Sepsis is the leading cause of death in critically ill patients in intensive care units. The pathophysiological features of sepsis subsequent to bacterial infection include organ failure and death caused by the dysregulation of the immune response and excessive oxidant and free radical production (1, 2). During sepsis, immune cells such as neutrophils and macrophages release superoxide and/or nitric oxide (NO); NO reacts with the free radical superoxide, leading to the formation of the potent oxidant peroxynitrite, which has a short half-life (3). Although peroxynitrite has numerous functions in host defense and is an important microbicidal compound, it may also have deleterious effects on host tissues (4). In fact, the well-studied adverse effects of excess NO generation on host tissues during the inflammatory response are caused by the formation of peroxynitrite rather than NO itself. Peroxynitrite reacts with a wide range of biological molecules, including amino acids, leading to changes in protein structure and function (4, 5). It also causes chemical cleavage of DNA and a reduction in host defenses by oxidation of cellular thiol pools, lipid peroxidation, and tyrosine nitration. As such, a causative role of peroxynitrite has been implicated in diseases such as asthma, acute lung injury, idiopathic pulmonary fibrosis, inflammatory bowel disease, septic shock, arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, Alzheimer disease, acute renal ischemia, and hyperlipidemia (4, 6). One approach to attenuate the toxic effects of peroxynitrite is the use of pharmacological strategies aimed at limiting tissue damage, that is, via inhibition and/or scavenging of peroxynitrite . Caffeic acid phenethyl ester (CAPE) is an active component of honeybee products and has established antimitogenic, anticarcinogenic, anti-inflammatory, and immunomodulatory activities in diverse systems (7, 8). However, most importantly, CAPE exhibits antioxidant activity and inhibits lipoxygenases, protein tyrosine kinases, and the activation of the transcription factor nuclear factor-κB (NF-κB). The additional biological effects of CAPE include the inhibition of arachidonic acid release from cell membranes and cyclooxygenase 1 (COX-1) and COX-2 activities (8, 9). In this study, we tested the ability of CAPE to scavenge peroxynitrite in murine macrophages and in two mouse models of sepsis to determine its potential as a therapeutic for inflammatory disorders.
MATERIALS AND METHODS
One hundred sixty male Balb/c mice weighing 20 to 25 g were obtained from the animal experimentation unit in the Faculty of Medicine, University of Malaya. Mice were kept in individual cages under standard conditions (12 h light and 12 h dark). The animals were fed a diet of Purina laboratory chow and given ad libitum access to water. The study was carried out in accordance with the guidelines for animal experimentation of the University of Malaya Animal Ethics Committee under the approved protocols and terms set out in project license ANES/14/07/2010/MKAK (R).
Cell culture and reagents
The murine macrophage cell line RAW 264.7 (ATCC no. TIB-71; American Type Culture Collection, Rockville, Md) was maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 100 U penicillin/mL, and 100 U streptomycin/mL in a humidified 37°C, 5% CO2 incubator. Phenol red–free DMEM, FBS, and antibiotics (penicillin, streptomycin) were purchased from Nacalai Tesque (Kyoto, Japan). Lipopolysaccharide, Escherichia coli 0111 B4, CAPE, the inducible NO synthase (iNOS) inhibitor 1400W, and interferon-γ (IFN-γ) were purchased from Sigma-Aldrich (St. Louis, Mo).
LPS/IFN-γ stimulation of RAW 264.7 cells
Murine RAW 264.7 macrophages maintained in 10% FBS-DMEM were seeded at a density of 2 × 106 cells/well in 24-well plates and incubated for 24 h at 37°C. The cells were subsequently washed with PBS and resuspended in fresh medium containing a range of concentrations of CAPE (0.125 – 1 μg/mL) or 100 μM of the iNOS inhibitor 1400W. Untreated cells were included as positive or negative controls in each experiment. After a 1-h incubation at 37°C in a 5% CO2 atmosphere, LPS/IFN-γ was added to the cultures at concentrations of 1 μg/mL and 35 ng/mL, respectively, to increase the induction of iNOS expression and produce high levels of NO, followed by a second incubation for 24 h under the previously described conditions (10). The cells were then processed to assess for viability or for NO or peroxynitrite detection as described later.
Measurement of viability
The viability of RAW 264.7 macrophages was determined in cultures that were either untreated or treated with a combination of LPS/IFN-γ and CAPE (0.125 – 1 μg/mL) or the iNOS inhibitor 1400W (100 μM). Viability was measured in terms of cellular respiration, as assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Briefly, the cells were cultured, stimulated with LPS, and treated with CAPE or 1400W as previously described, after which 100 μL of MTT (5 mg/mL) was added to each well for 1-h incubation under the same conditions. The MTT solution was then removed, and the cells were solubilized in 200 μL DMSO with shaking for 5 min. Absorbance was measured at 550 nm using a microplate reader (GloMax-Multi Microplate detection, Promega, Madison, Wis) (10). All experiments were repeated five times in triplicate.
