Cardiovascular disease (CVD) is the leading cause of deaths in the population of adults aged 85 years and older [
Due to the rapid induction and recovery from anesthesia, propofol, 2-6-diisopropylphenol, is extensively utilized in general anesthesia and sedation [
Forkhead box protein O1 (FoxO1) is one important forkhead transcription factor which plays a crucial role in cellular adaptation to oxidative stress through regulation of antioxidant genes [
The H9c2 cells, a cardiomyocyte cell line originally derived from the rat left ventricle, were purchased from the Cell Bank of Chinese Academy of Science (Shanghai, China). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum both from GIBCO-Invitrogen (Grand Island, NY). Cells were maintained in a humidified atmosphere consisting of 5% CO2 and 95% air at 37°C.
Cells were exposed to hypoxic conditions (oxygen deprivation, 0.5% O2) for 24 h in culture medium deprived of glucose and serum. After hypoxia, the cells were reoxygenated under normoxic conditions (reoxygenation) for 24 h in normal medium [
For cell viability experiments, cells were seeded in 96-well cell culture at 2 × 104 cells/well. After 24 h of culture, cells were treated with propofol or NAC for hypoxia-oxygenation, respectively. Cell viability was assessed by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Beyotime, Haimen, China). Then, 10
Cell death was assessed based on the amount of lactated hydrogenase (LDH), which was measured using a LDH Activity Assay Kit (Beyotime, Haimen, China). Culture medium was collected and transferred to a 96-well plate. LDH reaction mix was added to each well, and the plates were incubated for 30 min at room temperature (RT). The absorbance was read at 450 nm when the reaction was stopped.
Intracellular ROS levels were monitored by using 2′, 7′-dichlorofluorescin diacetate (DCFH-DA, Beyotime, Haimen, China), which forms the fluorescent compound dichlorofluorescein on oxidation with ROS. Cells were preloaded with 10
MDA assay was determined by lipid peroxidation MDA Assay Kit (Beyotime, Haimen, China). Cells were lysed and reacted with thiobarbituricacid (TBA). The product has an absorbance peak at 532 nm. MDA was calculated by using a standard curve according to the manufacturer’s data sheet.
SOD activity was assayed with a commercial kit purchased from Dojindo (Kumamoto, Japan). Cells were sonicated with ice-cold PBS buffer (PH 7.4). After protein concentration was determined, samples were reacted with WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl) 2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon reduction with a superoxide anion. After 20 min of incubation at 37°C, absorbance was measured at 450 nm with a microplate reader. Specific activity was calculated by using the equation according to the manufacturer’s data sheet.
The activity was measured by CAT Assay Kit (Beyotime, Haimen, China), using the peroxidation function of catalase for determination of enzyme activity, based on the enzyme reaction with methanol in presence of H2O2, to produce formaldehyde. The product has an absorbance peak at 520 nm. CAT was calculated by using a standard curve according to the manufacturer’s data sheet.
After treatment, cells were harvested and washed with cold phosphate buffered saline (PBS). Cells were lysed with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Roche, Germany) and centrifuged. The supernatants were collected and quantified for protein concentration with bicinchoninic acid (BCA) kit (Beyotime, Haimen, China) according to the manufacturer’s instructions, separated on 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA in TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated sequentially with primary antibodies at 4°C overnight. A variety of primary antibodies were used during these experiments: FoxO1 (Cell Signaling, Danver, MA, USA), p-AMPK (Cell Signaling, Danver, MA, USA), AMPK (Cell Signaling, Danver, MA, USA), SOD1 (Protein Tech Group, Chicago, IL, USA), CAT (Protein Tech Group, Chicago, IL, USA),
All assays were independently done three times. All data were presented as the mean ± standard error of mean (SEM). Quantitative data were analyzed by one-way analysis of variance (ANOVA). Student-Newman-Keuls test was used for post hoc analysis to identify significant differences between groups. Statistical significance was set at
Compared with control group, cell viability was significantly decreased during OGD/R insult in model group. Compared with model group, propofol treatments significantly inhibited the decrease of cell viability and LDH leakage induced by OGD/R (Figures
Protective effects of propofol on OGD/R induced cytotoxicity in H9c2 cells. (a) Cell viability was assessed by measuring the MTT reduction. Propofol (5, 10, 20, and 50
OGD/R is known to induce oxidative stress. In this experiment, OGD/R obviously elevated intracellular ROS levels compared with control. Interestingly, compared with OGD/R group, both propofol and NAC obviously inhibited ROS levels (Figure
CAT and SOD are endogenous antioxidative enzymes that protect against ROS-induced damage [
Enzymatic activity of SOD activity (a), CAT activity (b) and western blotting of SOD1 expression (c), and CAT expression (d) in H9c2 cells. For detecting protein expression, cells were treated with propofol (5, 10, and 20
We evaluated FoxO1 expression by using the ratio of FoxO1 and GAPDH in each group. Compared with the control group, the ratio of FoxO1 and GAPDH was significantly decreased during OGD/R insult in model group and the levels of FoxO1 in propofol groups were increased in a dose-dependent manner (Figure
Western blotting of FoxO1 and AMPK expression in H9c2 cells. (a) The effects of propofol on FoxO1 expression. The levels of FoxO1 were determined by western blotting and GAPDH was used as positive control. (b) The effects of propofol on AMP activated protein kinase expression. The levels of phospho-AMPK and AMPK were determined by western blotting; at this time,
In our research, the propofol emulsion we used contains 10% soybean oil, and the final concentration of oil in the culture media was less than 0.1% in the following experiments. The lipid control group showed no significant effect on cell viability compared with the control group (the data were not shown). In addition, other research shows that nonlipid nanoemulsion propofol and propofol emulsion were equivalent concerning effectiveness, safety, and adverse effects in the doses used [
Oxidative stress has been implicated as a major aspect of the pathophysiology of ischemic myocardial injuries, in which reactive oxygen species generated during the reperfusion period induce a variety of cellular damages, which is an important risk factor in the pathogenesis of cardiovascular ischemic diseases [
Under physiological conditions, cytoplasmic reactive oxygen species are scavenged by the antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as other small molecular antioxidants, such as glutathione, ascorbic acid, and
SODs are a class of enzymes that catalyze the dismutation of two superoxide radicals to form hydrogen peroxide and molecular oxygen [
The FoxO subfamily of forkhead transcription factors plays a crucial role in cellular adaptation to oxidative stress through regulation of antioxidant genes as well as by transactivating ROS-detoxifying enzymes such as superoxide dismutase and catalase [
Activity of FoxO was regulated by AMPK in different cell lines during oxidative stress [
In summary, propofol promotes cell survival through improving the activities and protein expressions of antioxidant enzymes against oxidative stress injury in H9c2 cells
All the authors have no conflict of interests to declare.
This project was supported by the Scientific Research Foundation of Shanghai Jiao Tong University School of Medicine (Grant no. 12XJ3008), Scientific Research Foundation of Shanghai General Hospital (Grant no. 11B24), and National Natural Science Foundation of China Youth Project (no. 81300090).