Primary cell cultures
Early postnatal P0-P1 Wistar rat pups’ cortices were used for neuron-astrocyte co-cultures. The animal protocol was approved by the Experimental Animal Care Committee of the Second Hospital of Hebei Medical University. The cultures were prepared according to previous report [24]. Briefly, cerebral cortices were isolated and dissociated by 2 mg/ml papain and 2 mg/ml deoxyribonuclease (Pro. No. DN25, Sigma, USA) for 30-min at 37℃. To terminate enzyme activity the tissue was rinsed with DMEM-F12 (Cat. No. 11320033, Gibco, China) containing 10% FBS (Cat. No. 10099141C, Gibco, China) twice gently. Cortical tissues were carefully triturated with a fire-polished pipette ten times in plating medium to dissociate the cells. Cells were plated at 500,000/ml onto 24-well or 6-well dishes which were pre-coated with 0.1 mg/ml poly-L-lysin and incubated at 37℃ with a gas mixture containing 5% CO2 and 95% O2. After 3-hour incubation, the medium was replaced completely with neurobasal-A (Cat. No. 10888022, Gibco, China) maintenance medium containing 2% B27. Thereafter, the medium was refreshed in half amount every three days. After pre-cultures for 7–9 days, healthy cultures were used for the study.
Reagent preparation
Abeta1-42 (Abeta, Cat. No. HY-P1363, MCE, China) was suspended in hexafluoro propanol (HFIP, Pro. No. 105228, Sigma, USA) to an initial concentration of 1 mM on ice. The suspension was incubated at room temperature for one hour, and then was aliquoted. HFIP was allowed to evaporate overnight in the hood. The dried peptide film was resuspended in DMSO (Pro. No. D2650, Sigma, USA) to a stock concentration of 5 mM. Before administration, the oligomers were diluted in cold PBS to a working concentration of 100 µM. The solution was incubated at 4℃ for 24-hour and centrifuge at 14,000 × g for 10 min at 4℃ to remove insoluble aggregates. The supernatant was soluble Abeta oligomers.
Sulbactam sodium (Cat. No. HY-B0334A, MCE, China) and glutamate (Pro. No. G5889, Sigma, USA) was dissolved in sterile PBS to stock solutions at concentrations of 200 mM and 30 mM, respectively.
Grouping and treatments
Cells were divided into control (CTL), Abeta, glutamate (Glu), Abeta + Glu and sulbactam (Sul) + Abeta + Glu groups. The final working concentrations in cultures were 10 µM for Abeta, 60 µM for glutamate, and 250 µM, 500 µM and 1000 µM for sulbactam. For Sul + Abeta + Glu group, cells were first incubated with sulbactam in above doses for 48-hour. Then, the culture medium was renewed and Abeta was added into the cultures. After 24-hour incubation in the presence of Abeta, the cultures were added with glutamate and incubated for 30 min. For Abeta, glutamate and Abeta + Glu groups, Abeta and glutamate were added to the cultures in the corresponding time points to the Sul + Abeta + Glu group. After the above administration and incubation, the maintenance medium in each group was renewed, and cells were incubated in the renewed maintenance medium for 24-hour. Thereafter, the cells were harvested for assays.
Hoechst-Propidium Iodide double stain
Neuronal death induced by Abeta and glutamate was analyzed by Hoechst 33258 (HO, Pro. No. 14530, Sigma, USA) and Propidium Iodide (PI, Pro. No. P4170, Sigma, USA) double stain. Cultures were carefully washed twice with sterile PBS and stained in situ with HO and PI at same concentration of 10 µg/ml at 37℃ for 15 min. Viable cells were stained nuclei as dark blue (weak HO positive). Dead cells were appeared nuclei as light blue (intensive HO positive) or red (PI positive). Images were captured with an inverted microscope camera (ZEISS Axiocam 503 color, Germany). Neurons were identified and distinguished from the glial cells by their cell bodies and neurites based on the merged images of bright fields and fluorescent images. At least five views for each well were captured, and each group were repeated five times. The cell count and calculation of neuronal death rate was performed by an investigator who was blinded to the experimental design and grouping.
CCK8 assay
The formazan dye of CCK8 solution generated by the dehydrogenase in living cells is directly proportional to the number of viable cells. CCK8 (Cat. No. HY-K0301, MCE, China) solution was added to each well in the volume of 10% maintenance medium. After two-hour incubation, the absorbance of each well was measured at 450 nm by a microplate reader.
