Na-K-ATPase/GLT-1 interaction participates in EGCG protection against cerebral ischemia-reperfusion injury in rats.

BACKGROUND

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.

PURPOSE

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.

STUDY DESIGN

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.