Sun, Jian-Guo Xu, Department of Anesthesiology, Jinling
Hospital, College of Medicine, Nanjing University, Nanjing 210002,
Jiangsu Province, ChinaEnzyme-linked
immunoadsordent assay (ELISA)
Xiao-Dong Wang, Department of Surgery, Jinling Hospital,
College of Medicine, Nanjing University, Nanjing 210002, Jiangsu
Hong Liu, Department of Chest Surgery, Jinling Hospital,
College of Medicine, Nanjing University, Nanjing 210002, Jiangsu
Correspondence to: Professor Jian-Guo Xu, Department of
Anesthesiology, Jinling Hospital 305 East Zhongshan Road, Nanjing
210002, Jiangsu Province, China.
AIM: To investigate the protective effect of ketamine on the
endotoxin-induced proinflammatory cytokines and NF-kappa B
activation in the intestine.
METHODS: Adult male Wistar rats were randomly divided into 6 groups:
(a) normal saline control, (b) challenged with endotoxin (5 mg/kg)
and treated by saline, (c) challenged with endotoxin (5 mg/kg) and
treated by ketamine (0.5 mg/kg), (d) challenged with endotoxin (5
mg/kg) and treated by ketamine (5 mg/kg ), (e) challenged with
endotoxin (5 mg/kg) and treated by ketamine (50 mg/kg), and (f)
saline injected and treated by ketamine (50 mg/kg). After 1, 4 or 6
and IL-6 mRNA were investigated in the tissues of the intestine
(jejunum) by RT-PCR. TNF-a
and IL-6 were measured by ELISA. We used electrophoretic mobility
shift assay (EMSA) to investigate NF-kappa B activity in the
RESULTS: NF-kappa B activity, the expression of TNF-a
and IL-6 were enhanced in the intestine by endotoxin. Ketamine at a
dose of 0.5 mg/kg could suppress endotoxin-induced TNF-a
mRNA and protein elevation and inhibit NF-kappa B activation in the
intestine. However the least dosage of ketamine to inhibit IL-6 was
5 mg/kg in our experiment.
CONCLUSION: Ketamine can suppress endotoxin-induced production of
proinflammatory cytokines such as TNF-a
and IL-6 production in the intestine. This suppressive effect may
act through inhibiting NF-kappa B.
Sun J, Wang XD, Liu H,
Xu JG. Ketamine suppresses intestinal NF-kappa B activation and
proinflammatory cytokine in endotoxic rats. World J Gastroenterol
2004; 10(7): 1028-1031
Gram-negative bacteria caused sepsis remains an important cause
of morbidity and mortality in septic and endotoxemic patients.
Lipopolysaccharide (LPS), or endotoxin, a major component of the
outer surface of Gram-negative bacteria, is a potent activator of
cells of the immune and inflammatory systems, including macrophages,
monocytes and endothelial cells, and contributes to
the systemic changes seen in septic shock[1-4]. The
endotoxic shock syndrome is characterized by systemic inflammation,
multiple organ damage, circulatory collapse and death[1,2].
The important role of the intestinal mucosa in the
inflammatory and metabolic responses to sepsis, severe injury and
other critical illnesses has been increasingly recognized during the
last decade. Thus, there is evidence that the gut mucosa becomes the
site for production of various inflammatory cytokines[5,6]
and other yet unidentified substances that may influence not only
the mucosa itself but also the function and integrity of remote
organs and tissues[7,8]. Indeed, the gut mucosa has been
proposed to be the “motor” of multiple organ failure in critical
illness. Besides, sepsis and severe injury are also
associated with loss of mucosal integrity, resulting in increased
permeability and bacterial translocation. These changes may
accelerate the development of multiple organ failure.
an intravenous anesthetic, has been advocated for anesthesia in
septic or severely ill patients because of its cardiovascular
stimulating effects[11,12]. And several previous studies
reported that ketamine could suppress LPS-induced tumor necrosis
factor alpha (TNF-a)
production in the serum and reduced mortality in carrageenan-sensitized
endotoxin shock mice[13,14]. However, few studies were
undertaken to investigate the protective effect of ketamine on the
inflammatory response in the intestine during septic shock in
vitro. Since local produced cytokines were regarded as the
contributing factors in tissue damage during sepsis[15-17].
