Cell culture
SH-SY5Y-APP cells were kindly donated by Zhifang Dong (Chongqing Medical University, China). Briefly, SH-SY5Y-APP cultures were maintained at 37°C in a humidified atmosphere (5% CO2) in low-glucose Dulbecco’s minimal essential medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1% penicillin/streptomycin antibiotics (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). When confluence was 80–90% and passaged to new culture dishes, 6-well plates or 96-well plate Glucose. After passage by trypsinization, cells were placed in the incubator for 24 h before experimental interventions. DEX (Sigma–Aldrich, USA), rapamycin (RAPA), 3-methyladenine (3-MA) and compound C (CC) (MCE, USA) were dissolved in the culture medium.
Experimental Groups
SH-SY5Y-APP cells were cultured in mediums containing different concentrations (5.5, 50, 60, 70, 80, 90, 100 mM) of glucose for different times (12, 24, 48 h) to establish the high-glucose injury model. SH-SY5Y-APP cells were cultured in low-glucose medium containing different six concentrations of DEX (0, 0.1 1, 10, 100, 200 µM) to determine the safe concentration of DEX. To investigate the optimal therapeutic concentration of DEX, SH-SY5Y-APP cells were cultured in HG medium containing different concentration levels of DEX (0, 0.1, 1, 5, 10, 20, 50, and 100 µM).
To explore the protective effect of DEX on HG-induced SH-SY5Y/APP cell injury, cells were divided into the control, DEX, HG, and HG + DEX groups. SH-SY5Y/APP cells were maintained in drug-free and low-glucose medium. Cells in the DEX group were cultured in low-glucose (5.5 mM) medium containing 1 µM DEX for 24 h. SH-SY5Y/APP cells in the HG group were cultured in high glucose (80 mM) for 24 h. Cells were maintained in high-glucose (80 mM) medium containing 1 µM DEX for 24 h in the HG + DEX group,
To investigate whether DEX improves SH-SY5Y-APP cell damage in a high-glucose environment by regulating autophagy, cells were divided into the control, HG, HG + DEX, HG + RAPA, HG + DEX + RAPA, HG + 3-MA, and HG + DEX + 3-MA groups. Cells in the HG + RAPA group were maintained in high-glucose medium supplemented with 200 nM RAPA. SH-SY5Y-APP cells in the HG + DEX + RAPA group were maintained in high-glucose medium containing 1 µM DEX and 200 nM RAPA for 24 h. In the HG + 3-MA and HG + DEX + 3-MA groups, SH-SY5Y-APP cells were pretreated with 5 mM 3-MA for 1 h. To explore the pathway by which DEX regulates autophagy, cells were divided into the control, HG, HG + DEX, and HG + DEX + CC groups. Cells in the HG + DEX + CC group were pretreated with 10 mM CC (AMPK inhibitor) for 1 h and then cultured in high-glucose medium containing 1 µM DEX for 24 h.
Assessment Of Cell Viability
SH-SY5Y-APP cells were cultured in 96-well plates and differentiated with RA for 5 days. After being exposed to different treatments for 1 day, cell viability was assessed by the Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China) assay. Briefly, the culture media was absorbed and replaced with 10 µl of CCK-8 solution and cells were incubated at 37°C for 1 h. After that the absorbance was measured at 450nm optical density with a microplate reader.
Cell Morphology Observation
Cells were seeded in 6-well plates. at a density of 5 × 105 cells/well in a final volume of 3 mL/well and incubation at 37°C for 24h.by a phase-contrast light microscope (Leica DMI8, Germany, 20 × objective), morphological changes characteristic of SH-SY5Y-APP cells were observed. Microphotographs in each group were taken randomly with software LAS X.
Western Blot Analysis
For western blot experiments, we referenced the previous experimental protocol[19]. SH-SY5Y-APP cells were cultured as described in the previous section in 6-well plates. After the medium in each group was discarded, SH-SY5Y-APP cells were washed with phosphate-buffered saline (PBS) three times. And then 200 µl of 2% sodium dodecyl sulfate (SDS) was added to each well. RIPA lysis buffer (Beyotime Biotechnology, China) containing phenylmethanesulfonylfluoride (PMSF) were used to prepare total proteins. Protein samples were electrophoresed on an SDS gel (Beyotime, China) and transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Millipore, USA). After being blocked with 5% nonfat milk in PBST (PBS plus 0.2% Tween-20) for 1 h at room temperature, the membranes were incubated with the primary antibody in antidilution buffer (Beyotime, China) at 4°C overnight. The membrane was incubated with multiple primary antibodies as follows: LC3 (1:1000, #4108), Beclin-1 (1:1000, #3738), Bax (1:1000, #2772), AMPK (1:1000, #5831), p-AMPK (1:1000, #2535), mTOR (1:1000, #2972), p-mTOR (1:1000, #2971), β-Actin (1:1000, #4970) (from Cell Signaling Technology, USA), cleaved caspase3 (1:500, ab49822), Bcl-2 (1:1000, ab196495), p62 (1:3000, ab109012), and GAPDH (1:10000, ab37168) (from Abcam, USA). After being washed with TBST three times, the membranes were incubated with the HRP-conjugated secondary antibodies (1:20000, Bioworld China) for 1 h and then washed with PBST three times. ECL western blotting detection reagents (Thermo, 32209) were used for the detection step, and the bands were analyzed using ImageJ software.
Flow Cytometric Analysis Of Apoptosis
The different groups of cells were stained with FITC-Annexin V and propidium iodide (PI) in the dark with the Annexin V-FITC/PI Apoptosis Detection Kit (BD556547, BD Bioscience, USA) according to the manufacturer’s instructions. Then, each sample was washed and analyzed with an Accuri™ C6 flow cytometer (BD Bioscience, USA).
Assessment Of Autophagy By Monodansylcadaverine (Mdc) Staining
MDC is a fluorescent dye that stains autolysosomes, and it was used to observe cellular autophagy. SH-SY5Y-APP cells were seeded in 12-well plates with cover slips. After incubation with 10 µM MDC (G0170, Solarbio, China) for 45 min in the dark, the cells were washed once with PBS and visualized by fluorescence microscopy (Leica DMI8, Germany, 20 × objective) with an excitation wavelength of 355 nm and emission wavelength of 512 nm. LAS X software was used to acquire images. In order to measure the mean fluorescence intensity of MDC, Image ProPlus software (Version 6.0, Media Cybernetics, Bethesda, FL, USA) was used to quantitative analysis.
Statistical analysis
All experiments in this study were repeated at least three times for each condition. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software Inc. La Jolla, CA, USA). The data were analyzed by one-way analysis of variance (ANOVA) followed by the multiple comparisons test. The data are expressed as the mean ± SD, and the p value threshold was set at ≤ 0.05.