Nitric oxide has a short half-life of only a few seconds because it is quickly converted to nitrate (NO3) and nitrite (NO2). These products can be measured using the colorimetric Griess reaction to indirectly determine the NO concentration. RAW 264.7 cells were cultured and treated as previously described, after which 100 μL of the culture was placed in a 96-well plate with an equivalent amount of the Griess reagent (50 μL of 1% sulfanilamide in 5% concentrated H3PO4 and 50 μL of 0.1% naphthylethylenediamine dihydrochloride in distilled water). The reaction between the Griess reagent and the NO2 present in the supernatant yields a pink derivative that can be spectrophotometrically quantified from a standard concentration curve prepared from an NO2 standard (10).
Dihydrorhodamine-123 oxidation assay
Three models of dihydrorhodamine-123 (DHR-123) were used in this study: incubation with SIN-1 (3-morpholinosydnonimine) as a peroxynitrite donor, incubation directly with peroxynitrite, and incubation with LPS/IFN-γ for live cell stimulation. All models have been previously described (10).
DHR-123 oxidation using SIN-1
The oxidation of DHR-123 in the presence of SIN-1 spontaneously releases NO and superoxide under physiological conditions. At pH 7.4, SIN-1 is converted to SIN-1A via a base-catalyzed ring opening, during which oxygen undergoes a univalent reduction to the radical O2 -. SIN-1A then releases NO and is converted to the stable metabolite SIN-1C, whereas the O2 - reacts with NO to form peroxynitrite (ONOO-). The oxidation of DHR-123 by peroxynitrite results in the formation of fluorescent rhodamine-123, which can be measured by fluorometric analysis (GloMax-Multi Microplate) at an excitation wavelength of 460 to 530 nm and an emission wavelength of 530 to 590 nm. In experiments examining the effects of CAPE (0.125 – 1 μg/mL) on peroxynitrite scavenging, 100 μM SIN-1 was used and the reactions were carried out in PBS, with incubation of the samples for 2 h at 37°C (11). The amount of NO in 100 μL of the supernatant was measured as previously described for the NO assay.
DHR-123 oxidation using peroxynitrite
The ability of peroxynitrite to oxidize DHR-123, resulting in its conversion to fluorescent rhodamine-123, was measured directly as previously described (4). Briefly, 10 μM peroxynitrite was mixed with PBS containing 20 μM DHR-123 in the presence or absence of either CAPE (0.125 – 1 μg/mL) or 100 μM of the iNOS inhibitor 1400W. After a 15-min incubation at room temperature, the fluorescence of the rhodamine-123 reaction product was measured (GloMax-Multi Microplate) at an excitation wavelength of 460 to 530 nm and an emission wavelength of 530 to 590 nm.
DHR-123 oxidation by LPS/IFN-γ–treated RAW 264.7 cells
Cells were cultured, treated with CAPE or 1400W, and stimulated with LPS/IFN-γ, as previously described for RAW 264.7 cells but in the presence of 10 μM DHR-123. After 24 h, 100 μL of the culture suspension was removed and the amount of rhodamine-123 was determined fluorometrically; an additional 100 μL was used for NO measurement, as previously described for the NO assay.
Mice were randomly assigned to eight groups (10 mice per group) and injected intraperitoneally with a lethal dose of LPS (25 mg/kg in a final volume of 100 μL). An equivalent volume of saline was administered to the negative control group (12). Treated mice received either 5 or 25 mg/kg of CAPE (total volume, 200 μL), whereas the positive or negative controls were administered 200 μL of saline. Both CAPE and the saline control were administered the day before and 2 h before LPS administration by intraperitoneal injection. Endotoxemia was confirmed in the LPS-treated mice at 7 h after the injection. Blood was collected from the four groups (treated and control groups) by cardiac puncture, and the amount of 3-nitrotyrosine, a marker of peroxynitrite, was measured in the serum. Surviving mice (four groups) were administered CAPE or saline for 3 days after LPS administration. Viability was monitored every 12 h for 7 days.