And nuclear factor kappa B (NF-kappa B) was verified to be an
inducible transcription factor that was required for the
transcription of some proinflammatory cytokines such as TNF-a,
interleukin 6 and interleukin 8 (IL-6 and IL-8). Our
previous study indicated that ketamine could inhibit endotoxin
induced NF-kappa B and TNF-a
in vitro. Therefore, this study was to
investigate whether ketamine could suppress endotoxin-induced
NF-kappa B activation and proinflammatory cytokines in the intestine
in vitro in order to define a possible mechanism of the
anti-inflammatory effect of ketamine.
Animals and treatment
Adult male Wistar rats (250-300 g body mass) used in this
experiment were purchased from Shanghai Animal Center, Shanghai,
China. The rats were exposed each day to 12 h of light and darkness
respectively. The experimental protocol followed the institution’s
criteria for the care and use of laboratory animals in research.
Further, all animals received humane care in compliance with
Institutional Animal Care Committee.
The Wistar rat endotoxemia model was established by
injection with a dose of LPS (5 mg/kg, Escherichia coli O111: B4)
(Sigma Chemical Co., USA) via the tail vein. Then all animals were
immediately treated with different doses of ketamine (0.5, 5, 50
mg/kg) or normal saline (10 mL/kg) intraperitoneally (ip). Endotoxin
and ketamine were diluted with normal saline at different
concentrations so as to inject them into the rats at the same volume
(10 mL/kg ). After 1, 4 or 6 h, animals were killed, and tissues
from the intestine was removed and kept in liquid nitrogen for later
use. We used six rats in every time point of each group.
Electrophoretic mobility shift assay (EMSA)
extracts of the intestine tissue was prepared by hypotonic lysis
followed by high salt extraction[20-22]. EMSA was
performed using a commercial kit (Gel Shift Assay System; Promega,
Madison, WI) as previously described. The NF-kappa B oligonucleotide
probe, (5’-AGTTGAGGGGACT TTCCCAGGC-3’), was end-labeled with [g-32P]
ATP (Free Biotech, Beijing, China) with T4-polynucleotide kinase.
Nuclear protein (80 mg)
was preincubated in 9 mL
of a binding buffer, consisting of 10 mmol/L Tris-Cl, pH 7.5, 1 mmol/L
MgCl2, 50 mmol/L NaCl, 0.5 mmol/L EDTA, 0.5 mmol/L DTT,
40 mL/L glycerol, and 0.05 g/L of poly-(deoxyinosinic deoxycytidylic
acid) for 15 min at room temperature. After addition of the 1 mL
32P-labeled oligonuleotide probe, the incubation was
continued for 30 min at room temperature. Reaction was stopped by
adding 1 mL
of gel loading buffer, and the mixture was subjected to
non-denaturing 40 g/L polyacrylamide
gel electrophoresis in 0.5×TBE
buffer. The gel was vacuum-dried and exposed to X-ray film (Fuji
Hyperfilm) at -70 °C
polymerase chain reaction (RT-PCR)
RNA was extracted with TriPure Isolation Reagent (Roche Molecular
Biochemicals, Switzerland) and quantified by absorption at 260 nm.
Reverse-transcription (RT) was implemented using Reverse
Transcription System (Promega, WI, USA) according to the protocol.
