Section of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.
Respir Res. 2012 Jan 18;13(1):4. doi: 10.1186/1465-9921-13-4.
Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.
C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.
Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.
These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.
脯氨酸丰富的酪氨酸激酶 2（Pyk2）在中性粒细胞脱粒和体外趋化作用中是必不可少的。然而，其在脂多糖诱导的急性肺损伤（ALI）过程中对肺炎症和水肿形成的影响尚不清楚。本研究的目的是确定抑制 Pyk2 对 LPS 诱导的急性肺炎症和损伤的体内影响。
C57BL6 小鼠经气管内给予 10mg/kg LPS 或生理盐水。在挑战前 1 小时通过腹腔内给予 TAT-Pyk2-CT 来抑制 Pyk2。在 LPS 处理后 18 小时，通过支气管肺泡灌洗分析细胞计数、肺组织学和 BAL 中的蛋白浓度进行分析。通过小鼠细胞因子多重试剂盒测量 BAL 中的 KC 和 MIP-2 浓度。通过计算机控制的小动物呼吸机使用压力-容积曲线测定静态肺顺应性。通过分光光度法测定肺匀浆中渗出的 Evans 蓝浓度。
气管内滴注 LPS 可诱导中性粒细胞明显浸润肺间质和肺泡腔，TAT-Pyk2-CT 预处理可减轻这种浸润。TAT-Pyk2-CT 预处理还可减轻 1）肺组织中的髓过氧化物酶含量，2）肺中 Evans 蓝染料渗出的血管渗漏和支气管肺泡灌洗中蛋白浓度的增加，以及 3）肺顺应性的降低。在每种模式下，用对照蛋白 TAT-GFP 处理均没有阻断作用。相比之下，TAT-Pyk2-CT 并未减少支气管肺泡灌洗中的中性粒细胞趋化因子 MIP-2 和角质细胞衍生趋化因子的产生。Western blot 分析证实，TAT-Pyk2-CT 预处理可将 LPS 刺激的肺中 Pyk2 的酪氨酸磷酸化降低至对照水平。
这些结果表明，Pyk2 在小鼠急性肺损伤的发展中起重要作用，而 Pyk2 的药理学抑制可能为有发生急性肺损伤风险的患者提供潜在的治疗策略。
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