Sepsis is the leading cause of death in intensive care patients and it can cause persistent and uncontrolled release of proinflammatory cytokines (1, 2). This severe immune response induces multiple organ failure. Lipopolysaccharide (LPS) administration to animals under anesthesia is a strategy for inducing an inflammatory response (1). However, the anesthesia model has its drawbacks. First, the exposure of laboratory animals to the anesthetic agents might change their immune function (3, 4), including the production of cytokines (5-10) and the reduction in the activity of natural killer cells (10-12). Second, hemodynamic changes after anesthesia enhance coagulation (10, 13, 14). Because most animal studies were performed under anesthesia, their conditions are different from the clinical cases in which patients are in a conscious state. Therefore, the results obtained from anesthetized animals need to be re-examined. It has been demonstrated that such anesthetics may influence the immune response (3, 4, 15-17), but pentobarbital has not been discussed yet, which is an anesthetic used mainly in animal studies. In this present report, we compared the results of endotoxemia between pentobarbital-anesthetized and conscious animals. Surprisingly, the results suggest that pentobarbital not only reduces systemic tumor necrosis factor-α (TNF-α) release, but also decreases the degree of tissue damage under LPS administration. These results indicate that the medical effects of certain drugs, which were performed on pentobarbital-anesthetized animals, might have resulted from the synergistic effect of pentobarbital. This study provides a view to probe into the medical effects of anesthetics besides their anesthetic effects.
MATERIALS AND METHODS
The phosphorylated nuclear factor-κB (pNF-κB)/human recombinant green fluorescent protein (hrGFP) and phosphorylated activator protein 1 (pAP-1)/hrGFP plasmids containing the NF-κB and AP-1 transcription binding site, respectively, followed by a hrGFP reporter gene, were purchased from Stratagene, USA. The higher the activity of transcription factors in a cell is, the higher the expression of hrGFP is.
The HEK 293 cell line and P338D1 cell line were obtained from the Biosource Collection and Research Center (Food Industry Research and Development Institute, Taiwan, China) and cultured with Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum.
Animals were purchased from the National Laboratory Animal Center (Taipei, Taiwan, China). C57BL/6JNarl mice were 6-week female mice. Wistar-Kyoto rats were 16-week male rats. The study was approved by our Institutional Animal Care and Use Committee.
It was purchased from MTC Incorporation (Cambridge, Ontario, Canada), whose trade name is Somnotol.
Spleen preparation and culture
C57BL/6JNarl mice were killed by carbon dioxide asphyxiation. The spleen was taken, minced with Dulbecco modified Eagle medium, and filtered with a mesh. Soup was centrifuged at 1,200 rpm at 12°C for 5 min. The supernatant was removed and 5 mL ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2-EDTA) was added. After 5-min incubation, the mixture was centrifuged (at 1,200 rpm at 12°C for 5 min) and washed twice with phosphate-buffered saline (PBS) to remove ACK buffer. Five milliliters of RPMI-1640 was added to resuspend the cells. A total of 2 × 106 cells were then incubated in four conditions, as follows: (1) with RPMI-1640 (containing 10% fetal bovine serum and 1% phosphatidylserine) (the control group), (2) with RPMI-1640 and 14 μg/mL of LPS (Sigma Chemical Co, St Louis, Mo) (the LPS group), (3) with RPMI-1640 and pentobarbital (the pentobarbital group), (4) with RPMI-1640, LPS, and pentobarbital (the LPS + pentobarbital group). Each mixture was collected after 48 h and stored at −80°C.
One hundred microliters of capture antibody (0.8 μg/mL) was added into each well of an enzyme-linked immunosorbent assay (ELISA) plate (Costar, USA), and the plate was incubated overnight. Wash buffer (0.05% Tween 20 in PBS, pH 7.2~7.4) was applied three times. Three hundred microliters of block buffer was added, and the plate was incubated for 1 h at room temperature. Wash buffer was applied three times. One hundred microliters of samples were added into each well and incubated at room temperature for 2 h. The plate was then washed with wash buffer three times. One hundred microliters of detection antibody (150 ng/mL) was added into each well. Samples were incubated at room temperature for 2 h and then washed three times with wash buffer. One hundred microliters of tetramethylbenzidine substrate (Clinical, USA) was added into each well, and the plate was incubated at room temperature for 20 min. To stop the reaction, 50 μL of stop solution (1N HCl) was added and the quantification was determined by the ELISA reader (Sunrise, Switzerland) at the absorbance wavelength of 450 nm.
