Drug and cells culture treatment
SLI, provided by Tianjin Tasly Pharmaceutical Co, Ltd (Tianjin, China), is approved by CFDA (China Food and Drug Administration, GuoYaoZhunZi-Z20110011) in 2011. The fingerprint chromatogram of SLI contains five major bioactive components, namely Sal B, Sal D, Sal Y, alkannic acid and RA [25]. Mouse neuroblastoma N2a cells (ATCC, Manassas, USA) were cultured in Minimum Essential Medium (MEM; Hyclone, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), and antibiotic-antimycotic (Gibco) at 37 °C temperature in a humidified incubator under 5% CO2 and 95% air. The cells were divided into control group, oxygen-glucose deprivation/reoxygenation (OGD/R) group, and SLI groups with different concentration gradients (10, 25, or 50 µg/ml). The OGD/R model was performed as our previously described [26]. N2a cells were cultured in balanced salt solution and then transferred to a special chamber that was closed and included a gas mixture with consisting of 95% N2 and 5% CO2. The special chamber was placed in an incubator at 37 °C for 4 h. After OGD, N2a cells were maintained in complete medium in a normoxic incubator for 4, 6, or 24 h. Control cells were treated identically without undergoing OGD conditions.
Cell Viability And LDH Release Analysis
Cell viability was analyzed using CCK-8 assay. N2a cell suspension (100 µl/well, 1.0 × 106/ml) was cultured in 96-well microplates until the cells adhered to the wells and grew evenly, which can be used in experiments. After cell grouping treatment, remove the cell supernatant, add 100 µl MEM containing 10% CCK-8 (Dojindo, Japan) into each well of the culture plate, and incubate at 37℃ for 30 min. The absorbance was measured at 450 nm using a microplate reader (TECAN, Switzerland).
Lactate dehydrogenase (LDH) release assay was detected by an LDH Cytotoxicity Assay Kit (Dojindo, Japan). After the cells were incubated in different groups, 25 µl cell supernatant was collected from each well and transferred to the new 96-well microplates and added CytoTox-ONETM Reagent, which were prepared in advance. The new 96-well plate was incubated 20℃ for 30 min, then 50 µl of stop solution was added to the wells, and mixed for 30 s while shaking. The wavelength absorbance at 560/590 nm was measured for the cell viability test (TECAN, Switzerland).
Detection Of Apoptosis By Flow Cytometry
Apoptosis was evaluated by Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen, USA). After treatment, N2a cells were trypsinized, washed with cold PBS, and then resuspended in 150 µl binding buffer containing 5 µl Annexin-V-FITC and 1 µl propidium iodide (PI). After incubation at 37 °C for 15 min in dark, the fluorescence level was quantified by flow cytometry (BD Biosciences, USA).
Determination Of Cytochrome C Release
Cytochrome c (Cyt-c) release was examined by using the Cytochrome-c ELISA Assay Kit according to manufacturer’s instructions (JingKangBio, Shanghai, China). After drug intervention, N2a cells were collected and washed twice. 50 µl of samples and standards were added to the wells of microplate, which were pre-coated with cytochrome c specific antibody. Then 100 µl of a streptavidin-HRP conjugate is added to all wells and incubated at 37 °C for 60 min. Washed with washing buffer and added chromogenic agent A (50 µl) and B (50 µl) into the microwells, and then incubated at 37 °C in the dark for 15 min. After adding stop solution (50 µl), the absorbance was measured using a microplate reader (TECAN, Switzerland).
Western Blotting Analysis
The cells were lysed in cell lysis buffer (Beyotime, Shanghai, China). After centrifugation of 12000 g for 10 min, the supernatant was obtained and used for western blot analysis. Detection of protein concentration was measured using BCA assay kit (Beyotime, Shanghai, China). Subsequently, protein was separated on 10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After that, the membrane was incubated with primary antibodies against Cleaved-Caspsase-3 (CST, 9664), Bcl-2 (Abcam, ab27795), LC3B (Abcam, ab48394), Beclin-1 (CST, 3738), p62 (Abcam, ab56416), p-Akt (CST, 4060), Akt (CST, 4685), mTOR (CST, 2983), β-actin (CST, 4970) at 4 °C overnight. After the last wash, the membranes incubated with goat anti-mouse or anti-rabbit peroxidase-conjugated secondary antibody (ZSGB-Bio, Beijing, China) for 1 h. Then, immunoreactive proteins were visualized using chemiluminescence reagent (ECL; Millipore, USA). The staining intensity of each band was evaluated by densitometry and quantified using ImageJ software.
Statistical analysis
The data were analyzed by SPSS 23.0 statistical software, which were expressed as mean ± S.D. The comparison between different groups was analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison tests. The differences were statistically significant at P < 0.05.