We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as
normalization control. The sequences of the primers were: TNF-a
(sense) CACCACGCTCTTCTGTCTACTGAAC, (antisense)
CCGGACTCCGTGATGTCTAAGTACT; IL-6 (sense) GACTGATGTTGTTGACAGCCACTGC, (antisense)
TAGCC ACTCCTTCTGTGACTCTAACT; GAPDH (sense) CACGGCAAGTTCAATGGCACA, (antisense)
GAATTGTGAGGGAGAGTGCTC. A total volume of 100 mL
reaction contained 2 mL
of RT product, 1.5 mmol/L MgCl2, 2.5 U Taq DNA polymerase,
primer and 1×Taq
DNA polymerase magnesium-free buffer (Promega, WI, USA). Then the
reaction mixture was overlaid with two drops of mineral oil (Sigma
Chemical Co., USA) and incubated in thermocycler (MiniCycler PTC
150, MJ Research Inc, USA) programmed to pre-denature at 95 °C
for 2 min, denatured at 95 °C
for 1 min, annealed at 60 °C
for 1 min and extended at 72 °C
for 2 min for a total of 30 cycles. The last cycle was followed by a
final incubation at 72 °C
for 5 min and cooled to 4 °C
. The polymerase chain reaction products were 546 bp (TNF-a),
509 bp (IL-6) and 970 bp (GAPDH) respectively. Then they were
electrophoresed on a 15 g/L agarose gel stained with ethidium
bromide. The gel was captured as a digital image and analyzed using
Scion Image software (Maryland, USA). Values in each sample were
normalized with GAPDH control.
and IL-6 in the intestine were measured using commercially available
enzyme-linked immunoassay kits (Diaclone USA for TNF-a;
Biosource USA for IL-6) according to the test protocol. Values were
expressed as pg per milligram protein (pg/mg prot).
Statistics and presentation of data
Data were expressed as mean±SE. Statistical significance
was determined by one-way ANOVA using SPSS 10.0. A value of P<0.05
was considered significant.
NF-kappa B activation in the intestine
experiments were performed to examine the effect of ketamine on the
activation of NF-kappa B induced by endotoxin. As shown in Figure 1,
NF-kappa B activation in the intestine was increased after endotoxin
challenge as compared with unstimulated group. The activity of
NF-kappa B was in a time dependant manner after endotoxin injection.
Ketamine inhibited NF-kappa B activation at three (0.5, 5, and 50
mg/kg) dosing levels (P<0.05, as compared with endotoxin
group) (Figure 1).
1(PDF) Activation of
NF-kappa B in the intestine. Normal saline treatment (Lane 1). 1, 4,
6 h after endotoxin challenge (Lane 2, 3, 4), endotoxin plus
ketamine (0.5, 5, 50 mg.kg-1) (Lane 5, 6, 7), ketamine
only (50 mg.kg-1) (Lane 8) aP<0.05
vs Lane 1; bP<0.01 vs Lane 1; dP<0.01
vs Lane 2.
Expression of TNF-a
in the intestine. Normal saline treatment (Lane 1). 1, 4, 6 h after
endotoxin challenge (Lane 2, 3, 4), endotoxin plus ketamine (0.5, 5,
50 mg.kg-1.) (Lane 5, 6, 7), ketamine only (50 mg.kg-1)
(Lane 8); bP<0.01 vs Lane 1; dP
<0.01 vs Lane 2.
mRNA expression by endotoxin challenge and the protective effect of
sustained a baseline level in normal rats. Endotoxin caused a
transient elevation of TNF-a
mRNA in the intestine. This activity increased with time reaching a
maximum 1 h after sepsis. Ketamine was administered
intraperitoneally soon after endotoxin challenge. TNF-a
gene expression was analyzed 1 h later since TNF-a
could reach the maximum level about 1 h later. Ketamine suppressed
expression in a dose dependent manner. We found that ketamine at a
dose of 0.5 mg/kg could suppress TNF-a
expression significantly. This dosage was far below clinical
anesthetic level (Figure 2).
of IL-6 in the intestine. Normal saline treatment (Lane 1). 1, 4, 6
h after endotoxin only (Lane 2, 3, 4), endotoxin plus ketamine (0.5,
5, 50 mg/kg) (Lane 5, 6, 7), ketamine only (50 mg/kg) (Lane 8); bP<0.01
vs Lane 1; cP<0.05 vs Lane 3.
Protective effect of ketamine on the TNF-a
production in the intestine. All of the values were obtained 1 h
after sepsis. Lane 1 normal saline; Lane 2 endotoxin (5 mg/kg); Lane
3 endotoxin (5 mg/kg) plus ketamine (0.5 mg/kg); Lane 4 endotoxin (5
mg/kg) plus ketamine (5 mg/kg); Lane 5 endotoxin (5 mg/kg) plus
ketamine (50 mg/kg); Lane 6 ketamine only (50 mg/kg) bP<0.01
vs Lane 2.
