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

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).