LDH assay
LDH in the cytoplasm releases when cellular membrane is compromised. The concentration of LDH in the culture medium is proportional to the number of dead cells in cell cultures. Briefly, the supernatant from each well was transferred into a 96-well plate correspondingly. Each well was triplicate. The LDH substrate (Ser. No. A0202-1-2, Nanjing Jiancheng Bioengineering, China) was added according to the protocols and mixed with supernatant thoroughly without bubble. The supernatant was incubated at 37℃ for 15 min and the stop solution was added. The absorbance of each well was measured at 450 nm by a microplate reader.
Immunofluorescence stain
Primary neuron-astrocyte co-cultures were rinsed with 0.01 M PBS and fixed with 4% paraformaldehyde for 15 min at room temperature, and then the fixed cells were rinsed with 0.01 M PBS. Cells were permeabilized with 0.3% Triton X-100 for 10 min and rinsed with 0.01 M PBS. Non-specific bindings were blocked with 5% bovine serum albumin (Cat. No. 4240GR500, BioFroxx, China) at 37℃ for one hour. Cells were incubated with primary antibody against BCL2 (1:200, Cat. No. YT0470, Immunoway, USA), BAX (1:200, Cat. No. 60267-1-lg, Proteintech Group, China), Cleaved Caspase-3 (1:200, Cat. No. WL02117, Wanleibio, China), GLT1 (1:200, Cat. No. AB1783, Sigma, USA), GFAP (1:500, Cat. No. 16825-1-AP, Proteintech Group, China) or MAP2 (1:500, Cat. No. 17490-1-AP, Proteintech Group, China) at 4℃ overnight. Cells were rinsed with 0.01 M PBS and incubated with secondary antibody CoreLite 488 conjugated goat anti-mouse/rabbit IgG (1:500, Cat. No. SA00013-1/SA00013-2, Proteintech Group, China) or CoreLite 594 conjugated goat anti-mouse/rabbit IgG (1:500, Cat. No. SA00013-3/SA00013-4, Proteintech Group, China) at 37℃ for one hour. Images were captured with an inverted microscope camera (ZEISS Axiocam 503 color, Germany). At least five views for each well were imaged, and each group were repeated five or six times. The fluorescent density and co-localization were measured and analyzed with the mean grey value and Pearson’s R value by ImageJ. The analysis was performed by an investigator who was blinded to the experimental design and grouping.
Western blotting
Cells were collected with 1000 µl pipette tip by triturating them off the well. Total proteins were extracted in RIPA lysis buffer containing protease inhibitors (Cat. No. BC3710, Solarbio, China). The protein concentration was assayed with BCA method (Cat. No. PC0020, Solarbio, China). Twenty µg protein of each sample mixed with loading buffer was loaded in each lane. The samples were electrophoresed in 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Cat. No. 03010040001, Roche, USA). The nonspecific bindings were blocked in 5% bovine serum albumin (Cat. No. 4240GR500, BioFroxx, China) mixed in distilled water and incubate at 37℃ for one hour. The membranes were then incubated with primary antibodies against BCL2 (1:1000, Cat. No. YT0470, Immunoway, USA), BAX (1:1000, Cat. No. 60267-1-lg, Proteintech Group, China), Cleaved Caspase-3 (1:500, Cat. No. WL02117, Wanleibio, China), GLT1 (1:1000, Cat. No. AB1783, Sigma, USA), GAPDH (1:1000, Cat. No. 60004-1-Ig, Proteintech Group, China) at 4℃ overnight. The membranes were washed with TBST and incubated with secondary antibodies to either HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (1:1000, Cat. No. SA00001-1, Proteintech Group, China) for GAPDH and BAX, or HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:1000, Cat. No. SA00001-2, Proteintech Group, China) for BCL2 and cleaved caspase-3, or biotin labeled anti-guinea-pig immunoglobulin IgG (1:1000, Cat. No. 16-17-06, KPI, China) for GLT1. The immunoreactivities were detected by Enhanced Chemiluminescent kit (Cat. No. SW134-01, Sevenbio, China) and scanned with Amersham Imager 600 (UK). ImageJ was used to quantify band signal into integrated density. Experiments were repeated five or six times.
Statistical analysis
All statistical analysis was performed using GraphPad Prism 8.2.1. The data were presented as mean ± SD (standard deviation). Normality was examined by Shapiro-Wilk test, data passed normality test were analyzed with one-way ANOVA, and post hoc multiple comparisons were assessed with Tuckey test. P<0.05 was considered to be statistically significant.