Bax抑制因子1对人妊娠期肝内胆汁淤积症胎盘滋养细胞凋亡的影响及其机制

目的：探讨Bax 抑制因子1（BI-1）对人妊娠期肝内胆汁淤积症（ICP）胎盘滋养细胞凋亡的影响及其机制。方法：选取2018 年5 月至2019 年5 月新疆维吾尔自治区人民医院产科住院分娩的ICP 孕产妇15 例为研究对象，设为ICP 组，另取15 例正常孕产妇作为对照组，免疫组织化学显色检测ICP 组和对照组孕妇胎盘组织BI-1 的表达；体外培养滋养细胞系HTR8，应用不同浓度（10、50、100 μmol/L）的牛磺胆酸（TCA）进行刺激，RTPCR及免疫印迹检测HTR8细胞BI-1 mRNA 及蛋白的表达；用过表达BI-1 的慢病毒载体感染HTR8细胞，荧光显微镜及RT-PCR 检测感染效率后，将细胞分为对照组（NC组）、TCA处理的感染LV-NC 细胞组（TCA+LV-NC 组）、TCA处理感染LV-BI-1 细胞组（TCA+LV-BI-1 组）；分别用Annexin V-FITC、透射电镜及JC-1 流式线粒体膜电位检测试剂盒检测3 组滋养细胞凋亡、线粒体超微结构及线粒体膜电位；免疫印迹检测3 组细胞Cyt-C 及凋亡相关蛋白Bcl-2、Bax 及cleaved caspase-3 的表达。结果：与对照组相比，ICP 组胎盘组织BI-1 表达显著降低；体外实验结果显示，TCA能够显著降低HTR8细胞BI-1 mRNA及蛋白表达，且呈浓度依赖性；应用慢病毒成功建立过表达BI-1 的滋养细胞系。与NC组细胞相比，TCA+LV-NC 组与TCA+LV-BI-1 组细胞凋亡率和线粒体损伤水平均显著增加，线粒体膜电位明显降低；而与TCA+LV-NC 组相比，LV+BI-1 组细胞凋亡率与线粒体损伤水平均明显降低，线粒体膜电位显著增加；过表达BI-1 能够有效阻止TCA导致的滋养细胞Bcl-2/Bax 比值的降低，以及线粒体Cyt-C 的释放及cleaved caspase-3 表达的增加。结论：BI-1 在ICP 患者胎盘组织中的表达显著降低，而体外过表达BI-1 可能通过上调滋养细胞中Bcl-2/Bax 比值，抑制细胞线粒体膜电位降低及结构损伤，从而减少Cyt-C 的释放，最终降低凋亡蛋白caspase-3 活化而改善TCA诱导的凋亡作用。

Effect of over-expression of Bax inhibitor 1 on placental trophoblast cell apoptosis in intrahepatic cholestasis during pregnancy and its mechanism

Objective To explore the effect and mechanism of Bax inhibitor 1 （BI-1） on apoptosis of human placentaltrophoblast in intrahepatic cholestasis of pregnancy（ ICP）. Methods Fifteen patients with ICP who hospitalizedin Department of Obstetrics, People’s Hospital of Xinjiang Uygur Autonomous Region from May 2018 to May 2019were enrolled in this study and designed as ICP group. Meanwhile, fifteen healthy pregnant women were enrolledas control group. The expression of BI-1 in placenta of ICP pregnant women and normal control group was detectedby immunohistochemistry. The trophoblast line HTR8 was cultured in vitro and stimulated by taurocholate（ TCA）of different concentrations（ 0, 50, 100 μmol/L）. The mRNA and protein expression level of BI-1 in HTR8 weredetected by RT-PCR and Western blotting. HTR8 cells were infected with lentivirus to overexpress BI-1， and theinfection efficiency was detected by fluorescence microscopy and RT-PCR. The cells were divided into three groups negative control group（ NC）， TCA treated LV-NC group（ TCA+LV-NC）， and TCA treated LV-BI-1 group（ TCA+LVBI-1）. Annexin V-FITC， transmission electron microscopy and JC-1 flow cytometry were used to detect trophoblastapoptosis， mitochondrial ultrastructure and mitochondrial membrane potential. Western blotting was used to detect theexpression of Cyt-C and apoptosis related proteins Bcl-2， Bax and cleaved caspase-3 in TCA induced trophoblasts.Results Compared with the NC group， the expression of BI-1 in the placenta of ICP patients was significantlydecreased. In vitro experiments showed that TCA could significantly reduce the expression of BI-1 mRNA andprotein in trophoblasts in a concentration dependentmanner. A trophoblast cell line overexpressing BI-1was successfully established by lentivirus. Comparedwith NC group， TCA+LV-NC group and TCA+LV-BI-1 group significantly increased the apoptosis rate and mitochondrial damage level， and significantly decreased themitochondrial membrane potential. Compared with TCA+LV-NC group， the apoptosis rate and mitochondrial damagelevel of HTR8 cells in LV+BI-1 group were significantly decreased， and the mitochondrial membrane potential wassignificantly increased. Overexpression of BI-1 could effectively prevent the decrease of Bcl-2/Bax ratio， the releaseof Cyt-C in mitochondria and the expression of cleaved caspase-3. Conclusion The expression of BI-1 in placenta ofICP patients is significantly decreased， and over-expression of BI-1 in vitro may improve TCA induced apoptosis byupregulating the ratio of Bcl-2/Bax in trophoblast， inhibiting the decrease of mitochondrial membrane potential andstructural damage， thereby reducing the release of Cyt-C and ultimately reducing the activation of caspase-3.