Experimental animals and reagents
Male Sprague-Dawley rats (weighing 210–250 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China) and fed under a 12-h light/dark cycle, with no restrictions to food or water. Primary astrocytes were obtained from the cerebral cortex of newborn Sprague-Dawley rats. All rat care and use procedures were consistent with the regulations established by Chongqing Medical University, approved by the Institutional Animal Care and Use Committee, and performed in accordance with National Institutes of Health guidelines for the care and use of experimental animals.
Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Poly-l-lysine was purchased from Sigma-Aldrich (Milan, Italy). Trypsin and Hank’s solution were purchased from HyClone (Logan, UT, USA). Penicillin/streptomycin and phosphate-buffered saline (PBS) were obtained from Beyotime (Shanghai, China).
Primary culture of rat cortical astrocytes and oxygen-glucose deprivation/recovery (OGD/R) treatment
The method used for astrocyte culture was described in our previous study [7]. Briefly, cortical astrocytes were inoculated in 6-well plates at a density of 2.0 × 106 cells/well in DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin and then cultured in a 5% CO2 humidified incubator at 37 °C. After culture for 2–3 weeks, microglia and oligodendrocytes were removed from the plates, and the plates were shaken gently. The remaining astrocytes were separated using trypsin, and the digested cells were directly plated into 6-well plates. Astrocytes were used for subsequent experiments after adhering to and distributing on the bottom of the plate. The purity of astrocytes was detected by glial fibrillary acidic protein staining.
The method for OGD/R was described previously [7]. Briefly, astrocytes were established with glucose-free DMEM, which was previously equilibrated with 94% N2, 1% O2, and 5% CO2 for 6 h in an incubator at 37 °C. Then, glucose-free DMEM was replaced with normal medium (DMEM/F12 medium, containing 10% FBS and 1% penicillin/streptomycin) and placed in an incubator containing 5% CO2 for 24 h at 37 °C. The experimental animals were randomly divided into the following groups: control group, OGD/R group, scramble group (OGD/R + scrambled small interfering RNA [siRNA]), and SIRT1 siRNA group (OGD/R + SIRT1 siRNA).
Middle cerebral artery (MCA) occlusion (MCAO)/R model and groups
Ischemic stroke was induced by MCAO, as previously described [18]. Briefly, the tip of a 0.32-mm circular plug was inserted into the left internal carotid artery through the residual end of the common carotid artery and was then pushed gently to block the MCA, thereby inducing ischemia. The blood flow was recovered by pulling the plug out 70 min later. The same procedure was performed in sham operated animals to separate the left common carotid artery without occlusion. Rats with neurological deficit (neurological score = 2) immediately after reperfusion were excluded from the study. We established a transient MCAO reperfusion model and monitored cerebral blood flow, as previously described. The success rate of the MCAO model was evaluated by measuring the neurological deficit score. The operation method in the sham group was the same as that in the MCAO/R group, without blockage of the MCA. SIRT1 siRNA was purchased and used in accordance with our previous study [16]. Adeno-associated virus (AAV) was purchased from Neuron Biotech (Shanghai, China). One month prior to MCAO, AAV was injected into the left cerebral cortex. PGC-1α, PPARγ, and NRF2 siRNAs were designed and synthesized by GenePharma Corporation (Shanghai, China). MCAO was conducted 24 h after intraventricular injection of PGC-1α, PPARγ, and NRF2 siRNAs, as described previously [16], after overexpression of SIRT1.
All rats (n = 378) were randomized into two experiments. In the first experiment, we evaluated the effects of SIRT1 on oxidative stress after ischemic stroke, whereas in the second experiment, we evaluated the regulatory mechanisms of SIRT1 on the PGC-1α/PPARγ/NRF2 pathway. For the first experiment, the rats were randomly assigned to the following groups: sham group (n = 32, 2 died); MCAO/R group (n = 36, 6 died); MCAO/R + negative control siRNA group (n = 34, 4 died); and MCAO/R + SIRT1 siRNA group (n = 38, 8 died). For the second experiment, the rats were randomly assigned to the following groups: MCAO/R group (n = 35, 5 died); MCAO/R + negative control AAV group (n = 34, 4 died); MCAO/R + SIRT1 AAV group (n = 33, 3 died); MCAO/R + SIRT1 AAV + NC siRNA group (n = 35, 5 died); MCAO/R + SIRT1 AAV + PGC-1α siRNA group (n = 33, 3 died); MCAO/R + SIRT1 AAV + PPARγ siRNA group (n = 34, 4 died); and MCAO/R + SIRT1 AAV + NRF2 siRNA group (n = 34, 4 died).
