背景:急性肺损伤(ALI)及其更严重的形式,急性呼吸窘迫综合征(ARDS)是威胁生命的肺部疾病,我们现在缺乏有效的治疗方法。炎症反应对于启动ALI/ARDS至关重要。因此,改善炎性反应可能有益于疾病的治疗。有越来越多的数据表明磷酸二酯酶-4(PDE4)-选择性抑制剂,这可能会升高细胞环状腺苷3',5'-单磷酸(cAMP)水平,可以抑制炎症。然而,尚未确定它们是否可用于治疗IgG免疫复合物(IgG-IC)相关的ALI。
方法:ALI是通过用IgG免疫复合物的气道沉积处理小鼠来诱导的。通过用罗利普兰/罗氟司特处理小鼠或巨噬细胞来提高细胞cAMP浓度。肺损伤的程度由肺通透性反映,白细胞积累,组织学改变和促炎介质的表达。6-Bnz-cAMP和H-89用于调节蛋白激酶A(PKA)活性,和8-pCPT-2'-O-Me-cAMP用于激活由cAMP(Epac)直接激活的交换蛋白。通过实时PCR分析基因表达,ELISA或蛋白质印迹。CCAAT/增强子结合蛋白(C/EBP)和活化蛋白1(AP-1)转录活性通过测量荧光素酶的产生来估计。
结果:IgG-IC诱导的ALI被PDE4选择性抑制剂减毒,这是由于细胞因子和趋化因子的表达减少。有趣的是,我们发现cAMP下游效应分子PKA而不是Epac参与IgG-IC介导的促炎介质产生的负调控。机械上,cAMP-PKA信号轴的激活导致MAPK通路的失活,导致C/EBP和AP-1介导的促炎介质转录减少。
结论:我们的数据表明,第一次,cAMP-PKA信号通过下调MAPK激活参与IgG-IC相关炎症反应的下调,这对C/EBP和AP-1的转录活性至关重要。总的来说,本实验为PDE4选择性抑制剂应用于临床治疗IgG-IC相关急性肺损伤提供了理论基础。
BACKGROUND: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are life threatening pulmonary diseases, and we are now lack of effective therapeutic methods. Inflammatory responses are essential for initiating ALI/ARDS. Thus, ameliorating inflammatory reaction might be beneficial for treatment of the disease. There are increasing data that phosphodiesterase-4 (PDE4)-selective inhibitors, which may elevate cellular cyclic adenosine 3\', 5\'-monophosphate (cAMP) level, could suppress inflammation. However, whether they could be used to treat IgG immune complex (IgG-IC)-associated ALI has not been determined.
METHODS: ALI is induced by treating mice with airway deposition of IgG immune complexes. Cellular cAMP concentrations are elevated by treating mice or macrophages with Rolipram/Roflumilast. The degree of pulmonary injury is reflected by lung permeability, leukocyte accumulation, histological change and expressions of pro-inflammatory mediators. 6-Bnz-cAMP and H-89 are used to regulate protein kinase A (PKA) activity, and 8-pCPT-2\'-O-Me-cAMP is applied to activate exchange proteins directly activated by cAMP (Epac). Gene expressions are analyzed by real-time PCR, ELISA or Western blot. CCAAT/enhancer binding protein (C/EBP) and activation protein 1 (AP-1) transcription activities are estimated by measuring the luciferase productions.
RESULTS: IgG-IC-induced ALI is attenuated by the PDE4-selective inhibitor, which is due to reduced expressions of cytokine and chemokines. Interestingly, we find that cAMP downstream effector molecule PKA but not Epac is involved in negative regulation of IgG-IC-mediated pro-inflammatory mediators\' productions. Mechanistically, activation of cAMP-PKA signal axis leads to inactivation of MAPK pathway, resulting in a decrease in C/EBP- and AP-1-mediated transcriptions of pro-inflammatory mediators.
CONCLUSIONS: Our data demonstrate, for the first time, that cAMP-PKA signal is involved in down-regulation of IgG-IC-associated inflammatory responses via down-regulating MAPK activation, which is critical for transcriptional activities of C/EBP and AP-1. Collectively, our experiments provide theoretical base for the potential application of PDE4-selective inhibitor to clinic for treatment of IgG-IC-related acute lung injury.