Protective effect of ketamine on the IL-6 production in the
intestine. All of the values were obtained 4 h after sepsis. Lane 1
normal saline; Lane 2 endotoxin (5 mg/kg); Lane 3 endotoxin (5
mg/kg) plus ketamine (0.5 mg/kg); Lane 4 endotoxin (5 mg/kg) plus
ketamine (5 mg/kg); Lane 5 endotoxin (5 mg/kg) plus ketamine (50
mg/kg); Lane 6 ketamine only (50 mg/kg) bP<0.01
vs Lane 2.
IL-6 expression in intestine by endotoxin challenge and the
protective effect of ketamine
The IL-6 expression of the small intestine is shown in Figure 3.
Endotoxin also enhanced IL-6 expression in the intestine. However
the peak time was 4 h after endotoxin challenge. We observed the
protective effect of ketamine at this peak time. Ketamine suppressed
IL-6 expression in a dose dependent manner. Unlike TNF-a,
the minimal dosage at which ketamine could suppress IL-6
significantly was 5 mg/kg. This was within clinical anesthetic range
of ketamine on TNF-a
and IL-6 production in intestine homogenates after endotoxin
Ketamine suppressed endotoxin-induced TNF-a
and IL-6 production in a dose dependent manner. Ketamine beyond the
concentration of 0.5 mg/kg could inhibit TNF-a
production, however the minimal dosage at which ketamine suppressed
IL-6 significantly was 5 mg/kg. This was within clinical anesthetic
range (Figures 4 and 5).
Our laboratory and others have demonstrated that ketamine could
suppress endotoxin-induced some proinflammatory cytokines in
vitro. However it is to be determined in complex in
vitro studies. We assessed the cytokines and transcriptional
factor NF-kappa B in the intestine because of the important status
of the intestine in sepsis or systemic inflammation reaction
syndrome (SIRS). The intestine was not only the passive organs
injured by sepsis but participation in the pathogenesis of SIRS[5,6].
is regarded as the most important proinflammatory cytokine, which is
released early after an inflammatory stimulus. And
IL-6, which is elevated after TNF-a,
contributes to both morbidity and mortality in conditions of
“uncontrolled” inflammation. Among the cytokines
produced in the intestinal mucosa during inflammation, TNF-a
and IL-6 are particularly important because of its multiple
biological effects both in the intestine and in other organs and
tissues. In this study, we demonstrated that ketamine suppressed
both endotoxin-induced TNF-a
and IL-6 expression and production in the intestine. TNF-awas
the first cytokine expressed after endotoxin stimulation and later
IL-6, which was consistent with several previous reports[24,25].
Studies had demonstrated that ketamine could suppress endotoxin-induced
cytokines in vitro. However, proinflammatory cytokines just
and IL-6 were not merely stimulated by endotoxin in vitro.
Therefore, our experimental protocol was more physiological and
closer to clinical condition.
NF-kappa B is associated in the cytoplasm with its inhibitory
subunit, inhibitory kappa B (IkB),
which prevents NF-kappa B from translocating into the nucleus.
Endotoxin can induce the phosphorylation and degradation of IkB.
Many effector genes including those encoding cytokines (TNF-a
and IL-6) are in turn regulated by NF-kappa B. To
determine whether ketamine could inhibit NF-kappa B activation, we
did EMSA to detect NF-kappa B activity in the intestine. We found a
constitutive activation of NF-kappa B in intestine. Endotoxin could
enhance NF-kappa B activation in the intestine and it was most
significant 1 h later. Although we had previously demonstrated that
ketamine could inhibit NF-kappa B activation in peripheral blood
mononuclear cell (PBMC) after endotoxin challenge in vitro.
It was to be studied whether ketamine had this effect in vitro.
