小窝蛋白1（Caveolin-1）通过NF-kB信号通路介导高氧肺损伤的作用机制研究

Hyperxia-induced lung injury is one of major complications in severe premature infants, and may lead to bronchopulmonary dysplasia. The mechanisms of lung injury induced by hyperoxia are not yet fully elucidated. Caveolin-1(CAV1), as an important structural protein and regulatory protein, is wildly expressed on lung epithelial cells, vascular endothelial cells, fibroblasts and so on. Our previous results found that Cav1 promoted the production of peroxide in the hyperoxic environment, regulated oxidative stress in mice, induced inflammation of lung, and accelerated lung injury. Studies have showed that CAV1 might mediate lung injury under the condition of hyperoxia by regulation multiple signaling pathways, and NF-kB signaling pathway may be one of the significant signaling pathways. Therefore, we think CAV1 is possible to enhance inflammatory response of lung in the hyperoxic environment through NF-kB signaling pathway. Based on that, by using Cav1 gene knockout mice and mimicking hyperoxic environment, the study is to identify the phenotypes of lung injury in mice, detect oxidative stress of lung vascular endothelial cells and the proteins and cytokines relating to NF-kB signaling pathway. The project studies at the tissue, cellular and molecular levels. Thus we want to reveal one of possible mechanisms of lung injury regulated by CAV1 in the hyperoxic environment, which may provide the theoretical foundation for elucidating the pathogenesis of lung jury with hyperoxia.

高氧肺损伤是危重早产儿的主要并发症之一，后期可能发展为支气管肺发育不良。目前，高氧肺损伤的机制尚未完全阐明。小窝蛋白1(Caveolin-1，CAV1)是重要的结构蛋白和调节蛋白，广泛表达于肺泡上皮细胞、血管内皮细胞、成纤维细胞等。我们前期实验结果提示，CAV1促使高氧环境下过氧化物的生成，调控小鼠的氧化应激反应，参与肺部炎症反应，促进肺损伤。有研究发现，CAV1可能调控多个信号通路参与高氧肺损伤，其中NF-kB信号通路可能是重要的调控通路之一。因此，我们认为CAV1可能通过NF-kB信号通路促进高氧环境下肺部炎症反应。基于此假设，本项目采用Cav1基因敲除小鼠，模拟高氧环境，从组织、细胞、分子水平研究，检测肺损伤表型、肺血管内皮细胞的氧化应激反应、NF-kB信号通路相关蛋白和细胞因子。从而揭示高氧环境下CAV1参与肺损伤的可能作用机制之一，为阐明高氧肺损伤的发病机理提供理论基础。

高氧急性肺损伤（HALI）是早产儿常见并发症之一，可能发展为支气管肺发育不良（BPD），其机制尚未完全阐明。近年研究发现，小窝蛋白-1（Cav-1）可通过调控多种信号转导途径影响肺损伤的发生发展。本研究构建HALI大鼠，检测不同时间点肺组织中Cav-1的mRNA和蛋白及磷酸化（P-Cav-1-Y14）水平变化，构建针对大鼠Cav-1的腺病毒载体（Ad-Cav-1），通过H2O2对A549细胞株处理、建立体外氧化损伤细胞模型，探讨Cav-1通过调控PI3k/Akt/NF-κB通路活性引起Th17/Treg失（regulatory T）衡在高氧性肺损伤中的作用。结果显示：HALI大鼠肺组织中Cav-1 mRNA显著下降，Cav-1表达及磷酸化显著下降。通过气管插管将Ad-Cav-1滴入大鼠肺组织后，大鼠Cav-1的表达及磷酸化显著上升，PI3k/Akt/NF-κB通路蛋白PI3k p85α、Akt表达显著下降，磷酸化水平下降。Ad-Cav-1滴入大鼠肺组织后Foxp3阳性率增加，IL-17阳性率下降，肺组织中RORγt因子转录水平显著下降，流式细胞术检测Th17/Treg比例显著下降。MTT检测体外氧化损伤模型细胞活力下降，Ad-Cav-1可使细胞活力上升，Cav-1、PI3k/Akt/NF-κB等相关因子表达及磷酸化均相比模型组有改善，PI3k/Akt/NF-κB通路激动剂可减缓Ad-Cav-1对细胞的作用。研究提示高氧可通过下调肺组织中Cav-1及其磷酸化的表达，引起肺组织炎症损伤；增加Cav-1可减少肺组织释放PI3k、Akt、NF-κB等炎症因子，平衡Th17/Treg，保护HALI。同时收集2016年至2020年NICU胎龄小于32周早产儿的临床资料，分析不同程度BPD的高危因素，得出肠外营养时间是中、重度BPD的危险因素，动脉导管未闭和氨茶碱是重度BPD的危险因素，咖啡因在BPD严重程度中的作用不确定，小于胎龄与BPD严重程度无关。BPD生物信息学分析提示CD74、HLA-DRA、HLA-DPA1、HLA-DMA、HLA-DRB5、HLA-DOB、HLA-DQA1、HLA-DPB1、HLA-DMB、HLA-DQB2均为上调基因，这10个核心基因可能通过调节炎症反应导致人BPD的发生。该研究探讨HALI发病机制，以期为治疗提供靶点，对临床上防治BPD提供依据。