巨噬细胞中 SIRT1 / Notch 调控应答在烧伤脓毒症中的作用及机制研究

Sepsis is a challenging problem in critical care medicine research. Excessive activation of macrophages is the key link in the development and progression of sepsis. Notch pathway is closely related to the polarization of macrophages and the release of inflammatory cytokines. Previous study has showed that Notch pathway in macrophages was over activated while the expression of SIRT1 was significantly suppressed during sepsis after burns. SIRT1 agonist could remarkably reduce the mortality of mice suffered from sepsis after burns and degrade the excessive inflammatory reaction. Meanwhile, the activation of Notch pathway was significantly suppressed and the viscera function was improved. However, the mechanism of SIRT regulation in macrophages in sepsis is still unclear. Accordingly, based on the previous research, we are planning to explore the interaction of SIRT1-Notch pathway in regulate the function and state of macrophages in sepsis through gene knockout and molecular biology methods. It is critical to clarify the SIRT1-Notch pathway interaction in macrophages. The study is expected to provide new theoretical basis and therapeutic targets to reduce uncontrolled inflammation and improve the survival rates in sepsis.

脓毒症是烧、创伤外科及危重病医学面临的难题之一，巨噬细胞过度活化是其发生、发展的根本环节，而Notch信号通路与巨噬细胞的极化、炎症因子释放密切相关。项目组前期实验结果显示：烧伤脓毒症发生时，巨噬细胞内Notch信号通路处于过度活化状态，SIRT1的表达受到明显抑制。给予SIRT1激动剂可显著降低烧伤脓毒症小鼠的死亡率及过度炎症反应，抑制Notch信号通路活化，明显改善脏器损伤。然而，烧伤脓毒症时SIRT1在巨噬细胞中的具体调控机制，及其与Notch信号通路的调控应答机理尚不明确。据此，本研究拟在前期研究基础上，从调节巨噬细胞的功能着手，通过基因敲除，定点突变等分子生物学方法，探讨SIRT1/Notch调控应答对巨噬细胞状态及功能的影响，并重点阐述脓毒症时巨噬细胞中SIRT1/Notch信号通路的调控应答机制，为减轻失控性炎症反应，提高脓毒症的生存率提供新的理论基础及治疗靶点。

脓毒症是烧、创伤及危重病医学面临的难题，巨噬细胞过度活化是其发生发展的根本环节，而Notch信号通路与巨噬细胞的极化、炎症因子释放密切相关。课题组以SIRT1调控巨噬细胞状态及功能、介导烧伤脓毒症的发生为关键点，通过脓毒症动物模型及LPS活化的M1巨噬细胞模型进行了系统研究。1）发现随着M1型巨噬细胞炎性相关分子的表达增多，巨噬细胞Notch信号通路活化，SIRT1表达显著下调，并具有显著相关性。体外实验中，活化或过表达SIRT1可显著抑制Notch 信号通路，抑制M1巨噬细胞炎性因子的表达，缓解炎症反应。动物模型观察到，SIRT1不仅可显著提高脓毒症小鼠的存活率，还可显著抑制小鼠血清中炎症因子的释放，减轻脓毒症小鼠的脏器损伤。2）揭示了SIRT1介导巨噬细胞内Notch信号通路的调控机制：SIRT1可通过促进NICD的去乙酰化抑制Notch信号通路。3）通过基因敲除鼠模型证实抑制巨噬细胞Notch信号通路，可拮抗NF-κB通路的活性，进而影响脓毒症炎症反应的发生。4）明确了SIRT1抑制NF-κB/p65 乙酰化水平，进而抑制NF-κB/p65入核，直接调控NF-κB信号通路的活化从而负性调控炎症反应的作用模式。5）在以上结果的基础上，围绕巨噬细胞SIRT1/Notch调控轴，确定了3个以SIRT1为靶点的靶向干预分子（miRNA-138, miRNA-9, miRNA-34a），1个以Notch为靶点的靶向干预分子（miRNA-146a）。6）以本课题为依托结合表观修饰实验，课题组对脓毒症时，SIRT1调控巨噬细胞的乙酰化位点进行了鉴定。7）创新性的提出SIRT1可通过琥珀酰化修饰调控巨噬细胞介导炎症反应的新机制，并进行了探索性研究。这些结果的获得进一步明确了SIRT1参与调控脓毒症失控性炎症反应的发生发展、维持机体内稳态的新理论。