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From the Department of Anesthesiology, Anesthesiology Research Laboratories, Renmin Hospital of Wuhan University, People's Republic of China.
Address correspondence and reprint requests to Zhengyuan Xia, MD, PhD, Anesthesiology Research Laboratory, Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, 430060 People's Republic of China. Address e-mail to zhengyuan_xia{at}yahoo.com.
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Abstract |
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cellular toxicity, which might be reversed by propofol, an anesthetic with antioxidant properties, in human vascular endothelial cell line ECV304. Cultured ECV304 were either not treated, treated with 10 µM of hydrogen peroxide (H2O2), treated with TNF- (40 ng/mL) alone, TNF- in the presence of 10 µM of H2O2 (H+T), or propofol plus H2O2 for 24 h. Cell viability was measured by lactate dehydrogenate (LDH) assay. Cell apoptosis was assessed by flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling. The antiapoptotic Bcl-2 and pro-apoptotic Bax protein expressions were measured by immunocytochemical analysis. Increases in apoptosis, Bax, lipid peroxidation product malondialdehyde, LDH, and decreases in Bcl-2, superoxide dismutase, and glutathione peroxidase were observed in TNF-–treated cells. H2O2 10 µM did not cause significant lipid peroxidation (0.75 ± 0.03 nmol/mg of malondialdehyde protein) as compared with control (0.70 ± 0.04 nmol/mg of malondialdehyde protein) (P > 0.05) but further enhanced TNF-–induced lipid peroxidation, upregulated Bax, and down-regulated Bcl-2 expression and enhanced TNF-–induced cell apoptosis (P < 0.05). Propofol 50 µM attenuated TNF- and H2O2-induced cell apoptosis, accompanied by decreases in malondialdehyde and LDH production and restoration of Bcl-2 expression. Propofol exerts protective effects against H2O2-enhanced TNF- cell toxicity by reducing oxidative injury. |
Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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) and reactive oxygen species (ROS), which are increased during myocardial ischemia reperfusion injury and atherosclerosis, promote cardiomyocyte apoptosis subsequent to the induction of endothelial cells apoptosis (2). Thus, inhibition of TNF-– and ROS-induced endothelial cells apoptosis may represent an effective therapy for myocardial ischemia reperfusion injury.
Propofol, an IV anesthetic with potential antioxidant property, has a chemical structure similar to that of phenol-based free-radical scavengers, such as vitamin E, and reduces free radicals (3). Propofol has been shown to attenuate hydrogen peroxide (H2O2)-induced mechanical and metabolic derangements in the isolated rat heart (4). Our study showed that propofol can dose-dependently reduce TNF-–induced human umbilical vein endothelial cells (HUVECs) apoptosis in vitro, and the effect was more profound at concentrations 
50 µM (5). TNF- and ROS may work synergistically in inducing endothelial cell apoptosis. It is unknown, however, whether ROS enhancements of TNF- cellular toxicity was mediated through enhanced lipid peroxidation or primarily through the modulation of pro- and antiapoptotic proteins.
We hypothesize that oxygen free radicals, the production of which is increased in patients undergoing cardiac surgery using cardiopulmonary bypass (6) and often accompanied by increases in TNF- (7), could enhance TNF-–induced cell injury, even at a "trace" concentration that does not cause significant lipid peroxidation per se and that propofol can attenuate TNF- and H2O2 cellular toxicity. The hypothesis was tested using human vascular endothelial cell line ECV304, and the possible associations between cell apoptosis, anti- and pro-apoptosis protein Bcl-2 and Bax, intracellular antioxidant enzyme glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were investigated.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Confluent cultures of ECV304 cells were preincubated for 24 h in Dulbecco Modified Eagle Medium containing 1% serum before experiments. Cells were then randomly divided into five study groups: control, H2O2 (H), TNF- (T), H2O2 plus TNF- (H+T), and propofol plus H2O2 and TNF- (P+H+T). To assess the effect of propofol and H2O2 on TNF-–induced apoptosis, ECV304 cells from different study groups were further incubated for 24 h either with culture media only (control group) or in the presence of H2O2 (10 µM; Group H), TNF- (40 ng/mL; Group T), H2O2 plus TNF- (Group H+T), or TNF- plus H2O2 and 50 µM of propofol (Group P+H+T). In group P+H+T, cells were pretreated with propofol for 30 min before the addition of TNF- and H2O2. All experiments were repeated in seven different cell cultures.
