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

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

doi:10.3389/fphys.2021.719753

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 BKCa (mitoBKCa) 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 mitoBKCa 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 BKCa 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 mitoBKCa channel. Low-dose ethanol pretreatment significantly attenuated the OGD/R-induced neuronal apoptosis mediated by the mitoBKCa channel which modulated the mitochondrial function by impeding the uncontrolled opening of mitochondrial permeability transition pore (MPTP).

Highlights

Contributions of ischemia-reperfusion (I/R) to morbidity and mortality of ischemic strokes have been well documented (Yan et al, 2015)
The NPo of BKCa was decreased under oxygen-glucose deprivation and reperfusion (OGD/R), while low-dose EtOH-PC counteracted the effect of OGD/R on current, conductance, close time, and NPo of BKCa (Figure 2)
Our findings suggested that OGD/R-induced neuronal apoptosis was mediated by BKCa channel

Summary

Introduction

Contributions of ischemia-reperfusion (I/R) to morbidity and mortality of ischemic strokes have been well documented (Yan et al, 2015). The oxygen-glucose deprivation and reperfusion (OGD/R) was used as a well-established in vitro model to induce neuronal apoptosis under pathological conditions and to study the neuroprotective effect of the pharmacological intervention (Alluri et al, 2015; Zhang et al, 2016). The intrinsic pathway was mediated by the mitochondrial depolarization and the subsequent release of cytochrome C and other large molecules, leading to the activation of caspases and the formation of apoptotic bodies (Burg et al, 2006). Pharmacological intervention, such as metformin or neuroserpin, might protect neurons from OGD/R-induced damage (Meng et al, 2016; Yang et al, 2016)

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Conclusion