PC12 cells were obtained from the Cell bank of the Chinese Academy of sciences.
Genechem company (Shanghai, China) constructed all plasmids including wild type ERα, wild-type SIRT1 and SIRT1 siRNA. NLRP3 antibody was purchased from Adipogen (San Diego, USA). IL- 1β, IL- 18, β-actin and Caspase–1 p10 antibodies were purchased from Santa Cruz Biotechnology (Dallas, USA). Fu GENE HD, FCM Assay System Kit was obtained from Roche (Diagnostics, Indianapolis).
The Laboratory Animal Center of Hubei Province supplied the female Sprague-Dawley (SD) rats with weights ranging between 280 to 320g. Rats were kept in pathogen-free environments and separated into sham, M&OVA, MCAO and M&E2 groups.10% chloral hydrate anesthesia was used prior to bilateral ovariectomization of the M&OVA group by insertion of nylon monofilament into the anterior cerebral artery. Group prior to MCAO, M&OVA groups: 7 days before OVA, the rats were administered estrogen (0.5mg/kg) intraperitoneally before MCAO. Numbers of animals utilized and animal suffering was kept to a minimum. The protocol was approved by the Institutional Animal Care and Use Committee and the Medical ethics committee of wuhan university of science and technology (No:201961).
Middle cerebral artery occlusion/reperfusion model (MCAO)
350mg/kg i.p. of 10%chloral hydrate was used to anesthetize rates by insertion of nylon monofilament (0.25–0.28mm diameter) into the anterior cerebral artery via the external carotid artery for 2h prior to recovering perfusion. Focal cerebral ischemia was induced by occlusion of the right MCAO with a nylon monofilament (diameter criteria,0.25–0.28mm) with a heparin-coated tip (Huang et al., 1994).This coated filament was introduced into the internal carotidartery through the external carotid artery, up to the origin of the anterior cerebral artery, and occluded the MCA andanterior cerebral artery for 2 h. Rats with a neurological score <3 were removed from the study. All rats were euthanized at ischemic 2h-reperfusion 24h. After the MCAO building, once rats lack the alertness of physical or mental that result from the symptoms of unconscious or bleeding, rats would be euthanasia by an overdose of pentobarbital (120 mg/kg intraperitoneal injection) to produce unconsciousness and death [25,26]. The humane method we chosen is to induce a rapid, painless, and distress-free death.
Infarct volume evaluation
Coronal sectioning was performed on brains followed by TTC (Sigma, USA) staining for quantification by incubation in 2% TTC solution at 37°C for 20min prior to paraformaldehyde (4%) fixation. Infarct volume was calculated by totaling infarct area per section and multiplying it through the distance between section as indicated by image analysis software (Version 1.61, NIH image).
Cell culture and Treatment
PC12 cells were cultured in Ham’s F12K Medium supplemented with FBS (2.5%), horse serum (15%), penicillin and streptomycin (100μg/ml) and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Wild-type ERα, SIRT1 or SIRT1 siRNA plasmids were used to transfect PC12 cells following their seeding and coating with poly-L-Lysine. Transfection was performed in Ham’s F12K-Medium with DNA (0.5μg) and Fu GENE (2.5μl) prior to adding culture medium consisting of 400μM hydrogen peroxide.
Brain pieces were fixed in Bouin’s solution following washing in physiological saline. They were then dehydrated using increasing ethanol concentrations, paraffin embedded and cut to 2mm. Brain sections were hematoxylin-eosin (HE) stained and examined under the light microscope.
PC12 cells’ viability was examined using flow cytometry (FCM) following PI and Annexin V-FITC staining.
Detection of LDH release
The LDH-release kit (Nanjing Jincheng Bioengineering Institute) was used to quantify LDH as per manufacturer’s instructions.
Western blot analysis
A cell pellet containing 50μg cells or cerebral cortex was collected and electrophoresed in sodium dodecyl sulfate-polyacrylamide gel (15%) prior to membrane probing with antibodies (NLRP3, caspase–1 p10, IL- 1β, IL- 18, ERα and SIRT1 antibody) and incubated overnight at 4°C. An ECL chemiluminescence system was used to visualize signals using a computerized image processing software (Sunnyvale, CA).
Assays were repeated three to five times and represented as mean + standard deviation (S. D.). ANOVA (analysis of variance) was used to examine significant differences of varied groups. A P-value below 0.05 was considered statistically significant.