Adult male Sprague–Dawley rats weighing 200 to 300 g (Experimental Animal Center of Chongqing Medical University) were housed in a specific-pathogen-free facility with a 12-h light/dark cycle and were allowed free access to standard chow and water for 1 week. Then, rats were randomly divided into 2 groups: control (n = 10), which remained on a standard diet until the end of the experiment, and HG diet, which was fed regular chow and had continuous free access to water containing 50% glucose (n = 10). Rats were sacrificed by intraperitoneal injection of pentobarbital after a 40-day experimental procedure. Brains were removed for analysis, and their remaining carcasses were transferred to the animal care center.
Glucose tolerance test
After a 40-day experimental procedure, blood glucose was measured using a glucometer (Roche Diagnostics Scandinavia AB, Bromma, Sweden). Rats were fasted overnight for 16 h. The plasma glucose concentration of tail blood was measured before and 2 h after intragastric injection of 50% glucose (2 g/kg body weight).
Antibodies and chemicals
Anti-tyrosine hydroxylase (TH) (ab112, Abcam, Cambridge, UK); Anti-TH (sc-25,269, Santa Cruz, Santa Cruz, USA); anti-cleaved Caspase 3 (9664; CST, Danvers, USA); Anti-Fyn (ab184276, Abcam, Cambridge, UK); anti-p-Y416 (6943; CST, Danvers, USA); anti-mTOR (2972; CST, Danvers, USA), anti-p-mTOR (Ser2448) (5536; CST, Danvers, USA); anti-p70 S6 Kinase (9202; CST, Danvers, USA); anti-p-S6K(Thr389) (97,596; CST, Danvers, USA); anti-β-actin (20536–1-AP, Proteintech, Wuhan, China), FITC-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China); FITC-conjugated donkey anti-mouse (Proteintech, Wuhan, China); Alexa Fluor 555-conjugated donkey anti-rabbit (Beyotime, Haimen, China); PP1 (HY-13084,MCE,New Jersey, USA); rapamycin (HY-10219, MCE, New Jersey, USA); all-trans retinoic acid (RA) (R2625, Sigma–Aldrich, Missouri, USA); glucose (15,023,021,Thermofisher, Waltham, USA).
Human SH-SY5Y neuroblastoma cell culture, differentiation and treatment protocol
SH-SY5Y cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in MEM/F12 containing 10% fetal bovine serum (FBS), 25 mM glucose and 1% penicillin-streptomycin (100 units/mL) (Thermo Fisher, Waltham, USA). After plating for 24 h, media containing 10 μM RA was used to induce differentiation on day 1 and was continued for 5 days. On day 4, the medium was replaced with MEM/F12 medium containing 1% FBS and 10 μM RA. On day 6, differentiated cells were maintained in MEM/F12 medium containing 75 mM glucose or 69.5 mM mannitol plus 5.5 mM glucose for another 6 days. All drugs were dissolved in DMSO. Cells were pretreated with PP1 (10 μM) or rapamycin (100 nM) for 1 h before HG (75 mM) treatment and continued with the following procedures. Media was replaced every 2 days.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
TUNEL staining of brain sections in the substantia nigra from rats was performed using the One Step TUNEL Apoptosis Assay Kit (C1090, Beyotime, Haimen, China). Dopaminergic neurons were labeled with an anti-TH antibody and a related FITC-conjugated secondary antibody. Images were acquired using a laser scanning confocal microscopy (Nikon 1R, Japan).
Cell viability measurement
MTS analysis was performed using a Celltiter 96 AQueous One Solution Assay Kit (Promega, Madison, USA) to measure cell viability. Briefly, SH-SY5Y cells were seeded into 96-well plates and differentiated with RA for 5 days. After further treatment with drugs (detailed in the cell culture, differentiation and treatment protocol section), cells were incubated with 100 μL of newly added media containing 10 μL of MTS reagent at 37 °C for 4 h. The absorption was subsequently measured at 490 nm using a microplate reader (MultiSkan, GO, Thermo Fisher, Waltham, USA).
Measurement of apoptotic death
After treatment with drugs, differentiated SH-SY5Y cells were collected and resuspended in phosphate buffer solution. An Annexin V-FITC apoptosis analysis kit (Sungene Biotech, Tianjin, China) was used to identify apoptotic cells. Next, the apoptotic rate was determined using CytoFLEX (Beckman Coulter, California, USA).
