2.1 Cell culture
The hippocampal neuronal HT22 cell line of mouse were purchased from the American Type Culture Collection (ATCC). HT22 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) containing 10% foetal bovine serum (Gibco, Grand Island, USA) and 1% penicillin-streptomycin solution (Life Technologies, Carlsbad, ON, Canada) at 37°C in a humidified incubator with 5% CO2. The medium was completely changed every 72 h.
All HT22 cell lines were divided into three groups. The control group, sevoflurane treatment group and sevoflurane-WNK-463 group. The cell culture was exposed in an airtight plastic chamber with inlet and outlet connectors. The inlet port of the chamber was used to adjust the concentration of sevoflurane (AbbVie Inc., North Chicago, IL, USA), which was connected to a sevoflurane vaporizer. The outlet of the chamber was used to monitor sevoflurane concentration through a gas monitor (PM 8060, Drager, Lübeck, Germany) until the target concentration was reached.
In the control group, the cell culture was grown in the chamber with humidified atmosphere with 5% CO2 (95% air/5% CO2) at 37°C for 6 h.
In the sevoflurane group, the chamber was gassed with a concentration of sevoflurane (4.1%) in the carrier gas (95% air/5% CO2) for 30 min as described previously . And the cell culture was put in the chamber and then kept tightly sealed for 6 h at 37°C.
The WNK1 inhibitor WNK-463 was dissolved in dimethyl sulfoxide (DMSO) and diluted with sevoflurane-treated medium. In the sevofluren-WNK-463 group, the cell culture was pre-treated with WNK-463 in the medium for 30mins, and then treated with sevoflurane (4.1%) in the carrier gas (95% air/5% CO2) for 6 h.
2.2 MTS assay
The CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega Corporation, Madison, WI, USA) is a colourimetric method for determining the number of viable cells and screening of optimum drug concentration (WNK-463) in proliferation in cytotoxicity assays according to a previous study . We added WNK-463 at a certain concentration gradient of 0, 0.5, 1.0, 2.5, 5.0 µmol, in order to find the optimum concentration of WNK-463 through the results of MTS. Move 20µl of CellTiter 96® AQueous One Solution Reagent into each well of the 96-well assay plate containing the samples in 100µl of culture medium. The culture wells with MTS reagent was placed in an incubator at 37℃for 2 hours and then recording the absorbance at 490 nm with a 96-well plate reader. The results of the control group were set at 100% and the survival rates of the other two groups were calculated. From the MTS results (absorption was detected at 490 nm using a microplate reader), we found that with the increase in WNK-463 concentration, the protective effect was gradually enhanced and reached a peak at 1.0 µmol before it decreased which was used in our subsequent experiments (P < 0.01, see supplementary file, S-Figure 1).
2.3 TUNEL assay
The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) method was performed to label the 3’-terminus of the fragmented DNA of apoptotic cells according to the manufacturer’s protocol  (One Step TUNEL Apoptosis Assay Kit, Beyotime, China). Phosphate-buffered saline (PBS) containing 4% paraformaldehyde was added to the three groups of cells and incubated for 1 hour and washed with 1x PBS 3 times for 5 min. After permeabilization with 1x PBS containing 0.1% Triton X-100 for 2 min on ice, the cell slices were incubated with a TUNEL working solution for 1h at 37°C in darkness. The TUNEL-positive cells were labelled by Cy3 and were examined with fluorescence microscopy (Olympus IX710 Camera, Japan). The cells with green fluorescence were considered apoptotic cells. To count the number of apoptotic cells, we selected the same-sized areas of each cell slice for analysis with Image Pro Plus software.
2.4 Measurement of intracellular calcium levels ([Ca2+]i)
According to before the treatment method of cells after collecting, learned that, the supernatant fluid to join without phenol red culture medium, Hank’s balanced salt solution (HBBS) wash three times, to join the Fluo-4, AM concentration of 5 umol/L working liquid Beyotime biological co, LTD(Shanghai), incubation in 37 ℃ for 30 min, using HBSS flushing cells 3 times, and continue in HBSS incubation for 30 min, under laser confocal microscope test, excitation wavelength of 494 nm, emission wavelength of 516 nm.
2.5 Western blot analysis
The proteins including caspase-3, WNK1, NKCC1 were detected by western blot analysis. The proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentrations were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Equal amounts of protein were resolved over 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. The primary antibodies were caspase-3 antibody (#9662, 1:1000 dilution), WNK1 antibody (NB600-225, 1:500 dilution) and NKCC1 antibody (#14581,1:500 dilution). The membranes were then incubated with the secondary antibodies (Abcam) for 1 h at room temperature. Positive signals were visualized by enhanced chemiluminescence (ECL) reagent (GE Healthcare, Little Chalfont, UK). The intensity of the bands was quantified using Image LabTM Software (Bio-Rad, CA, USA).
2.6 Statistical analysis
All experiments were repeated three times. The results of multiple experiments are presented as the means ± standard deviations (SD). Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). The P-values were calculated using one-way variance analysis (ANOVA). A P-value of <0.05 was considered to indicate statistical significance.