肺成纤维细胞和平滑肌细胞中HDAC4在哮喘气道重塑过程中的作用机制研究

Our previous work found that histone deacetylase 4 (HDAC4) expression was significantly decreased in lung fibroblasts and airway smooth muscle cells in E3 rat asthma models, which implied HDAC4 may be involved in the asthmatic airway remodeling process. Therefore, the project first screen the HDACs expression profile in lung tissue by real-time PCR, Western blotting and immunohistochemistry. And we found HDAC4 may be a key enzyme regulating the development of airway remodeling. Then we will test and verify the expression of HDAC4 in samples from asthma patients. Second, we will screen the regulation factors of HDAC4 by Human Cytokine Antibody Array, and using the stimulating factors in vitro or in vivo to validate the exact factors which induce the changing of HDAC4 in airway remodeling. Next, we will detect the target gene of HDAC4 by upregulating or downregulating HDAC4 expression by gene clone or siRNA in vitro and in vivo. In addition, we use co-immunoprecipitation assay and confocal fluorescence to implore the co-localization and the interaction between the transcription factors which interacted with HDAC4, and the signal pathway which HDAC4 involved in lung fibroblasts and smooth muscle cells. In this project, we will reveal the the HDAC4 function in airway remodeling in lung fibroblasts and smooth muscle cells in asthma, and provide the significant theoretical basis for the research of asthmatic pathogenesis, and offer new targets for the medicine research and development.

我们发现在E3大鼠哮喘模型中，参与气道重塑的肺成纤维细胞和平滑肌细胞中，组蛋白去乙酰化酶4（HDAC4）随着疾病的进展，表达明显降低，提示着HDAC4可能参与气道重塑的调控。该项目首先拟检测哮喘模型肺组织及原代成纤维细胞和气道平滑肌的HDACs表达谱，进一步确定HDAC4是气道重塑的关键酶，并在人群样本中观察HDAC4与气道重塑和哮喘疾病表型的关系；同时，采用细胞因子列阵分析、生物信息学分析及已知信号通路分析等明确模型中HDAC4表达降低的上游调控机制；继而通过细胞水平的干预实验筛选并验证HDAC4下游调控的重塑表型相关分子表达变化情况，分析并鉴定气道重塑过程中HDAC4在肺成纤维细胞和平滑肌细胞中参与的相关信号通路及相互作用的转录因子，阐明其详细的分子调控机制。实验结果将揭示HDAC4转录后调控对气道重塑的影响及可能调控机制，为哮喘气道重塑防治新途径提供理论依据和实验基础。

该项目拟阐明肺成纤维细胞中组蛋白去乙酰化酶HDAC4在哮喘气道重塑过程中发挥作用的机制，发现: （1）在E3大鼠哮喘模型急性和慢性肺组织中中，组蛋白去乙酰化酶4（HDAC4）随着疾病的进展，表达明显降低，且主要表达在上皮下组织，主要包括肺成纤维细胞和平滑肌细胞；（2）在空气污染物、炎症因子等作用下，气道上皮结构功能发生异常并持续性活化，可释放多种炎症因子，促进气道重塑的过程，选用IL-4刺激BEAS-2B细胞培养上清 (ISEM)刺激肺成纤维细胞HLF-1， HDAC4的表达明显降低（3）将ISEM和对照的正常上皮细胞培养上清NEM送检做细胞因子列阵系列（Human Cytokine Antibody Array），结果显示ISEM较NEM中有多重炎症及重塑相关的细胞因子和趋化因子分泌增加；（4）结合细胞因子列阵分析及生物信息学的分析，用多个细胞因子刺激成纤维细胞，筛选出能够下调HDAC4表达的细胞因子IL-6，在成纤维细胞系HFL-1中IL-6通过诱导SOCS3抑制HDAC4基因的表达；（5）细胞因子IL-1β在成纤维细胞HFL-1中可以通过诱导HDAC4表达增加调控气道重塑相关基因COX2等的表达。实验结果将揭示HDAC4转录后调控对气道重塑的影响及可能调控机制，为哮喘气道重塑防治新途径提供理论依据和实验基础。