Department of Neurology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2022 Oct 28;47(10):1332-1344. doi: 10.11817/j.issn.1672-7347.2022.220162.
Cerebral infarction is a subtype of stroke with high incidence and disability rate. Ischemia reperfusion injury (IRI) is the key point of cerebral infarction treatment. UbiA prenyltransferase domain containing 1 (UBIAD1) is a kind of enzyme with various biological functions including electron transport in mitochondrial respiratory chain, lipid metabolism, and oxidative stress which are related to IRI. The purpose of this study aims to determine the neuroprotective effects and the underlying mechanisms of UBIAD1 in cerebral IRI.
We employed oxygen-glucose deprivation/reoxygenation (OGD/R) model in mouse neuroblastoma Neuro2a (N2a) cells to mimic cerebral IRI. Lentivirus vector over-expressed UBIAD1 was transfacted into N2a cells to maintain high and stable expression of UBIAD1. In the first part of the experiment, N2a cells were divided into 5 groups: A non-OGD (N2a cells without exposure to OGD) group, groups of reoxygenation 0, 4, 12 and 24 h after 4 h of OGD, respectively. In the second part of the experiment, N2a cells were divided into 6 groups: A Con (normal cell)+non-OGD group, an EV (cell transfected with empty vector)+non-OGD group, an OE (over-expressed UBIAD1)+non-OGD group, a Con+OGD/R group, an EV+OGD/R group, and an OE+OGD/R group. In the third part, the N2a cells were divided into 8 groups: A Con+non-OGD group, an OE+non-OGD group, a Con+non-OGD+nNOS inhibitior 7-nitroindazole (7-NI) group, an OE+non-OGD+7-NI group, a Con+OGD/R group, an OE+OGD/R group, a Con+OGD/R+7-NI group, and an OE+OGD/R+7-NI group. The morphological changes of Golgi apparatus were observed under the confocal laser scanning microscope. The mRNA and protein levels of , secretory pathway Ca-ATPase isoform 1 (), and were determined by real-time PCR and Western blotting, respectively. Cell apoptosis rate was detected with flow cytometry; cell viability was detected with MTT assay, and NO release was determined with Griess assay.
Compared with the non-OGD group, the expression levels of mRNA and protein in N2a cells in the groups of 0, 4, 12 and 24 h reoxygenation after OGD 4 h decreased significantly (<0.05 or <0.01), and the longer the reoxygenation time, the more significant the reduction of expression. Compared with the Con+OGD/R group and the EV+OGD/R group, mRNA and protein levels of and were increased (<0.05 or <0.01), the apoptosis rate was decreased (all <0.01), and the cell viability was increased (all <0.01) in the OE+OGD/R group. The Golgi fragmentation was less in the OE+OGD/R group than that in the Con+ OGD/R group and the EV+OGD/R group. The mRNA and protein levels of endothelial NOS () and neuronal NOS () were decreased (<0.05 or <0.01), and the level of NO was decreased (all <0.01) in the groups over-expressed UBIAD1 (OE+non-OGD group vs Con+non-OGD group, OE+OGD/R group vs Con+OGD/R group). The level of NO and apoptosis rate of N2a cells were decreased (all <0.01) in the the groups pretreated with 7-NI (Con+OGD/R+7-NI group vs Con+OGD/R group, OE+OGD/R+7-NI group vs OE+OGD/R group).
UBIAD1 may exerts protective effects on OGD/R induced N2a cells by ameliorating Golgi apparatus dysfunction via the nNOS/NO pathway.
脑梗死是一种具有高发病率和高致残率的中风亚型。缺血再灌注损伤（IRI）是脑梗死治疗的关键。泛 A prenyltransferase 结构域包含 1（UBIAD1）是一种具有多种生物功能的酶，包括线粒体呼吸链中的电子传递、脂质代谢和氧化应激，这些都与 IRI 有关。本研究旨在确定 UBIAD1 在脑 IRI 中的神经保护作用及其潜在机制。
我们使用氧葡萄糖剥夺/再氧合（OGD/R）模型在小鼠神经母细胞瘤Neuro2a（N2a）细胞中模拟脑 IRI。过表达 UBIAD1 的慢病毒载体转染 N2a 细胞，以维持 UBIAD1 的高且稳定表达。在实验的第一部分，将 N2a 细胞分为 5 组：非 OGD（未暴露于 OGD 的 N2a 细胞）组、OGD 后再氧合 0、4、12 和 24 h 组，分别。在实验的第二部分，将 N2a 细胞分为 6 组：Con（正常细胞）+非 OGD 组、EV（转染空载体的细胞）+非 OGD 组、OE（过表达 UBIAD1）+非 OGD 组、Con+OGD/R 组、EV+OGD/R 组和 OE+OGD/R 组。在实验的第三部分，将 N2a 细胞分为 8 组：Con+非 OGD 组、OE+非 OGD 组、Con+非 OGD+nNOS 抑制剂 7-硝基吲唑（7-NI）组、OE+非 OGD+7-NI 组、Con+OGD/R 组、OE+OGD/R 组、Con+OGD/R+7-NI 组和 OE+OGD/R+7-NI 组。通过共聚焦激光扫描显微镜观察高尔基体形态变化。实时 PCR 和 Western blot 分别检测 、分泌途径 Ca-ATPase 同工型 1（）和 的 mRNA 和蛋白水平。用流式细胞术检测细胞凋亡率；用 MTT 法检测细胞活力，用 Griess 法检测 NO 释放。
与非 OGD 组相比，OGD 4 h 后再氧合 0、4、12 和 24 h 组 N2a 细胞中 mRNA 和蛋白表达水平明显降低（<0.05 或 <0.01），再氧合时间越长，表达降低越明显。与 Con+OGD/R 组和 EV+OGD/R 组相比，OE+OGD/R 组 、 mRNA 和蛋白水平升高（<0.05 或 <0.01），细胞凋亡率降低（均<0.01），细胞活力增加（均<0.01）。OE+OGD/R 组高尔基体片段化较 Con+OGD/R 组和 EV+OGD/R 组少。OE+非 OGD 组和 OE+OGD/R 组内皮型一氧化氮合酶（）和神经元型一氧化氮合酶（）mRNA 和蛋白水平降低（<0.05 或 <0.01），NO 水平降低（均<0.01）。与 Con+OGD/R 组相比，7-NI 预处理组（Con+OGD/R+7-NI 组与 Con+OGD/R 组、OE+OGD/R+7-NI 组与 OE+OGD/R 组）中 N2a 细胞的 NO 水平和凋亡率降低（均<0.01）。
UBIAD1 可能通过 nNOS/NO 途径改善高尔基体功能障碍对 OGD/R 诱导的 N2a 细胞发挥保护作用。
