MCP preparation
MCP (purity≥99%) was extracted from M. charantia by water extraction and alcohol precipitation, followed by the elimination of proteins and starch. The extraction method was provided by Professor Min Wang from China Pharmaceutical University. The detail procedure was described as our previous publication [11].
Antibody and reagents
The following primary antibodies were used in this study: mouse anti-SIRT1 antibody [19A7AB4] (1 µg/ml, ab110304, Abcam), rabbit anti-β-catenin antibody [E247] (1:5,000; ab32572, Abcam), mouse anti-beta III Tubulin antibody [TUJ-1] (1:1000, ab14545, Abcam), mouse MAb anti-GFAP antibody (1:1,000; ab106509, Abcam),mouse anti-CNPase antibody [11-5B] (5 µg/ml, ab6319, Abcam), mouse anti-acetyl Lysine antibody [1C6] (1:1,000; ab22550, Abcam), rabbit monoclonal anti-Lamin B1 antibody (1:3000, #13435, Cell Signaling Technology, USA), β-actin (1:5000, #4970, Cell Signaling Technology, USA). The secondary antibodies used in our experiment were goat anti-mouse IgG (1:10000) and goat anti-rabbit IgG (1:10000), which were purchased from Sigma-Aldrich (St. Louis, MO, USA). SIRT1 siRNA (#5239398) was obtained from Life techenologies (USA). Resveratrol was obtained from Sigma-Aldrich (St. Louis, MO, USA). Nicotinamide was acquired from Beyotime biotechnologies (USA).
2.2. Cell culture and treatment
C17.2 cells, an immortalized NSC line, were kindly provided by Professor Jiangang Shen (School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, SAR, China). C17.2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS, Gibco) and 2 mM L-glutamine at 37°C in humidified incubators with a 5% CO2 atmosphere and passaged at 50% confluence every two days. In the experiment, C17.2 cells were incubated with glutamate (100 μM) for 30 mins and then were reperfused for different period of times to mimic ischemic insults in vitro. Subsequently, each experimental group of C17.2 cells were removing glutamate prior to treatment with MCPs. C17.2-NSCs were exposed to Resveratrol (25 μM), Nicotinamide (Sirtuin inhibitors, 25 μM) or MCPs (5, 50 μg/ml) for 24 h. Resveratrol and Nicotinamide were added 24 h before glutamate treatment.
Primary cortical neural stem cells (E16-NSC) culture
All procedures involving the use of animals were approved by the local ethical review committee. Primary cortical neural stem cells (E16-NSC) were isolated from cerebral cortexes of Sprague-Dawley rat embryo on E16. Briefly, whole cerebral neocortices were removed from the rat fetuses, then mechanically dissociated. The single-cell suspension was transfected to 6cm culture dishes and cultured as floating neurospheres in a humidified atmosphere with 5% CO2 at 37°C. Neurospheres were maintained in neural-basal medium (DMEM/F-12, 1:1, Hyclone), which was supplemented with B27-supplement, 20 μg/l basic bFGF and 20 ng/ml EGF (both from Invitrogen), 2 mmol/l glutamine (Invitrogen), 10,000 U/l penicillin and 10 mg/l streptomycin (both from Hyclone). After 5-7 days, the neurospheres were trypsinised (0.05% trypsin with 0.02% EDTA,Sigma–Aldrich) as single-cell and seeded in poly-D-lysine-treated culture plates at a density of 5 × 104 cells. then cells were cultured for 2 to 3 days at 37 °C in a 7.5% CO2 atmosphere and used for in vitro experiment.
siRNA transfection
Small interfering RNAs (siRNAs, 5239398) targeting sitr1 were obtained from Life techenologies. 6-well plates were plated with C17.2-NSCs for transfection, and when cells reached 60% to 70% confluence, the lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) were used to transfected cells with either SIRT1-specific small interfering RNA (siRNA) (50 nM) or negative control siRNA (50 nM) mixed with Lipofectamine RNAiMAX (all from Life Technologies) according to the manufacturer’s instructions. All transfected cells were harvested at 48 h after transfection for subsequent experiment.
