Hypoxia-induced oxidative stress and apoptosis of trophoblast are involved in the pathogenesis of preeclampsia (PE). Extensive research reports that the principal vagal neurotransmitter acetylcholine (ACh) shows anti-oxidative and anti-apoptotic effects in various diseases models. However, the role of ACh in hypoxic trophoblast remains unknown. Here, we examined the apoptotic levels of human placenta and explored the role(s) of ACh on cobalt chloride (CoCl2)-treated (trophoblast-derived) HTR-8/SVneo cells for mimicking hypoxic injuries. Cell counting kit-8 (CCK-8), dihydroethidium (DHE) probe, western blotting, immunofluorescence staining, migration and invasion assay were employed in the current study. Our data showed that placentas from PE women exhibited increased level of reactive oxygen species (ROS) and apoptotic index than those in normal pregnancy. Our in vitro study showed that CoCl2 enhanced ROS generation and apoptosis in HTR-8/SVneo cells through the activation of the p38 mitogen-activated protein kinase (p38 MAPK)/nuclear factor-κB (NF-κB) pathway. ACh significantly decreased hypoxia-induced ROS generation and the resulting apoptosis, accompanied by lowered phosphorylation of p38 MAPK and NF-κB. Western blotting analysis further confirmed that ACh decreased the ratio of pp38 MAPK/p38 MAPK, p-NF-κB/NF-κB, Bax/Bcl-2 and cleaved Caspase-3/Caspase-3. Besides, ACh promoted cell invasion and migration ability under hypoxic conditions. Atropine, the muscarinic receptor antagonist, abolished ACh’s effects mentioned above. Overall, our data showed that ACh exerted protective effects on hypoxia-induced oxidative stress and apoptosis in trophoblast cells via muscarinic receptors, indicating that improved vagal activity may be of therapeutic value in PE management.

中文翻译：

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乙酰胆碱通过 p38 MAPK/NF-κB 通路改善缺氧诱导的滋养层细胞氧化应激和凋亡

缺氧诱导的氧化应激和滋养层细胞凋亡参与了先兆子痫（PE）的发病机制。大量研究报告称，主要的迷走神经递质乙酰胆碱 (ACh) 在各种疾病模型中显示出抗氧化和抗凋亡作用。然而，ACh 在缺氧滋养层中的作用仍然未知。在这里，我们检测了人胎盘的凋亡水平，并探讨了乙酰胆碱对氯化钴 (CoCl2) 处理（滋养层衍生）HTR-8/SVneo 细胞模拟缺氧损伤的作用。本研究采用细胞计数试剂盒 8 (CCK-8)、二氢乙锭 (DHE) 探针、蛋白质印迹、免疫荧光染色、迁移和侵袭测定。我们的数据显示，与正常妊娠相比，PE 女性的胎盘表现出更高的活性氧 (ROS) 水平和凋亡指数。我们的体外研究表明，CoCl2 通过激活 p38 丝裂原活化蛋白激酶 (p38 MAPK)/核因子-κB (NF-κB) 通路来增强 HTR-8/SVneo 细胞中 ROS 的产生和凋亡。ACh 显着降低了缺氧诱导的 ROS 生成和由此产生的细胞凋亡，同时降低了 p38 MAPK 和 NF-κB 的磷酸化。Western印迹分析进一步证实ACh降低了pp38 MAPK/p38 MAPK、p-NF-κB/NF-κB、Bax/Bcl-2和切割的Caspase-3/Caspase-3的比率。此外，乙酰胆碱促进细胞在缺氧条件下的侵袭和迁移能力。毒蕈碱受体拮抗剂阿托品消除了上述 ACh 的作用。全面的，