CYLD调节巨噬细胞坏死性凋亡在脓毒症治疗中的作用机制研究

Sepsis is one of the world’s deadly diseases. Treatment is still a problem. Antibiotic and immune systems are at a loss for germs hiding in phagocytes because of the incomplete phogocytosis, which lead to the deterioration of infection. It was recently found that inhibition of necroptosis signal molecules can increase the germ numbers and decrease the survival rate. We also found that inhibition of necroptosis by Nec-1 decreased the mouse survival rate. It was further proved recently that ubiquitin enzyme CYLD is an important molecule in regulating macrophage necroptosis. We also proved that macrophage CYLD was up-regulated during sepsis, and the phosphorylated RIPK1, one of the necroptosis proteins, decreased, which indicated that CYLD is closely related to necroptosis. We hypothesize that CYLD is up-regulated during sepsis to promote macrophage necroptosis. The germs are released from the macrophages, which gives antibiotics and immune cells the opportunity to kill the germs. CYLD will be the new therapeutic target. We will study the mechanism of CYLD regulating macrophage necroptosis in sepsis therapy in the molecule and cell levels and in vivo, and try to reveal the sepsis essence to find the new therapeutic target. These studies will find the new theoretical evidence and therapeutic strategy, and have important theoretical and clinical significance.

脓毒症是世界致死性疾病之一，而治疗仍是难题。由于不完全吞噬，抗生素和免疫系统对躲藏在吞噬细胞内的病菌束手无策，导致感染恶化。新近发现，抑制坏死性凋亡信号分子可升高病原菌载量、降低生存率，我们前期也证实抑制坏死性凋亡，显著降低脓毒症小鼠生存率。最近发现泛素化酶CYLD是巨噬细胞坏死性凋亡的重要调节分子，我们前期也发现脓毒症时巨噬细胞CYLD显著上调，敲除CYLD后坏死性凋亡蛋白RIPK1磷酸化水平明显下降，提示CYLD与坏死性凋亡紧密联系。我们设想：脓毒症时通过进一步上调CYLD，促进巨噬细胞坏死性凋亡的发生，释放细胞内“吞而不死”的病菌，使免疫系统和抗生素有机会对其发动清除作用，将是脓毒症治疗的新靶点。我们将在分子、细胞和在体水平，研究CYLD调控巨噬细胞坏死性凋亡在脓毒症治疗中的作用，阐明脓毒症本质，发掘治疗新靶点，为治疗脓毒症提供新的理论根据和治疗策略，具有重要的理论和临床意义。

脓毒症相关性脑病（Sepsis associated encephalopathy，SAE）是指脓毒症发生后宿主反应失调引起的弥漫性脑功能障碍，出现谵妄、情感功能及认知功能障碍等临床症状，具有较高的致死率与致残率。据报道，全球每年有5000万脓毒症患者，其中SAE发病率高达76%，一旦发生SAE，死亡率高达70%；即使幸存，亦有很大一部分患者遗留长期认知功能及情感功能障碍，给家庭和社会带来沉重负担。然而，目前SAE长期症状发病机制尚不明确，无法提供有效治疗措施。因此，深入探讨SAE的发病机制，寻找有效干预靶点是减轻SAE损伤、改善患者预后的关键。研究发现，SAE患者与小鼠均存在明显脑萎缩，以海马尤为严重；病理结果提示海马神经元死亡是其脑萎缩的主要原因。大量研究显示，SAE病理过程中海马可出现凋亡、坏死性凋亡、焦亡等多种程序性细胞死亡（Programmed cell death，PCD）方式。Cell（2020, 2021）、Nature（2021）等杂志发表的一系列外周感染性疾病及肿瘤研究结果显示，同时抑制多种PCD能有效减少细胞死亡。然而，其在SAE中的作用及分子机制尚不明确，寻找干预多种PCD的策略有望有效减轻SAE。髓样分化因子2（Myeloid differentiation factor 2，MD2）是一种小分泌糖蛋白，我们前期的研究证实MD2是调控凋亡及坏死性凋亡的枢纽分子，抑制MD2可以有效减少脑卒中模型神经元丢失，发挥脑保护作用。然而，MD2在SAE中的作用尚未见报道；阐明MD2通过诱导多种PCD诱发SAE的内在机制有望为治疗SAE提供新思路。本实验利用盲肠结扎穿孔术（Cecal ligation and perforation，CLP）建立脓毒症相关性脑病小鼠模型，观察其海马神经元PCD通路相关蛋白的表达；并干预细胞死亡，观察对其行为学的影响。同时，利用MD2条件敲除小鼠，MD2特异性拮抗多肽等方法，深入研究MD2对多种PCD的调控作用，及阻断MD2对脓毒症小鼠脑功能紊乱的作用。