钠钾ATP酶配体转录后精密调控TH1细胞因子mRNA稳定性的分子机制及其在脓毒症中的应用价值

The acquired immunosuppression, or immunoparalysis is an important pathological mechanism for the high mortality of sepsis; however, effective therapies for immunoparalysis are not clinically available. By drug screening, we found that Na+,K+-ATPase ligand, such as ouabain can effectively reverse immunoparalysis, and further, this effect was related to the increased TH1 cytokines mRNA stability by ouabain-induced HuR nuclear export, especially for TNF α. Unexpectedly, ouabain also increased miR181s expression to destabilize TNFα mRNA, and this effect was similar as that of LPS. To explain the contradictory result, we found the binding site of miR181s on the 3'-UTR of TNFα mRNA locates exactly within HuR binding sites. Therefore, we presume in this study that the induction of miR181s by ouabain belongs to feedback protective mechanism, the purpose of which is to suppress the overproduction of TNFα and the resultant inflammatory injury. However, miR181s-mediated TNFα mRNA degradation also leads to the enhancement of immunoparalysis. To circumvent the potential immunosuppressive effect of miR181s, ouabain treatment-induced HuR export can compete with miR181s on the shared target of TNFα mRNAs, or recruit TNFα mRNAs to stress granules in case of being degraded by miR181s, thereby leading to stabilization of TNFα mRNA and the reversal of immunoparalysis.

获得性免疫抑制，或免疫麻痹是脓毒症高死亡率的重要病理机制，但临床治疗手段匮乏。通过药物筛选，我们发现钠钾ATP酶配体如哇巴因能有效逆转脓毒症免疫麻痹，且该作用与哇巴因诱导HuR出核，增强TH1细胞因子，尤其是TNFαmRNA稳定性相关。意外的是，哇巴因也能刺激miR181s表达降解TNFαmRNA，且该作用与内毒素颇为相似。为解释该矛盾结果，我们发现miR181s在TNFαmRNA 3′-UTR上的结合位点恰好位于HuR结合位点内部，由此推测哇巴因或内毒素诱导的miR181s表达属于机体反馈性保护机制，旨在抑制TNFα过度积累导致的炎性损伤，但也会因为抑制TNFα表达加剧免疫麻痹。为规避miR181s潜在免疫抑制作用，在哇巴因作用下，HuR能与miR181s竞争结合TNFαmRNA，或形成压力颗粒将TNFαmRNA包裹在内，躲避miR181s的降解，最终稳定TNFαmRNA，逆转免疫麻痹。

脓毒症发生率高、病情凶险、病死率高，对人类健康造成巨大威胁，免疫麻痹是重症脓毒症死亡的重要病因，但病理机制不详，临床治疗药物缺乏。利用HPLC-MS/MS检测方法，我们发现重症脓毒症病人血浆中内源性哇巴因大量表达。为探索作用机制，我们分别建立脓毒症炎症模型与能够模拟临床脓毒症免疫麻痹的小鼠盲肠穿孔结扎+沙门氏菌二次打击模型，发现哇巴因在脓毒症炎症模型上加速小鼠死亡，但在脓毒症免疫麻痹模型上却很显著性地改善小鼠存活率，由此推测内源性哇巴因具有调节脓毒症免疫状态新功能。为探索作用机制，我们发现哇巴因能对抗LPS诱导的单核细胞内毒素耐受。在脓毒症免疫麻痹状态下，LPS诱导的miR181d表达能促进TH1细胞因子（TNF-α、GM-CSF、IFN-γ）的快速降解，诱导内毒素耐受，使单核细胞对病原微生物的二次进攻失去响应。此时哇巴因的存在能刺激单核细胞核释放RNA结合蛋白HuR，HuR一方面与miR181d竞争在TNF-α mRNA 3’-UTR上结合位点；另一方面形成压力颗粒(stress granules)将TNF-α mRNA包裹在内,避免其被核酸酶降解,最终稳定TNF-α mRNA。通过诱导HuR出核，哇巴因也同时稳定了GM-CSF与IFN-γ等TH1炎症因子mRNA，最终实现在脓毒症免疫麻痹状态下对TH1细胞因子表达重新编程，逆转脓毒症免疫麻痹，增强机体在脓毒症免疫麻痹状态下抗病原微生物二次感染的能力。该结果发表于EMBO MOL MED（IF>10), 被审稿人认为是一项很有创新性工作。.鉴于内源性哇巴因在脓毒症免疫麻痹中的重要作用，我们测定了100多例脓毒症病人内源性哇巴因含量，发现它的含量能在临床上很好地用于脓毒症免疫状态的分型，与临床其它指标一致率>90%。因此，内源性哇巴因很可能是脓毒症免疫分型的重要标志物之一。相关研究内容获得多项国内专利授权，研究成果被《Critical Care》、《J Immuno》等免疫学权威期刊引用。 该项目发现脓毒症免疫麻痹新机制，发现哇巴因在免疫调节中的新功能与临床应用价值，为临床脓毒症免疫治疗提供新的理论依据。发表SCI论文17篇，中文核心1篇，申请专利5项，授权专利5项。培养博士生3名，硕士生2名。