Cell viability
We used Cell Counting Kit 8 (CCK-8) assays to evaluate the activity of astrocytes. Astrocytes were cultured on 96-well plates. Lentivirus-infected astrocytes were cultured for 3 days before OGD/R. After OGD/R, CCK-8 solution (10 µL/mL) was added to 96-well plates. Astrocytes were cultured at 37 °C for 2 h, and a micro flat-panel reader (Bio-Rad, Foster City, CA, USA) was used to determine the absorbance at 450 nm.
Lactate dehydrogenase (LDH) assay
LDH released from astrocytes was evaluated using an LDH test kit (Nanjing Jiancheng Bioengineering Research Institute), according to the manufacturer's instructions. We used a microplate reader (Bio-Rad) to assess the optical density at 450 nm.
Assessment of malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity
Cells were collected after OGD/R, and brain tissues were collected after the 24 h reperfusion. Cells and brain tissues were gently homogenized. SOD activity and MDA contents were measure using xanthine oxidase and thiobarbituric acid, respectively. All assays were performed with kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing) [31]. A lipid peroxidase MDA detection kit (Jiancheng Biotechnology, Nanjing, Jiangsu, China) was used to determine the MDA content, and a micro flat-panel reader was used to determine the absorbance at 530 nm. Additionally, a SOD activity test kit (Jiancheng Biotechnology) was used to determine SOD activity, and a micro flat-panel reader was used to determine the absorbance at 450 nm.
Immunocytochemistry and immunohistochemistry
Cell slides and frozen sections were fixed with 4% paraformaldehyde and then washed with PBS. Next, 3% FBS/0.01% Triton X-100 was used to seal in PBS at 37 °C for 1 h, and samples were then incubated overnight at 4 °C with the following primary antibodies: anti-SIRT1 (1:150; cat. no. 25122; SAB), anti-PPARγ (1:50; cat. no. sc-271392; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-PGC-1α (1:100; cat. no. NBP1-04676; Novus). Samples were then incubated for 30 min at 37 °C with secondary antibodies tagged with either DyLight 488 (anti-mouse; green; cat. no. A23210; Abbkine) or DyLight 549 (anti-rabbit; red; cat. no. A23320; Abbkine) at 1:200. A laser-scanning confocal microscope was used to observe the sections and glass slides.
Infarct volume analysis
The area of cerebral infarction was evaluated by triphenyltetrazolium chloride (TTC) staining. The infarct area of each coronary section was measured using Image Pro Plus software. The following formula was used to calculate the corrected volume: percentage of infarct volume (%) = (total infarct volume – [left hemisphere volume – right hemisphere volume]) / right hemisphere volume × 100% [19].
Evaluation of neurological deficit score
After MCAO/R, the neurological deficit score of the experimental group was evaluated by blind examiners using a modified scoring system developed by Longa et al. [20].
Brain water content assay
We used the previously described ratio of wet weight (WW) to dry weight (DW) to assess brain edema [21]. After MCAO/R, the rats were sacrificed, and the contralateral and ipsilateral cerebral hemispheres were rapidly removed for measurement of WW. The cerebellum was evaluated as an internal control. The tissues were dried in an oven at 100 °C for 24 h and then weighed to obtain the DW. The following formula was used to calculate tissue water content: (WW – DW) / WW × 100%.
Tissue preparation and histological evaluation
The rats were sacrificed after MCAO/R. Next, 0.9% sodium chloride and 4% paraformaldehyde was used for intraperitoneal perfusion. The brain was embedded in paraffin after dissection and immersed in paraformaldehyde for 24 h. Then, 5-µm-thick coronal sections 1.2 mm anterior to and 3.6 mm posterior to the bregma were stained with hematoxylin and eosin (H&E) or 0.1% cresyl violet (Nissl staining) according to standard procedures and prepared for microscopic examination.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining
TUNEL staining was performed using a kit (cat. no. 11684817910; Roche Molecular Biochemicals, Inc., Mannheim, Germany) according to the manufacturer’s instructions. Briefly, protease K (20 mg/mL, 15 min) and 0.3% H2O2 were used for 30 min to treat sections. Next, sections were incubated with terminal deoxyribonucleotide transferase at 37 °C for 1 h and then with peroxidase-binding antibodies at 37 °C for 30 min. The sections were reacted with 3, 30-diaminobenzidine hydrochloride solution at room temperature for 10 min. A fluorescence microscope (BX51; Olympus Japan) was used to identify TUNEL-positive cells with fluorescein isothiocyanate green fluorescence. TUNEL-positive cells were counted in 10 microscope fields in each section at 400 × magnification.