Quantitative real-time polymerase chain reaction (qRT–PCR)
Total RNA from the substantia nigra of brain tissues from rats after a 40-day experimental procedure and drug-treated differentiated SH-SY5Y cells were extracted using RNAiso plus (Takara, Shiga, Japan) and transcribed into complementary DNAs using HiScript 21II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) on the Applied Biosystems Veriti-Well Thermal Cycler (Thermo Fisher, Waltham, USA). qRT–PCR was performed using ChamQ Universal SYBR qPCR MasterMix (Vazyme, Nanjing, China) on the CFX96 Real-Time System (Thermo Fisher, Waltham, USA). Relative gene expression levels were calculated using the 2−ΔΔCt method . The primer sequences were as follows: Human Fyn: forward: 5′-GGTGTGAACTCTTCGTCTCATA-3′; reverse: 5′-TGTCCGTGCTTCATAGTCATAA-3′. Rat Fyn: forward: 5′-AGCGAAACTGACGGAGGAGAGG-3′; reverse: 5′-GTGCTGAGGGGTGGGGTCTG-3′. Actb2 was used as a housekeeping gene for qRT–PCR in this study.
Total proteins were extracted from substantia nigra brain tissues of rats after a 40-day experimental procedure and differentiated SH-SY5Y cells with respective treatments using RIPA (Beyotime, Haimen, China). Centrifuged protein lysates were mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and boiled for 10 min. Equal amounts of protein were separated by 8–12% SDS–PAGE, gels were cut and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked with QuickBlock Blocking Buffer (Beyotime, Haimen, China) for 30 min at room temperature and then incubated with primary antibodies overnight at 4 °C. β-actin was used as a housekeeping gene. After washing in Tris-buffered saline with Tween-20 (TBST), membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (A21020 Abkine, Wuhan, China) for 1 h at room temperature. Proteins on the PVDF membranes were visualized using a chemiluminescent HRP substrate (WBKLS0100, Merck Millipore, Darmstadt, Germany) and scanned using a Fusion-FX7 image analysis system (Vilber Lourmat, Collégien, France).
Protein extracts from differentiated SH-SY5Y cells treated with HG or not for 6 days were diluted in cell lysis buffer for immunoprecipitation (Beyotime, Haimen, China) and incubated with 2 μg of rat IgG (Beyotime, Haimen, China), anti-Fyn antibody, or anti-mTOR antibody overnight at 4 °C. Then, Protein A/G agarose (Beyotime, Haimen, China) was added to the mixtures and rotated at 4 °C for 2 h. The mixtures were centrifuged at 1000 g for 5 min, and the precipitates were washed with lysis buffer 5 times. Next, the immunoprecipitates were dissolved in 1× SDS loading buffer and boiled for 10 min. Western blots were performed as described above to verify the interactions between proteins.
Fixed brain tissues were dehydrated in 30% sucrose and sliced into 10-μm-thick frozen sections that were immersed in acetone for 15 min at 4 °C and permeabilized in 0.4% Triton X-100. Then, the sections were blocked with normal donkey serum at room temperature for 1 h after antigen retrieval. Sections were incubated with TH and Fyn, mTOR or cleaved caspase 3 antibody at 4 °C overnight. The sections were then washed with PBS again and incubated with the corresponding secondary fluorescent antibody in darkness at room temperature for 1 h. Then, the sections were stained with DAPI at 37 °C for 10 min and mounted with antifade mounting media. Images were acquired using laser scanning confocal microscopy (Nikon 1R, Japan).
All data were assessed for distribution, and if the data were normally distributed, they are presented as the means±standard deviation (SD). At least three replicated independent experiments were performed. Data analysis and graph creation were performed using SPSS 20.0 software (IBM, Armonk, USA) and GraphPad Prism 6.01 (GraphPad software, La Jolla, USA). The differences between 2 groups were analyzed using Student’s t-test. For comparisons of 3 or more groups, one-way ANOVA with Bonferroni’s or Dunnett’s T3 post hoc analysis was used. p < 0.05 was considered statistically significant.