sRAGE纠正mRAGE/sRAGE失衡在肺间质纤维化中的保护作用及分子机制

The receptor for advanced glycation end-products (RAGE) is highly expressed in the lung. RAGE is involved in the pathogenesis of pulmonary fibrosis, however, it remains unclear about the role and mechanism of the different kinds of RAGE. We found that RAGE deficent mice protected from bleomycin-induced lung fibrosis, and we also found that lower serum level of endogenous secretory RAGE in the patients with IPF than in health. Therefore, we propose that lung injury polarizes the expression of RAGE toward membrane RAGE (mRAGE) in the lung, which leads to the decreased expression of soluble RAGE (sRAGE). Subsequently, the imbalance of mRAGE/sRAGE leads to accelerate the occurrence of pulmonary fibrosis. To clarify the loss of sRAGE is the pathogenesis of fibrosis, we will evaluate the expression of mRAGE and sRAGE in bleomycin-induced lung fibrosis in mice. To evaluate the protective effect of sRAGE on bleomycin-induced fibrosis, BAL fluid will be collected for cell counting and measurements of lung permeability, and profibrotic cytokines of TGF-beta and PDGF after intratracheal bleomycin in the mice with or without intraperitoneal administration of sRAGE. To evaluate the inhibit effect of sRAGE on epithelial-mesenchymal transtion process, alveolar type II epithelial cells will be isolated from mice and exposes to HMGB1 with or without sRAGE then will be checked the changes of cell phenotypes. This study will demonstrate that sRAGE plays a protective role in pathogenesis of bleomycin-induced fibrosis and will explore the underlying mechanism. sRAGE is expected to be a new biomarker of fibrosis and a therapeutic target for pulmonary fibrosis.

晚期糖基化终产物受体(RAGE)主要在肺部表达,以细胞膜型(mRAGE)和可溶型(sRAGE)形式存在。RAGE与肺间质纤维化密切相关，然而RAGE的作用尚不清楚。我们既往的研究发现RAGE基因敲除鼠没有出现博莱霉素诱导的肺间质纤维化，而IPF患者血清内源性分泌的sRAGE减少。由此我们提出肺损伤后，肺内RAGE表达向mRAGE偏移，sRAGE减少，导致mRAGE/sRAGE失衡，促使肺间质纤维化发生的假说。为验证这一假说，我们将在博莱霉素肺纤维化小鼠模型上，观察肺组织mRAGE和sRAGE的动态变化,并予以sRAGE干预，证明肺纤维化时mRAGE/sRAGE失衡，纠正sRAGE丢失对肺纤维化的抑制作用。结合sRAGE对促纤维化因子变化的影响以及对原代肺泡II型上皮细胞EMT变化的影响，探讨sRAGE对肺纤维化保护作用机制。为建立肺间质纤维化的预测指标并为提供新的治疗靶点奠定理论基础。

晚期糖基化终产物受体（receptor for advanced glycation end products，RAGE）在肺以外的组织中传导炎症信号，健康成人的肺部却大量表达RAGE。 既往的研究发现，在博莱霉素（BLM）诱导的肺纤维化模型中，RAGE基因敲除小鼠的肺损伤受到保护。然而可溶性晚期糖基化终产物受体（soluble　RAGE，sRAGE）在肺纤维化小鼠模型中的作用需要进一步阐述，本研究将阐明sRAGE在小鼠肺纤维化模型中的作用并解明其作用机制。 C57BL/6小鼠气管内注入BLM，用建立肺纤维化模型，并分为对照组（control组）和sRAGE干预组（sRAGE组），sRAGE组经腹腔注射sRAGE，control组注射PBS。在第0、3、7、21天观察肺组织学变化，肺泡灌洗液(BALF)中炎症细胞和蛋白变化，TGF-beta1、 HMGB-1的水平。我们将分离培养原代小鼠II型肺泡上皮细胞建立HMGB-1 诱导的EMT变， 加入sRAGE后观察细胞的上皮细胞、间质细胞标志物的变化，评价sRAGE对EMT的影响．3天后sRAGE组肺组织中炎症细胞浸润明显轻于对照组，21天后sRAGE组的肺纤维化较对照组减轻。sRAGE组BALF中炎症细胞总数较对照组低，其中主要的炎症细胞为中性粒细胞。发现sRAGE组BALF中蛋白总含量较对照组低, sRAGE干预组BALF的TGF-beta1、HMGB-1的水平较对照组减少。在分离培养的原代小鼠II型肺泡上皮细胞中，加入HMGB-1后激活RAGE/HMGB-1通路，检测并发现间质细胞标志物mRNA alpha-SMA水平升高，上皮细胞标志物mRNA E-cadhrin的水平降低， 提示激活RAGE/HMGB-1通路诱导II型肺泡上皮-见皮细胞转化（EMT）。在这个体系中加入sRAGE后发现上皮细胞标志物E-cadhrin的mRNA水平没有明显丢失，间质细胞标志物mRNA alpha-SMA水平没有明显升高，提示sRAGE可能阻断了RAGE/HMGB-1轴诱导的EMT。结论以及意义： sRAGE减轻BLM诱导的小鼠肺纤维化。其可能的机理是sRAGE减轻了博莱霉素诱导的急性期肺损伤，并通过阻断RAGE/HMGB-1信号通路作用于前纤维化生长因子，同时sRAGE抑制肺泡上皮细胞EMT，从而抑制了肺纤维化的发生。