Tumor necrosis factor-α mRNA expression assay
P338D1 cells (107) were cultured and treated with LPS, and coincubated without or with pentobarbital (final concentration, 12.5 μg/mL) for 6 h. The treated cells were harvested, and total RNAs were extracted by phenol/chloroform method as described (18). Complementary DNAs were reverse-transcribed from total RNA by SuperScript First-Strand Synthesis SuperMix kit (Invitrogen, USA), and the TNF-α complementary DNA was amplified by polymerase chain reaction (PCR) with the primer pairs (mouse TNF-alpha 5′: ATgAgCACAgAAAgCATgATCCgCgA; mouse TNF-alpha 3′: TCACAgAgCAATgACTCCAAAgTAgAC). The products of reverse transcription-PCR were analyzed by agarose electrophoresis, and the results were photographed.
Transcription factor activity assay
According to the manufacturer's instruction, pNF-κB/hrGFP and pAP-1/hrGFP were transfected by Lipofectamine 2000 (Invitrogen) into Balb/3T3 cells seeded in the 6-well plate, respectively. Twenty-four hours later, cells were passaged by versene (0.2 g EDTA-4 Na/L in PBS) and seeded into a 24-well plate. The transfectants were treated with LPS (14 μg/mL) and coincubated without or with pentobarbital (12.5 μg/mL) for 16 h, respectively. The transfectants were harvested and analyzed by flow cytometer. Specific FL-1 fluorescent intensities, representing the activities of the transcriptional factors, were calculated. In each plate, control plasmid phosphorylated cytomegalovirus/hrGFP was transfected into the target cells to measure the transfection efficiency, which was approximately 60%.
p38 Mitogen-activated protein kinase expression assay
As previously described, P338D1 cell lines were treated with LPS and coincubated with or without pentobarbital. The cells were harvested and mixed with the sample buffer (62.5 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 5% β-mercaptoethnol, 10% glycerol, 0.01% bromophenol blue). After the samples boiled, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and the products were transferred to a polyvinylidene fluoride membrane. The samples were probed with rabbit antimouse p38 polyclonal antibody (Santa Cruz, Europe) or mouse anti-β-actin monoclonal antibody (Biovision, USA). After washes, the membranes were reprobed with goat antirabbit immunoglobulin G, horseradish peroxidase conjugated (MP Biomedicals, USA) or rabbit anti-mouse immunoglobulins/horseradish peroxidase polyclonal antibody (DakoCytomation, Ely, Denmark). Finally, the blots were washed, developed, and visualized by enhanced chemiluminescence detection according to the manufacturer's instructions (Pierce, USA).
Tumor necrosis factor-α cytotoxicity assay
Target cells were seeded into the 96-well plate. Twelve hours later, pentobarbital at different concentrations was applied or not applied, with or without 2,500 pg/mL TNF-α (the control group, the TNF-α group, the pentobarbital group, the TNF-α + pentobarbital group). The supernatant was removed after 16 h. One hundred microliters of fresh medium was added with 20 μL MTS (CellTiter 96 Aqueous One Solution cell proliferation assay, Promega, USA). Cells were cultured in a carbon dioxide incubator at 37°C for 4 h. The absorbance was detected at 492 nm wavelength by an ELISA reader (Sunrise). Relative cell survival (%) = Sample absorbance/Control absorbance × 100%. The control group was cultured in normal growth medium, and its relative cell survival is equal to 100%.
Cell apoptosis assay
A total of 293 cells (2 × 106) in 3 mL growth medium were treated with 10 mM deferoxamine mesylate (DFO; Sigma) for 16 h. The cells were harvested and suspended into 100 μL staining solutions (20 μL Annexin V-fluorescein isothiocyanate (FITC) labeling reagent and 20 μL propidium iodide (PI) in 1 mL binding buffer). The mixture was incubated for 15 min and analyzed by flow cytometer. FL-1 represents Annexin V-FITC staining (apoptosis), and FL-3 represents PI staining (dead cells). The relative apoptosis index = the fluorescent intensity of samples/the fluorescent average of the negative control × 100%.