ECV304 cell apoptosis was assessed according to the percentage of cells with hypodiploid DNA using the propidium iodide staining technique, as previously described (8). Briefly, cells (106) were harvested by brief centrifugation and washed once with phosphate buffed saline. They were then fixed in 70% cold methanol on ice for 24 h and resuspended in 1 mL of hypotonic fluorochrome solution (50 µg/mL of PI, 3.4 mM of sodium citrate, 1 mM of Tris, 0.1 mM of EDTA, and 0.1% Triton X-100) and incubated in the dark at 4°C for 1 h before analyzed by flow cytometer (Beckman Coulter). A minimum number of 5000 events was collected and analyzed on the software. Apoptotic ECV304 nuclei were distinguished by their hypodiploid DNA content from the diploid DNA content of normal nuclei. All measurements were performed under the same instrument settings.
Apoptosis was also detected using DNA in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) staining, according to the manufacturer's protocol (Boster Biological Technology CO Ltd, Wuhan, China), as previously described (5). In brief, after equilibration, cells were end labeled with digoxigenin-11-dUTP by TdT enzyme in buffer for 1 h at 37°C in a humidifying chamber. After treatment with stop-wash buffer, slides were incubated with antidigoxigenin antibody–peroxidase conjugate, rinsed, and stained with diaminobenzidine tetrahydrochloride. Negative controls were incubated with phosphate buffed saline instead of TdT enzyme, and positive controls were treated with DNase1. Sections were counterstained with Mayer's hematoxylin and mounted and processed, as described (5).
Cytotoxicity was quantified by measuring lactate dehydrogenate (LDH) release in the medium during the exposure to different reagents. LDH release was determined using a cytotoxicity detection LDH kit (Nanjing Jiancheng Biological Product, China) according to the manufacturer instructions. In this colorimetric assay, h-nicotinamide adenine dinucleotide was reduced to NADH through the conversion of lactate to pyruvate by LDH, and then NADH reduced tetrazolium dyes to formazan dyes in the presence of diaphorase. In brief, 25 µL of culture supernatants were mixed with 75 µL of the LDH substrate mixture in a 96-well plate. After incubation for 1 h at room temperature, the reaction was stopped by adding 100 µL of 1 M HCl, and the absorbance was read at 570 nm. Values of LDH level were expressed as units per liter.
Bcl-2 and Bax protein expression were assessed by immunocytochemical staining, as previously described (5). In brief, slides with cells were blocked with 5% bovine serum albumin in 2% normal goat serum and then incubated with primary polyclonal antibodies against Bcl-2 (1:50; Boster) or Bax (1:200; Boster), respectively. Immunosignals were detected by the avidin biotin peroxidase complex assay kit (Boster) method with diaminobenzidine as chromogen, according to the manufacturer's instructions. To semi-quantify immunocytochemical staining, random fields (20–30 cells per slide) from 3 to 4 slides per group were analyzed by means of computer-assisted image analyzing system (IBAS-2000 Kontron, Germany). The slides were coded, and the investigator was blinded in regard to the groups before the completion of the assays. The density of Bcl-2 and Bax protein expression were expressed in arbitrary units.
Upon completion of the incubation studies, cells were removed and immediately mixed with cell lysis buffer and then sonicated for 10 s. Protein concentration was measured using Coomassie Brilliant Blue protein reagent (Nanjing Jiancheng Biological Product). Aliquots were then collected and quickly frozen at –70°C until assayed for malondialdehyde (MDA) concentrations, SOD, and GSH-Px activity.
Lipid peroxidation was determined by the thiobarbituric acid reaction using a commercial kit (Nanjing Jiancheng Biological Product), according to the manufacturer's recommendations, as previously described (9). Briefly, 100-µL cell lysates were incubated with 1.5 mL of reaction buffer and 1.4 mL of 0.2 M Tris–0.16 M KCl (pH value of 7.4) at 37°C for 30 min, followed by the addition of 1.5 mL of thiobarbituric acid reagent. The mixture was then heated in a boiling water bath for 10 min. After cooling with ice, 3.0 mL of pyridine:n-butanol (3:1 vol/vol) and 1.0 mL of 1 M NaOH were added and mixed by shaking. The absorbance was read at 548 nm. Values of MDA level were expressed as nanomoles per milligrams of protein.