Cecal ligation and puncture
Mice were randomly divided into eight groups (10 mice per group), and cecal ligation and puncture (CLP) was performed as described (13) with a few modifications. Briefly, the mice were anesthetized (10 per group) with ketamine (70 mg/kg, i.m. injection) and xylazine (10 mg/kg). An abdominal midline incision was then performed, and the cecum was isolated. After ligation of the cecum at 7 mm from the cecal tip, distal to the ileocecal valve, the ligated cecal stump was perforated by two “through and through” punctures (21-gauge needle). The cecum was then returned to its normal position, and the abdomen was closed with a suture thread. Mice were resuscitated by injecting s.c. 2 mL of prewarmed 0.9 saline solution using a 25-gauge needle. To analyze the CLP, the mice in the four groups (treated and control groups) were killed at 7 h after the procedure. Treated mice received either 5 or 25 mg/kg of CAPE (total volume, 200 μL), whereas the positive or negative controls were administered 200 μL of saline. Both CAPE and the saline control were administered by intraperitoneal injection the day before and 2 h before the CLP procedure. Surviving mice (four groups) were further treated with CAPE or saline for 3 days after treatment. Viability was monitored every 12 h for 7 days.
Measurement of 3-nitrotyrosine levels in RAW 264.7 cells
Lysates were prepared from RAW 264.7 cells collected after treatment as previously described for the stimulation experiments, and the level of 3-nitrotyrosine, a marker of peroxynitrite, was measured. The harvested cells were placed on ice and immediately lysed in RIPA buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail and then centrifuged at 16,000g for 15 min at 4°C. The supernatants (lysates) were transferred to precooled tubes and stored at −80°C until analysis. 3-Nitrotyrosine concentrations in the lysates were measured with a nitrotyrosine enzyme-linked immunosorbent assay kit (Cell Biolabs, Inc., San Diego, Calif) according to the manufacturer’s instructions and normalized to the amount of protein in milligrams. Protein concentrations were measured using the Bradford method.
Detection of 3-nitrotyrosine levels in serum
Blood was collected after 7 h from all groups of mice (CLP and endotoxemia), and the concentration of 3-nitrotyrosine in the serum was measured as previously described.
Data were analyzed using GraphPad prism statistical software (version 6; GraphPad Software Inc., La Jolla, Calif). Data are expressed as mean ± SEM. Tukey multiple comparisons test was used to determine the statistical significance of differences between the experimental and control groups. Kaplan-Meier analysis was used to compare survival rates. Statistical significance was set at P ≤ 0.05.The half maximal inhibitory concentration (IC50) was calculated using sigmoidal dose-response nonlinear regression using the same software.
CAPE inhibits peroxynitrite production in vitro
Caffeic acid phenethyl ester inhibited peroxynitrite generation from the synthetic substrate SIN-1 with an IC50 of 0.037 μg/mL, whereas the effect of 1400W (100 μM) was similar to that of the negative (untreated) control (Fig. 1A). Neither CAPE nor 1400W (100 μM) reduced the accumulation of nitrite produced from SIN-1 (Fig. 1B). Moreover, direct incubation of CAPE and peroxynitrite for 15 min inhibited DHR-123 oxidation with an IC50 of 0.05 μg/mL, whereas the results achieved with 1400W were the same as those with the untreated control (Fig. 1C). Taken together, these results suggest that CAPE acts by scavenging peroxynitrite rather than by inhibiting its synthesis.
CAPE inhibits intracellular peroxynitrite production
None of the doses of CAPE and 1400W used in this study was found to be cytotoxic to murine RAW 264.7 macrophages based on a comparison of the viability of the cells after a 24-h incubation with these substances with that of untreated cells (Fig. 2A). In contrast, CAPE and 1400W significantly protected cell viability from the negative effects of the immune response induced with LPS/IFN-γ. In cells induced with LPS/IFN-γ and treated with CAPE and 1400W, viability was more than 88% compared with the less than 74% viability seen in the control cells induced with LPS/IFN-γ but untreated with CAPE (P < 0.001; Fig. 2B).
Both CAPE and 1400W (100 μM) inhibited peroxynitrite production in response to the LPS/IFN-γ induction of RAW 264.7 cells (IC50: CAPE, 0.4 μg/mL; 1400W, 100 μM), as shown by the reduced DHR-123 fluorescence in these cells as compared with that in the untreated cells (Fig. 2C). Moreover, the CAPE reduction of the LPS-induced immune response–mediated accumulation of nitrite was dose dependent. In the positive control, 1400W also blocked nitrite accumulation, whereas no such response was seen in untreated LPS-induced cells (Fig. 2D). After a 24-h incubation of RAW 264.7 cells with LPS/IFN-γ, CAPE and 1400W blocked intracellular peroxynitrite formation, as confirmed by a reduction in the level of 3-nitrotyrosine, an intracellular marker of ONOO- (P < 0.001; Fig. 3).
