组蛋白乙酰化修饰调控TLR信号通路负性调节分子表达介导脓毒症二次感染急性肺损伤的机制研究

Acute lung injury (ALI) is the most common cause of death in sepsis secondary infection. Recent studies have found that a large number of neutrophils accumulated in the lung, secreted amounts of TNF-α and lead to acute lung injury during sepsis secondary infection. However, the mechanism modulating the up-regulation of TNF-α in neutrophils is still undefined. Our preliminary data showed that during sepsis secondary infection, a series of acetylation-related genes were altered significantly in neutrophils and HDAC inhibitor could reduce the mortality rate of septic mice. Meanwhile, the negative regulatory molecules in TLR signalling pathway (IRAK-M、SOCS1 and SHP-1) downregulated significantly and might the targets of acetylation. Therefore, we hypothesized that HDAC might inhibit the expression of the above negative regulatory molecules by inducing the histone deacetylation in their promoter region, promote the secretion of granulocyte-derived TNF-α indirectly, and lead to sepsis-induced ALI accordingly. The applicant plans to employ secondary infection septic mice models to isolate the neutrophils, detect the expressions and activities of negative regulatory molecules in TLR4 signalling pathway, identify the specific HDAC molecules and reveal the relationship of HDAC and its targets (inhibitory regulators) by ChIP-PCR. Hereby, the epigenetic regulation mechanism leading to neutrophil over-activation will be revealed during secondary infection of sepsis. Our study will provide new theoretical basis and experimental evidences for the prevention and clinical therapy of sepsis secondary infection induced ALI.

急性肺损伤（ALI）是脓毒症继发二次感染患者最常见的致死原因。二次感染时粒细胞分泌TNF能力显著增加，成为导致ALI的重要效应细胞，但TNF上调机制不清。申请人发现：脓毒症二次感染时，组蛋白去乙酰化酶（HDAC）抑制剂可显著降低小鼠死亡率；粒细胞组蛋白乙酰化修饰相关基因变化明显；TLR通路负性调节蛋白可能是乙酰化调节的重要靶点。据此提出：组蛋白乙酰化水平降低引起TLR通路负性调节分子表达下降，间接促进粒细胞TNF分泌。申请人拟采用脓毒症二次感染ALI模型，检测粒细胞TLR4通路负性调节分子表达水平和活性，分析其与TNF分泌及ALI程度的关系；通过分析HDAC家族分子表达/酶活性与负性调节分子的相关性，明确关键HDAC亚型；采用ChIP-PCR技术分析HDAC靶点，明确组蛋白乙酰化修饰对负性调节分子的转录调控作用，揭示粒细胞功能转化机制，为临床预防和治疗二次感染ALI提供理论和实验依据。

急性肺损伤(ALI)是脓毒症继发二次感染患者最常见的致死原因。二次感染时粒细胞分泌TNF-α能力显著增加，成为导致ALI的重要效应细胞，但TNF-α上调机制不清。既往研究证实，固有免疫细胞如单核巨噬细胞和NK细胞在受到初次感染时，出现免疫训练(Trained immunity)现象，表现出对二次感染的反应增强，其在初次受到刺激后相关基因的表观遗传学调控发生改变，并在二次刺激中维持了激活/抑制的修饰特性，致使二次感染发生后这些基因能够迅速表达或维持抑制，最终使免疫应答反应得到增强。本研究首次发现并提出了脓毒症二次打击情况下粒细胞免疫训练的概念，表现为其分泌促炎因子TNF-α增强，及清除病原菌能力增强。机体因粒细胞过量分泌TNF-α，导致炎症风暴，发生ALI而死亡。本研究进一步发现，脓毒症诱导的粒细胞染色质松散且异染色质蛋白表达减低，其TNF-α基因处染色质开放，易于转录；转录组学分析提示：TLR4通路负性调控因子IRAK-M，SHP-1 及 SOCS1 表达显著下降，其组蛋白乙酰化修饰降低，且在二次感染时维持低水平，进而蛋白水平表达亦降低，导致TLR4通路负反馈机能减退，过度活化进而造成TNF-α表达水平升高。HDAC抑制剂（HDACi） 能够逆转该现象，恢复三个负性调控蛋白乙酰化水平，提高蛋白表达水平，并最终降低TNF-α分泌能力，提高模型小鼠的生存率。本研究从表观遗传学调控机制的角度，对脓毒症二次打击情况下，粒细胞TLR4信号通路、组蛋白修饰调控进行了深入研究，寻找到潜在的粒细胞免疫训练调控机制及针对过量TNF-α分泌的药物干预靶点-HDACi。

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