All animal experiments were approved by the Committee on Animal Experimentation of The Affiliated Hospital of Putian University (Putian, China; approval no. 2018-0023) and performed in compliance with the Guidelines for the Care and Use of Laboratory Animals (17). A total of 36 4-month-old adult male Sprague-Dawley rats (weight, 180-250 g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were divided into six groups (n=3). The rats were kept in an animal breeding room for 2 weeks under the same conditions of temperature (22±2˚C), humidity (50±10%) and controlled habitat with a 12-h light/dark cycle (lights were on between 8:00 a.m. and 8:00 p.m.). Rats were administered food and water ab libitum.

Primary astrocytes were used in the present study, which were isolated as follows: Rats were anesthetized with different concentrations of sevoflurane (cat. no. 1612540; Millipore Sigma) as follows: 0, 1, 2, 4, 8 or 16%, from 9:00 a.m. to 3:00 p.m. Specifically, rats were housed in a chamber (length, 50 cm; width, 30 cm; height, 30 cm) containing 30% O₂, 68% N₂ and the corresponding concentrations of sevoflurane for 6 h. Immediately after anesthesia, the animals were euthanized via cervical dislocation and the heads were removed. The isolated brains of rats were sterilized with PBS containing 10% penicillin and streptomycin (cat. no. 10378016; Gibco; Thermo Fisher Scientific, Inc.) and placed in precooled sterile calcium- and magnesium-free (CMF) Hank's Balanced Salt Solution (HBSS) (cat. no. H9394; Sigma-Aldrich; Merck KGaA). All the cerebral hemispheres were collected in a petri dish on ice containing 5 ml CMF-HBSS, and the collected cortical tissue was minced as finely as possible. The tissue was transferred into a 15 ml tube with 12 ml CMF-HBSS. A total of 1.5 ml 2.5% trypsin and 10 mg/ml DNase solution were added, and the mixture was incubated at 37˚C for 15 min. The tissue was triturated 10-15 times using a glass Pasteur pipette and the mixture was further incubated at 37˚C for 5 min. The supernatant was filtered using a 100-µm cell strainer. The suspension was centrifuged at 130 x g for 6 min at 4˚C and the supernatant was carefully discarded. It was ensured that the pelleted cells at the bottom were not disturbed. Every group of samples was mixed together with up to 20 ml of astrocyte medium (cat. no. A1261301; Thermo Fisher Scientific, Inc.) to resuspend the combined dissociated glial cells, which were subsequently cultured in a flask in a humidified incubator at 37˚C with 5% CO₂. The astrocyte medium was changed on the next day. A total of 4 days after seeding the cells, the flask was vigorously shaken 5-10 times to remove the unwanted cell types, and the supernatant was discarded. A total of 10 ml fresh CMF-HBSS was added in each flask, which was vigorously shaken 5-10 times and the supernatant was again discarded. A total of 20 ml fresh astrocyte medium was added into each flask. The confluent cells were the desired astrocytes. The astrocytes were maintained in high glucose DMEM (cat. no. 11965084) containing 10% FBS (cat. no. 16141061), and 1% penicillin and streptomycin (cat. no. 10378016; all from Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37˚C with 5% CO₂. The astrocytes were then used for the following experiments.

Astrocytes were grouped into four groups: Anti-miR-negative control (NC) [infected with lentivirus (LV)-miR-367-3p inhibitor negative control], anti-miR-367-3p (infected with LV-miR-367-3p inhibitor), miR-NC (infected with LV-miR-367-3p mimic negative control), miR-367-3p (infected with LV-miR-367-3p mimic). The lentiviral miRNA inhibitor vector was pCLenti-U6-shRNA-CMV-EGFP-WPRE, and the lentiviral miRNA overexpression vector was pCLenti-U6-miR30 (miRNA)-CMV-EGFP-F2A-Puro-WPRE, which were purchased from OBiO Technology (Shanghai) Corp., Ltd. miR-367-3p inhibitor (5'-AGTAATGGCCATCACCATTGCT-3'), inhibitor-NC (5'-CCATTACTAGCAATGGTGATGG-3'), miR-367-3p mimic (5'-CCATTACATGCAATGGTGATGGAU-3') and mimic-NC (5'-AUCCATCACCATTGCTAGTAATGG-3') were constructed by Sangon Biotech Co., Ltd. Lentiviral particles were packaged by co-transfection with lentivirus and pHelper 1.0 and 2.0 vectors carrying the miR-367-3p inhibitor, inhibitor-NC, miR-367-3p mimic or mimic-NC into 293T cells (Procell Life Science & Technology Co., Ltd.). In brief, a total of 20 µg lentiviral plasmids, including pHelper 1.0: pHelper 2.0: pLVX (LV-miR-367-3p inhibitor or NC; LV-miR-367-3p mimic or NC)=7.5 µg: 2.5 µg: 10 µg, were added into 500 µl serum free DMEM (Solution A). A total of 60 µl Lipofectamine^® 3000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) was added into 500 µl serum free DMEM (Solution B). Solution B was added drop by drop into solution A, mixed gently and incubated for 20 min at room temperature to prepare solution C. Solution C was added into 293T cells drop by drop and mixed gently, and 293T cells were cultured in a 37˚C incubator for 16 h. Subsequently, serum free DMEM was changed into complete DMEM medium with 10% FBS. A total of 48 h after transfection at 37˚C, lentivirus particles were harvested from the cell culture supernatant and concentrated to 10⁸ transduction units/ml.

