游离血红蛋白对一氧化氮信号调控在储存红细胞回输不良反应中的作用机制研究

The putative “red blood cell (RBC) hypothermic storage lesion during ex vivo storage” is the focus of intense interest and investigation in recent years. However, the clinical significance of transfusion of “old blood” remains unclear. Recently, several meta-analysis indicate that certain pathologic conditions sensitize the recipients, including those undergoing cardiac surgery, trauma victims, and critically ill patients, to the old blood transfusion related adverse effects (TRAE). These susceptible hosts are endothelial dysfunctional with nitric oxide (NO) reduction considered to be the hallmark. This raises fundamental question regarding the actual risk associated with transfusion of stored blood: If endothelial dysfunction is expected to promote TRAE. In the previous study, we reveal that syngeneic transfusion of stored mouse RBCs induces TRAE in mice suffering endothelial dysfunction (HFD and db/db mice), whereas such response are not evident in wild-type (healthy) mice. In addition, our previous study also implicates extracellular free hemoglobin as the offending molecule. Extracellular free hemoglobin avidly scavenges NO and thus reduces NO bioavailability. Furthermore, it has recently been proved Hb also regulates NO diffusion from endothelium to smooth muscle via Hbα expressed in endothelial cells and at the myoendothelial junction. The hypothesis that will be testified in this project is that the increased free Hb after stored blood transfusion down-regulates NO signaling through scavenging NO and decreasing NO diffusion from endothelium to smooth muscles, and thus cause adverse reactions to old blood transfusion. This grant application proposes studies to investigate the underlying mechanisms of TRAE and meanwhile to explore simple methods to prevent TRAE by reducing Hb-NO interaction via treating stored blood in vitro before transfusion. Learning which patient populations and why they are at great risk of, and finding a effective method to alleviate the adverse responses to transfusion of stored blood would be a major advance allowing better utilization of blood bank resources.

储存红细胞（RBC）回输不良反应（TRAE）是目前研究热点，但其发生机制尚不明确。我们在前一研究中发现，以一氧化氮（NO）生物利用率降低为特征的内皮功能障碍动物，对TRAE尤为敏感。通过建立小鼠RBC储存模型，我们初步证实游离血红蛋白（Hb）对NO的清除在TRAE发生过程中发挥了重要作用。在此基础上结合最新研究进展，本项目大胆假设，Hb对NO弥散功能的调节也参与了TRAE的发生，力图在完善Hb对NO信号调节理论的同时，系统阐明TRAE发生的确切机制。同时本项目还创新性的提出在体外直接对储存RBC进行NO负载的方法，力争在增加NO含量的同时，将RBC中的游离Hb氧化为高铁Hb，使其丧失对NO的清除能力，从而实现减少TRAE发生的目的。这些研究可以拓展对TRAE发生机制的认识，并在此基础上开发具有临床推广前景的潜在治疗方法，为增强临床用血的安全性和有效性提供理论支持和实验依据。

储存红细胞（RBC）在体外发生储存损伤导致回输后不良反应（TRAE）的发生是目前研究热点，但其发生机制尚不明确。我们研究发现，以一氧化氮（NO）生物利用率降低为特征的内皮功能障碍动物对TRAE尤为敏感。在Toapload和失血性休克模型中，我们都发现存在内皮功能障碍的HFD或db/db小鼠输入储存RBC后的血管收缩反应和组织损伤严重程度加剧。通过建立小鼠RBC储存模型，我们证实游离血红蛋白（Hb）对NO的清除是TRAE发生的关键。游离Hb脱离红细胞膜的束缚后通过双加氧反应快速清除NO，导致微循环障碍、组织氧化损伤、炎症反应等NO生物利用度下降为主要表现的TRAE发生。通过吸入NO或给予游离Hb的中和蛋白Hp等手段，将游离Hb氧化成高铁Hb或与游离Hb形成复合物，减少游离Hb对NO的清除，能有效缓解TRAE的发生。但以上治疗效应在存在内皮功能障碍的动物发生钝化，说明此类动物对NO生物利用度降低更为敏感，NO信号调节发生变化是影响治疗效应的关键所在。吸入NO整个治疗过程须进行严密的监测，这在一定程度上限制了吸入NO的临床应用。本研究创新性通过在体外直接对储存RBC进行NO负载处理，证实负载NO的储存RBC回输后TRAE发生率显著降低 。这一方法较吸入NO更为安全易行，具有更广阔的临床应用前景。同时将动物研究的成果积极实现临床转化，在临床工作中观察到体外循环下心脏手术患者术后急性肾脏损伤的发生率很高，其发生机制与体外循环过程中大量产生的游离Hb密切相关，给予长时间体外循环的心脏多瓣膜置换手术患者在体外循环开始的时候给予吸入80 ppm的NO，发现吸入NO可通过降低游离Hb对NO的清除降低术后急性肾脏损伤的发生率，减少慢性肾功能不全的发生率，减少严重肾脏不良事件的发生率。本研究在深入探讨TRAE发生机制的同时努力寻找减轻TRAE的更为安全有效的潜在治疗方法，为提升临床用血的安全性提供了理论支持和实验依据。