以外泌体为载体的 miR-223通过调节SERCA2a和iNOS表达改善脓毒症心功能障碍的机制研究

Sepsis-induced cardiac dysfunction results in an increased mortality by 20%-40%. Hence, to clarify the mechanisms by which sepsis induces cardiac dysfunction is crucial to improve the survival rate of sepsis. It has been reported that the levels of miR-223 in deadpatients with sepsis were significantly lower than those in survivors, suggesting that miR-223 may be a key molecule in the pathological processes of sepsis. Moreover, recent study has suggested that the binding of TNF-α with TNFR1 resulted in a decrease in myocardial contractility in septic mice through up-regulating inducible nitric oxide synthase (iNOS) and down-regulating sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a). Our preliminary studies showthat there was a reduced myocardial contractility in miR-223 knockdown septic cell. Furthermore，exosome-mediated entry of miR-223 could bind to TNFR1 in cradiomyocytes. Therefore, we hypothesized that exosomal miR-223 acts as an antagonist to inhibit TNF-α mediated up-regulation of iNOS and/ordown-regulation of SERCA2a, thereby to protect against sepsis-induced cardiac dysfunction. We will produce sepsis models in miR-223 KO，miR-223 TG，and TNFR1 KO mice combined with molecular biology and high resolution image analysis to elucidate the mechanisms by whichexosomal miR-223 improves cardiac function in sepsis. The results obtained by the proposed project may provide insights into sepsis-induced cardiac dysfunction.

脓毒症致心功能障碍使脓毒症患者死亡率上升20%-40%，脓毒症死亡患者血清中miR-223水平显著低于存活者。细胞脓毒症模型中miR-223水平降低促进细胞凋亡，提示miR-223是脓毒症转归的重要分子；脓毒症中TNF-α与TNFR1结合介导iNOS上调和SERCA2a下调可能导致脓毒症小鼠心肌收缩力下降。我们发现，脓毒症心肌细胞模型中敲除miR-223后，细胞钙浓度及单个心肌细胞收缩力显著下降；心肌细胞中经外泌体导入外源性miR-223与TNFR1结合。因此提出假设：脓毒症中经外泌体导入的miR-223竞争性与TNFR1结合，抑制TNF-α介导的iNOS上调和SERCA2a下调，改善心脏功能。我们将在miR-223敲除/过表达、TNFR1敲除脓毒症小鼠中，应用分子生物学、高清影像学等手段揭示miR-223对改善脓毒症心脏功能的作用和机制，为脓毒症心脏功能障碍治疗提供新思路。

脓毒症患者常伴有明显心功能障碍，并且脓毒症心功能障碍可使脓毒症患者病死率显著增加。但目前缺乏特异性的biomarkers，临床主要治疗手段包括：容量复苏、正性肌力药物和血管活性药物等，但这些方法都没有被证明治疗有效。miRNAs在几乎所有的心血管过程中发挥强大的调控作用，同时研究发现miRNAs在脓毒症诱导的心功能障碍中发挥了关键作用。本研究通过小动物超声心动、PEG法提取外泌体、酶联免疫吸附法、TUNEL、miRNA高通量测序、全转录组测序、超高效液相色谱-串联飞行时间质谱联用仪进行非靶向代谢组学分析等方法，探究了脓毒症心功能障碍时心脏组织中包括miRNAs、LncRNAs、CircRNAs在内的非编码RNA的变化以及心脏组织中代谢产物的变化，这将为脓毒症心功能障碍biomarkers的寻找，以及临床脓毒症心功能障碍的治疗提供新的研究方向和理论依据。