低白蛋白血症引发中性粒细胞NETs形成加重脓毒症凝血功能障碍的机制研究

Coagulation dysfunction is a key point in the development and prognosis of sepsis. Recent studies have found that neutrophil extracellular NETs (NETs) are closely related to the occurrence of coagulation dysfunction in sepsis, but the mechanism of the formation of NETs in sepsisis still unclear. Previous studies have found that hypoalbuminemia plays an important role in inducing the formation of NETs and participating in the occurrence of coagulation dysfunction in sepsis.Through literature reading, previous published studies and preliminary experimental results, We sugested that type A scavenger receptor (SR-A) was activated, and then NETs were formed by TNF-α-ROMO1-mROS axis in septic hypoalbuminemia. Firstly the effect of SR-A activation on NETs formation induced by low-albumin was verifIed by in vitro cell experiments using inhibitors, siRNA interference or plasmid overexpression and detected by laser confocal, immunoprecipition, quantitative PCR and other experimental methods. Furthermore, it was verified whether SR-A regulated NETs formation through the TNF-α-romo1-mROS axis. Finally, SR-A knockout mice were used to establish sepsis model, together with albumin intervention, the formation of NETs and Coagulation related molecules in vivo were detected. Finally, the specific mechanism of septic hypoalbuminemia mediated NETs formation and its role in the occurrence of coagulation dysfunction were clarified.

凝血功能障碍是脓毒症发生发展的关键环节。最近研究发现中性粒细胞胞外诱捕网（NETs）的形成可加重脓毒症凝血功能障碍，然而NETs形成机制尚不清楚。课题组前期研究发现脓毒症低白蛋白血症在诱导中性粒细胞NETs形成具有重要作用。结合文献和前期工作提出低白蛋血症通过A型清道夫受体（SR-A）活化，进而启动TNF-α-ROMO1-mROS轴介导NETs形成的假说。为此，本课题拟首先通过体外细胞实验使用抑制剂干预，siRNA干扰或质粒过表达等实验手段验证SR-A活化在低白蛋白诱导NETs形成的作用。进而验证SR-A是否通过TNF-α-ROMO1-mROS轴调控NETs形成的机制。最后使用SR-A敲除小鼠建立脓毒症模型，并使用白蛋白干预，对体内NETs形成及凝血相关分子进行检测。最终阐明脓毒症低白蛋白血症介导NETs形成加重凝血功能障碍的相关机制。

凝血功能障碍是脓毒症发生发展的关键环节。最近研究发现中性粒细胞胞外诱捕网（NETs）的形成可加重脓毒症凝血功能障碍，然而NETs形成机制尚不清楚。课题组前期研究发现脓毒症低白蛋白血症在诱导中性粒细胞NETs形成具有重要作用。通过本课题研究，我们发现低白蛋白可导致中性粒细胞胞内钙离子浓度显著上升，随着白蛋白的加入胞内钙离子逐渐恢复至正常水平。使用不同浓度的白蛋白与培养基中的钙离子共培养，随着白蛋白浓度增加培养基中的游离钙逐渐减少。流式检测白蛋白同样可使无血清培养时胞内的钙离子显著降低。使用胞外钙离子螯合剂或胞内钙离子清除剂均可抑制无血清诱导的胞内钙离子增加和NETs形成，因此白蛋白可通过与钙离子结合导致胞外游离钙减少。综上所述，本课题阐明了低白蛋白导致胞外游离钙增加，游离钙进入胞内促进线粒体活性氧形成最终导致NETs形成的机制。同时在脓毒症模型中我们验证了低白蛋白对于NETs的形成发挥了重要作用，而NETs形成进一步加重脓毒症凝血功能障碍。本课题的研究成果有望为阐明脓毒症凝血功能障碍的发生奠定新的实验和理论基础，同时为临床上使用白蛋白治疗脓毒症提供新的理论依据。

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