2.3. Cellular immunofluorescence staining
For differentiation detection, the expression of Tuj1, GFAP, and CNP were analyzed by cellular immunofluorescence staining. The cells were seeded on glass coverslips in DMEM supplemented with 10% FBS. Coverslips were rinsed with PBS and then fixed with 4% paraformaldehyde solution for 10 minutes at room temperature (RT). After fixation, the cells were incubated in blocking buffer (1× PBS with 10% normal goat serum and 0.3% Triton X-100) for 1 hour at RT. Then cells were rinsed three times with 0.01 M PBS (pH 7.4), incubated with the primary antibody (anti-Tuj1, anti-GFAP, or anti-CNP), overnight at 4°C, washed with PBS for three times, and then incubated with the secondary antibody (Alexa Fluor 488–conjugated goat anti-mouse antibody ,1:200, Invitrogen) for 2 hour at RT, and DAPI (10 mg/mL) was added 15 minutes to display the nuclei. Images of 10 randomly selected fields of view were captured under a fluorescence microscope.
Protein Sample preparation
The cells were washed three times with iced PBS and lysed using 300~400 μL lysis buffer (containing 50 mM Tris-HCl, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, 1 mM Na3VO4, 1 mM p-nitrophenyl phosphate, 0.5 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 10 µg/mL pepstatin) on ice for 30 minutes before collection. The mixture was then centrifuged to isolate the supernatant, which contained the cytoplasmic protein. The pellet was washed by adding 400 μL of lysis buffer and then centrifuged to discarde the supernatant. The pellet was re-suspended with 100 μL of RIPA lysate (containing 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 1 mM p-nitrophenyl phosphate, 0.5 Mm PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 10 µg/mL pepstatin). After three times ultrasonic treatments for 5 seconds at intervals of 5 seconds and centrifugation at 12,000g for 20 minutes at 4°C, the supernatant was the nuclear protein.
Immunoprecipitation
Coimmunoprecipitation (co-IP) was performed according to the following procedures. Briefly, protein samples were precleared for 1 h at 4°C using 20 µl of protein A-Sepharose CL-4B (Amersham Biosciences, Uppsala, Sweden) to remove nonspecific proteins. After cetifugation, supernatants were incubated with 1 µg of primary antibodies overnight at 4°C. Targeted immune complexes were captured with 2 h incubation of Protein A. Samples were eluted three times with immunoprecipitation buffer. Targeted proteins were eluted by boiling at 100°C for 5 min in SDS-PAGE loading buffer and then isolated by centrifugation. Then immunoprecipitates were subjected to Western blot analysis.
Immunoblotting
Western blot analysis was performed according to the standard protocol. Briefly, Proteins were separated by by 4-12 % SDS-PAGE and transferred to PVDF) membranes (Merck KGaA, Darmstadt, Germany) for 90 min at 15 V. The membranes were then blocked with 5 % non-fat dry milk in Tris-Buffered Saline and Tween 20 (TBST) and then incubated with appropriate primary antibodies at 4℃ overnight, followed by washing with TBST, then incubating with horseradish peroxidase-conjugated secondary antibodies in TBST for 1 hour at RT. Then the bands were visualized by ECL (Pierce) and detected by BioRad digital imaging system (Bio-Rad Laboratories, Inc). For quantification, the density of the bands on the membrane were quantified using Image J 1.48 software.
Assessment of SIRT1 activity
Assessment of SIRT1 activity was performed according to the manufacturer's instruction in CELL SIRT1 COLORIMETRY ASSAY KIT (GenMed Scientifics Inc., U.S.A).
Data analysis and statistics
All values are presented as the means ± S.D. Statistical analysis of the results was carried out by Student’s t-test or ANOVA, followed by Duncan’s new multiple range method or the Newman-Keuls test. P-values < 0.05 were considered be statistically significant. All experiments were repeated at least thrice in an independent manner.