Cell separation and treatment
As described previously , Sprague Dawley (SD) rats aged three weeks were purchased from the Experimental Animal Center of Xiangya Hospital of Central South University. All rats were bred in a specific pathogen-free environment in 12-h light-dark cycle and fed with rodent diet and water. All rats were anaesthetized with inhaling isoflurane (2%, CAS NO. 64181101, Lunan Pharmaceutical Co., LTD. Shandong, China) and sacrificed by cervical dislocation. The whole brain was removed after opening the cranial cavity. High-purity brain microvascular fragments were obtained, followed by homogenization and fractionation. Microvessels were subjected to incubation at 37 ℃ and 5% CO2 in a tissue culture flask coated by collagen which contained FBS (10%), heparin (100 milligram per milliliter) and basic fibroblast growth factors (10 nanogram per milliliter). RBMVECs (the rat brain microvascular endothelial cells) were observed using microscopy, and factor VIII-related antigen expression was detected. RBMVECs were transferred into an anaerobic incubator configured to an atmosphere of 5% CO2, 94% N2, 1% O2, 98% humidity and 37°C to induce oxygen and glucose deprivation (OGD). The cells were reperfused by returning them to the incubation medium containing different concentrations of glucose (5, 10, 30, 45 and 75 mM) for 24 h of culture. The RBMVECs cultured in a medium with 5 mM glucose served as the control. In addition, Erastin and RSL3, purchased from Sigma-Aldrich (St. Louis, MO, USA), were dissolved into DMSO with different concentrations. Erastin (2, 5 10 μm and RSL3 (60, 120, 180 μm) were used to treat RBMVECs in vitro for 24 h prior to OGD induction.
All experiments were approved by Institutional Animal Care and Use committee in Central South University. The protocol for the use of rats followed the guidelines of the Care and Use of Laboratory animals.
LncRNA-MEG3-siRNA, p53-siRNA and their negative controls were constructed by Genepharm Co., (Shanghai, China). In addition, the sequence of p53 was inserted into a pcDNA3.1 vector to generate the recombinant plasmid of p53. LncRNA-MEG3-siRNA (20μm), p53-siRNA (25μm) and recombinant plasmid of p53 (50μm) were transiently transfected into RBMVECs with the help of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The expression of lncRNA-MEG3 and p53 was detected 48 h after transfection.
Lactate dehydrogenase (LDH) and cell viability assay
Using a commercial CCK-8 (cell counting kit-8; CK04, provided by Dojindo, Tokyo, Japan), cell viability was assessed. Briefly, after the indicated experiments, a CCK-8 working solution was added to each well and incubated for two hours at thirty-seven centigrade degree. The absorbance values were determined at 450 nm using a microplate spectrophotometer (Bio-Tek, Winooski, VT, USA). A LDH detection kit (purchased from Roche, Basel, Switzerland) was used as previously described [29, 30], and OD (optical density) values were recorded at 450 nm. Cell survival rates and the values of LDH release were standardized to the values in the control group and expressed as percentages.
Real-time quantitative polymerase chain reaction (PCR)
After the indicated experiments, the total RNA was extracted by using Trizol (provided by Invitrogen, Carlsbad, CA, USA) according to the product instructions. PCR was performed with specific primers and a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The 2-ΔΔCt method was used to calculate the relative levels of lncRNA-MEG3, p53, FTH1, GPX4 and ACSL4 . The primer sequences are shown in Table-1.
As described previously , cells were lysed by a lysis buffer. SDS-PAGE (12%, w/v) was used to separate the proteins, which were then transferred onto PVDF membranes (provided by Thermo, USA). Primary antibodies (monoclonal) against GPX4 (cat. no. ab125066, dilution 1:1000, Abcam UK), p53 (cat. no. ab131442, dilution 1:1000, Abcam UK), FTH1 (cat. no. ab240277, dilution 1:1000, Abcam UK), ACSL4 (cat. no. ab227256, dilution 1:1000, Abcam UK) and β-actin (cat. no. ab179467, dilution 1:2000, Abcam, UK), along with peroxidase-conjugated rabbit anti-IgG secondary antibodies (cat. no. A2074, dilution 1:4000, Sigma-Aldrich, USA), were used in the Western blot analysis.
Iron concentration assay
After indicated treatments, RBMVECs were immediately homogenized in PBS (phosphate-buffered saline). The supernatant was collected after centrifugation. The iron concentration was measured by using an Iron Assay Kit (ab83366, Abcam, UK) according to the product instructions. Iron concentrations were expressed as increments compared with the values of the control group (100%).
Lipid reactive oxygen species (ROS) measurement
After experiments, RBMVECs were incubated with C11-BODIPY 581/591 (D3861, Invitrogen). Then, the transplanted RBMVECs were cultured with the reagent at a working concentration of 2.5 µM for 30 min in an incubator. Using flow cytometry (provided by Becton Dickinson FACS Canto TM, USA), cellular fluorescence intensity was analyzed and cell images were acquired through a fluorescence microscope (IX81; Olympus). Lipid ROS production was expressed as increments compared with the values of the control group (100%).
Assessment of MPO (myeloperoxidase) and GSH (glutathione) expressions
RBMVECs were homogenized and the supernatant was collected for the analysis of MPO and GSH expressions by using a MPO assay kit (A044, Jiancheng Bioengineering Institute, Nanjing, China) and a total GSH/oxidized GSH assay kit (A06, Jiancheng Bioengineering Institute, Nanjing, China), respectively. The ratio of GSH/GSSG was calculated. The levels of MPO and GSH were standardized to the values of the control group and were expressed as percentages.
Detection of cell apoptosis
The apoptosis of RBMVECs was detected using an Annexin V-FITC/PI apoptosis detection kit (Sigma-Aldrich Trading Co., Shanghai, China) following the manufacturer’s instructions. Apoptotic cells were quantified using a FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
ChIP (Chromatin immunoprecipitation) assay
Sonicated nuclear lysates were purified, and using a ChIP assay kit (P2078, Beyotime), immunoprecipitation was performed according to the method described in a previous study of the authors in this research .
All data were presented as mean ± SD. One-way analysis of variance was applied with a Bonferroni post hoc test or Student’s t-test. Data were analyzed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). P< 0.05 indicated significant difference.