Preparation of animals
Sixteen-week-old male Wistar-Kyoto rats were purchased from the National Animal Center and housed in the university animal rooms under a 12-h light/dark cycle. Food and water were provided ad libitum. Animals were anesthetized with ether inhalation for about 10 min. During the period of anesthesia, a femoral artery was cannulated and connected to a pressure transducer to record the arterial pressure and the heart rate on a polygraph recorder (PowerLab, AD Instruments Co, Mountain View, Calif). A femoral vein was catheterized for the i.v. administration of drugs. The operation procedure was completed within 15 min, and the section wound was smaller than 0.5 cm2. After the operation, the animal was placed on a metabolic cage (17). The rat awoke soon after the operation. During the experiment, the body temperature was measured rectally by a digital thermometer (HR 1300 thermometer, Yokogawa, Japan) for every minute.
Lipopolysaccharide shock was induced by slow i.v. infusion of 10 mg/kg of LPS (Sigma) in 20 min. The infusion started 24 h after the operation. The drug was dissolved in sterile physiological saline solution immediately before use. All invasive procedures were performed under aseptic conditions. After LPS administration, animals were observed for 48 h (19).
Animals were divided into the NS, LPS, and Pento groups (n = 8). The NS group received a 1-mL injection of isotonic sodium chloride solution. The LPS group received 10 mg/kg of LPS (diluted in 1 mL) infusion. The Pento group received continuous infusion of pentobarbital at 10 mg/kg per h after LPS. The blood samples were collected before isotonic sodium chloride solution and LPS and at 0.5, 1, 3, 6, 9, 12, 18, 24, 36, and 48 h after the administration of saline or the drug.
Blood sample analyses
Blood samples for the measurement of white blood cells, lymphocytes, and platelets (Micro OT, Roche Co, Mannheim, Germany) were taken and immediately centrifuged at 3,000g for 10 min. The supernatant was collected for nitrate/nitrite measurement with high-performance liquid chromatography (ENO-20, AD Instruments Co, Mountain View, Calif). Enzyme-linked immunosorbent assay was performed for TNF-α measurement.
Blood biochemical analyses
The plasma samples were diluted by 1:100 with distilled water before measurements. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH), creatine kinase (CK), serum urea nitrogen (SUN), and amylase were measured with an autoanalyzer (Vitros 750, Johnson-Johnson Co, Rochester, NY) for evaluating various organ functions. The ALT and AST are for the liver function, the LDH and CK are for the heart and other possible organ (such as muscle) functions, the SUN is for the renal function, and the amylase is for the pancreatic function.
Data of in vivo experiments are expressed as mean ± SE. Multiple analysis of variance and Scheffé test were used to compare the difference between and among groups (n = 8 in each group). P < 0.05 was considered to be significant. All in vitro data were compared by Student t test, and P < 0.05 was considered to be significant. The data of the transcriptional factor activity assay were obtained from three independent experiments and duplicated in each group (n = 6). The data of the in vitro cytokine assay were obtained from three independent experiments and duplicated in each group (n = 6). The data of TNF-α cytotoxic assay were obtained from four independent experiments (n = 4 each group). The data of the apoptosis assay were obtained from three independent experiments and duplicated in each group (n = 6).
Pentobarbital lowers the TNF-α concentration in serum in the presence of LPS in vitro and in vivo
Pentobarbital was assumed to be able to modify the inflammatory effects of LPS in endotoxemia. The results showed that pentobarbital significantly lowered TNF-α release from P338D1 cells (mouse macrophage cells) under LPS stimulation (Fig. 1A). Moreover, pentobarbital also decreased TNF-α expressions of splenocytes in the presence of LPS (Fig. 1B). In addition, the expression of TNF-α mRNA was reduced after pentobarbital treatment in the presence of LPS (Fig. 2). These results indicate that pentobarbital has anti-inflammatory ability in vitro.
