Department of Medical Biology, Faculty of Medicine, Aydin Adnan Menderes University, Aydin, Turkey.
Department of Medical Biology, Faculty of Medicine, Aydin Adnan Menderes University, Aydin, Turkey.
Placenta. 2023 Aug;139:200-211. doi: 10.1016/j.placenta.2023.07.007. Epub 2023 Jul 11.
Placental inflammation is implicated in the pathophysiology of many pregnancy complications, including fetal growth restriction, preeclampsia, gestational diabetes, and choriocarcinoma. Mitochondrial dysfunction, one of the outcomes of placental inflammation, is characterized by loss of membrane potential, accumulation of oxygen radicals, mitochondrial protein folding defects, and disturbances in mitochondrial dynamics. Protein kinase R (PKR) is stimulated by double-stranded RNA and bacterial endotoxins in the presence of pathogens and is a critical immune response enzyme. PKR is also correlated with the cell death response during endoplasmic reticulum stress. In this study, we aim to investigate the effects of PKR activity stimulated by lipopolysaccharide (LPS), and double-stranded RNA analog (Poly I:C) on mitochondrial unfolded protein response (mtUPR), mitochondrial membrane potential, apoptosis, and oxidative stress in placental trophoblasts.
We applied LPS and Poly I:C to BeWo cells to induce PKR activation. In addition, cells were treated with 2-aminopurine (2-AP) to inhibit the kinase activity of PKR. Protein levels of ATP-dependent Clp protease proteolytic subunit (CLPP) and heat shock protein 60 (HSP60) were determined after treatments. Apoptotic markers were detected by real-time PCR and flow cytometry. PKR-induced reactive oxygen radicals (ROS) accumulation and mitochondrial membrane potential change were assessed by flow cytometry.
It was determined that PKR activation-induced apoptosis in BeWo cells by reducing the levels of mtUPR proteins (CLPP and HSP60) and caused a decrease in mitochondrial membrane potential. PKR inhibition was sufficient for decreases in apoptotic markers and caused a reduction in the ratio of depolarized and ROS (+) cells.
Our results showed that LPS and Poly I:C administration stimulated PKR in BeWo cells in vitro. Furthermore, PKR activation is correlated with the levels of proteins involved in mitochondrial homeostasis and apoptosis. Our findings will contribute to understanding the role of PKR activation in placental inflammation and related diseases.
胎盘炎症与许多妊娠并发症的病理生理学有关，包括胎儿生长受限、子痫前期、妊娠期糖尿病和绒癌。胎盘炎症的后果之一是线粒体功能障碍，其特征是膜电位丧失、氧自由基积累、线粒体蛋白折叠缺陷以及线粒体动力学紊乱。蛋白激酶 R（PKR）在病原体存在下被双链 RNA 和细菌内毒素刺激，是一种关键的免疫反应酶。PKR还与内质网应激过程中的细胞死亡反应有关。在这项研究中，我们旨在研究脂多糖（LPS）和双链 RNA 类似物（Poly I:C）刺激 PKR 活性对胎盘滋养层细胞线粒体未折叠蛋白反应（mtUPR）、线粒体膜电位、凋亡和氧化应激的影响。
我们应用 LPS 和 Poly I:C 诱导 BeWo 细胞中 PKR 的激活。此外，还通过 2-氨基嘌呤（2-AP）处理来抑制 PKR 的激酶活性。处理后测定 ATP 依赖性 Clp 蛋白酶解亚基（CLPP）和热休克蛋白 60（HSP60）的蛋白水平。通过实时 PCR 和流式细胞术检测凋亡标志物。通过流式细胞术评估 PKR 诱导的活性氧自由基（ROS）积累和线粒体膜电位变化。
确定 PKR 激活通过降低 mtUPR 蛋白（CLPP 和 HSP60）水平诱导 BeWo 细胞凋亡，并导致线粒体膜电位降低。PKR 抑制足以降低凋亡标志物并降低去极化和 ROS（+）细胞的比例。
我们的结果表明，LPS 和 Poly I:C 给药在体外刺激 BeWo 细胞中的 PKR。此外，PKR 激活与参与线粒体动态平衡和凋亡的蛋白质水平相关。我们的发现将有助于理解 PKR 激活在胎盘炎症和相关疾病中的作用。
