M1-M2混合型肺泡巨噬细胞高表达HB-EGF促进肺损伤修复的作用及机制研究

Acute respiratory distress syndrome (ARDS) is a serious respiratory disorder with significant mortality in patients. The lung injury and repair in ARDS are closely related to alveolar macrophage (AM). It is known that AMs are activated to M1 phenotype at the acute phase of ARDS, resulting in exacerbation of lung injury. While at the resolving phase of ARDS, AMs are polarized to M2 phenotype contributing to repair lung tissue. So in the development of lung injury, AMs can undergo a M1-to-M2 transition in which they exhibit a M1/M2 intermediate polarization (M1-M2) phenotype. However, the special feature and function of M1-M2 AMs are unknown. Our preliminary study showed that M1-M2 AMs expressed a high level of HB-EGF, a kind of epidermal growth factor. In addition, we also found that HB-EGF in lung tissue was significantly increased on the 2th day after the onset of lung injury. It has been reported that HB-EGF can promote tissue repair and stimulate alveolar epithelial cell type Ⅱ (AEC 2) proliferation. Therefore, we establish the hypothesis that M1-M2 AMs can promote AEC 2 proliferation and lung tissue repair through highly expressing HB-EGF in lung injury development. In this study, we would apply the in vivo model of endotoxin-induced acute lung injury in mouse, together with the in vitro model of isolated AMs and AEC 2, to investigate the following questions: (i) whether M1-M2 AMs secrete a high level of HB-EGF in ARDS development; (ii) whether HB-EGF plays a key role in AEC 2 proliferation, alveoli regeneration and lung tissue repair; (iii) whether epidermal growth factor receptor (EGFR) and ERBB4, the receptor of HB-EGF, mediate the repairing effect of HB-EGF in lung injury. In summary, the present study will not only identify a new repairing function of AMs in the transition from M1 to M2, but also provide novel therapeutic strategies for treatment of ARDS.

急性呼吸窘迫综合征（ARDS）死亡率高，其肺组织损伤修复与肺泡巨噬细胞（AM）密切相关：AM在ARDS急性期极化为M1型加重肺损伤，在恢复期转化为M2型促进肺修复。随着肺损伤发展，AM会经历亚型转化表现为M1-M2混合型，但这类AM有何特殊作用还不清楚。我们预实验发现：M1-M2型AM可高表达上皮生长因子HB-EGF；肺损伤后2-5天，肺组织HB-EGF量显著增加。HB-EGF能促进组织修复和Ⅱ型肺泡上皮细胞（AEC2）增殖。因此推测：肺损伤中M1-M2型AM可能通过高表达HB-EGF促进AEC2增殖而启动肺组织修复。本项目拟从细胞及整体动物水平，研究肺损伤中M1-M2型AM是否大量表达HB-EGF，HB-EGF能否促进肺损伤后AEC2增殖、肺泡再生与肺组织修复，其受体EGFR与ERBB4是否介导该作用。本项目将阐释AM由M1向M2转化时促进肺损伤修复的新作用，为ARDS治疗提供新策略。

研究背景：急性呼吸窘迫综合征（ARDS）死亡率高，其肺组织修复与肺泡巨噬细胞（AM）密切相关：在ARDS急性期，AM极化为M1型加重肺损伤，在恢复期转化为M2型促进肺修复。随着肺损伤发展，AM会经历亚型转化表现为M1-M2混合型，但这类AM在肺损伤发生发展过程中发挥何种作用尚不清楚。我们前期研究发现M1-M2混合型AM高表达一种表皮生长因子——肝素结合型表皮生长因子（HB-EGF）。HB-EGF能促进肺组织生长与肺泡II型上皮细胞（AEC2）增值，但HB-EGF对肺损伤修复有何作用至今也不清楚。.研究内容：使用细菌内毒素制作小鼠ARDS模型，检测ARDS后肺组织中的HB-EGF表达情况。收集小鼠AM制作M1-M2极化模型，检测HB-EGF表达量。使用免疫荧光技术检测损伤肺组织中HB-EGF与AM标志物（Iba-1）的共表达情况。使用外源性HB-EGF干预ARDS小鼠，检测HB-EGF对损伤肺组织修复影响。使用AG1478特异性抑制表皮生长因子受体（EGFR）活性，检测EGFR对HB-EGF功能的影响。.实验结果：与对照组相比，肺组织中的HB-EGF含量在ARDS发生后逐渐增高，在第7天达到高峰（386.58±27.11pg/ml vs. 236.34±44.95pg/ml，p＜0.001）；去除AM后，ARDS肺组织中HB-EGF含量显著降低。在ARDS后第7天肺组织中，Iba-1与HB-EGF共表达。在体外实验培养的M1-M2混合型AM中，HB-EGF表达量显著升高。使用重组HB-EGF蛋白处理明显减少了第3、5、7天损伤肺组织中的中性粒细胞数量、肺水蛋白含量，减少了炎症因子TNF-α、IL-6与IL-1β的浓度，促进了肺泡结构重建与AEC2增殖。ARDS后3、5、第7天，肺组织中EGFR磷酸化水平显著升高。使用AG1478可明显抑制HB-EGF促进肺损伤修复的效果。.科学意义：本课题揭示了HB-EGF在ARDS发生发展过程中促进损伤肺组织修复的新作用，为ARDS的治疗提供了新策略。