The total SOD activity in cell lysates was assayed using a reagent kit (Nanjing Jiancheng Biological Product) according to the manufacturer's recommendations. The assay was based on the inhibition by SOD of NADPH oxidation by molecular oxygen in the presence of EDTA, manganese chloride, and 2-mercaptoethanol and used under the same conditions described by Paoletti and Mocali (10). One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of NADPH oxidation of the control by 50% at 25°C. SOD activity was expressed as units per milligrams of protein.
The GSH-Px activity in cell lysates was determined with an assay kit (Nanjing Jiancheng Biological Product) according to the manufacturer's recommendations. Briefly, 200 µL of sample lysate was mixed with 2.68 mL of 0.05 M phosphate buffer (pH value of 7.0) containing 5 mM of EDTA, followed by the addition of 0.1 mL of 8.4 mM NADPH, 0.01 mL of glutathione reductase, 0.01 mL of 1.125 M NaN2, and 0.1 mL of 0.15 M glutathione. The enzymatic reaction was initiated by the addition of 0.1 mL of 2.2-mM H2O2. The conversion of NADPH to NADP was followed by continuous recording of the changes in absorbance at 340 nm between 2 and 4 min after initiation of the reaction. One GSH-Px unit was defined as the enzyme activity required to convert 1 mmol of NADPH to NADP per milligram tissue protein and the result was expressed as units per milligrams of protein.
Results are expressed as mean ± sem. Biochemical assays for LDH, SOD, MDA, and GSH-Px were performed in duplicate or triplicate for each specific sample. Therefore, all the data points are the mean of numbers that themselves are the mean of duplicate or triplicate measurements for these parameters. Significance was evaluated using analysis of variance (GraphPad Prism 4) followed by Tukey post hoc test. The correlation relationships were evaluated by the Pearson test. P < 0.05 was considered statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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, which increased ECV304 cells AI by approximately 24-fold (Fig. 1c) (P < 0.001; T versus C). Stimulation of cells concomitantly with H2O2 and TNF- led to a approximately 30-fold increase in cell AI as compared with the control (Fig. 1d) (P < 0.001; T+H versus C; P < 0.05; T+H versus T; P < 0.001; T+H versus H), showing apparent synergistic effects. However, apoptosis caused by TNF- and H2O2 were reduced significantly by the addition of propofol (Fig. 1e) (P < 0.001; T+H+P versus T+H). Results from TUNEL staining (Fig. 2) were similar to that obtained by flow cytometry.
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As shown in Fig. 3, LDH release was minimal in both the control and the H2O2-treated groups. Stimulation of cells with TNF- resulted in a dramatic increase in LDH release, and the LDH release was further significantly increased with the addition of H2O2 (P < 0.001; T+H versus T or H). Propofol significantly reduced TNF-– and H2O2-induced LDH release.
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Treatment with TNF- led to a significant reduction in Bcl-2 protein expression and a significant increase in Bax protein expression as compared with untreated controls (Fig. 4; P < 0.05; T versus C). Stimulation of cells concomitantly with H2O2 and TNF- led to a further decrease in Bcl-2 protein expression (Fig. 4A; P < 0.05; T+H versus T). It is of interest that H2O2 did not exaggerate a TNF-–induced increase of Bax protein expression (Fig. 4B). The ratios of Bcl-2 expression over Bax expression (Bcl-2-Bax) in Groups T and T+H were also significantly reduced as compared with the control (data not shown). In the presence of propofol, the increase in Bax and decrease in Bcl-2 was significantly attenuated (P < 0.05; T+H+P versus T+H), resulting in an increase of Bcl-2-Bax ratio (data not shown).
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The effects of different culture conditions on MDA concentrations are shown in Figure 5. The MDA concentration was small in the control group. H2O2 10 µM did not cause an increase in MDA production (P > 0.05; H versus C). But MDA concentration increased significantly in cells exposed to TNF- (P < 0.001; T versus C), and it was further increased when cells were exposed to both TNF- and H2O2 (P < 0.05; T+H versus T; P < 0.001; T+H versus H). Propofol decreased the MDA concentration induced by TNF- and H2O2 (P < 0.05; T+H+P versus T+H).
