- MCPs preparation
MCPs (purity≥99%) was extracted from M. charantia by water extraction and alcohol precipitation, followed by the elimination of proteins and starch. Studies in our team [11] have identified MCPs composition by high performance liquid chromatography (HPLC) and mass spectrometry (MS). HPLC-MS/MS analysis revealed that galacturonic acid was the major ingredients of MCP accounted for 93.7% and rhamnose, xylose, mannose, glucose, galactose were in the molar ratio of 0.06%, 0.18%, 0.03%, 0.8% and 0.28% respectively. The extraction method was provided by Professor Min Wang from China Pharmaceutical University.
- 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), rabbit anti-BrdU antibody (1:1000, B35138, Life technologies), mouse anti-beta III Tubulin antibody [TUJ-1] (1:1000, ab14545, Abcam), mouse anti-MAP2 antibody (1:1,000; ab5392, 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). Normal goat serum stock solution was purchased from ZSGB-BIO (#ZLI-9021, China).
- Middle Cerebral Artery Occlusion (MCAO) Model
Adult male Sprague-Dawley rats (240~280 g) were obtained from Shanghai Experimental Animal Center of the Chinese Academy of Science. All the surgical procedures were in accordance with the institutional guidelines, and the experimental procedures were approved by the Animal Ethics Committee of Xuzhou Medical University (Approval ID: SCXK (SU) 2010-0003). The rats were maintained in standard cages in a controlled environment (temperature: 24 ± 1℃; relative humidity: 50~60%; light period: 06:00~18:00) and given access to food and water ad libitum. The rats were used in the study after three days of acclimatization. The middle cerebral artery occlusion (MCAO) stroke model was performed with 5 h of MCAO plus reperfusion 14 d. Briefly, rats were anesthetized with 4% isofluorane and maintain1ed at 2% isofluorane via inhalation. In MCAO group, a silicon-coated suture with a tip diameter of 0.38 mm (Doccol, Redlands, CA, USA) was inserted from the external carotid artery (ECA) into the internal carotid artery (ICA), until its tip occluded the origin of the left middle cerebral artery (MCA). In Sham group, rats underwent the same anesthesia and surgical procedure without MCA occlusion.
- Experimental groups and drug administration
All treatments were administered in a blinded manner. Rats were randomly divided into 3 groups with six mice per group (Fig. 1): (1) sham operation rats; (2)MCAO rats; (3) MCAO rats treated with MCPs (200 mg/kg) after MCAO. The rats treated with MCPs were subjected to intragastric administration of MCPs (200 mg/kg) every 24 h for 2 week after MCAO.
- Brain section Immunofluorescence labeling
Anesthetized rats were perfused through the left ventricle with ice saline followed by ice 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Brains were quickly removed and fixed with the same fixation solution overnight at 4℃. The tissue were gradient dehydrationed with 20% and 30% sucrose for 24-48 h. 20-μm coronal sections were taken from fresh frozen mouse brains. After washing with 0.01 M PBS, the slices were incubated with blocking buffer (PBS with 10% goat serum and 0.5% Triton X-100) for 2 hours at RT. For in situ hybridization, double immunofluorescence was used to co-localize BrdU plus Tuj1 (MAP2, GFAP or CNP) in the same slices overnight at 4℃. Then, appropriate fluorescent antibody was added and incubated at room temperature for 1 hour followed by staining of the nuclei with DAPI (10 mg/mL). Images were obtained with a digital microscope (olympus CKX53, Japan) and analyzed by Image-Pro Plus 6.0 software.
- Cell culture
C17.2 cells are an immortalized NSC line originally derived from external germinal layer of mouse cerebellum. For the unique ability to self-renew and generate both neurons and glia in vitro, this cell line is predominately used as a model for multipotent stem cells[35]. In the present study, C17.2 cells 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.
- Establishment of ischemia-reperfusion cell model and drug treatment
Glutamate is the major excitatory neurotransmitter in the central nervous system. Early events following ischemic damage include reactive oxygen species (ROS)-mediated oxidative stress and glutamate-induced excitotoxicity, both of which contribute to rapid cell death within the infarct core. Exposure to glutamate, implicated in damage induced by oxidative stress, is usually used as an in vitro model of ischemia/reperfusion-induced cell death. For in vitro mimic these damage, the model of injured NSCs by glutamate was built up. The detail method were described as previously reported [36.37]. Briefly, for C17.2 cells, glutamate-mediated neurotoxicity was induced by 30 min exposure to 100 μM glutamate in the normal feeding medium. For restoration, cultured C17.2 cells were rinsed twice with PBS and the original feeding medium was restored for different period of times (0d,1d,3d,4d,5d,14d) to establish glutamate-induced insults in vitro.
For drug treatments, MCPs at various concentrations (1.0, 2.0, 3.0, 5.0, 10.0 & 20.0 µg/ml) were added into cell cultures for 24h after the end of glutamate exposure. To activate or inhibit Sirt1, C17.2 cells were pre-incubated with Resveratrol (Sirt1 activators, 25 μM) or Nicotinamide (Sirt1 inhibitors, 25 μM) for 24 h prior to glutamate exposure.
- 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.
- 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 5% normal goat serum stock solution) 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 and Subcellular 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
Sirt1 deacetylase activity was determined with the CELL SIRT1 COLORIMETRY ASSAY KIT ( GenMed Scientifics Inc., U.S.A) based on FluordeLys-SIRT1 substrate peptide. Protein extracts from C17.2 cells were incubated with the fluorogenic acetylated peptide substrate. The reaction was carried out at 37°C for 1h, and the fluorescent signal was measured at 360nm excitation and 460nm emission on a fluorescence plate reader. Results were expressed as percentage of the control.
- Data analysis and statistics
All values are presented as the means ± SD. Statistical analysis of the results was carried out by one-way or two-way ANOVA, followed by Duncan’s new multiple range method or the Newman-Keuls test. P-values < 0.05 were considered be statistically significant. All of the experiments were repeated at least three times in an independent manner.