In our experiment, we found ketamine could inhibit NF-kappa B
activation. However it was not in a dose dependent manner. We did
not found any NF-kappa B activity changes in the group administered
ketamine (50 mg/kg ) only, which excluded ketamine itself had any
effect on NF-kappa B activity.
rats were not anesthetized during the whole experiment, we did not
monitor the arterial pressure and pulse rate to confirm septic
shock. Because the drug studied in our investigation was ketamine,
just an anesthetic drug. To exclude any other anesthetic drug
disturbance, we had to give up that monitoring. However, this septic
model was successfully used in many other researches[27-29].
In addition, we did find that the rats were dispirited with
piloerection and diarrhea, which indicated the septic shock
dosage of ketamine used in this study was from 0.5 to 50 mg/kg,
which covered the clinical range. Roytblat et al.
reported that a single dose of ketamine 0.25 mg/kg administered
before cardiopulmonary bypass suppressed the increase in serum IL-6
during and after coronary artery bypass surgery. However, other
studies demonstrated such a small dose of ketamine did not suppress
IL-6 production. The reason was not clear. In this
study, only ketamine reaching a dose of 5 mg/kg could suppress IL-6
production in the intestine. There were perhaps some differences
between human being and animals or between in vitro and in
vitro studies. We found 0.5 mg/kg
ketamine suppressed TNF-a
production, which was in accordance with those in vitro
conclusion, we demonstrated that ketamine could suppress endotoxin-induced
and IL-6 expression and production in the intestine. And this
suppressive effect might act through inhibiting NF-kappa B. Further
study is required to elucidate the mechanism of ketamine action.
We thank Dr. Genbao Feng for his technical assistance.
Opal SM, Cohen J. Clinical Gram-positive sepsis: does it
fundamentally differ from Gram-negative bacterial sepsis? Crit
Care Med 1999; 27: 1608-1616
Rietschel ET, Brade H, Holst O, Brade L, Muller-Loennies S,
Mamat U, Zahringer U, Beckmann F, Seydel U, Brandenburg
K, Ulmer AJ, Mattern T, Heine H,
Schletter J, Loppnow H, Schonbeck U, Flad HD, Hauschildt S, Schade
UF, Di Padova F,
Kusumoto S, Schumann RR. Bacterial
endotoxin: chemical constitution, biological recognition, host
immunological detoxification. Curr
Topics Microbiol Immunol 1996; 216: 39-81
Wenzel RP, Pinsky MR, Ulevitch RJ, Young L. Current
understanding of sepsis. Clin Inf Dis 1995; 22: 407-412
Huemann D, Glauser MP, Calandra T. Molecular basis of
host-pathogen interaction in septic shock. Curr Opin Microbiol
Huang L, Tan X, Crawford SE, Hsueh W. Platelet-activating
factor and endotoxin induce tumor necrosis factor gene
expression in rat intestine and
liver. Immunology 1994; 83: 65-69
Meyer TA, Wang J, Tiao GM, Ogle CK, Fischer JE, Hasselgren
PO. Sepsis and endotoxemia stimulate interleukin-6
production. Surgery 1995; 118:
Magnotti LJ, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric
lymph but not portal blood increases endothelial cell
permeability and promotes lung injury
after hemorrhagic shock. Ann Surg 1998; 228: 518-527
Sambol JT, Xu DZ, Adams CA, Magnotti LJ, Deitch EA.
Mesenteric lymph duct ligation provides long term protection
against hemorrhagic shock-induced
lung injury. Shock 2000; 14: 416-419
Langkamp-Henken B, Donovan TB, Pate LM, Maull CD, Kudsk KA.
Increased intestinal permeability following shock and
penetrating trauma. Crit Care Med
1995; 23: 660-664
Swank GM, Deitch EA. Role of the gut in multiple organ
failure: bacterial translocation and permeability changes.
J Surg 1996; 21: 411-417
Lippmann M, Appel PL, Mok MS, Shoemaker WC. Sequential
cardiorespiratory patterns of anesthetic induction with
ketamine in critically ill patients.
Crit Care Med 1983; 11: 730 -734
Yli-Hankala A, Kirvela M, Randell T, Lindgren L. Ketamine
anaesthsia in a patient with septic shock. Acta Anaesthsiol
Scand 1992; 36: 483-485
Takenaka I, Ogata M, Koga K, Matsumoto T, Shigematsu A.
