Mitochondrial BKCa Mediates the Protective Effect of Low-Dose Ethanol Preconditioning on Oxygen-Glucose Deprivation and Reperfusion-Induced Neuronal Apoptosis

Fang Su; Huajun Yang; Anchen Guo; Zhengyi Qu; Jianping Wu; Qun Wang

doi:10.3389/fphys.2021.719753

Mitochondrial BK_Ca Mediates the Protective Effect of Low-Dose Ethanol Preconditioning on Oxygen-Glucose Deprivation and Reperfusion-Induced Neuronal Apoptosis

Front Physiol. 2021 Oct 25:12:719753. doi: 10.3389/fphys.2021.719753. eCollection 2021.

Authors

Fang Su^{1

2

3}, Huajun Yang², Anchen Guo^{3

4}, Zhengyi Qu¹, Jianping Wu⁵, Qun Wang^{2

3

4}

Affiliations

¹ Department of Neurology, The Fourth Hospital of Harbin Medical University, Harbin, China.
² Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
³ China National Clinical Research Center for Neurological Diseases, Beijing, China.
⁴ Collaborative Innovation Center for Brain Disorders, Beijing Institute of Brain Disorders, Capital Medical University, Beijing, China.
⁵ Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, China.

Abstract

Ischemia-reperfusion (I/R) injury contributes to the morbidity and mortality of ischemic strokes. As an in vitro model, oxygen-glucose deprivation and reperfusion (OGD/R) exposure induces neuronal injury. Low-dose ethanol preconditioning (EtOH-PC) was reported to alleviate neuronal apoptosis during OGD/R. However, whether the mitochondrial BK_Ca (mitoBK_Ca) channel is involved in the neuroprotective effect of EtOH-PC during OGD/R is not clearly defined. This study attempts to explore the mediation of the mitoBK_Ca channel in the neuroprotective effect of EtOH-PC on OGD/R-induced neuronal apoptosis and the underlying mechanisms. OGD/R model was established using primary cortical neurons that were preincubated with ethanol. Subsequently, the cell viability was measured by CCK-8 assay, and the apoptotic cells were determined by TUNEL assay. Annexin V/7-AAD staining and mitochondrial membrane potential using JC-10 were detected by flow cytometry. Western blot analysis was performed to check the apoptosis-related proteins. In the mixed primary culture, 95% neurofilament-positive cells were cortical neurons. Low-dose EtOH-PC (10 mmol/L) for 24 h significantly attenuated the OGD2h/R24h-induced neuronal apoptosis through activating the BK_Ca channel. Further investigations suggested that ethanol pretreatment increased the mitochondrial membrane potential (MMP) and downregulated the production of cleaved caspase 3 in OGD/R-injured neurons by activating the mitoBK_Ca channel. Low-dose ethanol pretreatment significantly attenuated the OGD/R-induced neuronal apoptosis mediated by the mitoBK_Ca channel which modulated the mitochondrial function by impeding the uncontrolled opening of mitochondrial permeability transition pore (MPTP).

Keywords: apoptosis; ethanol preconditioning; ischemia-reperfusion (I/R) injury; mitoBKCa channel; oxygen-glucose deprivation and reperfusion (OGD/R); stroke.