外源性一氧化碳经sGC/cGMP/Class I PI3K/mTOR途径调控脓毒症时中性粒细胞自噬失稳态的机制研究

Autophagy is a vital homeostatic process triggered by starvation and other cellular stresses, in which cytoplasmatic cargo is targeted for degradation in specialized structures termed autophagosomes. Many results have revealed that autophagy is involved in clearance of intracellular and extracellular pathogens and plays key roles in innate immune and adaptive immune. Autophagy adjustment disorders during sepsis are important factors leading to dysfunction of Organ dysfunction, the detail mechanisms, however, are not completely elucidate. This project employs gene knock-out animal models and CRISPR-Cas9 gene editing system to examine the dynamic state of polymorphonuclear neutrophils (PMN) autophagy during sepsis under the intervention of the exogenous CO liberated by CO-releasing molecules. The important factors will be investigated in the molecular and cellular levels through applying updated molecular biological techniques including gene analysis, mass spectrometric analysis, measurement of protein expressions, which investigate the dynamic state of PMN autophagy, the dynamic changes of expressions of autophagy related gene and proteins during sepsis. Furthermore, this project also focuses on the important influences of the adjustment disorder of sGC/cGMP/Class I PI3K /mTOR signal transduction to PMN autophagy. Therefore, we are in attempt to elucidate the disorder of PMN autophagy during sepsis to determine whether sGC/cGMP/ Class I PI3K /mTOR signal transduction will be the novel targets of exogenous CO, in order to elucidate molecular mechnisms (even partly) responsible for the regulatory effects of CO on abnormal PMN autophagy and to provide theoretical basis and new idea for the research and treatment of sepsis.

脓毒症时中性粒细胞自噬失稳态是导致机体天然免疫系统失衡、器官功能不全的重要因素。我们的前期研究显示，内毒素刺激后PMN自噬过程紊乱，自噬相关基因和蛋白表达异常，使用外源性一氧化碳干预后自噬紊趋于稳态，但详细机制不清。本研究采用基因敲除动物模型、CRISPR-Cas9系统基因编辑细胞分子敲除模型，以外源性一氧化碳进行干预，运用基因分析、蛋白质定位定量测定等手段，在分子、细胞和整体等不同层面上，研究脓毒症时中性粒细胞自噬失稳态的动态变化、sGC/cGMP/Class I PI3K/mTOR信号通路对自噬过程的调控机制，并利用原子力显微技术结合透射电镜，精确观察脓毒症时PMN表型和自噬体形成，旨在阐明PMN自噬失稳态的重要特征，探明外源性CO通过sGC/Class I PI3K/mTOR途径调节脓毒症时中性粒细胞自噬紊乱的（部分）分子学基础，为发现脓毒症干预新靶点提供新的思路。

脓毒症时中性粒细胞自噬失稳态是导致机体天然免疫系统失衡、器官功能不全的重要因素。本研究采用相关细胞模型、WGCNA基因分析、单细胞测序、荧光多色染色空间定位分析手段，在分子、细胞和整体等不同层面上，研究脓毒症时自噬失稳态对中性粒细胞功能以及T淋巴细胞功能的影响。结果表明：1.通过加权基因共表达网络分析，从基因层面验证了中性粒细胞自噬与脓毒症时中性粒细胞的功能改变密切相关。2.通过细胞实验进行进一步的验证，从基因和蛋白层面同步证明脓毒症早期中性粒细胞自噬有明显的上调。3.基因测序和动物实验同步证明了自噬标志分子LC3的表达与免疫抑制分子PD-L1的表达有明显的相关性。细胞和动物实验同步验证了促进自噬能够抑制脓毒症时中性粒细胞PD-L1的表达。4.脓毒症时，中性粒细胞的凋亡功能收到了明显的抑制，而促进自噬能够对中性粒细胞的延迟凋亡有所改善。5.脓毒症时，自噬的改变能够调控中性粒细胞免疫抑制分子PD-L1的表达。我们通过单细胞测序发现一群具有免疫抑制功能的中性粒细胞亚群（PD-L1阳性的中性粒细胞亚群），该亚群的存在可以促进Treg的形成、T淋巴细胞凋亡的升高以及活化的降低，从而促进机体的免疫抑制。本课题初步阐明了脓毒症时中性粒细胞自噬功能失稳态的重要性，为临床脓毒症治疗提供了新思路。