Isolation of the primary neurons from Sprague-Dawley rat fetuses (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) has been described previously (Liu Y. et al., 2012) with minor modification. All the experiments have been approved by the Ethics Committee of the Beijing Tiantan Hospital of Capital Medical University. The bilaterally cortical brain tissue was collected and minced, followed by digestion with 0.125% trypsin EDTA supplemented with 0.5 mg/ml DNase for 15 min at 37°C, and were then terminated by mix with complete medium containing DMEM/F12 with 10% fetal bovine serum and 5% horse serum. Single-cell suspension was then isolated by centrifugation followed by resuspension with culture medium (DMEM/F12, 10% FBS, 5% HS, 0.5 mmol/L L-glutamine, and 1% penicillin/streptomycin) into the plates coated with poly-L-lysine (0.1 g/L). After 4 h incubation, the medium was changed with a medium composed of neurobasal-R medium, 2% B27, 1% BSA, and 1% penicillin/streptomycin. The cells were maintained at 37°C in an incubator supplemented with 5% CO₂.

Immunofluorescence was performed to confirm the purity of the isolated neuron cells as previously described (Su et al., 2017). Briefly, cells were first fixed with 4% paraformaldehyde after culturing for 7 days, and then, they were stained with anti-neurofilament antibody and anti-glial fibrillary acidic protein antibody to label the endogenous expression of both proteins at 4°C overnight (1:500, Beijing GuanXing Yun Science and Technology Co., Ltd., Beijing, China). The next day, the unbind primary antibodies were removed by washing, and then, the cells were incubated with secondary antibodies at room temperature for about 1 h. The unbind secondary antibodies were removed as previously described, and then, the DNAs were stained with DAPI for 2 min at room temperature. Cell images were collected using a microscope from Olympus.

To establish the OGD/R mode, neuron cells were first challenged with glucose-free DMEM (Thermo Fisher Scientific, Inc., Waltham, MA, United States) and a hypoxic condition with 5% carbon dioxide, 2% oxygen, and 93% nitrogen at 37°C for 1, 2, and 3 h, respectively, and then, they were recovered with culture medium with normal glucose as well as normoxic condition with 5% carbon dioxide at 37°C for 24 h (reoxygenation period).

Cells were first treated with paxilline at 5 μmol/L for 10 min, and then, they were incubated with 10 mmol/L ethanol (Sigma-Aldrich, St. Louis, MO, United States) for 24 h followed by OGD/R.

The cell viability was measured by Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) Briefly, 20 μl of the CCK-8 reagent was mixed with the culture medium and then incubated at 37°C for about 4 h. The absorbance at OD 450 was measured by the Molecular Device M5.

Borosilicate glass patch pipettes for single-channel recordings had a resistance of 3–5 MX when filled with an internal solution. Recordings were made using a patch clamp amplifier and patch master 2.73 amplifier (Heka, Lambrecht, Pfalz, Germany). The single-channel recordings were filtered at 1–5 kHz and digitized at 20 kHz. All experiments were performed at room temperature (22–25°C). Fitmaster software (Heka, Lambrecht, Pfalz, Germany) was used for data analysis. Open probability is expressed as channel open probability (NPo), where N represents the number of single channels present in the patch, and Po is the open probability of a single channel. NPo was calculated as follows: NPo = (A₁ + 2 A₂ + 3 A₃ + n An)/(A₀ + A₁ + A₂ + …An), where A₀ is the area below, the plot of the amplitude histogram corresponds to the closed state, and A₁–An represents the area of the histogram, reflecting different open current levels from 1 to n channels in the patch.

TUNEL assay for the detection of apoptosis was performed according to the instruction of the manufacturer (Thermo Fisher Scientific, Waltham, MA, United States). Apoptotic cells were pictured by microscope (Olympus), and counted and analyzed by Graphpad Prism 6.0 from five independent fields.

Apoptosis was determined using the PE Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) and detected by flow cytometry (BD C6; BD Biosciences, Franklin Lakes, NJ). Briefly, the cells were collected by trypsinization and then washed with PBS three times to remove the debris and complete medium. Subsequently, cells were stained with 5 μl of Annexin V-PE and 5 μl of 7-AAD at dark for 15 min at 23–25°C. For the analysis of the apoptotic cells at different stages as well as the non-apoptotic cells, the early apoptotic cells were defined as positive for PE-Annexin-V and negative for 7-AAD, whereas the late-stage apoptotic cells were defined as positive for PE-Annexin V as well as 7-AAD.

After treatment, the neuronal cells were collected by trypsinization and then labeled with JC-10 (Beyotime Biotechnology, Shanghai, China) at 37°C for 30 min. Next, the cells were centrifuged at 1,000 rpm for 4 min, followed by washing with PBS three times. The cells were then suspended with 400 μl flow buffer and were analyzed by flow cytometry (BD C6; BD Biosciences, Franklin Lakes, NJ).

