Chapter 13 Figures (2)
III. Publishing
1. Yang FL, Li CH, Hsu BG, Tsai NM, Lin SZ, Harn HJ, Chen HI, Liao KW, Lee RP. Pentobarbital reducing TNF-alpha release and the tissues damage in
experimental endotoxemia model. Shock. 2007 Sep;28(3):309-16. (Equal contributed to Yang FL)
2.Shih-Han Kao, Ching-Yi Lin, Cheng-Yu Chen, Chi-Han Li, Chueh-Jen Tsai,
Yuan-Ting Hsieh, Shang-Chih Yang, Chang-Jer Wu, Wei-Sheng Chung, Ru-Ping Lee, Kuang-Wen Liao. The Enhancement of Anti-tumor Immunity by a SARS Fragment Fusion to a Low-immunogenic Tumor-derived Peptide. J Gene Med (Submitted)
3. Chi Han Li; Fwu Lin Yang; Ching Yi Lin; Bang Gee Hsu; Tseng Feng Jen; Yu Cheng Chen; Ru Ping Lee; Kuang Wen Liao. The Treatment of Propofol Induced the TGF-β1 Expression in Trauma Patients Sera: Propofol-Induced TGF-β1 in Human Endothelial Cells Suppress Endocytosis Activities of Monocytes in vitro. Manuscript in preparation. (Equal contributed to Yang FL)
Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited.7
THE REDUCTION OF TUMOR NECROSIS FACTOR-! RELEASE AND TISSUE DAMAGE BY PENTOBARBITAL IN THE EXPERIMENTAL
ENDOTOXEMIA MODEL
Fwu Lin Yang,*†‡Chi Han Li,§Bang Gee Hsu,‡kNu-Man Tsai,¶ Shinn Zong Lin,*† Horng Jyh Harn,*†Hsing I. Chen,* Kuang Wen Liao,§and Ru Ping Lee***
*Institute of Medical Sciences, Tzu Chi University;†Neural Medicine Science Center and‡Division of Surgical Critical Care Unit, Tzu Chi Hospital, Hualien;§Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu;kDepartment of Nephrology, Tzu Chi Medicine Center Hospital, Hualien;¶School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung; and
**Department of Nursing, Tzu Chi University, Hualien, Taiwan, Republic of China
Received 17 Jul 2006; first review completed 9 Oct 2006; accepted in final form 18 Dec 2006
ABSTRACT—Sepsis is the leading cause of death for intensive care patients. Lipopolysaccharide (LPS) administration to animals under anesthesia is a strategy for the study of uncontrolled release of proinflammatory cytokines. Anesthetics have been indicated that they can specially affect immune responses, such as the inflammatory response. Pentobarbital is an anesthetic used mainly in animal studies. Thus, the effect of pentobarbital on tumor necrosis factor-! (TNF-!) release was determined. The results revealed that pentobarbital suppressed the expression of TNF-! mRNA and its proteins, which may result from the decrease in the activities of nuclear factor-JB and activator protein 1 and the reduction of the expression of p38 mitogen-activated protein kinase by pentobarbital. After the inhibitory activity of the pentobarbital for TNF-! release was proven in vivo, the cytotoxic effects of LPS were examined in vivo with or without pentobarbital treatments. In vivo results indicated that plasma levels of alanine aminotransferase, aspartate aminotransferase, lactic dehydrogenase, creatine kinase, serum urea nitrogen, and amylase decreased dramatically in the anesthetic group with pentobarbital administration. Finally, the effect of pentobarbital on TNF-!Yrelated cell death was monitored in vitro, and the results indicated that pentobarbital could directly enhance the viabilities of cells under the treatment of TNF-! and protected cells from apoptosis induced by deferoxamine mesylateYinduced hypoxia. These results suggest that pentobarbital significantly influences the LPS-induced inflammatory responses and protects cells from death directly and indirectly induced by TNF-!. The information provides a perspective to re-evaluate the results of the experiments in which animals were anesthetized with pentobarbital. The anti-inflammatory effects of the drugs may have been caused by the synergistic effect of pentobarbital.
KEYWORDS—Pentobarbital, LPS, conscious rats, organ injury, TNF-!
INTRODUCTION
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 anes-thesia 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 (5Y10) and the reduction in the activity of natural killer cells (10Y12). 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, 15Y17), 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-! (TNF-!) 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 Plasmid
The phosphorylated nuclear factor-.B (pNF-.B)/human recombinant green fluorescent protein (hrGFP) and phosphorylated activator protein 1 (pAP-1)/hrGFP plasmids containing the NF-.B 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.
Cell lines
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.
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SHOCK,Vol. 28, No. 3, pp. 309Y316, 2007
Address reprint requests to Ru Ping Lee, PhD, Laboratory of Physiological Nursing Research, Department of Nursing, Tzu Chi University, No. 701, Section 3, Chung Yang Rd, 97004 Hualien, Taiwan. E-mail: [email protected].
Supported in part by the National Science Council (grant no. 93-2314-B-320-010) and Mike Biological Technologies (Hualien, Taiwan) Ins. for assistance of equipment.
Dr. Fwu Lin Yang and Chi Han Li contributed equally to this study. Dr.
Kuang Wen Liao and Dr. Ru Ping Lee contributed equally to this study.
DOI: 10.1097/shk.0b013e31803dd04d Copyright* 2007 by the Shock Society
Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited.7 Mice
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.
Pentobarbital
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 Na2YEDTA) 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 106cells 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 142g/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 j80-C.
