补肾安胎法调控PIEZO1介导MAPK/Akt/Notch通路交互作用治疗自然流产的机制研究

Maternal fetal interface remodeling angiogenesis is an important mechanism of spontaneous abortion. It has been proved that Bushen Antai medicine can improve the expression of VEGF, the function of angiogenesis and integrity of organelle in endometrium. Our research group has proved that MAPK / AktAK / Notch is involved in the development of SA. Bushen Antai medicine can regulate the proliferation function of decidual cell and increase the expression of hormone receptor through MAPK pathway, and regulate the expression of PR and VEGF through Notch pathway. The latest literature showed that the absence of Piezo1 can cause miscarriage due to vascular remodeling in pregnant rats. The applicant collected villi from patients with SA and normal early pregnancy and the low expression of Piezo1 in SA was confirmed by PCR. According to bioinformatics analysis and literature studies, Piezo1 regulated the relationship between MAPK and Akt pathways. Based on this, a scientific hypothesis was put forward: Bushen Antia Jianwei Shoutai Pill may up-regulate the expression of Piezo1 and mediate the MAPK/Akt/Notch pathway crosstalk to promote angiogenesis at the maternal and fetal interface of SA, therefore it may play a role in miscarriage prevention. Based on the rat model of in vivo abortion and the model of trophoblastic cells in vitro, through clinical sample validation-in vitro mechanisms-in vivo validation. Using integrated molecular biology, Chinese medicine pharmacology, electrophysiology and imaging techniques, the specific mechanism of Piezo1 mediated MAPK/Akt/Notch pathway was expounded from tissue, serum, gene, protein and cell signal system. To elucidate the mechanism of regulating Piezo1 to promote the function of angiogenesis at maternal and fetal interface.

母胎界面血管生成重塑是自然流产(SA)发病的重要机制。课题组研究表明补肾安胎法能提高子宫内膜VEGF表达，改善血管生成功能；MAPK/Akt，AKT/Notch均参与SA的发生，补肾安胎法能通过MAPK通路调节蜕膜细胞增殖，通过Notch通路调节PR和VEGF表达。最新文献表明Piezo1缺失可致妊娠大鼠因血管重建障碍导致流产，前期验证在SA绒毛组织中Piezo1呈低表达。根据生信分析和文献研究提示Piezo1介导MAPK、Akt通路。故提出：减味寿胎丸可能上调Piezo1介导MAPK/Akt/Notch通路crosstalk促进SA母胎界面血管生成功能起到治疗作用。拟用流产大鼠和滋养细胞模型，从临床验证-体外机制-体内验证，整合分子生物学、电生理学、影像学等阐释Piezo1调控MAPK/Akt/Notch的机制，阐明补肾安胎法调控Piezo1介导母胎界面血管生成功能的具体分子机制。

母胎界面血管生成障碍是自然流产(SA)发病的重要机制。课题组前期研究表明MAPK/Akt /Notch通路均参与SA的发生，补肾安胎代表组方减味寿胎丸能通过Notch通路调节VEGFA表达，改善血管生成功能。故本项目从临床验证-体外机制-体内验证三个方面阐明减味寿胎丸调控Piezo1介导MAPK/Akt/Notch保护母胎界面血管生成功能的具体分子机制。①临床验证：经临床40对样本验证发现Piezo1在自然流产患者绒毛组织中呈高表达，提示Piezo1可能为SA发病潜在的关键靶点，同时，通过荧光共定位发现Piezo1在绒毛组织中内皮细胞及滋养细胞均表达，故分别研究滋养细胞与内皮细胞的Piezo1在母胎界面中的作用机制。②体外机制：分别以人原代滋养细胞、HTR8/SVneo滋养细胞系、Huvec内皮细胞系为载体，发现自然流产原代滋养细胞的Piezo1钙流减弱，并阐明Piezo1具有调控滋养细胞侵袭、迁移及体外管形成能力；MAPK/Akt/Notch通路在内皮细胞体外管形成中均具有促进作用，Notch通路同时受ERK/MAPK通路负向调控，本研究中Notch及PI3K通路在介导母胎界面血管生成中未见交互作用；Piezo1通过正向调控Notch通路介导内皮细胞的管形成能力；减味寿胎丸能增强SA原代滋养细胞侵袭、迁移及体外管形成的能力，并能增强siPiezo1 HTR8/SVneo侵袭、迁移及体外管形成的能力；同时，明确减味寿胎丸能增强siPiezo1 内皮细胞体外管形成能力。③体内验证：减味寿胎丸能通过Piezo1介导VEGFA/VEGFR2促进母胎界面血管生成，增加肾虚-黄体抑制病证结合流产大鼠模型母胎界面血流供应，改善局部血氧饱和度，增强母胎界面营养物质交换，从而降低流产率达到安胎的治疗效果。. 本研究发现Piezo1可能为自然流产发病的关键潜在分子，Piezo1促进滋养细胞侵袭、迁移及体外管形成，通过Notch通路介导VEGFA表达，促进内皮细胞体外管形成能力。阐明了补肾安胎代表组方减味寿胎丸可通过调控Piezo1改善SA滋养细胞侵袭、迁移及体外管形成能力，促进siPiezo1内皮细胞体外管形成。本研究进一步阐明减味寿胎丸通过调控Piezo1促进母胎界面血管生成，改善母胎界面血流供应，从而达到补肾安胎的作用，进一步丰富“肾主生殖”的科学内涵。