丹参酮ⅡA通过GSK-3β信号通路调控胎盘中水通道蛋白表达与羊水量异常关系的研究

Amniotic fluid volume abnormality is common complication during pregnancy. Aquaporins (AQPs) are a kind of glycoprotein that may mediate water transport across biological membrane. Our previous study that had published in Placenta found AQPs were expressed in the human placenta, which was related to pathogenesis of abnormal amniotic fluid. Our previous study found salvia miltiorrhiza treating oligohydramnios may regulate the expression of aquaporin 3 in the human amnion cells. Tanshinone ⅡA is main active ingredient of salvia miltiorrhiza. Our study found through Human Phospho-MAPK Antibody Array, the correlated signal transduction pathway with Tanshinone ⅡA regulating AQPs proteins expression in human amniotic epithelial WISH cells was glycogen synthetic kinase 3β (GSK-3β). Therefore, this study use primary cultured amniotic epithelial cells of term pregnant women with amniotic fluid volume abnormality and polyhydramnios model of AQP1 knock out mice to further investigate the relation of Tanshinone ⅡA regulate the expression of AQPs in placenta and Abnormal amniotic fluid on a cellular and animal level respectively, and explore feasibility for Tanshinone ⅡA treating abnormal amniotic fluid. Guide for GSK-3β signal transduction pathways in regulating a AQP expression and development of new drugs for the treatment of abnormal amniotic fluid and eventually reduce perinatal morbidity and mortality.

羊水量异常是妊娠期常见的并发症，水通道蛋白（AQP）是一类介导水跨生物膜转运的糖蛋白，我们的前期研究（发表在Placenta杂志）发现，AQP在人足月胎盘中表达与羊水量异常的发病机制密切相关，治疗羊水过少的复方丹参能够调节人羊膜上皮细胞中AQP3的表达，丹参酮ⅡA是复方丹参中的主要有效成份，通过MAPK信号转导通路抗体芯片筛查，我们已经发现丹参酮ⅡA调节人羊膜上皮WISH细胞中水通道蛋白表达，与糖原合成酶激酶-3β信号通路关系密切；因此,本课题取羊水量异常足月孕妇羊膜上皮细胞进行原代培养，同时，我们利用AQP1敲除小鼠建立的羊水过多模型，分别在细胞和动物水平上进一步探讨了丹参酮ⅡA调节胎盘中水通道蛋白的表达与羊水量异常的关系，探索丹参酮ⅡA治疗羊水量异常的可行性，引导针对GSK-3β信号转导通路调控某种AQP表达而治疗羊水量异常的新药开发，最终降低围产儿发病率和死亡率。

羊水量异常是妊娠期常见的并发症，水通道蛋白（AQP）是一类介导水跨生物膜转运的糖蛋白，AQP在人足月胎盘中表达与羊水量异常的发病机制密切相关，治疗羊水过少的复方丹参能够调节人羊膜上皮细胞中AQP3的表达，丹参酮ⅡA是复方丹参中的主要有效成份，通过MAPK信号转导通路抗体芯片筛查，我们已经发现丹参酮ⅡA调节人羊膜上皮WISH细胞中水通道蛋白表达，与糖原合成酶激酶-3β信号通路关系密切。 因此,本课题取羊水量异常足月孕妇羊膜上皮细胞进行原代培养，同时，我们利用AQP1敲除小鼠建立的羊水过多模型，分别在细胞和动物水平上进一步探讨了丹参酮ⅡA调节胎盘中水通道蛋白的表达与羊水量异常的关系，结果发现AQP1、AQP3、AQP8和AQP9蛋白均在羊水量正常组的羊膜上皮细胞、绒毛膜滋养细胞和胎盘滋养细胞的细胞质和（或）细胞核中表达，与羊水量正常足月产妇组相比，AQP1和AQP9蛋白在羊水过少组的羊膜上皮细胞、绒毛膜和胎盘滋养细胞中表达均显著降低；丹参酮IIA通过GSK-3β信号通路调节了正常羊水量组的人羊膜上皮细胞AQP1和AQP3的蛋白表达，而不是AQP8和AQP9的蛋白表达。AQP1敲除降低了雌鼠的见栓率和成功妊娠率。与野生型孕鼠相比，AQP1敲除妊娠孕鼠，随着孕周的增加，胎鼠的数目减少，羊水量增加，胎鼠体重和胎盘的重量无变化，羊水的成分Ca2+及渗透压降低，而Na+，K+，Cl-，尿素等浓度无明显变化。在不同孕周，AQP1敲除对胎盘和胎膜上AQP3、AQP8和AQP9 mRNA和蛋白的表达影响各不相同。GSK-3β蛋白抑制剂LiCl减少了孕鼠的羊水量，AQP1敲除导致的羊水过多与GSK-3β信号通路密切相关，其抑制剂或者激动剂有可能成为治疗羊水量异常的新靶点。本研究成果深化了对羊水平衡调节和母儿液体交换的分子机制的理解，明确了原因不明羊水量异常相关的发病机制；为治疗羊水量异常的新药开发提供了实验依据，最终将降低羊水量异常的围产儿发病率和死亡率，减少围产儿羊水量异常的发生，提高围产医学质量。