黄芪当归药对通过lncRNA BANCR调控ZEB1的DNA甲基化抑制特发性肺纤维化EMT的机制研究

The latest study shows that the epithelial to mesenchymal transition (EMT) plays a key role in the early progression of idiopathic pulmonary fibrosis (IPF). In addition to lung transplantation, there is no effective treatment for idiopathic pulmonary fibrosis. Astragalus and angelica mixture is a traditional prescription with proven clinical benefits for treating pulmonary fibrosis in Chinese medicine. The applicant has confirmed that astragalus and angelica mixture can reduce the expression of ZEB1. It may be related to modulate ZEB1 DNA methylation by lncRNA BANCR in improvement of epithelial to mesenchymal transition. But its molecular mechanism for treating pulmonary fibrosis is still unclear. Based on a large number of preliminary experimental results, the following scientific hypothesis is proposed: Astragalus and angelica mixture can down-regulate the expression of long non-coding RNA BANCR in lung epithelial cells, and reverse lncRNA BANCR to block the ZEB1 promoter region by forming a complex with DNA methyltransferase Methylation modification, thereby down-regulating the expression of ZEB1, inhibiting the epithelial to mesenchymal transition and fibrosis process of lung epithelial cells, thereby preventing and treating pulmonary fibrosis. This study will apply the combination vivo with vitro methods. In vivo experiment, the applicant is going to establish the IPF rat model. After intervention of astragalus and angelica mixture, the applicant wants to observe the influence of astragalus and angelica mixture on the expression of rat phenotypes and related cytokine to prove the effect of astragalus and angelica mixture on IPF. In vitro experiment, the applicant is going to knock down and overexpress lncRNA BANCR and ZEB1, respectively, to verify the molecular mechanism. We will further validate the regulatory mechanism through functional recovery experiments, MSP experiments, FISH experiments, ChIP experiments, RIP-qPCR experiments and EMSA experiments. The study illustrates the influence of astragalus and angelica mixture on the improvement of IPF, which provided the scientific pursuant of treating IPF by traditional Chinese medicine.

上皮间充质转化（EMT）增加导致的肺间质细胞过度增殖是特发性肺纤维化（IPF）发病的基础和关键。目前除肺移植外，尚无有效的治疗药物。黄芪当归药对是治疗IPF的经典药对，临床疗效确切。前期实验表明芪归药对可降低ZEB1的表达，可能与通过lncRNA BANCR调控ZEB1甲基化，抑制EMT有关。故提出假说：芪归药对可下调lncRNA BANCR的表达，促进ZEB1启动子区域的DNA甲基化，下调ZEB1的表达，抑制肺上皮细胞EMT，最终达到治疗IPF的效果。本项目拟体内体外实验结合，体内建立IPF大鼠模型，芪归药对干预后，分析大鼠表型和相关因子变化，验证芪归药对改善EMT的治疗效应和机制；体外分别敲减和过表达lncRNA BANCR和ZEB1，结合MSP、FISH、ChIP、RIP-qPCR、EMSA和挽救实验进一步验证该科学假说。阐明芪归药对治疗IPF的机理，为中医药防治IPF提供科学依据。

上皮间充质转化（EMT）增加导致的肺间质细胞过度增殖是特发性肺纤维化（IPF）发病的基础和关键。目前除肺移植外，尚无有效的治疗药物。黄芪当归药对是治疗IPF的经典药对，临床疗效确切。前期实验表明芪归药对可降低ZEB1的表达，可能与通过lncRNA BANCR调控ZEB1甲基化，抑制EMT有关。本课题首先通过生物信息学方法分析了ZEB1通过调控EMT影响IPF进程的潜在机制，验证发现在IPF大鼠肺组织中ZEB1以及上皮-间充质转化（EMT）相关标志物的蛋白相对表达量明显升高，而DNA甲基转移酶DNMT3a的蛋白表达显著降低。体外实验结果表明DNMT3a主要通过促进ZEB1启动子区甲基化抑制EMT，lncRNA BANCR能够与DNMT3a形成复合体，阻碍其对ZEB1启动子区的甲基化，黄芪当归药对能够下调BANCR的表达，从而起到抑制EMT，缓解IPF的作用。提示我们，临床上通过黄芪当归药对或能成为IPF的潜在治疗方法。