Luo Qingqing, Chai Xinqun, Xin Xiaoyan, Ouyang Weixiang, Deng Feitao
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Department of Obstetrics, The Affiliated Hospital of Southwest Medical University, Luzhou, China.
Diabetol Metab Syndr. 2023 Mar 17;15(1):49. doi: 10.1186/s13098-022-00974-y.
Gestational diabetes mellitus (GDM) is associated with retarded lung development and poor lung health in offspring. Mammalian target of rapamycin (mTOR) is a key regulator of vasculogenesis and angiogenesis. The aim of this study was to investigate the role mTOR plays in pulmonary vasculogenesis during fetal lung development under maternal hyperglycemia.
First, GDM was induced via streptozotocin injection in pregnant C57BL/6 mice before the radial alveolar count (RAC) in the fetal lungs was assessed using hematoxylin and eosin staining. The angiogenic ability of the cultured primary mouse fetal lung endothelial cells (MFLECs) was then assessed using the tube formation assay technique, while western blot and real-time polymerase chain reaction were performed to determine the expression of mTOR, regulatory-associated protein of mTOR (Raptor), rapamycin-insensitive companion of mTOR (Rictor), stress-activated protein kinase interacting protein 1 (Sin1), G protein beta subunit-like protein (GβL), Akt, tumor necrosis receptor associated factor-2 (TRAF2), and OTU deubiquitinase 7B (OTUD7B) in both the fetal lung tissues and the cultured MFLECs. Immunoprecipitation assays were conducted to evaluate the status of GβL-ubiquitination and the association between GβL and mTOR, Raptor, Rictor, and Sin1 in the cultured MFLECs.
The GDM fetal lungs exhibited a decreased RAC and reduced expression of von Willebrand factor, CD31, and microvessel density. The high glucose level reduced the tube formation ability in the MFLECs, with the mTOR, p-mTOR, p-Raptor, and TRAF2 expression upregulated and the p-Rictor, p-Sin1, p-Akt, and OTUD7B expression downregulated in both the GDM fetal lungs and the high-glucose-treated MFLECs. Meanwhile, GβL-ubiquitination was upregulated in the high-glucose-treated MFLECs along with an increased GβL/Raptor association and decreased GβL/Rictor and GβL/Sin1 association. Furthermore, TRAF2 knockdown inhibited the high-glucose-induced GβL-ubiquitination and GβL/Raptor association and restored the tube formation ability of the MFLECs.
Maternal hyperglycemia inhibits pulmonary vasculogenesis during fetal lung development by promoting GβL-ubiquitination-dependent mTORC1 assembly.
妊娠期糖尿病（GDM）与子代肺发育迟缓及肺健康状况不佳有关。雷帕霉素哺乳动物靶蛋白（mTOR）是血管生成和血管新生的关键调节因子。本研究旨在探讨mTOR在母体高血糖状态下胎儿肺发育过程中肺血管生成所起的作用。
首先，通过给怀孕的C57BL/6小鼠注射链脲佐菌素诱导GDM，然后用苏木精-伊红染色评估胎儿肺中的放射状肺泡计数（RAC）。接着，采用管形成试验技术评估培养的原代小鼠胎儿肺内皮细胞（MFLEC）的血管生成能力，同时进行蛋白质免疫印迹法和实时聚合酶链反应，以确定胎儿肺组织和培养的MFLEC中mTOR、mTOR调节相关蛋白（Raptor）、mTOR不敏感伴侣蛋白（Rictor）、应激激活蛋白激酶相互作用蛋白1（Sin1）、G蛋白β亚基样蛋白（GβL）、Akt、肿瘤坏死受体相关因子2（TRAF2）和OTU去泛素化酶7B（OTUD7B）的表达。进行免疫沉淀试验，以评估培养的MFLEC中GβL泛素化状态以及GβL与mTOR、Raptor、Rictor和Sin1之间的关联。
GDM胎儿肺的RAC降低，血管性血友病因子、CD31表达及微血管密度减少。高糖水平降低了MFLEC的管形成能力，在GDM胎儿肺组织和高糖处理的MFLEC中，mTOR、磷酸化mTOR、磷酸化Raptor和TRAF2表达上调，而磷酸化Rictor、磷酸化Sin1、磷酸化Akt和OTUD7B表达下调。同时，高糖处理的MFLEC中GβL泛素化上调，GβL/Raptor关联增加，GβL/Rictor和GβL/Sin1关联减少。此外，敲低TRAF2可抑制高糖诱导的GβL泛素化和GβL/Raptor关联，并恢复MFLEC的管形成能力。
母体高血糖通过促进GβL泛素化依赖性mTORC1组装，抑制胎儿肺发育过程中的肺血管生成。
