HMGB1通过PARP-1信号通路激活巨噬细胞介导呼吸机相关性肺损伤的机制研究

Ventilator induced lung injury (VILI) is a severe complication during mechanical ventilation. The positive correlations between HMGB1 and acute lung injury has already been approved in our previous study and the alveolar macrophages RAW264.7 were considered primarily responsible for the release of HMGB1 stimulated by LPS compared with alveolar epithelial cells, although the mechanism was still unknown. It has also been demonstrated that the PARP-1 played the important role in transferring of HMGB1 to cytoplasm. Based on these findings, we hypothesize that PARP-1 is activated by macrophages to stimulate the release of HMGB1; meanwhile, HMGB1 combines with its own receptors on the surface of macrophages. Such a positive feedback loop may aggravate acute lung injury. In this study, we aim to establish a VILI model to confirm the source of HMGB1 is principally from macrophages. The mechanism of the release of HMGB1 is to be illuminated and then verified by alveolar macrophages from TLR4-/- and RAGE-/- mouse. After establishing the main receptors, we intend to further verify in the knock-out mouse model of VILI. The object of the study is to illuminate the mechanism of the self-reactivity of macrophages. It is expected to establish a novel strategy targeting HMGB1 to prevent and treat the VILI, as well as provide the experimental and theoretical basis for clinical therapy.

呼吸机相关性肺损伤（VILI）是机械通气的严重并发症。我们在前期研究中发现HMGB1的表达与急性肺损伤存在正相关性，亦发现在LPS刺激下小鼠巨噬细胞RAW264.7相比肺泡上皮细胞分泌HMGB1显著增多，但具体机制不明。有研究认为PARP-1可介导HMGB1向胞浆的转移。因此，我们推测在VILI中巨噬细胞通过激活PARP-1，分泌HMGB1并结合自身表面受体，形成正向反馈环路，加重肺损伤。本项目拟通过建立体外VILI模型首先证明HMGB1的释放来源主要为巨噬细胞，进而阐明巨噬细胞分泌HMGB1的分子机制，并利用TLR4-/-和RAGE-/-小鼠的肺泡巨噬细胞进行验证，确定其主要受体。最后利用该受体敲除的小鼠，在体内VILI模型中进一步验证。本项目将阐明VILI中巨噬细胞利用HMGB1自身激活的机制，有望建立以HMGB1为靶点的VILI防治新策略，为临床治疗提供实验和理论基础。

呼吸机相关性肺损伤（VILI）是机械通气的严重并发症。本课题成功构建了小鼠VILI的模型，发现VILI组小鼠的血清与肺泡灌洗液中HMGB1的mRNA或蛋白含量增加。受机械牵张肺泡巨噬细胞的培养液中TNF-α等炎性因子增多，HMGB1蛋白表达增加。本课题证明了丙酮酸乙酯（EP）干预对VILI具有一定的保护作用。EP干预后，肺部中HMGB1的含量明显下降。细胞凋亡信号通路的关键蛋白Caspase3、 Caspase9和PARP三种蛋白在EP干预后均呈现上升趋势，说明EP干预可以激活细胞凋亡的信号通路。以此得出结论，EP对VILI的保护作用是通过抑制HMGB1的表达与激活细胞凋亡信号通路实现的。同时，在急性肺损伤（ALI）的基础研究方面，我们发现了糖皮质激素可以诱导M2的产生，抑制巨噬细胞向M1极化。在体外实验中发现，M1能诱导更多的Th17，而M2则可进一步诱导Treg。我们认为糖皮质激素在LPS诱导的急性肺损伤中发挥了“指挥棒”的作用，纠正了免疫状态的病态偏倚，而被纠正的M1/M2平衡又可进一步诱导适应性免疫中Treg的生成，进一步调节了Treg/Th17这对适应性免疫应答中的平衡。我们在研究中还发现CXCL16/CXCR6在LPS诱导的急性肺损伤中的重要作用。通过敲除CXCL16基因或者使用p38抑制剂进行治疗可以消除CXCL16的潜在效应，进一步研究证实CXCL16可能和ROS生成、肺泡上皮细胞屏障功能破坏有关。本研究从免疫学层间揭示了ALI的发病机制，阐明了HMGB1在VILI发病机制中的重要意义，有望建立以HMGB1为靶点的VILI防治新策略，为临床治疗提供实验和理论基础。