血红素通过miR-144-3p下调ALX表达：陈旧红细胞加重创伤后炎症失控的新机制

Transfusion is an essential measure of trauma therapy. However transfusion old stored blood has risk of aggravating progression of trauma. It is generally recognized that hemin released from old stored blood aggravats trauma process through stimulation of pro-inflammatory mediators and superimposing enhancement of post-traumatic inflammation. It is newly discovered that hemin can disturb imflammatory self-limiting process, which will follow by decreased expression of lipoxin A4 receptor (ALX), which is an inflammatory “stop”signal. Our data of pre experiment indicated that hemin regulated expression of ALX post-transcriptionally. Results of bioarray screen and bioinformatic analysis indicated that hemin could induce expression of miR-144-3p, which targeted ALX gene and down-regulated its expression. Therefore, hemin inhibited ALX expression through miR-144-3p and restrained inflammatory self-limiting process, which might be the new mechanism of aggravating trauma progression after transfusion old stored RBC. To test this hypothesis, firstly, this project will confirm that down-expression of ALX by miR-144-3p is the important mechanism of disturbance of self-limiting by hemin; secondly, molecular mechanism of hemin inducing miR-144-3p and miR-144-3p down-regulating ALX was investigated with molecular biotechnology methods such as chromatin immunoprecipitation (Chip-assay); finally, traumatic rat animal model which simulated clinical situation was used to validate the above mechanism. This project was aimed to discover the new mechanism of old stored RBC promoteing uncontroled inflammation after trauma.

输血是创伤救治的重要手段，但输注陈旧红细胞存在加重创伤病情的风险。一般认为陈旧红细胞释出的血红素通过刺激促炎介质表达、叠加增强创伤后炎症加重创伤进展，但新近发现炎症自限调节障碍才是创伤后炎症失控的关键。我们近期发现血红素可干扰炎症自限，这伴随炎症“刹车”分子ALX下调。预实验提示血红素在转录后水平调节ALX；芯片及生物信息学分析发现血红素可诱生miR-144-3p、而ALX可能是后者的抑制靶点。因而，血红素通过miR-144-3p下调ALX并干扰炎症自限可能是陈旧红细胞加重创伤病情的新机制。为证实该假说，本项目将首先证实miR-144-3p下调ALX是血红素干扰炎症自限的重要机制；进而通过染色质免疫共沉淀等方法揭示血红素诱生miR-144-3p及miR-144-3p下调ALX的分子机制；最后在模拟临床的创伤-输血动物模型中验证上述机制。本项目旨在揭示陈旧红细胞促进创伤后炎症失控的新机制。

输注红细胞是严重创伤大量失血患者的关键治疗手段，大量输注储存时间较长的陈旧红细胞可增加患者炎症损伤和死亡风险。游离血红素作为储存陈旧红细胞释放的重要损害相关分子模式通过增加炎性细胞因子表达、增强创伤后炎症加重创伤进展。新进研究表明炎症消退障碍是造成持续炎症反应的重要机制，血红素对炎症消退过程的影响及可能机制值得深入探究。本研究通过腹腔注射酵母聚糖A溶液构建具有典型“启动”-“终止”特征的自愈性局部炎症模型，研究血红素对炎症消退的影响。通过检测腹腔灌洗液炎症细胞因子水平、白细胞数量和比例并计算炎症消退指数明确了血红素显著延长了炎症消退时间，具有抑制炎症消退，促进持续炎症反应的作用。在机制层面，通过分离外周血中性粒细胞和诱导类中性粒细胞dHL-60明确了血红素刺激可显著下调炎症消退关键受体ALX/FPR2的蛋白表达而不影响mRNA的水平，由此推测血红素在转录后层面调控ALX/FPR2的蛋白表达。MicroRNA通过与靶基因3’-UTR区域结合抑制其翻译，是重要的转录后调控方式。随后通过线上工具TargetScanHuman和microRNA.org预测并筛选出最有可能与ALX/FPR2 3’UTR相结合的microRNAs，并在中性粒细胞和dHL60细胞中证实血红素刺激可显著上调mi-RNA-144-3p的表达。通过在细胞系HL-60中采用慢病毒感染和荧光素酶报告基因分析发现血红素对中性粒细胞ALX/FPR2蛋白表达的调控作用依赖miRNA-144-3p的表达上调，且证实了mi-RNA-144-3p与ALX/FPR2的3’-UTR相结合。经过上述动物实验和细胞实验证实了血红素通过mi-RNA-144-3p下调ALX/FPR2蛋白表达进而抑制炎症消退过程。此外，我们在脓毒症小鼠模型中发现游离血红素还具有协同放大炎症反应的作用，该作用与血红素刺激的巨噬细胞信号转导和转录活化因子（STAT3）的磷酸化升高呈正相关。.本研究发现了游离血红素通过上调mi-RNA-144-3p抑制炎症消退受体ALX/FPR2的蛋白表达，进而抑制炎症消退介质的促消退作用。该研究揭示了陈旧红细胞促进创伤后炎症失控的新机制，为降低严重创伤大量输血相关不良反应的防治提供了新的策略和干预靶点。