Reagents and antibodies
Dexmedetomidine was purchased from Yangtze River Pharmaceutical (Group) Co., Ltd., rabbit anti-ACSL4, rabbit anti-TFR1, and rabbit anti-mTOR were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-phospho-p-mTOR, anti-β-actin, and anti-PTGS2 antibodies were purchased from Abclonal, WuHan. Goat anti-rabbit immunoglobulin (IgG) horseradish peroxidase (HRP) secondary antibodies were purchased from Abclonal, (Wuhan, China). mTOR, TFR1, ACSL4, PTGS2, and GRAPDH primers were synthesized by Anhui tongyong Biological and Technological Company (Anhui, China).HT22 dedicated complete media was obtained from Procell Life Science&Technology Co., Ltd. (Wuhan, China).
Cell culture and treatment
The mouse hippocampal neuron cell line was purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China). The cell line was cultured in HT22 dedicated complete media in a cell incubator with 37℃，5%CO2 and 95% humidity. All neuronal cells were used within 20 generations. Selecting well-growing cells are used in experiments. Inoculate the cells in culture plate at appropriate cell density (in general converge to 80%-90%) and incubated a period of time in a cell incubator, and then subjected to treatments as described in each experiment.
Cell Viability Assay
Accordance with the reagent manufacturer’s protocol, we used CCK-8 Kit to detect the level of cell viability in different concentration of Dexmedetomidine at the same time, the absorbance values were measured at 450 nm using a microplate reader.
PI/Hoechst Fluorescence double-labeling method to detect cell apoptosis
Divided the HT22 cells into several groups as required and seeded cells on a six-well plate at a density of 1×105 cells/well. After culturing for 12 hours, added Hoechst 33342 and PI solution at a final concentration of 5mg/L to the medium. incubated for 10 minutes in the 37°C, 5%CO2 cell incubator. Used fluorescent microscope to observe and calculate the relative cell survival rate.
Malondialdehyde (MDA) Assay
Malondialdehyde (MDA), an end product of lipid peroxidation. Analysis of lipid peroxidation was executed by quantification of Lipid Peroxidation MDA Assay Kit with a specific colorimatria kit (cat #ab118970; Abcam) following reagent manufacturer's illustrations. Treated the cells according to the instructions, then the absorbance of cells was detected with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 532 nm, with MDA concentration calculated based on a standard curve.
Lipid ROS quantification
Configure PBS containing DHE fluorescent dye: add DHE fluorescent dye with a final concentration of 10μmol/L to PBS, inoculated HT22 cells in a six-well plate at an appropriate density as required, cultured for 12 hours, then added 200μM FAC to medium and subsequently incubated 24 hours, cells were stained with DHE for 30 min after washed twice with PBS. Used fluorescent microscope to observe and calculate the relative percentage of reactive oxygen cells.
Ferrous ion Assay
The level of Fe2+ was measured using Mito-FerroOrange (Dojindo Molecular Technologies, Tokyo, Japan). In brief, cells were seeded on Confocal small dishes and subjected to different treatments. The supernatant was discarded, and the cells were washed three times with HBSS. Then, the Mito-FerroOrange working solution (2 μL, 2mL) was added to the cells and incubated for 30 min. The cells were observed by confocal fluorescence microscopy.
Mitochondrial morphology and structure detection
Inoculate HT22 cells in a culture flask at an appropriate density, culture for 12h until the cells converge to 80%-90%, cells were incubated for 12 hours after indicated treatment and then extracted the electron microscope samples. Place samples under a microscope to observe the change of mitochondrial cristae, mitochondrial membrane density and cell nucleus of different groups.
The western blot Analyses
Protein expression was measured by Western blot in accordance with standard agreements. After indicated treatment and incubated, cells were acquired and washed twice with PBS and lysed in RIPA buffer including complete protease and phosphatase inhibitor. Following the instructions, the concentration of protein was quantified by the bicinchoninic acid protein assay kit. Boiled proteins were separated with 6%-10 % polyacrylamide gel electrophoresis (SDS-PAGE) and afterwards transferred onto polyvinylidene fluorides (PVDF) membrane through Semi-dry electroblotting. After blocking with 5 % skim milk, membranes were probed overnight at 4°C with specific primary antibodies. Subsequently, washing with TBST, membranes were incubated with an HRP-conjugated secondary antibody for 1h at room temperature. Enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA) was used to measure the expression of target bands. The primary antibodies used were as follows: anti-PTGS2, anti-p-mTOR, anti-mTOR, anti-TFR1.
GraphPad Prism software 8.0 were employed to statistical in this experiment. Data from three independent experiments and presented as mean ± SD. All data are depicted as the mean standard error of the mean (SEM). Differences between two groups and among groups were analyzed using Student’s t-test and one-way ANOVA. p<0.05 was regarded as statistically significant.