Laboratory of Neurochemistry and Cell Biology, Department of Biochemistry and Biophysics, Institute of Health Sciences, Federal University of Bahia - UFBA, Salvador, Bahia, 40110-902, Brazil.
School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michael's Building, White Swan Road, Portsmouth, PO1 2DT, UK.
BMC Complement Med Ther. 2023 May 11;23(1):154. doi: 10.1186/s12906-023-03959-0.
Stroke is a leading cause of death and disability worldwide. A major factor in brain damage following ischemia is excitotoxicity caused by elevated levels of the neurotransmitter glutamate. In the brain, glutamate homeostasis is a primary function of astrocytes. Amburana cearensis has long been used in folk medicine and seed extract obtained with dichloromethane (EDAC) have previously been shown to exhibit cytoprotective activity in vitro. The aim of the present study was to analyse the activity of EDAC in hippocampal brain slices.
We prepared a dichloromethane extract (EDAC) from A. cearensis seeds and characterized the chemical constituents by 1H and 13C-NMR. Hippocampal slices from P6-8 or P90 Wistar rats were used for cell viability assay or glutamate uptake test. Hippocampal slices from P10-12 transgenic mice SOX10-EGFP and GFAP-EGFP and immunofluorescence for GS, GLAST and GLT1 were used to study oligodendrocytes and astrocytes.
Astrocytes play a critical role in glutamate homeostasis and we provide immunohistochemical evidence that in excitotoxicity EDAC increased expression of glutamate transporters and glutamine synthetase, which is essential for detoxifying glutamate. Next, we directly examined astrocytes using transgenic mice in which glial fibrillary acidic protein (GFAP) drives expression of enhanced green fluorescence protein (EGFP) and show that glutamate excitotoxicity caused a decrease in GFAP-EGFP and that EDAC protected against this loss. This was examined further in the oxygen-glucose deprivation (OGD) model of ischemia, where EDAC caused an increase in astrocytic process branching, resulting in an increase in GFAP-EGFP. Using SOX10-EGFP reporter mice, we show that the acute response of oligodendrocytes to OGD in hippocampal slices is a marked loss of their processes and EDAC protected oligodendrocytes against this damage.
This study provides evidence that EDAC is cytoprotective against ischemia and glutamate excitotoxicity by modulating astrocyte responses and stimulating their glutamate homeostatic mechanisms.
中风是全球范围内导致死亡和残疾的主要原因。缺血后引起的脑损伤的一个主要因素是神经递质谷氨酸水平升高引起的兴奋性毒性。在大脑中，谷氨酸的动态平衡是星形胶质细胞的主要功能之一。安布瑞纳·塞亚内斯在民间医学中一直被使用，并且已经证明用二氯甲烷（EDAC）提取的种子提取物具有体外细胞保护活性。本研究的目的是分析 EDAC 在海马脑片中的活性。
我们从 A. cearensis 种子中制备了二氯甲烷提取物（EDAC），并通过 1H 和 13C-NMR 对其化学组成进行了表征。使用 P6-8 或 P90 Wistar 大鼠的海马脑片进行细胞活力测定或谷氨酸摄取试验。使用 P10-12 转基因小鼠 SOX10-EGFP 和 GFAP-EGFP 的海马脑片以及 GS、GLAST 和 GLT1 的免疫荧光，研究少突胶质细胞和星形胶质细胞。
星形胶质细胞在谷氨酸动态平衡中起着关键作用，我们提供了免疫组织化学证据表明，在兴奋性毒性中，EDAC 增加了谷氨酸转运体和谷氨酰胺合成酶的表达，这对于解毒谷氨酸至关重要。接下来，我们使用 glial fibrillary acidic protein (GFAP) 驱动增强型绿色荧光蛋白 (EGFP) 表达的转基因小鼠直接研究星形胶质细胞，并表明谷氨酸兴奋性毒性导致 GFAP-EGFP 的减少，而 EDAC 可防止这种减少。在缺血的氧葡萄糖剥夺 (OGD) 模型中进一步研究了这一点，其中 EDAC 导致星形胶质细胞突起分支增加，导致 GFAP-EGFP 增加。使用 SOX10-EGFP 报告小鼠，我们表明海马脑片中少突胶质细胞对 OGD 的急性反应是其过程的明显丧失，而 EDAC 可防止少突胶质细胞受到这种损伤。
本研究提供了证据表明，EDAC 通过调节星形胶质细胞反应并刺激其谷氨酸稳态机制，对缺血和谷氨酸兴奋性毒性具有细胞保护作用。
