血小板来源5-羟色胺抑制NETs形成与慢加急性肝衰竭“凝血稳态再平衡”的关系

Patients suffered with liver failure have significant coagulation disorders, which would be worsened by infections, frequently leading to acute bleeding including variceal and intracranial bleeding. Neutrophil extracellular traps (NETs) are constituted by released chromosomal DNA and other materials derived from primary and secondary particles of neutrophil. NETs can cause fatal coagulopathy including intravascular disseminated coagulation under septic conditions. Our retrospective investigation found that ACLF patients had greater neutrophil elastase levels and positively correlated with plasma D-dimer concentrations. The preliminary experiments in chronic liver injury mice model indicated that LPS increased intrahepatic NETs formation. Our in vitro experiments showed that the ability to inhibit NETs formation by platelets isolated from healthy controls was greater than liver failure cases, and fibrinogen was essential for this inhibition. The fibrinogen levels and PAC-1+ platelet percentages were both declined in liver failure patients compared to CHB. We found that liver failure patients had significant thrombocytopenia, and serotonin (5-HT) levels and its releasing efficiency under classical in vitro conditions were lower than CHB. Our in vitro experiments further presented that 5-HT inhibited NETs formations which is concentration-dependent with the minimal concentration at 10μM. Platelets are predominant carriers of peripheral 5-HT and taking accounts for the obvious high concentration of 5-HT to inhibit NETs formations, we speculated that such inhibition in vivo should occur under environments such as platelet and neutrophil aggregates. Based on our novel findings, we speculate that liver failure patients have less potent to control NETs. 5-HT can inhibit NETs formations, however liver failure patients have obvious thrombocytopenia and lower 5-HT contents in platelet, possible plus its impaired release. These patients had lower fibrinogen and fewer PAC-1+ platelets then possibly retard neutrophil and platelet aggregates, which is the context of 5-HT effects on NETs. In this proposal, we plan to perform kinds of in vitro experiments to clarify the mechanisms for 5-HT inhibition on NETs, and the effects of NETs contents on platelets activations including fibrinogen binding and whole blood coagulations will be observed. We plan to test the possible effects of NETs supernatants on 5-HT homeostasis, including 5-HT release and the levels, distributions and functions of several key molecules such as 5-HT transporter, VMAT2 and VAMP8/SNAP23. In mice model the bridge role of platelet to 5-HT inhibitions on NETs formation would be confirmed. We plan to establish a large liver failure cohort then try to build the possible associations between 5-HT homeostasis, NETs formation, clinical bleedings and survivals, which could help us to discover new biomarkers for prediction of major bleedings and deaths in such patients.

ACLF有明显凝血障碍、感染后恶化。感染时中性粒细胞形成NETs活化血小板。我们的回顾分析发现，ACLF血浆NETs水平升高、与D二聚体水平正相关；初步动物实验表明，LPS促进慢性肝损小鼠肝内NETs形成。我们的体外实验显示，纤维蛋白原存在时ACLF血小板抑制NETs形成能力下降；LPS增加血小板与中性粒细胞的聚集。我们发现ACLF血小板PAC1+比例降低、5-HT含量下降及释放减少。我们的体外实验进一步证实5-HT可抑制NETs形成、呈浓度依赖。假说：5-HT可能是在血小板与中性粒细胞聚集体内通过受体途径抑制NETs形成；感染时ACLF无法有效抑制NETs形成、致凝血障碍迅速恶化。拟通过多个实验阐明5-HT抑制NETs的机制、NETs对血小板5-HT稳态的影响；在小鼠模型及临床队列中建立血小板5-HT稳态与NETs形成、出血事件及临床预后的联系，寻找新指标预警ACLF出血、预测短期预后。

肝衰竭或终末期肝病患者易并发感染，导致组织损伤和器官功能衰竭。病原体结合并激活血小板，活化血小板与中心粒细胞结合诱导NETs形成, 其中活化血小板可释放多种活性物质。本研究围绕“5-HT在血小板-中性粒细胞聚集体微环境内通过受体途径抑制NETs形成”这一假说展开研究，以明确5-HT抑制NETs形成的机制及其在感染小鼠模型中对机体的影响。在体外研究中，我们发现5-HT抑制PMA诱导NETosis，并呈现出浓度梯度依赖性。5-HT的这一抑制效应是通过激活HTR2b而发挥作用的，并对Ionomycin诱导NETosis有着同样的抑制作用。接下来通过共孵育血小板和中性粒细胞模拟体内情况，进一步确认了阻断HTR2b可增加NETosis。在机制研究方面，我们发现5-HT对PMA诱导NETosis的抑制效应是通过抑制中性粒细胞中NOX活化（包括抑制gp91 phox 和p40 phox的共定位以及p40 phox磷酸化），减少其胞内ROS生成（包括胞内及线粒体内超氧阴离子生成的减少），并进一步抑制自噬发生、核膜降解和组蛋白瓜氨酸化及降解。5-HT对Iononmycin诱导NETosis的抑制可能也是通过抑制其胞内ROS生成发挥作用的，但其抑制的ROS来源仍不明确。我们发现虽然5-HT保护中性粒细胞使其保留一定的吞噬功能，但是在菌血症小鼠模型中，其总体效应表现为外源性5-HT造成肝内细菌聚集增多，这可能是由于5-HT抑制肝内NETs形成造成机体对细菌杀伤效应减少造成。以上研究结果表明：5-HT通过激活中性粒细胞HTR2b，抑制NOX活化，造成其胞内ROS生成减少，并抑制自噬发生、核膜降解和组蛋白瓜氨酸化及降解等下游一系列重要环节而抑制NETosis。在菌血症模型中，外源性5-HT抑制体内NETs形成，使得机体杀菌能力减弱，肝内细菌聚集增多。这一效应可能为肝衰竭或终末期肝病患者易并发感染机制之一。