Propofol reduces nitric oxide biosynthesis in lipopolysaccharide-activated macrophages by downregulating the expression of inducible nitric oxide synthase.

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M O L E C U L A R T O X I C O L O G Y

Propofol reduces nitric oxide biosynthesis in lipopolysaccharide-activated

macrophages by downregulating the expression of inducible

nitric oxide synthase

Received: 28 October 2002 / Accepted: 30 January 2003 / Published online: 12 March 2003 Springer-Verlag 2003

Abstract Nitric oxide is an active oxidant that

contrib-utes to the physiology and pathophysiology of

macro-phages. Propofol has been widely used in intravenous

anesthesia. It possess antioxidant and

immunomodu-lating eﬀects. This study aimed to evaluate the eﬀects of

propofol on nitric oxide production in

lipopolysaccha-ride-activated macrophages. Exposure of macrophages

to propofol (25, 50 and 75 lM), to lipopolysaccharide

(0.5, 1, 1.5 and 2 ng/ml) or to a combination of propofol

and lipopolysaccharide did not aﬀect cell viability.

However, propofol at 100 lM led to signiﬁcant cell

death (P<0.05). The levels of nitrite, an oxidative

pro-duct of nitric oxide, were increased in

lipopolysacchar-ide-treated macrophages in a concentration-dependent

manner (P<0.01), while propofol could

concentration-dependently decrease the lipopolysaccharide-enhanced

nitrite

levels

(P<0.01).

Immunoblotting

analysis

revealed that lipopolysaccharide increased the protein

level of inducible nitric oxide synthase (iNOS). The

co-treatment of propofol and lipopolysaccharide

sig-niﬁcantly reduced this lipopolysaccharide-induced iNOS

protein (357±49·10

3

versus 92±6·10

3

arbitrary units,

P<0.01). Analysis by reverse transcriptase-polymerase

chain reaction showed that lipopolysaccharide induced

mRNA of iNOS, but that the inductive eﬀect was

inhibited by propofol (95±7·10

2

_{versus 30±4·10}

2

arbitrary units, P<0.01). This study has demonstrated

that propofol, at therapeutic concentrations, could

suppress nitric oxide biosynthesis by inhibiting iNOS

expression

in

lipopolysaccharide-activated

macro-phages. The mechanism of suppression was at a

pre-translational level.

Keywords Propofol Æ Macrophages Æ Lipopoly

saccharide Æ Inducible nitric oxide synthase Æ Nitric

oxide

Introduction

Nitric oxide (NO) is a gaseous free radical synthesized

from

L

-arginine by calcium-dependent constitutive NO

synthase or calcium-independent inducible NO synthase

(iNOS) (Moncada et al. 1991). The diatomic free radical

is an important regulator of vasoconstriction, neuronal

transmission, immune response, and cell apoptosis

(Moncada et al. 1991; Horibe et al. 2000; Chen et al.

2002). NO can be either the mediator of non-speciﬁc

cellular immunity or the cause of autoimmune injury

during inﬂammation (Nathan 1992).

Lipopolysaccha-ride (LPS), a gram-negative bacterial outer membrane

component, has been identiﬁed as one of critical factors

involved in the pathogenesis of sepsis (Raetz et al. 1991).

In response to stimuli, LPS can bind to

membrane-localized Toll-like receptors, lead to the induction of

speciﬁc signal transduction pathways, and release large

amounts of NO into the general circulation to exhibit

systemic eﬀects (West et al. 1994; Schuster and Nelson

2000). In the pathophysiology of septic shock, the

ex-cessive production of NO following iNOS induction has

been proposed as a major factor involved in the tissue

damage (Lynn and Cohen 1995). The increases of NO in

macrophages could be modulated by a variety of drugs,

including anesthetic agents (Shimaoka et al. 1996; Chiou

et al. 2001).

As a safe and eﬀective intravenous anesthetic agent,

propofol (PPF, 2,6-diisopropylphenol) is widely used for

DOI 10.1007/s00204-003-0453-z

Ruei-Ming Chen Æ Gong-Jhe Wu Æ Yi-Ting Tai

Wei-Zen Sun Æ Yi-Ling Lin Æ Wen-Chi Jean

Ta-Liang Chen

R.-M. Chen Æ Y.-T. Tai Æ Y.-L. Lin Æ W.-C. Jean Æ T.-L. Chen (&) Department of Anesthesiology,

Graduate Institute of Medical Science, College of Medicine, Wan-Fang Hospital, Taipei Medical University,

No. 111, Sec. 3, Hsing-Lung Rd., 116 Taipei, Taiwan E-mail: tlchen@tmu.edu.tw

Tel.: +886-2-29307930 ext 2150 Fax: +886-2-86621150 G.-J. Wu

Department of Anesthesiology,

Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan W.-Z. Sun

Department of Anesthesiology, College of Medicine and Hospital,

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induction and maintenance of anesthesia in surgical

procedures, or for sedation in the intensive care unit

(Sebel and Lowden 1989; Young et al. 2000). In studies

on macrophages and neutrophils, PPF has been reported

to impair cell functions and may contribute to the

sup-pression of host immunity (Mikawa et al. 1998; Kotani

et al. 1998). Being similar in structure to

phenol-con-taining a-tocopherol and butylated hydroxytoluene,

PPF has the potential for antioxidation by directly

scavenging hydroxyl chloride, superoxide, hydrogen

peroxide and hydroxyl radical (Murphy et al. 1992;

Demiryurek et al. 1998). In addition to these oxidants,

our previous study has further shown that PPF can

protect macrophages from NO-induced cell death

(Chang et al. 2002).

