Liu Xin-Xin, Ke Xue-Ying, Jiang Chen, Bo Ling-Wei, Sun Nan, Li Lin-Lin, Qin Shi-Qi, He Jin-Chen, Ren Jia-Lin, Wu Qian-Qian, Li Shuai-Zhen, Yang Jia-Lei, Yu Lan-Ling, Lu Qi-Yong, Liu Li-Zhe, Li Wen-Ya, Xian Xiao-Hui, Zhang Li-Nan
Hebei Key Laboratory of Critical Disease Mechanism and Intervention, Department of Pathophysiology, Neuroscience Research Center, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang 050017, China; Department of Science and Education, Xingtai People's Hospital, 818 Xiangdu North Road, Xingtai 054001, China.
Basic Medical College, Hebei Medical University, Shijiazhuang 050017, China.
Phytomedicine. 2025 Jan;136:156349. doi: 10.1016/j.phymed.2024.156349. Epub 2024 Dec 27.
In China, stroke is the primary cause of adult death and disability. Because of the increased rate of blood vessel reperfusion, it is important to prevent cerebral ischemia-reperfusion injury, in which glutamate (Glu) excitotoxicity plays a critical role. The most important Glu transporter, GLT-1, is essential for the regulation of Glu, which is dependent on Na-K-ATPase (NKA)-induced ion concentration gradient differences. EGCG, a substance found in tea polyphenols, can reduce infarct areas in ischemia-reperfusion models, reduce stroke incidence, and prolong life in which NKA is involved.
In this study, we investigated the potential of EGCG in protecting against cerebral ischemia-reperfusion injury by regulating the interaction between NKA and GLT-1.
This study was designed to investigate the protective effects of EGCG against cerebral ischemia-reperfusion injury by modulating the interaction between NKA and GLT-1, utilizing both the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in co-cultures of rat hippocampal neurons and astrocytes.
The neuronal survival rate was assessed using CCK8, and the cerebral infarction area and neurological function were determined by TTC staining and neurological deficit scores. NKA activity was measured using an inorganic phosphorous detection method, and NKA and GLT-1 expression was detected using western blotting. The interaction between NKAα and GLT-1 was identified by co-immunoprecipitation (CoIP) assay, laser confocal microscopy, and Imaris 3D confocal rendering technology. An adenovirus vector with overexpression of NKAα was constructed, packaged, and injected into the rat lateral ventricle. Neurological function and the cerebral infarction area were identified, and the interaction between NKAα and GLT-1 was identified using CoIP assay.
EGCG reduced the infarction area and neurological deficit scores, restored NKA activity, alleviated the decrease in membrane NKAα and GLT-1 expression, and relieved the uncoupling of NKAα and GLT-1 in the hippocampal CA1 after rat MCAO/R injury. By promoting the coupling of NKAα and GLT-1 in rat MCAO/R models, overexpression of NKAα2 reduced the cerebral infarction area and neurological impairment scores.
EGCG improved cerebral ischemia-reperfusion injury by restoring NKA activity and increasing membrane GLT-1 expression due to NKA-GLT-1 interaction. For the first time, our findings demonstrate the critical role that NKA and GLT-1 colocalization plays in cerebral ischemia-reperfusion damage. Our findings provide new strategic directions for the pathogenesis and prevention of thrombolytic injury in the clinical treatment of stroke, while also serving as a basis for further development and utilization of EGCG.
在中国，中风是成人死亡和残疾的主要原因。由于血管再灌注率增加，预防脑缺血再灌注损伤很重要，其中谷氨酸（Glu）兴奋性毒性起关键作用。最重要的Glu转运体GLT-1对Glu的调节至关重要，这依赖于钠钾ATP酶（NKA）诱导的离子浓度梯度差异。表没食子儿茶素没食子酸酯（EGCG）是茶多酚中的一种物质，可减少缺血再灌注模型中的梗死面积，降低中风发生率，并延长涉及NKA的生存期。
在本研究中，我们研究了EGCG通过调节NKA与GLT-1之间的相互作用来预防脑缺血再灌注损伤的潜力。
本研究旨在利用大鼠大脑中动脉闭塞/再灌注（MCAO/R）模型以及大鼠海马神经元和星形胶质细胞共培养中的氧葡萄糖剥夺/复氧（OGD/R）模型，通过调节NKA与GLT-1之间的相互作用来研究EGCG对脑缺血再灌注损伤的保护作用。
使用CCK8评估神经元存活率，通过TTC染色和神经功能缺损评分确定脑梗死面积和神经功能。使用无机磷检测方法测量NKA活性，通过蛋白质免疫印迹法检测NKA和GLT-1的表达。通过免疫共沉淀（CoIP）分析、激光共聚焦显微镜和Imaris 3D共聚焦渲染技术鉴定NKAα与GLT-1之间的相互作用。构建、包装过表达NKAα的腺病毒载体并注入大鼠侧脑室。鉴定神经功能和脑梗死面积，并使用CoIP分析鉴定NKAα与GLT-1之间的相互作用。
EGCG减少了梗死面积和神经功能缺损评分，恢复了NKA活性，减轻了海马CA1区膜NKAα和GLT-1表达的下降，并缓解了大鼠MCAO/R损伤后NKAα与GLT-1的解偶联。通过促进大鼠MCAO/R模型中NKAα与GLT-1的偶联，NKAα2的过表达降低了脑梗死面积和神经损伤评分。
EGCG通过恢复NKA活性并由于NKA-GLT-1相互作用增加膜GLT-1表达来改善脑缺血再灌注损伤。我们的研究结果首次证明了NKA与GLT-1共定位在脑缺血再灌注损伤中起关键作用。我们的研究结果为中风临床治疗中溶栓损伤的发病机制和预防提供了新的战略方向，同时也为EGCG的进一步开发利用提供了依据。
