内质网蛋白ERp29激活p38MAPK信号通路诱导妊娠期肝内胆汁淤积症患者胎盘凋亡的机制研究

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disease which is potentially harmful to both the maternity and infant. Studies have shown that abnormal regulation of placental apoptosis is closely related with the molecular pathogenesis of ICP. Our previous study using an iTRAQ-based proteomics approach showed that ERp29 was overexpressed in the placenta tissue of ICP patients. ERp29 was a new endoplasmic reticulum protein which plays a key role in the regulation of p38 phosphorylation, and p38 was one of the important molecules in apoptosis regulation. Further studies showed that the apoptosis index in placenta from ICP patients was significantly increased. Moreover, our previous study in vitro showed that ERp29 expression and the apoptotic index were significantly increased in HTR-8/SVneo cells exposed to taurocholic acid (TCA); so were p-p38 and caspase-3 activity, compared with control groups. These findings suggested that ERp29 may play a key role in TCA-induced apoptosis in HTR-8/SVneo cells via activation of p38, which may participate in the pathogenesis of ICP. In this study, we would first apply overexpression, silent strategy and save test to verify that ERp29 is involved in the regulation of placental apoptosis in ICP patients by regulating p38 MAPK signaling pathway from pros and cons sides. We further demonstrate the role and apoptosis pathways which was mediated by ERp29 in ICP in vivo. The research will provide us new theoretical significance and a novel target for ICP treatment.

妊娠期肝内胆汁淤积症(ICP)是妊娠期严重危害母婴健康的并发症。胎盘滋养细胞凋亡调控异常在ICP发病中起关键作用。我们前期蛋白质组学研究发现ICP患者胎盘ERp29高表达，ERp29是一种能够调控p38磷酸化的新型蛋白，而p38是经典的凋亡调控分子之一。进一步研究发现：ICP患者胎盘凋亡增高，胆酸处理人HTR-8/SVneo细胞后，ERp29及p-p38表达均显著上调，细胞凋亡显著增高。基于上述发现我们提出假设：ERp29可能通过p38 MAPK信号通路调控胎盘滋养细胞凋亡参与ICP发病。本研究拟在细胞水平应用过表达、沉默策略及挽救实验从正反两方面论证ERp29通过调节p38 MAPK信号通路对胎盘滋养细胞凋亡的作用及途径，在体水平进一步阐释ERp29对ICP胎盘滋养细胞凋亡的作用与机制。本研究将为ICP的防治提供新的理论依据和分子靶点。

妊娠期肝内胆汁淤积症(ICP)是妊娠期严重危害母婴健康的并发症。胎盘滋养细胞凋亡调控异常在ICP发病中起关键作用。我们前期蛋白质组学研究发现ICP患者胎盘ERp29高表达，ERp29是一种能够调控p38磷酸化的新型蛋白，而p38是经典的凋亡调控分子之一。本研究在细胞水平应用过表达、沉默策略及挽救实验从正反两方面论证ERp29通过调节p38 MAPK信号通路对胎盘滋养细胞凋亡的作用及途径，应用动物实验进一步阐明ERp29对ICP胎盘滋养细胞凋亡的作用与机制。研究结果表明：1） HTR-8/SVneo细胞过表达ERp29后，细胞凋亡显著增高，ERp29、p-p38表达显著增高，凋亡相关指标BCL-2显著降低，Cyt-c、Caspase-9及Caspase-3显著增高，而各组ERK、JNK及Fas/FasL表达无显著差异，而干涉ERp 29表达后结果呈相反趋势；2）TCA（胆酸）处理HTR-8/SVneo细胞后细胞活力受抑制，而ERp29表达沉默通过影响p21，CyclinB1和Cdc2的表达而抑制TCA诱导的G2/M阻滞；3）我们应用免疫荧光方法证实ERp29和p38存在相互作用；4）HTR-8/SVneo细胞经TCA处理后，p-p38及p53表达水平升高，ERp29沉默后p-p38及p53表达水平下降，TCA诱导的细胞G2/M阻滞与凋亡也受到抑制。但是，挽救实验表明经MKK3（p38激酶激活剂）转染处理后，细胞G2/M阻滞与凋亡水平均上调，western blot结果显示G2/M阻滞相关指标p21，CyclinB1和Cdc2表达水平增高，凋亡相关指标BCL-2降低，Bax及Caspase-3升高，而HTR-8/SVneo细胞经TCA处理同时过表达ERp29时，应用SB203580处理（p38激酶抑制剂）呈相反趋势；5）ICP患者血清TBA、ALT及AST较健康人群均显著升高，免疫组化结果显示ICP患者胎盘组织ERp29蛋白表达水平较高且定位于胎盘组织滋养细胞胞浆，TUNEL结果显示ICP患者胎盘凋亡水平明显高于健康人群；6）动物实验结果显示ERp29敲减后孕鼠胎盘组织中ERp29表达水平降低，p-p38及p53表达也下调。本研究揭示了ERp29通过调节p38 MAPK信号通路对ICP患者胎盘滋养细胞凋亡的作用及途径。为ICP的防治提供新的理论依据和分子靶点。