CAPE inhibits peroxynitrite production in vivo
Caffeic acid phenethyl ester significantly inhibited peroxynitrite accumulation in the endotoxemia and CLP models at 7 h after LPS induction and postoperatively, respectively, by inhibiting 3-nitrotyrosine synthesis (P < 0.001 vs. the untreated control in both models; Fig. 4). Survival in the two sepsis models (endotoxemia and CLP) was significantly enhanced by CAPE treatment. Compared with the results for untreated mice, doses of 5 and 25 mg/kg of CAPE increased the survival of endotoxemic mice by 52% and 73%, respectively, and of CLP mice by 45% and 64%, respectively (Fig. 5).
Peroxynitrite is a strong oxidant and is therefore a potent inducer of cell death (4). Its breakdown or pharmacological inhibition is therapeutically beneficial in a variety of pathological conditions, for example, vascular diseases, ischemia-reperfusion injury, circulatory shock, inflammation, pain, and neurodegeneration (4, 14). The results of the current study clearly confirm that CAPE is a potent scavenger of peroxynitrite both in vitro and in vivo. Caffeic acid phenethyl ester treatment inhibited peroxynitrite activity in cultured RAW 264.7 cells stimulated with LPS/IFN-γ. Moreover, our results provide evidence that, at low concentrations, CAPE acts as a scavenger of peroxynitrite because its incubation with SIN-1 resulted in significantly lower levels of this oxidant. Similarly, the direct incubation of CAPE with peroxynitrite resulted in the inhibition of DHR-123 oxidation. The antioxidant activity of CAPE also caused a reduction in the level of 3-nitrotyrosine, a marker of peroxynitrite, in two animal models of sepsis, that is, CLP and endotoxemia. In addition, in septic mice treated with CAPE, the survival rate significantly increased. Scavengers and neutralizers of peroxynitrite are required for the treatment of many diseases as previously described. Various molecules (endogenous and exogenous compounds) can scavenge and react directly or indirectly with peroxynitrite in vitro and in vivo, for example, uric acid and ebselen (4). In vitro, the specific interaction between peroxynitrite and DHR produced a highly fluorescent oxidation product without interfering with NO and superoxide, providing an excellent indicator of peroxynitrite scavenging. The potency of peroxynitrite scavenging was inversely proportional to the amount of fluorescent product. Our in vitro results demonstrated that CAPE strongly scavenged peroxynitrite. In vivo, at physiological pH, peroxynitrite is protonated to generate peroxynitrous acid or can react with CO2 to form nitrosoperoxycarbonate. Both species can nitrate phenols such as tyrosine. The appearance of 3-nitrotyrosine is considered a biomarker of peroxynitrite-mediated protein oxidation and has been detected in organs after ischemia/reperfusion injury and sepsis (15, 16). In previous studies, scavengers and decomposition of peroxynitrite significantly reduced 3-nitrotyrosine levels in blood and organs (17). Our in vivo results showed that CAPE is a potent inhibitor of protein oxidation by peroxynitrite and reduces 3-nitrotyrosine levels in sepsis models. During sepsis, an increase in the levels of several proinflammatory cytokines and chemokines, reactive oxygen species (ROS), COX, and iNOS is seen, accompanied by the activation of the transcriptional activator NF-κB (18). In fact, ROS production is critical for NF-κB activation. Conversely, several antioxidants that downregulate NF-κB are protective in animal models of injury (19). Peroxynitrite activates NF-κB directly, that is, without preactivation by proinflammatory mediators such as tumor necrosis factor-α (TNF-α) and LPS, and it enhances the production of interleukin 8 and TNF-α via an NF-κB–mediated pathway in a variety of cells. The DNA-binding activity of NF-κB is also enhanced by peroxynitrite (20, 21). The mechanism underlying this interaction is thought to involve nitration of the tyrosine 181 residue of IκBα, an NF-κB inhibitor, leading to its dissociation and subsequent NF-κB activation (22). Persistent oxidative damage is caused by increased production of free radical species such as NO. These toxic compounds lead to severe cellular injury via a number of me chanisms, including the generation of reactive derivatives such as peroxynitrite (ONOO-). Among the numerous cellular targets of these agents are lipids, DNA, and proteins (23). Cellular exposure to high concentrations of peroxynitrite leads to rapid necrotic-type cell death because of acute and severe cellular energetic derangements (14, 24), whereas low concentrations of peroxynitrite result in programmed cell death (apoptosis) mediated by the activation of caspase-3, caspase-2, caspase-8, and caspase-9 (25, 26). Recent studies have demonstrated that peroxynitrite stimulates the release of mitochondrial apoptosis-inducing factor, which triggers DNA fragmentation processes (27), along with other mitochondrial proapoptotic factors. This, in turn, leads to cytochrome c–dependent apoptosis in the cytosol through the peroxynitrite-dependent oxidation of mitochondrial permeability transition pore components. The key role of peroxynitrite in promoting mitochondrial dysfunction is clearly exemplified in experimental sepsis, where peroxynitrite-mediated inhibition of mitochondrial respiration, in a process associated with mitochondrial protein nitration, is prevented by NOS inhibitors and Mn-porphyrin therapy (peroxynitrite decomposition) (28, 29). Furthermore, some of the physiological modulators of peroxynitrite reactivity have been shown to exert beneficial effects in animal models of inflammation and reperfusion injury (1, 5, 24, 30).