A total of 24 h before lentivirus transduction, astrocytes were seeded in 6-well plates with ~1x10⁵ cells to obtain ~30% confluency on the 2nd day. On day 2, 6.8 µl lentiviral particles (MOI=80) were added to each well. A total of 16 h later, the medium was replaced with lentivirus-free complete DMEM medium, and the astrocytes were cultured for another 48 h for transduction. Then, the lentiviral transduction efficiency was evaluated under a fluorescence microscope (Eclipse Ts2; Nikon Corporation).

For overexpression of BCL2L11, BCL2L11 open reading frame was inserted into pGV358-GFP vector (TsingKe Biological Technology) to construct pGV358-GFP-BCL2L11. pGV358-GFP was used as NC. The following primers (forward, 5'-TCAGTGCCTTCTCCAGACCAGACG-3'; and reverse, 5'-CATCAGAAGGTTGCTTGGCCAT-3') were designed to amplify the cDNA of BCL2L11. The lentiviral particles of pGV358-GFP-BCL2L11 were packaged following the procedures aforementioned. In brief, a total of 20 µg lentiviral plasmids including pHelper 1.0 and 2.0 vectors and pGV358-GFP-BCL2L11 were incubated with 60 µl Lipofectamine 3000 in 500 µl serum free DMEM for 20 min at room temperature and then added to 293T cells for co-transfection for 48 h in a 37˚C incubator. Subsequently, lentiviral particles were obtained from the cell supernatant and concentrated to 108 transduction units/ml. Astrocytes were then infected with the packaged pGV358-GFP-BCL2L11 lentiviral particles for 48 h, followed by adding G418 to the astrocytes for 4 weeks to obtain stable BCL2L11-overexpressing astrocytes.

For co-transfection with pGV358-GFP-BCL2L11 and LV-miR-367-3p mimic, stable BCL2L11-overexpressing astrocytes were used, and 6.8 µl LV-miR-367-3p mimic lentiviral particles (MOI=80) were added to each well of 6-well plate. A total of 16 h later, the medium was replaced with lentivirus-free complete DMEM, and the astrocytes were cultured for another 48 h.

As aforementioned, the hippocampal regions of the brain were minced and digested into cell suspensions. These cell suspensions were then added to a 96-well plate at 3x10³ cells per well in 100 µl complete DMEM medium, and cultured at 37˚C in a 5% CO₂ incubator for 6 h. A total of 10 µl CCK-8 reagent (cat. no. 96992; Sigma-Aldrich; Merck KGaA) was added to each well and cultured for 4 h. The absorbance was measured at 450 nm. Each sample was assayed in triplicate (18). The absorbance of the astrocytes sample from rats anesthetized with 0% sevoflurane was used as control group and calculated as 100% cell viability. The cell viability of the other groups was calculated as the absorbance of 1, 2, 4, 8 or 16% sevoflurane group/the absorbance of 0% sevoflurane group x100%.

Total protein was extracted from the astrocytes that had been previously digested from hippocampal tissue using trypsin. RIPA buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.) with PMSF (ratio, 1 ml:10 µl) was used to lyse the cells. Following homogenization, the supernatant was prepared by centrifugation at 1,000 x g at 4˚C for 20 min, and the transparent colloidal mucus was discarded. The protein was quantified using the BCA assay. A total of 20 µg of the total protein per lane was mixed with SDS loading buffer and proteins were denatured at 95-100˚C for 5 min. The proteins were separated by 10% SDS-PAGE, and the separated proteins were transferred to a PVDF membrane. The membrane was blocked using 5% skimmed milk containing 0.1% Tween-20 (TBST) at room temperature for 1 h, prior to incubation with the corresponding primary antibody at 4˚C for 12 h. The membranes were subsequently washed in TBST and incubated with the goat anti-rabbit IgG H&L (HRP) secondary antibody (1:10,000; cat. no. ab6721; Abcam) at room temperature for 2 h. The primary antibodies were as follows: i) Bax (1:5,000; anti-rabbit; cat. no. ab32503; Abcam); ii) BCL-2 (1:1,000; anti-rabbit; cat. no. ab32124; Abcam); iii) BCL2L11 (1:2,000; anti-rabbit; cat. no. ab32158; Abcam); and iv) GAPDH as a loading control (1:10,000; anti rabbit; cat. no. ab181602; Abcam). Bands were developed using Immobilon ECL Ultra Western HRP Substrate (cat. no. WBULS0100; MilliporeSigma) and the image were analyzed using ImageJ v1.8.0 (National Institutes of Health) (19).