Total cell lysate was prepared from neuronal cells with radioimmunoprecipitation assay buffer (RIPA) supplemented with protease inhibitor (Biochem, PA, United States). Cell concentrations were quantitated by BCA assay and were denatured with SDS-based sample buffer. In Western blot, equal amounts (30 μg) of protein were loaded and separated by SDS-PAGE and then transferred onto the PVDF membrane. The blot was first blocked with 5% non-fat milk and then incubated with primary antibody overnight at 4°C. Antibodies for cleaved caspase 3 (1:1,000; Cell Signaling Technology, MA, United States) and total caspase 3 (1:1,000; Cell Signaling Technology, MA, United States), anti-Drp1 (ab184247), and anti-Fis1 (ab156865) were purchased from Abcam, and anti-March 5 (19168) was purchased from Cell Signaling Technology. Next, the blots were washed with TBST and then incubated with secondary antibodies conjugated with HRP at room temperature for 1 h. Signals were collected by adding ECL solution and were captured by the FluorChem FC2 System (Cell Biosciences, Inc., Santa Clara, CA, United States), and images were analyzed using ImageJ software (NIH, United States).

All the cells were first collected by trypsinization and were washed with PBS two times, and then, they were resuspended with buffer containing Calcein AM, quenching solution. The concentration of the cells was 1×10⁶ ml^–1, and the cells were kept at 37°C for 30 min. The cells were centrifuged at 1,000 g for 5 min and then resuspended with detection buffer for flow cytometry analysis.

All the data were expressed as mean ± standard deviation (S.D.) and were analyzed with SPSS version 25.0. Significant differences were determined by ANOVA followed by Dunnett’s multiple comparison test, and the statistical significance was defined as P < 0.05.

To examine the purity of cortical neurons in the primary mixed culture, we stained the cells with anti-NF directed against the neurofilament protein and anti-GFAP directed against glia-specific glial fibrillary acidic protein and observed that 95% NF-positive cells were cortical neurons (Figure 1A). To determine the optimal OGD/R condition, we exposed the neurons to oxygen-glucose deprivation for 1, 2, or 3 h, respectively, and followed by reperfusion for 24 h. The neurons viability was decreased with an increase in the deprivation time, and we determined 2 h OGD as the optimal deprivation time (Figure 1B).

In view of our previous findings that 10 mmol/L ethanol treatment could activate BK_Ca channels at all membrane voltages (Su et al., 2017), we then investigated whether EtOH-PC reversed the OGD/R-induced neuronal injury through activating BK_Ca channel. We examined the current–voltage and conductance of BK_Ca channel using an inside-out patch after 10 mmol/L EtOH-PC followed by OGD2h/R24h. It suggested that ethanol increased the current and conductance of BK_Ca and decreased the close time of BK_Ca. OGD/R exposure markedly decreased the current and conductance of BK_Ca and increased the close time of BK_Ca. The NPo of BK_Ca was decreased under OGD/R, while low-dose EtOH-PC counteracted the effect of OGD/R on current, conductance, close time, and NPo of BK_Ca (Figure 2).

Since OGD/R decreased the neuronal viability and ethanol dramatically attenuated the OGD/R-induced neuronal injury, we next investigated whether apoptosis was involved in the decreased cell viability and whether ethanol could attenuate the OGD/R-induced neuronal apoptosis. As expected, OGD/R exposure led to apoptosis in neurons as demonstrated by the TUNEL assay. Furthermore, EtOH-PC had no effect on the normoxic neurons, but markedly reduced the apoptotic cells after OGD/R exposure. BK_Ca channel blocker paxilline preincubation counteracted the protective effect of ethanol (P < 0.01, Figure 3).

In addition, the neuron apoptosis was further confirmed by flow cytometry after staining with Annexin V-7-AAD, indicating that EtOH-PC dramatically reduced the apoptotic cells. Compared with EtOH + OGD/R group, BK_Ca channel blocker paxilline preincubation significantly counteracted the neuroprotective effect of EtOH-PC (Figures 4A,B).

Taken together, low-dose EtOH-PC protected against OGD/R-induced neuronal apoptosis through activating BK_Ca channel.

To determine whether the mitochondrial BK_Ca channel was involved in the protective effect of EtOH-PC, we examined the mitochondrial membrane potential using flow cytometry. Compared with the control group, OGD/R exposure partially opened MPTP and subsequently decreased the mitochondrial membrane potential. EtOH-PC closed MPTP and increased the MMP. However, BK_Ca channel blocker paxilline preincubation before ethanol significantly counteracted the effect of ethanol with the decrease of MMP (Figures 5A,B). In addition, a similar trend of regulation was further confirmed by the MPTP assay (Figure 6). It suggested that mitoBK_Ca mediated the protective effect of EtOH-PC.

When the mitochondrial membrane potential decreases, caspase 3 is cleaved and activated by the apoptosis-inducing factor released from the mitochondrial extracellular compartment and apoptotic protease activating factor-1 released from mitochondria. So, we examined the cleaved caspase 3 expression and found that OGD/R exposure upregulated the cleaved caspase 3 expression. EtOH-PC downregulated the cleaved caspase 3 expression. BK_Ca channel blocker paxilline significantly counteracted the downregulation effect of ethanol (Figure 7). In addition, markers (Drp1, Fis1, March 5) for the apoptosis pathway were analyzed by Western blot, and the results suggested that ethanol-mediated neuroprotective effect was dependent on the intrinsic apoptosis pathway, as can be seen from Figure 8. Collectively, our data demonstrated that mitoBK_Ca channel activation mediated the protective effect of EtOH-PC.