Department of Neurology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, Jiangsu 210008, PR China; Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu 210008, PR China.
Department of Neurology, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, Jiangsu 210008, PR China; Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu 210008, PR China.
Life Sci. 2018 Jul 1;204:46-54. doi: 10.1016/j.lfs.2018.04.056. Epub 2018 May 1.
Oxidative stress is one of the most important pathological mechanisms which could aggravate ischemic stroke injury. In order to seek for better treatment therapies to alleviate stroke injury, novel chemicals have been synthetized. In the present study, a new compound 4-((5-(tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2- hydroxybenzoic acid, named LX009, was used to determine whether it could reduce the oxidative stress caused by oxygen-glucose deprivation (OGD)/reperfusion (RP) and exert neuroprotective effect both in mouse Neuro 2A (N2A) neuroblastoma cells and mouse primary cortical neurons.
OGD/RP was performed as an in vitro model to mimic the pathologic process of ischemic stroke. We explored the anti-apoptosis effect of LX009 through CCK8 assay, calcein acetoxymethylester/propidium iodide (calcein-AM/PI) staining, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit, caspase-3 activity assay. Besides, the anti-oxidative stress effect of the drug was determined by intracellular reactive oxygen species (ROS) detection, nitrite analysis, measurement of mitochondrial membrane potential (MMP), intracellular catalase (CAT) and Mn-superoxide dismutase (Mn-SOD) activity.
Our results indicated that LX009 could alleviate OGD/RP-induced cell apoptosis. Furthermore, OGD/RP induced oxidative stress could be reserved by LX009, including measurements of intracellular ROS production, MMP, CAT and Mn-SOD activity. Mechanistically, the phosphorylation level of Akt, as well as the expression of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were elevated after LX009 treatment.
Our present study indicated that LX009 might have the potential to be an anti-oxidative stress agent in the future.
氧化应激是加重缺血性中风损伤的最重要病理机制之一。为了寻找更好的治疗方法来减轻中风损伤，人们合成了新型化合物 4-((5-(叔丁基)-3-氯-2-羟基苄基)氨基)-2-羟基苯甲酸，命名为 LX009。本研究旨在探讨 LX009 是否能减轻氧葡萄糖剥夺（OGD）/再灌注（RP）引起的氧化应激，并在小鼠神经母细胞瘤（N2A）和小鼠原代皮质神经元中发挥神经保护作用。
OGD/RP 作为体外模型模拟缺血性中风的病理过程。我们通过 CCK8 测定、钙黄绿素乙酰氧基甲酯/碘化丙啶（calcein-AM/PI）染色、膜联蛋白 V-荧光素异硫氰酸酯（FITC）/碘化丙啶（PI）凋亡试剂盒、半胱天冬酶-3 活性测定，探讨 LX009 的抗细胞凋亡作用。此外，通过检测细胞内活性氧（ROS）、亚硝酸盐分析、线粒体膜电位（MMP）、细胞内过氧化氢酶（CAT）和锰超氧化物歧化酶（Mn-SOD）活性，确定药物的抗氧化应激作用。
我们的结果表明，LX009 可减轻 OGD/RP 诱导的细胞凋亡。此外，LX009 还能减轻 OGD/RP 诱导的氧化应激，包括细胞内 ROS 产生、MMP、CAT 和 Mn-SOD 活性的测定。机制上，LX009 处理后 Akt 的磷酸化水平以及核因子-E2 相关因子 2（Nrf2）和血红素加氧酶-1（HO-1）的表达均升高。
本研究表明，LX009 将来可能具有成为抗氧化应激剂的潜力。
