MK2-HuR通路对炎症介质ICAM-1表达的调控作用及其对急性肺损伤的影响

A variety of pathogenic factors cause excessive expression of ICAM-1 in pulmonary microvascular endothelial cells. And the up-regulated ICAM-1 leads to massive neutrophil infiltration, increases vascular permeability, and exacerbates acute lung injury. Although over-expression of ICAM-1 has been implicated in the progression of acute lung injury, its regulatory mechanism is still largely unknown. Recently, we found that MK2 regulated the expression of ICAM-1 at the post-transcriptional level by causing HuR translocation from nucleus to cytoplasm to stabilize ICAM-1 mRNA. We therefore propose that MK2 regulates the expression of ICAM-1 through HuR in endothelial cell and MK2/HuR/ICAM-1 signaling cascade may play important role in acute lung injury. To verify this hypothesis, we would like to generate the MK2, HuR silencing/over-expressing human vascular endothelial cell lines respectively in dissection of the relationshhip between HuR and ICAM-1. We will then use vascular endothelial HuR gene knockout mice to illustrate the significance of HuR regulation on ICAM-1 in the development of acute lung injury. We anticipate that intervention of the MK2/HuR/ICAM-1 signaling pathway may become a new strategy for the treatment of acute lung injury.

各种致病因素引起肺微血管内皮细胞过度表达ICAM-1，导致大量中性粒细胞的肺部浸润、血管通透性增加，促进急性肺损伤的发展。虽然ICAM-1的异常表达参与急性肺损伤的发生，但其调控机制不明。最近我们发现MK2在转录后水平调控内皮细胞ICAM-1的表达；MK2活化导致HuR发生核-质转移，ICAM-1的mRNA水平也随之升高。于是我们认为：MK2通过HuR调控内皮细胞ICAM-1的表达，从而影响急性肺损伤的发生发展。为了验证这一假设，我们拟建立MK2,HuR沉默/高表达人血管内皮细胞株，从蛋白水平及转录后水平分析HuR调控ICAM-1的方式；然后我们使用血管内皮细胞HuR基因敲除小鼠，构建急性肺损伤模型，分析血管内皮细胞HuR敲除对于小鼠急性肺损伤的影响，以及HuR调控ICAM-1的机制。预期干预MK2/HuR/ICAM-1信号调节通路可成为治疗急性肺损伤的新策略。

ARDS是常见临床危重症，病死率高，治疗手段缺乏。严重感染和创伤等诱因导致肺微血管内皮细胞过度表达ICAM-1等促炎因子，ICAM-1等促炎因子可粘附活化中性粒细胞等多种炎症细胞，进而诱导更多炎症因子释放，形成失控的级联放大式炎症反应，最后导致大量炎症细胞在肺部聚集和活化，导致ARDS。虽然 ICAM-1 的异常表达参与ARDS的发生发展，但其调控机制仍不明确。在本研究中，我们利用肺微血管内皮细胞模型，通过基因沉默技术，分别抑制MK2和HuR表达，再用TNF或LPS刺激细胞，观察炎症反应变化。研究发现MK2通路可以影响RNA结合蛋白—HuR的细胞亚定位。在外界刺激时，HuR由细胞核转移到细胞质。此时HuR通过与ICAM-1 mRNA 3’UTR区域结合稳定ICAM-1的mRNA，进而导致ICAM-1 的过度表达，进而增强中性粒细胞对血管内皮细胞的粘附。我们接下来将体外实验中获得的发现在小鼠体内实验中反复进行验证。我们首先利用MK2抑制剂注射至小鼠体内，抑制内皮细胞MK2功能，然后利用LPS构建ARDS小鼠模型，观察ICAM-1表达及肺部中性粒细胞浸润和肺损伤情况。研究发现当小鼠体内MK2被抑制后，小鼠微血管内皮细胞表达ICAM-1减少，从而导致肺部中性粒细胞浸润减轻，肺损伤也明显减轻。接着我们又构建了选择性血管内皮细胞HuR基因敲除小鼠。利用基因敲除小鼠和对照小鼠构建了ARDS模型。观察两种小鼠肺部炎症反应差异。研究发现，HuR敲除小鼠肺微血管内皮细胞ICAM-1表达降低，肺部中性粒细胞浸润减少，肺损伤明显减轻。由此我们得出课题研究结论：MK2-HuR通路在调控ARDS关键促炎因子ICAM-1的表达中起重要作用，从而影响中性粒细胞的肺部浸润和ARDS的发生发展。这一研究发现，为深入阐明ARDS病理生理过程提供了新的证据，为临床防治感染导致的 ARDS 提供治疗靶点。