芒柄花素-7-丹酚酸酯的制备及其抗肺纤维化作用机制的研究

Pulmonary fibrosis is one of interstitial lung diseases with unknown mechanism. Until now there has been no effective therapy. So it is important clinical value to research its pathogenesis and effctive treatment. In the process of extracting astragalus and salvia, we found that formononetin from astragalus extracts and danshensu from salvia extracts can reduce the collagen deposition in lung tissue and prevent the progress of fibrosis. But formononetin has a poor water solubility, low bioavailability and slow effect, and danshensu has a unstable chemical stability, oxidation and discoloration. To overcome these drawbacks, the project team product a new compound named formononetin -7 - sal ester (F-7-P) by natural medicine chemistry technology. Compared with formononetin and danshensu, the water-solubility and the stability are all improved. Pharmacological activity shows that the compound has a strong effect of anti-pulmonary fibrosis, and its mechanism of action relates to the inhibition of muscle fibroblast proliferation. The data shows that the long chain of non-coding RNA-MRAK053938 might be involved in its mechanism of anti-pulmonary fibrosis.The project aims to study the effect of F-7-P in anti-pulmonary fibrosis and the regulative mechanism of MRAK053938, and provide the data for developing new drugs and gene therapy for pulmonary fibrosis.

肺纤维化是一种机制未明的弥漫性肺间质疾病，近年由于雾霾的发生，其发病率逐年上升，目前仍无有效治疗方法。项目组在研究黄芪和丹参提取物的过程中发现，黄芪提取物中的芒柄花素和丹参提取物中的丹参素能减轻肺内胶原沉积，阻止纤维化进程。但芒柄花素水溶性差，生物利用度低、显效慢；丹参素化学稳定性差，易氧化、变色。为克服该缺陷，项目组运用天然产物化学技术制备得到新的化合物命名为芒柄花素-7-丹酚酸酯（简称SF），作为一种具有双前体药物性质的化合物，与芒柄花素相比水溶性增强；与丹参素相比稳定性增加。药理活性显示该化合物具有良好的抗肺纤维化作用，初步发现其作用机制与抑制肌成纤维细胞的分化、增殖和迁移相关。项目组研究发现长链非编码RNA-MRAK053938能够参与调控肌成纤维细胞的功能，本项目拟研究SF抗肺纤维化的作用及其MRAK053938参与的调控机制，为开发治疗肺纤维化药物和潜在的基因靶点提供理论依据。

本项目以芒柄花素和丹参素为原料，运用天然产物化学技术制备得到新的化合物命名为芒柄花素-7-丹酚酸酯，克服了芒柄花素水溶性差，生物利用度低、显效慢以及丹参素化学稳定性差、易氧化、变色等的缺陷。通过博来霉素诱发的实验性小鼠肺纤维化模型和TGFβ1诱发的细胞模型系统探讨芒柄花素-7-丹酚酸酯的抗肺纤维化作用及表观遗传学机制。结果表明芒柄花素-7-丹酚酸酯能增强纤维化小鼠的肺功能，改善肺组织结构，抑制肌成纤维细胞的增殖、迁移、胶原分泌，减缓纤维化进程；且药理活性明显优于芒柄花素和丹参素。通过Cignal Finder Pathway System和转录组测序筛选出芒柄花素-7-丹酚酸酯的靶基因和靶通路-转录因子Mef2c介导的信号通路。实验结果显示，干扰或过表达Mef2c能够调控肌成纤维细胞的功能且能介导芒柄花素-7-丹酚酸酯对抗肺纤维化活性。在明确芒柄花素-7-丹酚酸酯能以非编码RNA-MRAK053938作为分子靶点的基础上并结合miRNA表达谱芯片数据，阐释MRAK053938的下游调控网络（miR-708-3p-ADAM17-STAT3/GATA）。PCR结果表明，miR-708-3p在IPF患者、肺纤维化体内外模型中均显著下调表达，且与肺纤维化的严重程度成负相关。且其表达受TGFβ1-smad3依赖的转录水平调控。TGFβ1能够抑制其启动子的活性。miR-708-3p mimic能增强E-cadherin表达，抑制α-SMA、波形蛋白、Snail、I 型和III型胶原等纤维化指标的表达，从而抑制肺纤维化的进程。纤维化小鼠中增强miR-708-3p表达具有显著的治疗作用；软件分析、萤光素酶报告实验和rescue实验发现靶基因ADAM17介导miR-708-3p的抗肺纤维化作用。 Cignal Finder 45-Pathway筛选发现miR-708-3p能够显著抑制转录因子STAT3、GATA报告基因的活性。该项目的研究为肺纤维化的治疗提供有效的药物-芒柄花素-7-丹酚酸酯和新的分子靶点：TGFβ1-MRAK053938-miR-708-3p-ADAM17-STAT3/GATA通路。

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