DNA修复酶APE1乙酰化修饰对特发性肺纤维化进程的影响及机制研究

The inhibition of autophagy in idiopathic pulmonary fibrosis (IPF) leads to the formation of extracellular matrix (ECM). Our preliminary experiment demonstrated that the protein expression level of DNA repair enzyme APE1 is increased and reached its peak at the beginning stage and then decreased at end stage in blomycin-induced lung fibrosis of mouse model, and its enzyme activity was negatively correlated with autophagy, indicating that APE1 may plays an important role in lung fibrosis, but the specific mechanism remain elusive. Preliminary experiments have shown that valproate (VPA), which is considered as histone deacetylase inhibitor, can induce the acetylation modification of APE1, activate cell autophagy and reduce the expression of TGF-β in alveolar epithelial cells, which suggest that acetylated APE1 is involved in the regulation of autophagy, and is closely related to the function of base excision repair (BER). Based on above, we hypothesize: "The acetylated APE1 promotes the combination of APE1 and Bcl2, inhibits the combination of Bcl2 and Beclin1 by inhibiting BER activity, furthermore, activates autophagy, reduces the synthesis of ECM”. The study will use APE1 related cell lines, pulmonary fibrosis of mouse model and clinical samples from patients with IPF. We propose two inter-related specific aims:1) To determine how the acetylated APE1 regulates autophagy and affects the process of pulmonary fibrosis; 2) To determine whether the acetylated APE1 is associated with TGF-β signaling pathways to affect the process of epithelial to mesenchymal transition(EMT). If above-mentioned mechanism can be clarified effectively, and it will provide experimental data for the future treatment of IPF as a therapeutic target.

在特发性肺纤维化（IPF）进程中，自噬的抑制可促使细胞外基质（ECM）的形成。我们前期研究发现：在小鼠肺纤维化模型肺组织中DNA修复酶APE1表达呈先升高后降低的趋势，其内切酶活性与自噬呈负相关，提示APE1在肺纤维化中可能起重要作用，但具体机制不清楚。预实验显示：去乙酰化酶抑制剂丙戊酸钠可诱导肺泡上皮细胞中APE1乙酰化，激活细胞自噬，TGF-β表达下调，表明APE1乙酰化参与自噬调控，并与碱基切除修复（BER）功能密切相关。我们提出：“APE1乙酰化通过抑制BER活性，促进APE1和Bcl2结合，抑制Bcl2和Beclin1结合，从而激活自噬，减少ECM的合成”这一新的分子机制。本研究拟利用APE1相关工具细胞株及小鼠肺纤维化模型和IPF临床样本探讨：APE1乙酰化如何调控自噬和影响肺纤维化进程？其是否协同TGF-β信号通路影响EMT？该机制的阐明，可为开发IPF治疗靶点提供理论依据。

特发性肺纤维化（IPF）是一种病因和发病机制不清，预后很差的疾病，临床上至今上没有有效的药物可逆转或延缓该病的进程，亟需认识其关键机制，探索新的治疗。DNA修复酶脱嘌呤/脱嘧啶核酸内切酶（Apurinic/apyrimidinic endonuclease，APE1/Ref-1或缩写APE1）是DNA碱基切除修复（Base excision repair，BER）途径中的一种多功能蛋白酶，一直被认为是肿瘤治疗的药物靶点；同时，BER活性启动可造成细胞外基质相关基因表达上调，但其在特发性肺纤维化病中研究甚少。本项目基于去乙酰化酶抑制剂丙戊酸钠可诱导肺泡上皮细胞APE1乙酰化，激活细胞自噬的假设，以肺泡上皮细胞，肺成纤维细胞和动物肺纤维化模型为基础，检测APE1乙酰化，自噬和BER等指标，观察该变化与肺纤维化严重程度及成纤维细胞表型转化、胶原产生的关联。观察肺上皮细胞和肺成纤维细胞经VPA处理后，自噬水平和APE1乙酰化水平的变化对上皮间质转化和BER活性的影响，以证实APE1是肺纤维化的重要靶点，通过形成APE1-自噬反馈影响肺纤维化，VPA通过促进APE1乙酰化和自噬可减少肺纤维化。以小鼠肺纤维化模型验证VPA治疗对肺纤维化相关标志物表达影响的效应，提示其具有治疗效果。本项目已发表相关论文6篇，其中SCI论文1篇，中文期刊5篇，培养硕士研究生5名，待发表SCI论文1篇。