Jiang Hongxiu, Yu Wenjing, Tao Xingran, Yan Qiao, Zhou Guanlun, Chen Chao, Han Guorong
Department of Obstetrics and Gynecology, The Second Hospital of Nanjing, Affiliated to Nanjing University of Chinese Medicine, Nanjing, China.
Biomed Res Int. 2025 Jul 26;2025:3000226. doi: 10.1155/bmri/3000226. eCollection 2025.
The objective is to explore the potential pathogenesis and therapeutic mechanism of Yinchenhao decoction (YCHD) in intrahepatic cholestasis of pregnancy (ICP) by focusing on the regulatory role of exosomal miR-370-3p on target genes TM9SF4 and KIT. Exosomes were isolated from the serum samples of normal pregnant women (control), patients with ICP, HTR-8/SVneo cells, and Sprague-Dawley (SD) pregnant rats via differential centrifugation. Characterization of these exosomes was performed using electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. Quantitative reverse transcription PCR (qRT-PCR) and the bioinformatics tool starBase were used to identify miR-370-3p as a candidate miRNA. Dual-luciferase reporter assays were used to confirm that TM9SF4 and KIT are direct targets of miR-370-3p. An in vitro ICP cell model was established using HTR-8/SVneo cells to investigate the interactions between miR-370-3p and its targets. An animal model was established to validate the targeted regulation of miR-370-3p on TM9SF4 and KIT, as well as the therapeutic effect of YCHD . The exosomal miR-370-3p expression was significantly upregulated, whereas the TM9SF4 and KIT expressions were downregulated as demonstrated by qRT-PCR and western blot analyses. RNA pull-down assays confirmed a direct negative regulatory relationship between miR-370-3p and both TM9SF4 and KIT at the molecular level. Finally, the therapeutic potential of YCHD was verified by its ability to reverse the altered expression patterns of miR-370-3p, TM9SF4, and KIT in the animal ICP model. Our study demonstrates that YCHD protects against ICP through the miR-370-3p/TM9SF4/KIT axis, suggesting miR-370-3p as a potential therapeutic target for ICP.
目的是通过关注外泌体miR-370-3p对靶基因TM9SF4和KIT的调控作用，探讨茵陈蒿汤（YCHD）在妊娠期肝内胆汁淤积症（ICP）中的潜在发病机制和治疗机制。通过差速离心从正常孕妇（对照组）、ICP患者、HTR-8/SVneo细胞和Sprague-Dawley（SD）妊娠大鼠的血清样本中分离外泌体。使用电子显微镜、纳米颗粒跟踪分析（NTA）和蛋白质印迹对这些外泌体进行表征。采用定量逆转录PCR（qRT-PCR）和生物信息学工具starBase鉴定miR-370-3p为候选miRNA。采用双荧光素酶报告基因检测法证实TM9SF4和KIT是miR-370-3p的直接靶标。利用HTR-8/SVneo细胞建立体外ICP细胞模型，研究miR-370-3p与其靶标的相互作用。建立动物模型，验证miR-370-3p对TM9SF4和KIT的靶向调控以及YCHD的治疗效果。qRT-PCR和蛋白质印迹分析表明，外泌体miR-370-3p表达显著上调，而TM9SF4和KIT表达下调。RNA下拉实验在分子水平上证实了miR-370-3p与TM9SF4和KIT之间存在直接的负调控关系。最后，YCHD逆转动物ICP模型中miR-370-3p、TM9SF4和KIT表达模式改变的能力验证了其治疗潜力。我们的研究表明，YCHD通过miR-370-3p/TM9SF4/KIT轴预防ICP，提示miR-370-3p作为ICP的潜在治疗靶点。
