Roles of pulmonary and hepatic P4501A enzymes in hyperoxic lung injury in mice

肺和肝 P4501A 酶在小鼠高氧性肺损伤中的作用

• 批准号: 7624161
• 负责人: BHAGAVATULA MOORTHY
• 金额: $ 37.5万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-16 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624161
• 关键词: 4 hydroxynonenal; Acute; Adult; Adult Respiratory Distress Syndrome; Air; Animals; Biological Assay; Bronchopulmonary Dysplasia; CYP1A1 gene; CYP1A2 gene; Catalysis; Cell Culture Techniques; Cells; Clara cell; Cytochrome P450; Cytochromes; DNA; Development; Electron Transport; Electrophoretic Mobility Shift Assay; Elements; Enzymes; Exposure to; F2-Isoprostanes; Foundations; Functional disorder; Gene Expression; Genes; Genetic Transcription; Glutathione; Guanosine; Hepatic; Histology; Human; Hyperoxia; Image; Immunohistochemistry; In Vitro; Indium; Individual; Infant; Inflammation; Inflammatory; Injury; Interleukins; Isoprostanes; Knock-out; Knockout Mice; Ligands; Lipid Peroxidation; Liquid substance; Liver; Luciferases; Lung; Lung diseases; Malondialdehyde; Mass Fragmentography; Measures; Mediating; Metabolism; Microsomes; Molecular; Mus; NADP; Neutrophil Infiltration; Organ; Oxidants; Oxidative Stress; Oxidoreductase; Oxygen; Oxygen Therapy Care; Patients; Play; Predisposition; Premature Infant; Prevention; Prevention strategy; Production; Proteins; Pulmonary Edema; Pulmonary Valve Insufficiency; Reactive Oxygen Species; Reporter Genes; Research; Research Personnel; Respiratory distress; Reverse Transcriptase Polymerase Chain Reaction; Role; Running; Site-Directed Mutagenesis; Syndrome; Testing; Time; Transcriptional Activation; Transfection; Transgenic Mice; Transgenic Organisms; Western Blotting; Wild Type Mouse; adduct; chromatin immunoprecipitation; cytokine; detoxication; enzyme activity; in vivo; lung injury; male; neutrophil; novel strategies; overexpression; oxidation; promoter; protein expression; receptor; research study; response; transcription factor

DESCRIPTION (provided by applicant): Supplemental oxygen administration is used extensively in the treatment of pulmonary insufficiency that is commonly observed in patients with acute respiratory distress syndrome (ARDS) and in preterm and term infants. However, hyperoxia causes lung damage in animals and humans. The central hypothesis of the research proposed in this application is that hyperoxia induces cytochrome P4501A (CYP1 A) enzymes in vivo through mechanisms involving transcriptional activation of the corresponding promoters, and that the induced CYP1A enzymes play protective roles against hyperoxic lung injury by catalyzing the detoxication of ROS-generated endogenous molecules (e.g., F2isoprostanes and isofurans), whose levels are elevated in the lungs of hyperoxic animals. The specific aims are: (1) to test the hypothesis that hyperoxia induces CYP1A1 and 1A2 gene expression through mechanisms involving transcriptional activation of the CYP1A1 or 1A2 promoter, presumably by generating endogenous ligands for the Ah receptor (AHR). We will use a transgenic mouse carrying a 10 kb human CYP1A1 promoter or an 8 Kb mouse promoter and luciferase reporter gene. These mice will be maintained in room air or exposed to hyperoxia for selected time points, and luciferase activities will be determined by in vivo bioluminescent imaging. (2) To characterize the molecular mechanisms of modulation of CYP1A1 gene by hyperoxia in cultured mouse lung cells. Mouse transformed Clara cells (mtCC) will be exposed to hyperoxia and time dependent effects on CYP1A1 parameters' will be determined. (3) To test the hypothesis that mice lacking the gene for CYP1A1, CYP1A2, or both genes (CYP1A1/1A2 double knockouts) will be more susceptible to hyperoxia-induced lung injury than similarly exposed wild type mice, and that the CYP1A enzymes play ' protective roles against hyperoxic lung injury by catalyzing the detoxication of ROS-generated endogenous molecules (e.g., F2 isoprostanes) in the lungs of hyperoxic animals. The mice will be exposed to hyperoxia for selected time points, and parameters of lung injury and those of pulmonary and hepatic CYP1A1/1A2 expression (run-on transcription, real time RT-PCR/Northern, Western, enzyme activities) will be studied. Levels of F2 isoprostanes and isofurans will be measured in lung and liver by gas chromatography/ mass spectrometry (GC-MS). (4). to test the hypothesis that liver P450s contribute to hyperoxic lung injury in vivo. We will expose wild type, and liver- specific P450 reductase (CPR) null mice to hyperoxia, and determine if hyperoxic responses are altered in the CPR-null mice. The long-term of this proposal is to develop rational strategies for the prevention and/or treatment of chronic lung diseases in infants and adults suffering from respiratory distress and lung dysfunction.

描述（由申请方提供）：补充供氧广泛用于治疗肺功能不全，这在急性呼吸窘迫综合征（ARDS）患者以及早产儿和足月儿中常见。然而，高氧会导致动物和人类的肺损伤。本申请中提出的研究的中心假设是高氧通过涉及相应启动子的转录激活的机制在体内诱导细胞色素P4501 A（CYP 1A）酶，并且诱导的CYP 1A酶通过催化ROS产生的内源性分子（例如，F2异前列烷和异呋喃），其水平在高氧动物的肺中升高。具体目标是：（1）检验高氧通过涉及CYP 1A 1或1A 2启动子转录激活的机制诱导CYP 1A 1和1A 2基因表达的假设，推测是通过产生Ah受体（AHR）的内源性配体。我们将使用携带10 kb人CYP 1A 1启动子或8 Kb小鼠启动子和荧光素酶报告基因的转基因小鼠。将这些小鼠在室内空气中或暴露于高氧中选定的时间点，并通过体内生物发光成像测定荧光素酶活性。(2)探讨高氧对小鼠肺细胞CYP 1A 1基因表达的影响及其分子机制。将小鼠转化的Clara细胞（mtCC）暴露于高氧，并确定对CYP 1A 1参数的时间依赖性影响。(3)为了检验以下假设，即缺乏CYP 1A 1、CYP 1A 2或这两种基因的基因（CYP 1A 1/1A 2双敲除）的小鼠将比类似暴露的野生型小鼠更容易受到高氧诱导的肺损伤，并且CYP 1A酶通过催化ROS产生的内源性分子（例如，F2异前列腺素）在高氧动物的肺部中。将小鼠暴露于高氧持续选定的时间点，并将研究肺损伤参数以及肺和肝CYP 1A 1/1A 2表达的参数（连续转录、真实的时间RT-PCR/北方、西方、酶活性）。将通过气相色谱/质谱法（GC-MS）测量肺和肝脏中F2异前列烷和异呋喃的水平。（四）、以验证肝脏P450参与体内高氧肺损伤的假设。我们将野生型和肝特异性P450还原酶（CPR）缺失小鼠暴露于高氧，并确定CPR缺失小鼠中的高氧反应是否改变。该提案的长期目标是制定合理的策略，用于预防和/或治疗患有呼吸窘迫和肺功能障碍的婴儿和成人的慢性肺病。