1.1 Cell Culture and treatment
The Conservation Genetics of the Chinese Academy of Sciences Kunming Cell Bank provided SH-SY5Y cells which were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with high glucose, 1% penicillin/streptomycin solution and 10% fetal bovine serum (FBS) (all products from ThermoFisher Scientific, OR, USA) at 37 ℃ under 5% CO2. β-Amyloid (Aβ) [1-42] (Human) (Invitrogen Scientific, OR, USA) was first dissolved in ddH2O at 6mg/mL and diluted to 1mg/mL with PBS prior to undergoing a 6 hours incubation period at 37 ℃. 0.025% butylated hydroxytoluene enhanced tetrahydrofuran (Sigma-Aldrich, MO, USA) was used to dilute lycopene (Sigma-Aldrich, MO, USA) to form a 10 mM stock solution. PBS was used for further dilution to improve solution stability and uptake [17]. SH-SY5Y cells were subjected to a 2 h-treatment period in the absence or presence of lycopene (5 μM) before being further incubated for 24 h with Aβ1-42 (20 μM). Cellular products were then harvested for further investigation.
1.2 Animals and treatment
Four-month old wild-type mice along with age-matched transgenic APP/PS1 (APPswePSEN1dE9) mice from the same litter were procured from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). All mice were allowed water and food ad libitum and reared in standard laboratory conditions for one month before further treatment. Animal experimental protocols were created in compliance to the regulations of the Institutional Animal Care and Use Committee of Zhongshan School of Medicine, Sun Yat-sen University and were approved by the relevant ethics committee.
After a month of environmental adaptation, APP/PS1 mice or age-matched littermates were fed 2 mg/kg lycopene (Sigma, MO, USA) diluted in corn oil or pure corn oil once a day for 12 weeks via gavage. 0.025% butylated hydroxytoluene enhanced tetrahydrofuran (Sigma, MO, USA) was used to dilute lycopene in order to produce a 10 mM stock solution before the addition into corn oil. The control group received only THF via the same method.
1.3 Morris Water Maze
A computerized tracking system of the Morris water maze (MT-200, Chengdu, China) was performed 12 weeks later to determine mice memory and spatial learning abilities. A tank of 50-cm-height and 120-cm-diameter containing opacified water at a temperature of 24 ± 1 ℃ was used. A 10-mm platform located 1 cm underwater was centered in the tank target quadrant after its division into four equal quadrants. Mice was first allowed to explore the tank four times a day for 120 s per trial over six consecutive days in the task acquisition phase. The escape latency was documented during the initial part of the test. Mice which failed to find the platform in 120 s were manually placed on it and kept in place for 20 s. The probe trial took place 7 d post-platform removal. The mice were allowed to swim for 120 s. Time spent in the target quadrant and number of times crossing the original platform area were recorded.
1.4 Tissue preparation
All mice were sacrificed immediately after the behavioural test. 50mls of cold normal saline was transfused intracardially and mice brains were harvested. All right and left cerebrums were divided. 4% paraformaldehyde was used to fix one hemisphere for histological analysis. Liquid nitrogen was used to freeze the isolated cortex and hippocampus from the other hemisphere. Samples were preserved at -80℃ for further experiments.
1.5 Immunofluorescence
Paraformaldehyde-treated hemisphere was immersed in phosphate buffer saline (PBS) containing 30% sucrose for 2 days before a freezing microtome (Leica SM2000R) was used to produce 40 µm sections. Sliced sections were rinsed thrice with PBS before being immersed for 30 mins in 1% bovine serum albumin (BSA) at 37°C. Primary antibodies were then exposed overnight to the sections at 4°C. Immunofluorescent secondary antibodies was then added the next morning after rinsing the samples thrice with PBS for 1h at 37°C. Cell nuclei were counterstained for 30s with Hoechst solution. The primary antibodies incorporated comprised of mouse anti- Aβ (1:1000, A5213, Sigma-Aldrich), rabbit anti-Iba-1(1:500, 019-19741, Wako Chemical), rabbit anti-GFAP (1:100, G9269, Sigma-Aldrich), mouse anti-NeuN (1:1000, MAB377, Chemicon International). The fluorescent secondary antibodies included: Alexa Fluor 488 goat anti-mouse (1:200, SA00006-1, Proteintech), Alexa Fluor 594 goat anti-rabbit (1:200, SA00006-4, Proteintech).
Microscopic analyses of Aβ/Iba-1, Aβ/GFAP, NeuN signal were imaged using a Zeiss LSM 800 confocal laser scanning microscope. Microscopic analyses of Aβ deposition were imaged using a Zeiss Axio Scan Z1 automatic digital slide scanning system. The fluorescence of each special signal was captured using the same parameter. The percentages of specially stained areas were determined by imaging three coronal sections spaced 200μm apart through the regions of interest (hippocampus or cerebral cortex) per mouse. Images were converted to digital quantification using the Image J software.