Many of the natural flavonoids found in fruits, vegetables, and teas are potent antioxidants and NF-κB inhibitors. One of the most potent lipophilic antioxidants is CAPE, which is rapidly absorbed and metabolized by plasma esterases (7, 9, 31). Caffeic acid phenethyl ester is nontoxic, readily bioavailable, and has long been used in traditional medicine. Its activities have been described in several reports and include the inhibition of lipoxygenase, protein tyrosine kinases, and lipid peroxidation (7, 9). Thus, CAPE is an antioxidant, an anti-inflammatory agent in the prevention of reperfusion injury, an immunostimulant, and an antiatherosclerotic agent. In rats, CAPE pretreatment reduces brain infarction and cerebral vasospasm, which is consistent with its defensive role against neurovascular inflammation and oxidative stress that occur secondary to injury, as demonstrated in a neonatal brain hypoxic-ischemic injury model and a cardiac ischemia reperfusion injury model (32). Caffeic acid phenethyl ester also reduces mortality and protects against organ failure during sepsis in animals (33). Our results showed that CAPE might have prevented the reduced viability caused by LPS treatment and increases survival rate in sepsis models. These results are in agreement with previous in vitro and in vivo studies demonstrating the protective effect of CAPE against cell death and multiorgan failure. The mechanism underlying CAPE-mediated cytoprotection has been investigated in several systems, both in vitro and in vivo, particularly with respect to the oxidants NO and peroxynitrite. As determined in the present study and elsewhere, CAPE protects cells against these cytotoxic molecules by scavenging peroxynitrite and inhibiting iNOS (34). Peroxynitrite scavenging, such as that performed by CAPE, completely blocked the activation of NF-κB (34). Unlike other antioxidants, CAPE specifically suppresses NF-κB binding to DNA, without affecting IκBα degradation (34). Previous studies have shown that CAPE inhibits the activation of NF-κB induced by various ROS-producing agents in human histiocytic cells and coronary artery endothelial cells (35). By inhibiting peroxynitrite formation and, therefore, NF-κB activation, CAPE prevents the downstream expression of NF-κB–dependent genes, including adhesion molecules (intercellular adhesion molecule 1 and E-selectin), cytokines (TNF-α and interleukin-1β), and iNOS (8, 9). The reduction in cytokine levels is particularly important because cytokines play a fundamental role in macrophage activation and are the first mediators of the inflammatory cascade (7, 8). Caffeic acid phenethyl ester also inhibits apoptotic cell death and prevents the proliferation and apoptosis of colorectal cancer cells and cerebellar granule neurons in vitro (8, 9). The antiapoptotic effects of CAPE rely on its ability to block ROS formation and inhibit caspase activity (7–9). Previous studies have shown that CAPE has numerous other beneficial antioxidant effects; it blocks ROS production in human neutrophils and inhibits the xanthine/xanthine oxidase system and formation of lipoxygenase, an additional intracellular source of free radical generation that causes extensive oxidative damage in multiple organ systems (7–9).
Caffeic acid phenethyl ester, a potent scavenger of peroxynitrite in vitro and in vivo, protects cells from death caused by free radical (NO and peroxynitrite) production during the immune response as well as protects animals from sepsis. Based on the multiple beneficial activities of CAPE, including its natural antioxidant activity, further studies should be directed at developing its pharmacological properties for the treatment of a wide range of inflammatory conditions. Thus, administration of CAPE in sepsis patients might offer a suitable new therapeutic tool for the treatment of septic shock, multiple organ failure, and other microbe-mediated diseases in humans, in whom “out-of-control” inflammation often leads to fatal outcomes.
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Keywords:© 2014 by the Shock Society
Caffeic acid phenethyl ester; inflammation; sepsis; peroxynitrite; nitric oxide; macrophage; cell death