Department of Anatomy, Dongguk University College of Medicine, Gyeongju 38066, Republic of Korea; Department of Fisheries Biology and Genetics, Patuakhali Science and Technology University, Patuakhali 8602, Bangladesh.
Department of Anatomy, Dongguk University College of Medicine, Gyeongju 38066, Republic of Korea; Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.
Phytomedicine. 2021 Jan;81:153415. doi: 10.1016/j.phymed.2020.153415. Epub 2020 Nov 19.
Neuronal excitotoxicity induces a plethora of downstream signaling pathways, resulting in the calcium overload-induced excitotoxic cell death, a well-known phenomenon in cerebrovascular and neurodegenerative disorders. The naturally occurring phytosterol, stigmasterol (ST) is known for its potential role in cholesterol homeostasis and neuronal development. However, the ability of ST to protect against the induced excitotoxicity in hippocampal neurons has not been investigated yet.
The present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons.
After H/R, neurons were initially subjected to trypan blue exclusion assay for the assessment of cell viability. Live staining using fluorescence dyes namely JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide), DCFDA (2',7'-dichlorofluorescein diacetate) and FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) were used to measure MMP, ROS and synaptic vesicle pool size. Immunostaining was performed to analyze the expression levels of vesicular glutamate transporter 1 (VGLUT1), N-methyl-D-acetate receptor subunit 2B (GluN2B), LC3BII, p62, and PTEN induced protein kinase 1 (PINK1) in neuron after H/R. Western blotting was carried out to measure the protein expression of GluN2B. The molecular dynamics simulation was employed to elucidate the LXRβ agonistic conformation of ST.
Pre-incubation of neuronal cultures with ST (20 μM) protected against excitotoxicity, and attenuated reactive oxygen species (ROS) generation, double-stranded DNA break, and mitochondrial membrane potential (MMP) loss. ST treatment also resulted in the downregulation of the expressions of VGLUT1 and GluN2B and the reduction of the size of recyclable synaptic vesicle (SV) pool. Like LXRβ agonist GW3695, ST suppressed the expression of GluN2B. Furthermore, ST induced mitophagy through upregulating the expressions of LC3BII, p62, and PINK1. The molecular simulation study showed that ST interacted with the ligand binding domain of liver X receptor β (LXRβ), a known binding receptor of ST, through multiple hydrogen bonding.
Collectively, these findings revealed that ST exhibited a promising neuroprotective effect by regulating both pre- and post-synaptic events following H/R, particularly, attenuation of GluN2B-mediated excitotoxicity and oxidative stress, and induction of mitophagy, and suggested that ST might be a therapeutic promise against ischemic stroke and its associated neurological disorders.
神经元兴奋毒性诱导大量下游信号通路，导致钙超载诱导的兴奋毒性细胞死亡，这是脑血管和神经退行性疾病中的一个众所周知的现象。天然存在的植物甾醇stigmasterol（ST）因其在胆固醇稳态和神经元发育中的潜在作用而闻名。然而，ST 防止海马神经元兴奋毒性的能力尚未被研究过。
本研究旨在探讨 ST 是否能防止海马神经元缺氧/复氧（H/R）诱导的兴奋毒性。
在 H/R 后，神经元首先通过台盼蓝排除试验评估细胞活力。使用荧光染料 JC-1（5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基-碳氰化碘化物）、DCFDA（2',7'-二氯荧光素二乙酸酯）和 FM1-43（N-（3-三乙铵丙基）-4-（4-（二丁基氨基）-亚苄基）-4-（二丁基氨基）-亚苄基）-4-（二丁基氨基）-亚苄基）测量 MMP、ROS 和可回收突触囊泡池的大小。免疫染色分析神经元中 VGLUT1、N-甲基-D-天冬氨酸受体亚基 2B（GluN2B）、LC3BII、p62 和 PTEN 诱导蛋白激酶 1（PINK1）的表达水平。Western blot 用于测量 GluN2B 的蛋白表达。采用分子动力学模拟阐明 ST 的 LXRβ激动构象。
神经元培养物中 ST（20 μM）的预孵育可防止兴奋毒性，并减轻活性氧（ROS）生成、双链 DNA 断裂和线粒体膜电位（MMP）丧失。ST 处理还导致 VGLUT1 和 GluN2B 的表达下调和可回收突触囊泡（SV）池的大小减小。与 LXRβ 激动剂 GW3695 一样，ST 抑制了 GluN2B 的表达。此外，ST 通过上调 LC3BII、p62 和 PINK1 的表达诱导自噬。分子模拟研究表明，ST 通过多个氢键与已知 ST 结合受体肝 X 受体β（LXRβ）的配体结合域相互作用。
总的来说，这些发现表明 ST 通过调节 H/R 后突触前和突触后的事件显示出有希望的神经保护作用，特别是减轻 GluN2B 介导的兴奋毒性和氧化应激，并诱导自噬，表明 ST 可能是治疗缺血性中风及其相关神经障碍的有希望的药物。