TUNEL staining was carried out using Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay kit (cat. no. C10245; Invitrogen; Thermo Fisher Scientific, Inc.). The experiment was conducted following the manufacturer's protocol. In brief, a total of 2.5x10⁵ astrocytes were seeded on coverslips and cultured in 6-well plates (Corning, Inc.) for 24 h before TUNEL staining. The astrocytes were rinsed with PBS three times, and then fixed with 4% paraformaldehyde for 15 min at room temperature. A total of 100 µl 0.25% Triton X-100 was added to completely cover the coverslips and incubated for 20 min at room temperature. A total of 100 µl terminal deoxynucleotidyl transferase (TdT) reaction buffer was added on each coverslip and the solution was allowed to spread completely over the surface. The coverslips were incubated for 10 min at room temperature. Another 100 µl of the TdT reaction cocktail containing TdT reaction buffer, EdUTP (dUTP modified with an alkyne) and TdT, was added to each coverslip and incubated them for 60 min at 37˚C. The coverslips were washed twice with 3% BSA (cat. no. B2064; MilliporeSigma) in PBS. A total of 100 µl Click-iT reaction cocktail containing Click-iT reaction buffer and Click-iT reaction buffer additive was added to each coverslip and incubated them for 30 min at room temperature in the dark. The coverslips were washed twice with 3% BSA in PBS. The primary antibody solution was added as supplemented by the manufacturer and incubated for 6 h at 4˚C, protected from light. The coverslips were washed with 3% BSA in PBS twice, and subsequently incubated with the secondary antibody for 0.5 h at room temperature, protected from light, followed by washing with 3% BSA in PBS twice. A total of 100 µl of 1X Hoechst 33342 solution per coverslip was added for 15 min at room temperature in the dark. The coverslips were washed twice with PBS, and subsequently imaged coverslips under a fluorescence microscope (Nikon Corporation) with fluorescence excitation at 495 nm and emission at 519 nm. At least 5 fields were captured per coverslip and 3 coverslips per group were analyzed to calculate the TUNEL-positive cells (%).

TargetScan v7.2 software (http://www.targetscan.org/vert_72/) was used to predict the potential miRNAs interacting with the BCL2L11 3'-untranslated region (3'-UTR). Dual luciferase reporter assay was carried out using Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (cat. no. 16185; Thermo Fisher Scientific, Inc.). In brief, the reporter plasmids (80 ng) containing wild type (WT) or mutant (Mut) 3'-UTR of BCL2L11were co-transfected with miR-367-3p mimic (50 nM) or miR-NC (50 nM) into 293T cells using Lipofectamine 3000. miR-367-3p mimic (5'-CCATTACATGCAATGGTGATGGAU-3') and mimic-NC (5'-AUCCATCACCATTGCTAGTAATGG-3') were constructed by Sangon Biotech Co., Ltd.

A total of 48 h after transfection, the luciferase assay reagent was added and luminescence signals were read at 640 nm for firefly luciferase and at 525 nm for green Renilla luciferase. Firefly luciferase was an experimental reporter and Renilla luciferase was used as a normalization control.

Hippocampal tissue that had been previously digested into individual cells using trypsin was used for PCR. RNA was extracted from the cells using 1 ml TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and 200 µl chloroform according to the manufacturer's protocol, incubated on a shaker, and centrifuged at 10,000 x g at 4˚C for 15 min. The upper solution was transferred to a clean RNase-free centrifuge tube and mixed with an equal volume of isopropanol, incubated for 15 min on ice, and centrifuged at 10,000 x g at 4˚C for 15 min. The RNA quality was evaluated as the absorbance at 260/280 nm ratio being 1.8-2.1. The samples were reverse transcribed to cDNA with PrimeScript RT reagent kit (cat. no. RR036Q; Takara Bio, Inc.). The RT reaction was performed at 37˚C for 15 min followed by 85˚C for 5 sec and 4˚C on hold. The TB Green^® Premix Ex Taq™ II kit (cat. no. RR820A; Takara Bio, Inc. Takara) with an ABI 7500 real-time qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to conduct qPCR. The thermocycling conditions used were as follows: 95˚C for 30 sec; followed by 40 cycles of 95˚C for 5 sec and 60˚C for 10 sec. The 2^-IICq method was applied for calculating the relative quantification (20). U6 was used as reference control for miR-367-3p.

