C/EBPβ去乙酰化调控肺泡上皮-间充质转化参与肺纤维化的机制研究

It's widely accepted that the alveolar epithelium injury and abnormal repairing, resulting in epithelial-mesenchymal transition (EMT) play a central role in Idiopathic Pulmonary Fibrosis(IPF). α-SMA is known as a maker of EMT. As a transcription factor, acetylated-C/EBPβ controls cellular transition and function through downstream gene. Recently, our group has found that the C/EBPβ acetylation, closely related to α-SMA, was obviously in lung tissues of IPF patients. There were significant increasing of C/EBPβ acetylation and α-SMA in EMT from A549 cells stimulated by TGF-β1 in vitro. C/EBPβ acetylation induced by p300 heightens α-SMA expression through binding its promoter site. We have checked the site between α-SMA and acetylated C/EBPβ by ChIP. So, we hypothesized that acetylation and deacetylation of C/EBPβ would adjust the EMT in the development of lung fibrosis. Based on the previous work, the project aim to check the interference of C/EBPβ deacetylation on EMT and lung fibrosis, through the human lung tissue, cells in vitro and animals in vivo. In the present study, we will check the role of C/EBPβ in lung fibrosis. Moreover, we will explore the mechanism of EMT induced by C/EBPβ acetylation. Furthermore, we will demonstrate the negative role of C/EBPβ deacetylation on EMT and lung fibrosis. With the project developed, it will help to show a possible target for lung fibrosis treatment.

肺泡上皮-间充质转化(EMT)是特发性肺纤维化(IPF)的发病机制之一，α-SMA被认为是EMT的标志。C/EBPβ经乙酰化修饰，调控靶基因表达，调节细胞增殖、分化及功能。我们前期研究发现IPF患者肺组织存在C/EBPβ乙酰化增强，与α-SMA表达关联；在TGF-β1诱导A549细胞的EMT中，C/EBPβ乙酰化水平及α-SMA蛋白表达增强；通过p300诱导A549细胞C/EBPβ乙酰化，证实C/EBPβ与α-SMA基因启动子位点结合，增强α-SMA基因表达。我们推测C/EBPβ乙酰化和去乙酰化可能通过调控α-SMA基因表达，参与调节EMT过程。本课题拟在前期工作基础上，通过人体肺组织、细胞及动物实验，探讨C/EBPβ在肺纤维化中的作用，阐明C/EBPβ乙酰化调控EMT的分子机制，揭示C/EBPβ去乙酰化对EMT及肺纤维化的干预作用，为以C/EBPβ靶点干预肺纤维化提供理论基础。

肺泡上皮-间充质转化(EMT)是特发性肺纤维化(IPF)的发病机制之一，α-SMA被认为是EMT的标志。C/EBPβ经乙酰化修饰，调控靶基因表达，调节细胞增殖、分化及功能。我们通过与对照组相比较，IPF患者肺组织表达C/EBPβ mRNA和蛋白较对照组明显增高（P<0.05）。Western Blot证实IPF患者肺组织表达I型胶原蛋白和α-SMA蛋白明显高于对照组（P<0.05）。与对照组相比，IPF患者肺组织C/EBPβ乙酰化水平增强，C/EBPβ乙酰化与α-SMA蛋白表达密切相关。体外A549细胞实验，体外培养后经TGF-β1处理，发现C/EBPβ mRNA及蛋白表达明显增强，且C/EBPβ乙酰化加强，WB检测发现α-SMA蛋白及胶原-I上调表达下调，说明TGF-β1诱导A549细胞的EMT进程，促进胶原形成，且这一过程可能与C/EBPβ表达增强及乙酰化水平增强相关；IP/WB 证实A549细胞C/EBPβ乙酰化与表达α-SMA蛋白相关；体外通过siRNA处理A549细胞，发现经C/EBPβ siRNA的A549细胞经TGF-β1处理后α-SMA蛋白及胶原-I表达未见上调，说明C/EBPβ为TGF-β1诱导A549细胞EMT进程；通过P300诱导A549细胞C/EBPβ乙酰化，可出现α-SMA蛋白及胶原-I上调表现，且IP/WB 证实A549细胞C/EBPβ乙酰化与α-SMA蛋白表达相关；而p300诱导A549细胞C/EBPβ乙酰化，参与EMT进程，促进胶原生成的致纤维化作用被蛋白去乙酰化酶1(HDAC1)中和，解除C/EBPβ与α-SMA蛋白关联。通过博来霉素气管内滴注构建肺纤维小鼠模型，肺组织羟脯氨酸含量增高；Real Time-PCR发现肺组织C/EBPβ mRNA表达增高；Western Blot检测C/EBPβ乙酰化水平、I型胶原蛋白变化明显增高；免疫共沉淀观察C/EBPβ与α-SMA蛋白共表达。通过博来霉素处理C/EBPβ基因敲除小鼠，发现肺纤维化病变、I型胶原蛋白及α-SMA高表达均受到明显抑制。本课题通过人体肺组织、细胞及动物实验，探讨C/EBPβ在肺纤维化中的作用，阐明C/EBPβ乙酰化调控EMT的分子机制，揭示C/EBPβ去乙酰化对EMT及肺纤维化的干预作用，为以C/EBPβ靶点干预肺纤维化提供理论基础。