基于长链非编码RNA调控STAT6诱导巨噬细胞极化探讨寿胎丸治疗RSA作用与机制研究

Macrophage (Mϕ) polarization to M1/M2 plays an impartment role in the immune system and involves in the recurrent spontaneous abortion (RSA). However, its unclear regulation mechanism obstructs the development and optimization of RSA treatments with explicit targets. Shoutaiwan is the most preferred therapy for RSA in Chinese medicine. In the pilot experiments, we found Mϕ polarization in RSA mouse model is skewed, and Shoutaiwan reverse this abnormal shift by inducing M2 differentiation. High-throughput sequencing identified a long non-coding RNA (LncRNA-E330013P06) that interacted with STAT6, a M2 differentiation inducing factor. In vitro studies found that Shoutaiwan regulated the expressions of LncRNA-E330013P06 and STAT6, and the regulating effects are opposite. Combining both the experiment results and the latest progresses in epigenetic studies, we hypothesize that the pathologic mechanism of RSA is the abnormal Mϕ polarization caused by LncRNA-suppressed STAT6 expression. Shoutaiwan targets on LncRNA/STAT6 pathway to induce M2 differentiation, and thus, it reverses the abnormal shift of Mϕ polarization and shows its therapeutic effects on RSA. This study will demonstrates the underlying mechanism of RSA and help understand the therapeutic effects of Shoutaiwan. Establishment of the scientific theory of traditional Chinese medicine provides the foundation for the prevention, diagnosis and cure of RSA.

巨噬细胞（Mϕ）是免疫系统重要组成部分，其亚群（M1/M2）失衡参与了复发性自然流产（RSA）发生，但其调控机制尚不清楚，从而限制了调控靶点明确药物发掘和组方优化。寿胎丸是补肾安胎经典方剂，预实验发现RSA小鼠存在Mϕ极化失衡，寿胎丸能够诱导M2亚群分化；高通量测序发现差异长链非编码RNA（LncRNA-E330013P06），M2亚群分化诱导因子STAT6是其预测靶基因，并发现寿胎丸对两者有负向调控作用。基于以上研究基础，结合表观遗传调控最新进展，推测LncRNA抑制STAT6导致Mϕ极化失衡是RSA发生的重要原因，寿胎丸靶向调控LncRNA/STAT6通路，诱导M2亚群分化，逆转Mϕ极化失衡，进而治疗RSA。预期结果将进一步揭示RSA发病机理，阐释寿胎丸治疗RSA的关键靶点与机制，丰富中医学“肾主生殖”理论科学内涵，为经典方剂深入挖掘及组方优化提供科学依据。

寿胎丸是补肾安胎经典方剂，对于治疗复发性自然流产（RSA）疗效显著，但目前其作用机制尚不明确。本研究在国家自然科学基金青年基金项目“基于长链非编码RNA调控STAT6诱导巨噬细胞极化探讨寿胎丸治疗RSA作用与机制研究（81704116）”资助下，重点探讨了巨噬细胞（Mϕ）极化的表观遗传调控在RSA中的作用，以及寿胎丸靶向长链非编码RNA（LncRNA）/STAT6通路的干预机制。研究内容及创新点包括：1. RSA模型小鼠Mϕ极化偏移：M2比例降低、M1/M2比率升高。M2型细胞因子（如IL-4、IL-10）分泌减少，M2标记基因如 arginase (Arg)-1等的表达水平降低。同时，M2相关转录因子STAT6表达降低，LncRNA-E330013P06表达上升，两者存在相关性。2. LncRNA-E330013P06通过调控STAT6的表达，影响M2极化状态。3. 寿胎丸在体外实验中可以抑制LncRNA-E330013P06表达，提高STAT6表达水平，促进M2极化。4. 寿胎丸在体内实验中能有效降低RSA小鼠胚胎吸收率，对妊娠具有保护作用。其作用机制是靶向LncRNA-E330013P06/STAT6通路，促进M2极化，维持M1/M2平衡，重建母胎免疫耐受。. 研究结果进一步阐释了RSA发生的表观遗传学调控机制，为寿胎丸诱导母胎免疫耐受提供了实验依据。本研究发表SCI论文3篇，中文论文2篇，培养硕士研究生2名。以获得的实验数据为基础，获得山东省重点研发计划项目课题1项，申请国家发明专利1项，获得中华中医药学会科学技术奖二等奖1项。部分工作正在投稿或审稿中，后续工作将标注基金号并及时向基金委汇报。