The following primers were used: miR-367-3p forward, 5'-TTCTCCGAACTTTGCACGTTT-3' and reverse, 5'-ACGTGACACGTTCGGAGAATT-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'.

All experimental data are presented as the mean ± standard deviation. The data were analyzed using GraphPad Prism 6 statistical software (GraphPad Software, Inc.). Unpaired Student's t-test was used for comparisons between two groups (21) and one-way ANOVA followed by Bonferroni's correction was used for comparisons between multiple groups. All experiments were repeated three times (n=3). P<0.05 was considered to indicate a statistically significant difference.

Sevoflurane inhibited the viability of astrocytes in a dose-dependent manner. The results of the CCK-8 cell viability assay revealed that at ≥4% sevoflurane the cell viability decreased in a dose-dependent manner compared with 0% sevoflurane (Fig. 1A). Western blot analysis indicated that compared with 0% sevoflurane, the expression levels of Bax were gradually increased, whereas the expression levels of Bcl-2 were gradually decreased. When the concentration of sevoflurane was 16%, the expression levels of Bax reached a maximum, and those of Bcl-2 were the lowest (Fig. 1B). The results of TUNEL assays demonstrated that with the increase of sevoflurane concentration, the apoptosis of astrocytes increased. When the concentration of sevoflurane reached 16%, the apoptosis of astrocytes was the highest (Fig. 1C).

TargetScan software was used to predict the potential miRNAs interacting with the BCL2L11 3'-UTR, and miR-367-3p was identified (Fig. 2A). Luciferase reporter assay was used to analyze the binding ability between the WT or Mut 3'-UTRs of BCL2L11 and miR-367-3p. The results demonstrated that co-transfection of miR-367-3p and BCL2L11-WT 3'-UTR exhibited a lower luciferase activity compared with co-transfection of miR-NC and BCL2L11-WT 3'-UTR. Co-transfection of the miR-367-3p or miR-NC and BCL2L11-Mut 3'-UTR demonstrated no difference in the luciferase activity (Fig. 2B). RT-qPCR results revealed that with an increase in the concentration of sevoflurane, the expression levels of miR-367-3p in astrocytes gradually decreased, compared with 0% sevoflurane. When the concentration of sevoflurane reached 16%, the expression levels of miR-367-3p were the lowest, compared with 0% sevoflurane (Fig. 2C). Western blot analysis was used to detect the protein expression levels of BCL2L11 in astrocytes. The results demonstrated that with the increase of sevoflurane concentration, the expression levels of the BCL2L11 protein in hippocampal astrocytes were significantly increased. When the concentration of sevoflurane reached 16%, the expression of BCL2L11 protein was the highest, compared with 0% sevoflurane (Fig. 2D).

In order to detect the interaction between miR-367-3p and BCL2L11, lentiviral infection with anti-miR-367-3p or miR-367-3p into the astrocytes from rats not treated with sevoflurane was performed. RT-qPCR results indicated that transfection with anti-miR-367-3p significantly decreased the expression levels of miR-367-3p compared with the anti-miR-NC group, whereas transfection with miR-367-3p significantly increased the expression levels of miR-367-3p compared with miR-NC group (Fig. 3A). Western blot analysis revealed that the protein expression levels of BCL2L11 were significantly increased following transfection with anti-miR-367-3p, and were significantly decreased after transfection with miR-367-3p, compared with anti-miR-NC or miR-NC, respectively (Fig. 3B).

Western blot analysis revealed that under 8% sevoflurane treatment, the protein expression of BCL2L11 and Bax in the astrocytes transfected with anti-miR-367-3p was significantly increased, while the protein expression of Bcl-2 was decreased, compared with astrocytes transfected with anti-miR-NC. The protein expression of BCL2L11 and Bax in astrocytes transfected with miR-367-3p were significantly decreased, and the protein expression of BcL-2 were significantly increased, compared with astrocytes transfected with miR-NC (Fig. 4A). The results of TUNEL staining revealed that apoptosis of the anti-miR-367-3p-transfected astrocytes were significantly increased, and that of the miR-367-3p-transfected astrocytes were significantly decreased (Fig. 4B), compared with anti-miR-NC or miR-NC, respectively.

BCL2L11 and miR-367-3p were co-transfected into rat hippocampal astrocytes; BCL2L11-NC was used as the control group. Western blot analysis revealed that the expression levels of BCL2L11 and Bax in co-transfected BCL2L11 + miR-367-3p astrocytes were significantly lower compared with those transfected with BCL2L11 alone, whereas the expression levels of BcL-2 in co-transfected BCL2L11 + miR-367-3p astrocytes were significantly higher than those transfected with BCL2L11 alone (Fig. 5A). The results of TUNEL staining demonstrated that the apoptosis of co-transfected BCL2L11 + miR-367-3p astrocytes was lower than that of cells transfected with BCL2L11 alone (Fig. 5B).