OGG1协同Smad3蛋白在特发性肺纤维化肺泡上皮细胞-间质转化（EMT）中的分子机制

The pathogenesis of idiopathic pulmonary fibrosis (IPF), a serious disease which threats human health, has not been elucidated. However, there is no effective treatment for it. Recently, 8-oxoguanine DNA glycosylase（OGG1） is well known on the role of DNA repair, anti-apoptosis, and the regulation of the immune system, etc. Our preliminary results show that: OGG1 plays an important role in the regulation of pulmonary fibrosis in mouse and EMT in alveolar epithelial cells, and also impact on autophagy. However, the mechanism of OGG1 in EMT of pulmonary fibrosis is still unclear. We recently verified that OGG1 interacts with Smad3 protein using Yeast two-hybrids and GST-pull down. It was reported that TGF-β1/Smad3 signaling pathway are potential molecular targets on the therapy of pulmonary fibrosis. Based on the above results, we propose a new molecular mechanism: OGG1/Smad3, which activates the target gene transcription in the downstream of Smad3, plays an influence in EMT, autophagy and pulmonary fibrosis. We will explore this by experiments in pulmonary fibrosis model of OGG1 knockout mouse or EMT model of alveolar epithelial cells. We propose three inter-related specific aims: 1) To determine the specific binding sites and downstream target gene of OGG1 and Smad3 interaction; 2) To determine the function of OGG1/Smad3; 3) To determine how OGG1 affect the process of autophagy and EMT via cooperating TGF-β1 signaling pathway. Above-mentioned mechanism can be clarified effectively, and it will provide experimental basis for the future treatment of IPF as a therapeutic target.

特发性肺纤维化（IPF）发病机制尚未阐明，目前无有效防治措施，是严重威胁人类健康的重要疾病。8-羟基鸟嘌呤DNA糖苷酶（OGG1）因具有DNA修复、抗凋亡及调控免疫的作用而备受关注。我们前期研究表明：OGG1参与调控小鼠肺纤维化进程和肺泡上皮细胞EMT过程，并对自噬产生影响，但具体机制不清。我们通过酵母双杂交等发现OGG1与Smad3相互作用，而TGF-β1/Smad3是潜在治疗肺纤维化的分子靶标。因此，我们提出“OGG1可能通过结合Smad3，协同激活通路下游基因转录，影响EMT、自噬及肺纤维化发生”这一新的分子机制。本研究拟利用OGG1敲除小鼠肺纤维化模型和原代肺泡上皮细胞EMT模型探讨：1）OGG1/Smad3的结合位点和调控的靶基因；2）OGG1/Smad3的生物学功能；3）OGG1协同TGF-β1信号通路如何影响自噬和EMT。以上机制的阐明，为今后研发IPF治疗靶点提供理论依据。

特发性肺纤维化（IPF）发病机制尚未阐明，目前无有效防治措施，是严重威胁人类健康的重要疾病。8-羟基鸟嘌呤DNA糖苷酶（OGG1）因具有DNA修复、抗凋亡及调控免疫的作用而备受关注。本课题组已发现：OGG1参与调控小鼠肺纤维化进程和肺泡上皮细胞EMT过程，并对自噬产生影响，但具体机制不清。我们通过酵母双杂交等发现OGG1与Smad3相互作用，而TGF-β1/Smad3是潜在治疗肺纤维化的分子靶标。因此，我们提出“OGG1可能通过结合Smad3，协同激活通路下游基因转录，影响EMT、自噬及肺纤维化发生”这一新的分子机制。本研究拟利用OGG1敲除小鼠肺纤维化模型和原代肺泡上皮细胞EMT模型探讨：1）OGG1/Smad3的结合位点和调控的靶基因；2）OGG1/Smad3的生物学功能；3）OGG1协同TGF-β1信号通路如何影响自噬和EMT。通过以上内容的研究，我们得出重要结果：（1）OGG1蛋白通过与Smad7蛋白结合介导TGF-β/smad信号通路轴调控肺细胞EMT和ECM过程。（2）OGG1可通过结合RACK1调控肺上皮细胞和成纤维细胞内质网应激。且发现RACK1能够影响成纤维细胞迁移和胶原收缩能力。并通过OGG1敲除小鼠肺纤维化模型体内验证了OGG1对肺细胞内质网应激及RACK1蛋白的影响。（3）OGG1可通过结合APE1调节肺上皮细胞和成纤维细胞衰老。并通过OGG1敲除小鼠肺纤维化模型体内验证了OGG1对肺细胞衰老及APE1蛋白的影响。（4）OGG1与Smad7蛋白相互作用的具体结合位点；OGG1与RACK1蛋白相互作用的具体结合位点。（5）βOGG1过表达可通过JNK信号通路减少氧化应激对人支气管上皮细胞导致的细胞凋亡。（6）OGG1敲除肺纤维化小鼠RNA测序结果显示OGG1主要调控下游差异靶基因炎症基因和氧化应激基因参与肺纤维化进程。（7）OGG1作为靶点治疗肺纤维化实验。在该基金的资助下，以第一作者或通讯作者发表6篇SCI论文，总影响因子20分，还发表了6篇中文核心期刊论文和7篇会议论文。这些结果阐明了OGG1蛋白在IPF的作用机制和作为一个潜在的分子靶点，为肺纤维化疾病的预防和治疗提供了重要的实验依据。