Cytokine measurement
One hundred microliters of capture antibody (0.82g/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 tetramethylben-zidine substrate (Clinical, USA) was added into each well, and the plate was incubated at room temperature for 20 min. To stop the reaction, 502L 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-! mRNA expression assay
P338D1 cells (107) were cultured and treated with LPS, and coincubated without or with pentobarbital (final concentration, 12.52g/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-! complementary DNA was amplified by polymerase chain reaction (PCR) with the primer pairs (mouse TNF-alpha 5¶: ATgAgCACAgAAAgCAT-gATCCgCgA; mouse TNF-alpha 3¶: TCACAgAgCAATgACTCCAAAgTAgAC).
The products of reverse transcriptionYPCR were analyzed by agarose electro-phoresis, and the results were photographed.
Transcription factor activity assay
According to the manufacturer’s instruction, pNF-.B/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 EDTAY4 Na/L in PBS) and seeded into a 24-well plate. The transfectants were treated with LPS (142g/mL) and coincubated without or with pentobarbital (12.52g/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%
"-mercaptoethnol, 10% glycerol, 0.01% bromophenol blue). After the samples boiled, sodium dodecyl sulfateYpolyacrylamide 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 antiY"-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 instruc-tions (Pierce, USA).
Tumor necrosis factor-! 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-! (the control group, the TNF-! group, the pentobarbital group, the TNF-! + pentobarbital group). The supernatant was removed after 16 h. One hundred microliters of fresh medium was added with 202L 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 2L staining solutions (20 2L Annexin VYfluorescein isothiocyanate (FITC) labeling reagent and 202L propidium iodide (PI) in 1 mL binding buffer). The mixture was incubated for 15 min and analyzed by flow cytometer. FL-1 represents Annexin VYFITC 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 providedad libitum. Animals were anesthetized with ether inhalation for about 10 min. During the period of anesthesia, a femoral artery FIG. 1. The reduction of LPS-induced TNF-! from immune cells by
pentobarbital. A, P338D1 cells were treated with LPS (the LPS group). In the same condition, P338D1 cells were coincubated with pentobarbital (the LPS + 8.752g/mL and the LPS + 12.5 2g/mL groups). The growth medium of untreated P338D1 cells served as the control group. *PG 0.05 indicates a significant difference between the LPS plus 8.752g/mL group and the LPS group.†PG 0.01 indicates a significant difference between the LPS plus 12.5 2g/mL group the LPS group. B, Splenocytes were treated with LPS (the LPS group). In the same condition, splenocytes were coincubated with pento-barbital (the LPS + 12.52g/mL groups). The growth medium of untreated splenocytes served as the control group. *PG 0.05 indicates a significant difference between the LPS plus Pento group and the LPS group.
310 SHOCKVOL. 28, NO. 3 YANG ET AL.
Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited.7 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
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 admin-istration, animals were observed for 48 h (19).
Experimental design
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 Instru-ments Co, Mountain View, Calif). Enzyme-linked immunosorbent assay was performed for TNF-! measurement.
Blood biochemical analyses
The plasma samples were diluted by 1:100 with distilled water before measure-ments. 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.
Statistical analysis
Data ofin vivo experiments are expressed as meanT SE. Multiple analysis of variance and Scheffe´ test were used to compare the difference between and among groups (n = 8 in each group).PG 0.05 was considered to be significant. All in vitro data were compared by Student t test, and P G 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 thein vitro cytokine assay were obtained from three independent experiments and FIG. 2. The effect of pentobarbital on the expression of TNF-! mRNA.
P338D1 cells were treated with or without LPS and were coincubated with or without pentobarbital. The mRNA levels of TNF-! were shown by reverse transcriptionYPCR. "-Actin mRNA levels served as an internal control to normalize the sample loading.
FIG. 3. The effect of pentobarbital on the production of TNF-! in vivo. After LPS infusion, the levels of TNF-! (A) and NO (B) in sera of the mice were measured. The untreated mice served as the negative control (the NS group). *PG 0.05 indicates a significant difference between the Pento group and the NS group.†PG 0.05 indicates a significant difference between the Pento group and the LPS group. The body temperature (C) of the rats was measured rectally by a digital thermometer. The untreated mice served as the negative control (the NS group).
SHOCKSEPTEMBER2007 PENTOBARBITALREDUCESTNF-! RELEASE ANDTISSUEDAMAGE 311
Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited.7 duplicated in each group (n = 6). The data of TNF-! 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).
RESULTS
Pentobarbital lowers the TNF-! 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-! release from P338D1 cells (mouse macrophage cells) under LPS stimulation (Fig. 1A). Moreover, pentobarbital also decreased TNF-! expressions of splenocytes in the presence of LPS (Fig. 1B). In addition, the expression of TNF-! mRNA was reduced after pentobarbital treatment in the presence of LPS (Fig. 2). These results indicate that pentobarbital has anti-inflammatory abilityin 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-! in sera of conscious rats in vivo. However, pentobarbital treatment reduced the serum concentration of TNF-! 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-! 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 temper-ature 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-! release was observed. However, the body temperature was not signifi-cantly different at this stage between the two groups. There-fore, a decrease in TNF-! release by pentobarbital should not be caused by the change in the body temperature.
Pentobarbital suppresses the activities of NF-JB and AP-1 in the presence of LPS
Previous literatures have reported that LPS activates NF-.B and AP-1 pathways to enhance the TNF-! 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-.B 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-.B in cells (Fig. 4A) and slightly
Previous literatures have reported that LPS activates NF-.B and AP-1 pathways to enhance the TNF-! 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-.B 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-.B in cells (Fig. 4A) and slightly