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As shown in Figure 6, there were no significant alterations in SOD and GSH-Px activities in cells exposed to H2O2 as compared with the control group. Statistically significant decreases in SOD (P < 0.001; T versus C) and GSH-Px (P < 0.001; T versus C) activities were seen when ECV304 cells were exposed to TNF-. The addition of H2O2 profoundly enhanced TNF-–induced decreases in SOD (P < 0.05; T+H versus T) and GSH-Px (P < 0.05; T+H versus T). Propofol significantly attenuated the decrease of SOD and GSH-Px activities induced by TNF- and H2O2 (P < 0.001; T+H+P versus T+H).
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There was a highly significant inverse correlation between the GSH-Px activity and AI (from flow cytometry assay) (n = 35; r = –0.9363; 95% confidence interval [CI], –0.9695 to –0.8691; P < 0.0001) and between SOD activity and AI (n = 35; r = –0.9228, 95% CI, 0.9629 to –0.8426; P < 0.0001). In contrast, there was a significant positive correlation between MDA production and AI (n = 35; r = 0.8769; 95% CI, 0.7551–0.9401; P < 0.0001).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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in many tissue and cell types (11,12), plays an important role in inducing cell apoptosis. Studies have shown that both the application of exogenous antioxidants such as vitamin E and the over-expression of endogenous antioxidant proteins such as Bcl-2, SOD, catalase, GSH, or GSH-Px can attenuate apoptosis induced by lipopolysaccharide and cytokines such as TNF in different cell types (13,14). Propofol is a widely used anesthetic with antioxidant capacity. However, propofol failed to demonstrate a beneficial affect in cardiac surgical patients when administered at conventional clinical concentrations of 2–4 µg/mL (11–23 µM) as compared with the volatile anesthetic sevoflurane (15,16). In contrast, propofol, when given at 6–8 µg/mL (35–46 µM) during myocardial ischemia and the early phase of reperfusion, has been shown to attenuate free-radical–mediated and inflammatory components of myocardial reperfusion injury in patients undergoing elective coronary artery bypass graft surgery as compared with the volatile anesthetic isoflurane (17). This phenomenon suggests that the potential cardioprotective effect of propofol in patients is dose dependent.
A previous study conducted by our group indicated that propofol dose-dependently reduced TNF-–induced HUVECs apoptosis at concentrations 12.5 µM, and the effect was most profound at concentrations 50 µM (5), but the effect did not significantly further improve when the propofol concentration was increased from 50 µM to 100 µM (5). Also, it is worth noting that propofol administered at large concentration (67 µM) before and during ischemia, as well as during the early phase of reperfusion, reduced the formation of 15-F2t-isoprostane, a specific index of ROS-induced lipid peroxidation, and enhanced functional recovery of the isolated ischemic-reperfused rat heart (18–20). Based on these observations, and the documented effects of propofol in maintaining healthy endothelial cell and heart function (4,21), we used propofol at 50 µM. Propofol 50 µM is more than the conventional dose range but is clinically relevant (17,22). The application of large-dose propofol results in an average propofol concentration of 11 µg/mL (63 µM) during cardiac surgery (22).
We have found that (a) H2O2 enhanced TNF- cellular injury in inducing ECV304 cell apoptosis. Propofol could significantly attenuate ECV304 cells from TNF-– and H2O2-induced cell apoptosis; (b) cells incubated with TNF- and H2O2 markedly decreased the Bcl-2 protein and increased the Bax protein expression, which was significantly attenuated by propofol; and (c) propofol significantly increased intracellular SOD and GSH-Px activity and attenuated the lipid peroxidation product MDA, resulting in enhanced cellular viability, as evidenced by the significant attenuation of cellular LDH release in the presence of TNF- and H2O2.