1.6 Western blot
Western blot was used to assess protein levels. SDS-PAGE gels were used to separate protein samples prior to immunoblotting onto polyvinylidene difluoride (PVDF) membranes. This was followed by endogenous reaction blocking for an hour at room temperature using bovine serum albumin (BSA) or 5% non-fat dry milk in TBST. Primary antibodies were then added onto the membranes before they were left to incubate overnight. The next morning, species specific HRP-conjugated secondary antibody was exposed at room temperature to the samples for an hour. A chemiluminescence substrate was used to develop samples. The corresponding bands were visualized with GE AI600 Imaging System (GE, USA). The primary antibodies used included Keap1(1:1000, ab66620, Abcam), Nrf2(1:1000, 14596S, Cell Signaling Technology), Gclc(1:1000, ab207777, Abcam), Gclm(1:1000, ab124827, Abcam), GSK-3β(1:1000, 12456, Cell Signaling Technology), p-GSK-3β(Ser9)(1:000, 5558, Cell Signaling Technology), AKT(1:1000, 9272, Cell Signaling Technology), p-AKT(Ser473)(1:1000, 4058, Cell Signaling Technology).
1.7 Quantitative real-time PCR
qRT-PCR was used to assess relative gene expressions. The HiPure Total RNA Mini Kit (Magen, Guangzhou, China) allowed for total RNA extraction, while the PrimeScriptTMRT Master Mix (Takara, Shiga, Japan) was used to cDNA production. Quantitative real-time PCR was then performed with the TB GreenTM Premix Ex TaqTMⅡ(Takara, Shiga, Japan) in compliance to manufacturer protocols. The CFX96 Real-Time PCR Detection System (BIO-RAD, USA) with SYBR GreenⅠ Master (Takara, Shiga, Japan) was used to carry out all reactions. The ∆∆Cq method was used to assess all data collected using the Bio-Rad CFX manager 3.1 software. The chosen internal control was GAPDH.
1.8 MDA assay
The malondialdehyde (MDA) Assay Kit (Beyotime, China) was used for this experiment. Cells were harvested after treatment as described previously. The lysates were sonicated on ice and centrifuged for 10 min at 1600 rpm at 4°C. The Bicinchoninic Acid Protein Assay (BCA) kit (Beyotime, China) was used to quantify concentration of the supernatants. The MDA content in the supernatants was measured using a spectrophotometer at 532 nm (Tecan, Switzerland) prior to calculation of relative protein levels.
1.9 8-OHdG assay
An enzyme-linked immunosorbent assay kit (Cloud-Clone, USA) was used to determine 8-hydroxydeoxyguanosine (8-OHdG) levels. Cells were placed onto 6-well plates and treated as described previously. The supernatants were extracted from the differentially treated cells and were assayed following the supplier’s instructions. Supernatant 8-OHdG content was measured spectrophotometrically at 450 nm.
1.10 ROS assay
Reactive oxygen species (ROS) was analyzed using the ROS Assay Kit (Beyotime, China). Briefly, cells were planted onto 96-well plates and preincubated with lycopene at concentration of 5μM for 2 h, followed by a 24 h exposure to Aβ1-42. The cells were then rinsed with PBS before and incubation with ROS substrate solution for 30min at 37 ℃ under 5% CO2 protecting from light. The absorbance was read at 490nm using a multifunctional microplate reader (SpectraMax M5, USA).
1.11 SOD assay
SOD concentration was analyzed with the help of a SOD Assay Kit (Beyotime, China). Cells were harvested after treatment as described previously. The lysates were sonicated on ice and centrifuged at 1600 rpm for 10 min at 4°C. The harvested supernatant was subjected to analysis using the BCA kit (Beyotime, China). The SOD concentration in the supernatants was measured spectrophotometrically at 450 nm (Tecan, Switzerland) and analyzed in relation to the protein levels.
1.12 GSH and GSSH assay
Glutathione (GSH) and GSSH were analyzed using the GSH and GSSH Assay Kit (Beyotime, China). In short, 6-well plates were used to seed cells which were pretreated as described previously. After incubation, cells were collected and GSH and GSSH were detected follow the instruction of manufacturer. A microplate reader was used to assess absorbance at 412 nm (Tecan, Switzerland).
1.13 Statistical analysis
The SPSS 20.0 software (IBM, NY, USA) was used to carry out all data analysis. The two-way repeated-measures ANOVA, followed by LSD post hoc comparisons, allowed for the assessment of escape latency data. Comparisons between two or more groups were carried out using the one-way ANOVA test followed by LSD post hoc comparison. Mean±SEM were used to express data. Statistical significance was achieved when P<0.05.