To study the effect of pentobarbital in vivo, an animal model of conscious rats with LPS treatment was established and used. After LPS treatment with or without pentobarbital administration, blood samples in each group were collected to measure the levels of inflammatory substances. The LPS infusion caused a dramatic increase in TNF-α in sera of conscious rats in vivo. However, pentobarbital treatment reduced the serum concentration of TNF-α in the presence of LPS (Fig. 3A). The difference was observed within 12 h after LPS treatment, but no difference was detectable after 12 h. The other indicator of LPS-induced inflammatory response, nitric oxide (NO), increased in the conscious rat model after LPS treatment. In contrast, pentobarbital did not affect the expression of NO in sera (Fig. 3B). In vitro and in vivo results indicate that pentobarbital has the ability to reduce the TNF-α release from immune cells. Because the effects could be mediated through the reduction of the body temperature, it was measured after different treatments. The body temperature of the animals in the LPS group (n = 6) and the LPS plus Pento group (n = 6) decreased (Fig. 3C). The decrease in the body temperature should be caused by the LPS administration. The data of the two groups were not significantly different. Within 9 h after the LPS administration, a conspicuous inhibitory effect of pentobarbital on TNF-α release was observed. However, the body temperature was not significantly different at this stage between the two groups. Therefore, a decrease in TNF-α release by pentobarbital should not be caused by the change in the body temperature.
Pentobarbital suppresses the activities of NF-κB and AP-1 in the presence of LPS
Previous literatures have reported that LPS activates NF-κB and AP-1 pathways to enhance the TNF-α expression and release. Therefore, Balb/3T3 cells were transfected with plasmids containing the enhanced GFP reporter gene under minipromoter control (the minipromoters were composed of several copies of NF-κB or AP-1 transcriptional factor binding sites) to determine the effects of pentobarbital on these signaling pathways. In our experiments, LPS increased the activities of NF-κB in cells (Fig. 4A) and slightly enhanced the activities of AP-1 (Fig. 4B). However, pentobarbital reduced the activities of NF-κB and AP-1 in the presence of LPS (Fig. 4). The result also showed that pentobarbital alone decreased the activities of NF-κB and AP-1 in cells at a concentration of 12.5 μg/mL. In addition, because LPS-induced TNF-α release involves p38 mitogen-activated protein kinase (MAPK) signaling pathway, we tested whether pentobarbital could interfere with it. Our result indicated that pentobarbital could reduce the amount of p38 MAPK in the presence of LPS (Fig. 5).
Pentobarbital reduces tissue damages
Tumor necrosis factor-α is a potent cytotoxic cytokine that results in tissue damages. As indicated in the previous results, pentobarbital decreases the TNF-α expression in vitro and in vivo. Whether pentobarbital could protect tissues from LPS-induced tissue damages was analyzed in the animal model of conscious rats in vivo. The results showed that LPS injection caused dramatic increases in ALT, AST, LDH, CK, BUN, and amylase in sera (Fig. 6) when compared with those of the normal control group, indicating that LPS injection deteriorated the hepatic (Fig. 6, A and B), the heart or skeletal muscle (Fig. 6, C and D), the renal (Fig. 6E), and the pancreatic functions (Fig. 6F), as reflected by the changes of blood biochemical substances. However, pentobarbital infusion suppressed the increases in all bioindicators in the presence of LPS (Fig. 6), indicating that pentobarbital could protect tissue damages from LPS-induced cytotoxic effects.
Pentobarbital protects cells from apoptosis
Besides the reduction of TNF-α release, the protective effects of pentobarbital on the survival of TNF-α target cells also needed to be determined. The HEK 293 human kidney cells are susceptive to TNF-α. Our results showed that the survival rates of HEK 293 cells were less than 40% under 2.5 ng/mL of TNF-α treatment for 16 h (Fig. 7). Different concentrations of pentobarbital all increased the viabilities of cells in the presence of TNF-α, whereas pentobarbital alone did not significantly affect the survival rates of HEK 293 cells (Fig. 7).
Tumor necrosis factor-α can increase the expression of adhesion molecules on the surface of immune cells and endothelial cells to cause the stagnant blood capillary effect and result in tissue hypoxia. When such hypoxia occurs, cells will undergo apoptosis. Deferoxamine mesylate has been shown to be able to induce apoptosis by the same mechanism as hypoxia, and therefore it was used to cause cell apoptosis in our in vitro experiment. We found that DFO alone increased the proportions of apoptosis of HEK 293 cells (Fig. 8). Nevertheless, pentobarbital protected the cells from apoptosis in DFO-induced hypoxia (Fig. 8).