Western immunoblot analysis
RIPA buffer containing phenylmethylsulfonyl fluoride was added to total proteins extracted from astrocytes and brain tissues. Protein (45 µg/lane) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Next, membranes were blocked with 5% nonfat milk TBST for 2 h and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-SIRT1 (1:150; cat. no. 25122; SAB), anti-β-actin (1:3000; Proteintech), anti-PGC-1α (1:1000; cat. no. NBP1-04676; Novus), anti-PPARγ (1:500; cat. no. sc-271392; Santa Cruz Biotechnology), anti-HO-1 (1:500; cat. no. 10707-1-AP; Proteintech), anti-NQO1 (1:500; cat. no. 11451-1-AP; Proteintech), anti-Lamin B1 (1:800; cat. no. ab16048; Abcam, Cambridge, UK), anti-acetyl-lysine (1:800; cat. no. ab22550; Abcam), and anti-NRF2 (1:500; cat. no. mab3925; RD). The next day, the membranes were washed and then incubated with secondary antibodies at room temperature for 2 h. An imaging densitometer (Bio-Rad) was used to detect the density of bands. Additionally, ImageJ was used to quantify the gray value of each band. The relative expression quantity of protein was standardized to β-actin or Lamin B1.
Quantitative real-time polymerase chain reaction (qPCR)
RNA was obtained and purified from astrocytes and brain tissue as previously reported [22]. Reverse transcription (Bio-Rad) was then used to convert the RNA in cDNA. cDNA was then subjected to PCT using a PrimeScript RT reagent kit (TaKaRa Biotechnology, Dalian). The primer sequences of SIRT1 and β-actin were described in our previous study [16]. A Bio-Rad CFX96 Connect Real-Time system was used to analyze the data. The mRNA levels were normalized to the expression of β-actin.
Immunoprecipitation and immunoblot analyses
Brain tissue and cell lysates were obtained after OGD/R and MCAO/R. Specific antibodies and Protein A/G beads (MCE, NJ, USA) were incubated with brain tissues or cell lysates overnight. Lysis buffer was used to wash beads four times, and immunoprecipitates were separated using SDS-PAGE. For western blotting, anti-PGC-1α (1:1000; cat. no. NBP1-04676; Novus), anti-PPARγ (1:500; cat. no. sc-271392; Santa Cruz Biotechnology), and anti-acetyl-lysine (1:800; cat. no. ab22550; Abcam) antibodies were used.
Electrophoretic mobility shift assay (EMSA)
EMSA was conducted using a Biotin Light EMSA Kit (Exprogen, China). For EMSA, Biotin PPAR probes, i.e., Bio-5-CTCGGAACTAGGTCAAAGGTCATCCCCT-3 3-GAGCCTTGATCCAGTTTCCAGTAGGGGA-5-Bio, and cold oligo probes, i.e., 5-CTCGGAACTAGGTCAAAGGTCATCCCCT-3 and 3-GAGCCTTGATCCAGTTTCCAGTAGGGGA-5 were used. Proteins were incubated in binding buffer at room temperature for 20 min, and samples were separated by nondenaturing 6.5% polyacrylamide gel electrophoresis in 0.25 × TBE buffer. The gel was vacuum-dried and exposed to X-ray film (FujiHyperfilm, Tokyo, Japan) at 80 °C with an intensifying screen. Computer-assisted densitometric analysis was used to assess the levels of PPARγ DNA binding activity.
Statistical analysis
All data are shown as means ± standard errors of the means (SEMs), and statistical analyses were conducted using GraphPad Prism software (version 6.0). Statistical comparisons were assessed with one-way analysis of variance followed by Tukey’s test. Statistical significance were considered when p values were less than 0.05.