温肺化纤汤对肺间充质干细胞外泌体的影响及分子机制

Idiopathic pulmonary fibrosis is a disease with rapid progress and poor prognosis. The effect of Wenfeihuaxian formula is accurate. Previous studies showed that Wenfeihuaxian formula can promote the generation of more exosomes of lung mesenchymal stem cells, but its biological effects and molecular mechanisms are not yet clear. We put forward the scientific hypothesis that " Wenfeihuaxian formula may play a role in repairing lung injury by affecting the exosomes of the mesenchymal stem cells in the lung". To answer this scientific question, this study plan to use in vivo and in vitro experiments to study changes of number and microRNA of exosomes secreted by lung mesenchymal stem cells in pulmonary fibrosis environment, and the biological effects will be observed. The intervention of this process Wenfeihuaxian formula will also be observed. Then, the detailed molecular mechanism will be analyzed. The completion of this project will find new ways and targets of Wenfeihuaxian formula, providing data support for its clinical application, and exploring new strategies for drug intervention in idiopathic pulmonary fibrosis.

特发性肺纤维化是一种进展快、预后差的疾病，采用温肺化纤汤治疗效果确切。前期研究结果显示，温肺化纤汤可以促进肺间质干细胞产生更多的外泌体，但其生物学效应和分子机制尚不明晰。我们提出“温肺化纤汤可能通过影响肺间充质干细胞外泌体发挥修复肺损伤部位作用”的科学假说。为解答这一科学问题，本研究拟采用体内和体外实验相结合，研究肺纤维化微环境中肺间质干细胞分泌外泌体数量和内含物的变化，进而观察这个变化所带来的生物学效应的改变，以及温肺化纤汤对这个过程的干预，并分析详细分子机制。课题完成将发现温肺化纤汤的新作用途径和作用靶点，为其临床应用提供数据支撑，同时也探寻药物干预特发性肺纤维化的新策略。

本研究显示，温肺化纤汤干预能显著减轻博来霉素诱导的SD大鼠肺组织的炎性细胞浸润以及纤维组织增生，修复受损的肺泡结构，减少胶原纤维沉积。温肺化纤汤干预后的肺充间质干细胞与肺成纤维细胞共培养，能降低肺成纤维细胞的凋亡率，并下调肺成纤维细胞中α-SMA和ColⅠ蛋白的表达，并增加Ⅱ型肺泡细胞特异性标记表面蛋白C的荧光表达。课题组通过PKH-26细胞荧光染色证实温肺化纤汤可以增加靶细胞外泌体摄入，揭示了温肺化纤汤可能通过增加肺间充质干细胞外泌体摄入来发挥调控肺成纤维细胞和Ⅱ型肺泡细胞，最终发挥修复受损肺组织、逆转纤维化的作用。全转录组测序分析寻找差异mRNA，揭示温肺化纤汤的新作用途径和作用靶点。进一步深入研究发现：温肺化纤汤可通过靶向AMPK/SIRT3途径清除ROS，调控肺充间质干细胞氧化损伤和代谢失衡，改善细胞功能。本研究的完成从分子水平为中医药治疗特发性肺纤维化（IPF）提供了理论和实验的新依据。