&nbsp; found&nbsp; that&nbsp; MWCNTs-COOH administration&nbsp; led&nbsp; to&nbsp; synovial&nbsp; inflammation&nbsp; within&nbsp;&nbsp; knee&nbsp; joints,&nbsp; as&nbsp; evidenced&nbsp; by infiltration of pro-inflammatory&nbsp; cells in synovium and&nbsp; meniscus. Mechanistic studies manifested that&nbsp; MWCNTs-COOH stimulated&nbsp; pro-inflammatory effects&nbsp; by activating macrophages, and&nbsp; the secreted&nbsp; pro-inflammatory&nbsp; cytokines in&nbsp; the supernatant&nbsp; could prime the&nbsp; synoviocytes and chondrocytes,&nbsp; leading to enhanced&nbsp; production of&nbsp; a large array&nbsp; of&nbsp;&nbsp; enzymes&nbsp; involved&nbsp; in&nbsp;&nbsp; articular&nbsp; cartilage&nbsp;&nbsp; degeneration,&nbsp; including&nbsp;&nbsp; matrix metalloproteinase&nbsp;&nbsp; (MMP)&nbsp;&nbsp; members&nbsp;&nbsp; and&nbsp;&nbsp; cyclooxygenase&nbsp;&nbsp; (COX)&nbsp;&nbsp; members&nbsp; and increased enzymatic activity of&nbsp; MMPs were demonstrated. Blockade of the&nbsp; cytokines by antibodies significantly attenuated the production of these enzymes. This meant that exposure&nbsp; to carbon&nbsp; nanotubes&nbsp; may promote&nbsp; the&nbsp; immune sensitization&nbsp; of&nbsp; body&nbsp; and improve the risk of suffering from immunological disease.<br /> &nbsp;&nbsp;&nbsp; Graphene oxide&nbsp; (GO) is increasingly used&nbsp; in biomedical applications&nbsp; because it possesses not only the&nbsp; unique properties of graphene including&nbsp; large surface area and flexibility but&nbsp; also&nbsp; hydrophilicity and&nbsp; dispersibility&nbsp; in aqueous&nbsp; solutions.&nbsp; However,there are conflicting results on its biocompatibility and&nbsp; biosafety partially due to large variations&nbsp; in&nbsp; physicochemical&nbsp; properties&nbsp; of&nbsp; GO,&nbsp; and&nbsp; the&nbsp; role&nbsp; of&nbsp; these&nbsp; properties including lateral size in the biological or toxicological effects of GO is still&nbsp; unclear. In this study, we&nbsp; focused on the role&nbsp; of lateral size by&nbsp; preparing a panel of&nbsp; GO samples with&nbsp; differential&nbsp; lateral&nbsp; sizes&nbsp; using&nbsp; the&nbsp; same&nbsp; starting&nbsp; material.&nbsp; We&nbsp; found&nbsp; that&nbsp; in comparison to its smaller counterpart, larger GO showed a stronger adsorption onto the plasma membrane with less phagocytosis,&nbsp; which elicited more robust interaction with toll-like receptors (TLRs) and more potent activation of NF-&kappa;B pathways. By contrast,smaller GO sheets were more likely taken up by cells. As a result, larger GO promoted greater&nbsp; M1&nbsp;&nbsp; polarization,&nbsp;&nbsp; associated&nbsp; with&nbsp;&nbsp; enhanced&nbsp; production&nbsp;&nbsp; of&nbsp;&nbsp; inflammatory cytokines and&nbsp; recruitment of&nbsp; immune cells.&nbsp; The in vitro&nbsp; results correlated&nbsp; well with local&nbsp; and systemic&nbsp; inflammatory responses&nbsp; after&nbsp; GO administration&nbsp; into abdominal cavity,&nbsp; lung&nbsp; or&nbsp; blood&nbsp; stream&nbsp; through&nbsp; tail&nbsp; vein.&nbsp; Those&nbsp; findings&nbsp; are&nbsp; important&nbsp; for considering future biological applications of carbon nanomaterials