Our results show that TNF- 40 ng/mL induced cell death with typical apoptotic features in ECV304 cells, in keeping with our previous study (5). However, H2O2 10 µM induced only a small amount of cell apoptosis. The H2O2 concentration (10 µM) used in this study is in the smallest range that induces minimal endothelial cell apoptosis but not necrosis (23). Interestingly, H2O2, when used at such a small concentration (i.e., 10 µM), profoundly augmented TNF-–induced vascular endothelial cell apoptosis. The possible mechanism for this phenomenon is likely that application of H2O2 could result in increased intracellular Bax and decreased Bcl-2 levels, which may make the cells more vulnerable to TNF- (24). Propofol, which had been shown to regulate apoptosis-related proteins in our previous study, attenuated TNF-– and H2O2-induced cell apoptosis by increasing antiapoptotic Bcl-2 and decreasing pro-apoptotic Bax protein production. High LDH activity levels in the TNF- and H2O2 group may be interpreted as a progression of cell injury, and hence, the decrease of LDH release after propofol treatment may be indicative of decreased cell injury.
A novel finding of the current study is that H2O2, at a trace concentration (10 µM), did not cause significantly enhanced lipid peroxidation in ECV304 cells and markedly exacerbated TNF-–induced increases in ECV304 cell lipid peroxidation and pro-apoptotic Bax protein, leading to enhanced cell apoptosis. Propofol attenuated H2O2-mediated exacerbation of TNF- effects. An overexpression of Bax accelerates the apoptotic death of cells triggered by a certain apoptotic stimulus, and the ratio of Bcl-2-Bax determines their survival or death (25). Propofol attenuates neuronal damage with increased Bcl-2 and decreased Bax proteins in rats after cerebral ischemia and reperfusion (26), an event associated with enhanced oxygen free-radical-induced lipid peroxidation (27). It is possible that propofol neuronal protection may be derived, in part, from its attenuation in vascular endothelial cell apoptosis subsequent to the amelioration of oxygen free radical, in particular, H2O2-induced lipid peroxidation.
Excessive ROS results in lipid peroxidation, oxidation of proteins, and DNA damage. Cells are often equipped with several antioxidants for the prevention of free-radical damage. SOD and GSH-Px, along with other enzymatic and nonenzymatic antioxidants, play a pivotal role in preventing oxidative cellular damage. The combined action of GSH-Px and SOD provides a repairing mechanism for oxidized membrane components. In the present study, a significant decrease in SOD and GSH-Px activity was seen after ECV304 cells were exposed to TNF-, indicating impairment in antioxidant defenses. Indeed, an increased intracellular level of MDA was associated with an increase of cellular LDH release and cell apoptosis. TNF- in combination with H2O2 led to the most profound decrease in intracellular antioxidant enzyme activity and increase in MDA. Propofol 50 µM enhanced cellular SOD and GSH-Px levels, resulting in decreased MDA production. The tight inverse correlations observed between the GSH-Px and SOD activities and AI and the positive correlation between apoptosis and MDA jointly suggest that enhancement of endogenous antioxidant preservation and the subsequent attenuation of lipid peroxidation may represent a major mechanism of propofol's cellular protection.
There are some limitations in the present study. First, our study was performed on ECV304 cell lines that are readily available and represent an established in vitro model of endothelial cells; however, it may not well represent the characteristic of cardiac endothelial cells. Second, we used TNF- in our model of inflammatory cytokine-induced endothelial cell injury, whereas factors other than TNF- also contribute to the development of vascular endothelial cell injury and myocardial ischemia-reperfusion injury. Therefore, caution must be taken when extrapolating the findings in the current study to clinical situations. Several reports have demonstrated that propofol may enhance antioxidant capacity, and this property may have contributed to its cardioprotective effects (18–21). However, data on this subject are largely experimental, and few studies have demonstrated an overt beneficial effect of propofol in myocardial stunning in the clinical situation. Nevertheless, propofol has been shown to attenuate myocardial lipid peroxidation during coronary artery bypass grafting surgery (28) and to reduce the postischemic inflammatory response in patients (17), which are potentially beneficial. Therefore, further study in this area is merited.
In summary, results from the current study indicate that H2O2, even at a trace concentration, via mechanisms involving decreased antiapoptotic Bcl-2 protein expression and further enhanced lipid peroxidation, could enhance TNF-–induced vascular endothelial cell apoptosis. Propofol, an IV anesthetic often used during anesthesia and postoperative sedation, can attenuate TNF- and H2O2 cellular toxicity.
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Footnotes |
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Accepted for publication March 7, 2006.
Presented, in part, at the 61st Annual Meeting of Canadian Anesthesiologist's Society, Vancouver, June 17–21, 2005.
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