In this study, the results suggest that pentobarbital infusion attenuates the multiple organ dysfunctions induced by LPS (Fig. 6). First, pentobarbital reduces the expression of TNF-α in the presence of LPS (Figs. 1 and 2). Gao et al. (20, 21) have demonstrated that LPS injection produces a large increase in the plasma TNF-α. In general, TNF-α is considered to be a principal mediator of endotoxemia and organ failure (22). The TNF-α has been implicated as an important mediator of the lethal effect of endotoxin, which can cause hepatic failure, and so on. Several literatures have shown that inhibitors for reducing the activity or the expression of TNF-α significantly decrease the endotoxin-induced damages (22-24). In addition, numerous studies have shown that the function of blood capillary is impaired and adhesion molecules aggregate to vessel walls under the septic progress (1, 8, 19, 25). The TNF-α strongly induces the expression of intercellular adhesion molecule, vascular cell adhesion molecule, and P-selectin in endothelial cells (26-28), which causes blood cells to adhere to endothelial cells (29). The amount of TNF-α in serum can be associated with the degree of tissue damage because of the stagnant blood capillary. In fact, several anesthetic agents, including pentobarbital, have been demonstrated that they markedly suppress the TNF-α-induced neutrophil-venule adhesion (30). For these reasons, the protective effect of pentobarbital might be caused by the suppression of the systemic release of TNF-α.
In the second protective mechanism, pentobarbital directly protects tissue cells from the cytotoxic effect of TNF-α, which is a well-known cytotoxic cytokine for certain tissue cells (Figs. 6 and 7). The LPS raised the levels of several biophysical indicators (ALT, AST, LDH, CK, BUN, and amylase) in sera (Fig. 6), which reflected the LPS-inducing damages of organ tissues. Pentobarbital increased the viability of the cells at the presentation of TNF-α, demonstrating its role in cell protection (Fig. 7). Lipopolysaccharide stimulation can cause the stagnant blood capillary effect, resulting in hypoxia in tissues. When such hypoxia occurs, cells will undergo apoptosis. Deferoxamine mesylate can induce the same apoptosis as hypoxia and has been used to cause cell apoptosis in vitro (31, 32). Pentobarbital can decrease the percentage of cells undergoing apoptosis in the presence of DFO treatment (Fig. 8). A similar phenomenon has been reported that barbiturates have a protective effect against cerebral ischemia, and it has suggested that pentobarbital inhibits apoptosis to prevent ischemic neuronal death (33). Therefore, the information that pentobarbital should have the ability to protect tissue cells from the LPS directly or indirectly induced cytotoxicity of TNF-α.
In mammals, toll-like receptor 4 on macrophages sends signals in the presence of LPS by associating with CD14 to activate its NF-κB pathway (34). Besides NF-κB, previous literatures have also reported that LPS-induced endotoxemia can cause an increase in the p38 MAPK expression, which is important for the LPS-induced TNF-α release (35, 36) and AP-1 activities in cells (37). In this study, the results reveal that pentobarbital suppresses the expression of p38 MAPK (Fig. 5) and the activities of NF-κB and AP-1 (Fig. 4) in the presence of LPS. Changes in the intracellular signaling pathway should be responsible for the decrease in TNF-α mRNA (Fig. 2) and the reduction of TNF-α protein expression caused by pentobarbital (Fig. 1, A and B).
During endotoxemia, proinflammatory cytokines act both locally and systemically to aggravate the organ damage. Many investigations have shown that i.v. anesthetics have anti-inflammatory effects on endotoxemia both in vitro and in vivo (12, 38). According to the results in this study, pentobarbital not only has an anti-inflammatory activity, but also directly protects cells from apoptosis. Accordingly, pentobarbital may be beneficial in preventing organ dysfunction in endotoxemia or septicemia, but it should be used with caution regarding their potential immunomodulatory properties in critically ill patients.
The authors thank Ke W. Yang and Yu K. Hung for technical assistance.
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Pentobarbital; LPS; conscious rats; organ injury; TNF-α