Ketamine suppresses endotoxin-induced tumor necrosis
factor alpha production in mice.
Anesthesiology 1994; 80: 402-408
Koga K, Ogata M, Takenaka I, Matsumoto T, Shigematsu A.
Ketamine suppresses tumor necrosis factor-a
mortality in carrageenan-sensitized
endotoxin shock model. Circ Shock 1995; 44: 160-168
Cavaillon JM, Munoz C, Fitting C, Misset B, Carlet J.
Circulating cytokines: the tip of the iceberg? Circ Shock
1992; 38: 145-152
Beutler BA, Milsark IW, Cerami A. Cachectin/tumor necrosis
factor: production, distribution, and metabolic fate in vitro.
J Immunol 1985; 135: 3972-3977
Keogh C, Fong Y, Marano MA, Seniuk S, He W, Barber A, Minei
JP, Felsen D, Lowry SF, Moldawer LL. Identification of
a novel tumor necrosis factor alpha/cachectin
from the livers of burned and infected rats. Arch Surg 1990; 125:
Baldwin AS Jr. The NF-kB
proteins: new discoveries and insights. Ann Rev Immunol 1996; 14:
Yu Y, Zhou Z, Xu J, Liu Z, Wang Y. Ketamine reduces NF kappa
B activation and TNF alpha production in rat mononuclear
cells induced by lipopolysaccharide in
vitro. Ann Clin Lab Sci 2002; 32: 292-298
Gong JP, Liu CA, Wu CX, Li SW, Shi YJ, Li XH. Nuclear factor kB
activity in patients with acute severe cholangitis. World J
Gastroenterol 2002; 8: 346-349
Zhou W, Jiang ZW, Tian J, Jiang J, Li N, Li JS. Role of NF-kB
and cytokine in experimental cancer cachexia. World J
Gastroenterol 2003; 9: 1567-1570
Liu Z, Yu Y, Jiang Y, Li J. Growth hormone increases lung NF-kappaB
activation and lung microvascular injury induced by
lipopolysaccharide in rats. Ann Clin
Lab Sci 2002; 32: 164-170
Kawasaki T, Ogata M, Kawasaki C, Ogata J, Inoue Y, Shigematsu
A. Ketamine suppresses proinflammatory cytokine
production in human whole blood in
vitro. Anesth Analg 1999; 89: 665-669
Hesse DG, Tracey KJ, Fong Y, Manogue KR, Palladino MA Jr,
Cerami A, Shires GT, Lowry SF. Cytokine appearance in
human endotoxemia and primate
bacteremia. Surg Gynecol Obstet 1988; 166: 147-153
Damas P, Ledoux D, Nys M, Vrindts Y, Groote D, Franchimont P,
Lamy M. Cytokine serum level during severe sepsis in
human: IL-6 as a marker of severity.
Ann Surg 1992; 215: 356-362
Baeuerle PA, Baltimore D. NF-kappa B: ten years after. Cell
1996; 87: 13-20
Arya R, Grossie VB Jr, Weisbrodt NW, Lai M, Mailman D, Moody
F. Temporal expression of tumor necrosis factor-a
nitric oxide synthase 2 in rat small
intestine after endotoxin. Dig Dis Sci 2000; 45: 744-749
Secchi A, Ortanderl JM, Schmidt W, Walther A, Gebhard MM,
Martin E, Schmidt H. Effects of dobutamine and
dopexamine on hepatic micro- and
macrocirculation during experimental endotoxemia: an intravital
in the rat. Crit Care Med 2001; 29:
Taniguchi T, Shibata K, Yamamoto K. Ketamine inhibits
endotoxin-induced Shock in Rats. Anesthesiology
2001; 95: 928-932
Roytblat L, Talmor D, Rachinsky M, Greemberg L, Pekar A,
Appelbaum A, Gurman GM, Shapira Y, Duvdenani A. Ketamine
attenuates the interleukin-6 response
after cardiopulmonary bypass. Anesth Analg 1998; 87: 266-271
JZ and Xu FM