The eﬀects of PPF on NO biosynthesis are diﬀerent

among various cell types. In cultured porcine aortic

endothelial cells and rat ventricular myocytes, PPF has

been shown to enhance NO production and cause the

vasodilatation or negative chronotropy (Petros et al.

1993; Yamamoto et al. 1999). A study on canine

pul-monary arterial rings revealed that PPF selectively

at-tenuated acetylcholine-induced relaxation by inhibiting

NO synthesis (Beutler and Poltorak 2001). PPF is often

used for sedation of patients suﬀering from critical

ill-ness such as sepsis (Young et al. 2000). NO is an

im-portant eﬀector in LPS-involved septic pathophysiology

(Raetz et al. 1991; West et al. 1994). However, the role of

PPF in regulating NO biosynthesis in LPS-activated

macrophages is still unknown. This study aimed to

evaluate whether PPF could modulate NO synthesis in

LPS-activated macrophages, and to study the possible

mechanism of any such modulation.

Materials and methods

Cell culture and drug treatment

A murine macrophage cell line, RAW 264.7, purchased from American Type Tissue Collection (Rockville, MD, USA) was used in this study as an experimental model. Macrophages were cultured in RPMI 1640 medium (Gibco, BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum, L-glutamine, penicillin

(100 IU/ml), and streptomycin (100 lg/ml) in 75-cm2ﬂasks at 37C in a humidiﬁed atmosphere containing 5% CO2. The cells were

grown to confluence prior to PPF administration. PPF, a pure compound sponsored by Zeneca Ltd (Macclesfield, UK), was freshly prepared for each independent experiment by dissolution in dimethyl sulfoxide (DMSO). The DMSO concentration in the medium was less than 0.1 % to avoid its toxicity to macrophages. LPS was dissolved in phosphate-buffered saline (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4).

Determination of cell viability

To determine the appropriate concentrations of PPF and LPS that were not cytotoxic to macrophages, cell viability was assayed by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method as described previously (Liu et al. 2001). Ten-thousand macrophages were seeded in 96-well tissue culture clusters for overnight culture. After preincubation with PPF, LPS

and a combination of PPF and LPS, cells were cultured with fresh medium containing 0.5 mg/ml MTT for another 3 h. The blue formazan product in cells was dissolved in DMSO and measured spectrophotometrically at a wavelength of 550 nm.

Quantiﬁcation of nitrite

Nitric oxide has such a short half-life that it is easily oxidized to nitrite and nitrate (Moncada et al. 1991). In order to evaluate whether PPF could modulate cellular NO, the amounts of nitrite were determined according to the technical bulletin of Promega’s Griess reagent sys-tem (Promega Corp., Madison, WI, USA). After exposure to the drugs, the culture medium of macrophages was centrifuged, and the supernatant was collected. Following a reaction of the supernatant with sulfaniamide and N-1-napthylethylenediamine, a colorimetric azo compound was formed and quantiﬁed by an Anthos 2010 mi-croplate photometer (Anthos Labtec Instruments GmbH, Wals/ Salzburg, Austria). Preliminary studies revealed that exposure of macrophages to 1 ng/ml LPS for 6, 12, 16 and 24 h led to 0.5-, 5-, 10-and 18-fold increases of cellular nitrite. Thus, after treatment for 24 h, LPS resulted in a maximum increase in cellular nitrite. The time interval of 24 h was chosen for the subsequent experiments.

Immunoblotting analysis

The NO production in macrophages in response to LPS stimulation is due to de novo syntheses of iNOS protein (West et al. 1994). Thus, an immunoblotting analysis was carried out to determine whether PPF could modulate iNOS protein. After pretreatment with the drugs for 24 h, macrophages were washed with the phosphate-buf-fered saline, and the cell lysates were collected after dissolving the cells in 50 ll of ice-cold radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl pH 7.2, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, 1 mM ethylenediaminetetraacetic accid). To avoid the degradation of cytosolic proteins by proteinases, a mixture of 1 mM phen-ylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and 5 lg/ml leupeptin was added to the RIPA buffer. Protein concentrations were quantified by a bicinchonic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Cytosolic proteins (100 lg) were resolved on 12% polyacrylamide gels and electrophoretically blotted onto ni-trocellulose membranes. The membranes were blocked with 5% non-fat milk at 37C for 1 h. Immunodetection of cellular iNOS protein was carried out using a mouse monoclonal antibody against mouse iNOS protein (Transduction Laboratories, Lexington, KY, USA). Cellular b-actin was immunodetected by a mouse monoclonal anti-body against mouse b-actin (Sigma, St. Louis, MO, USA) as an internal standard. Intensities of the immunoreactive bands were determined using the digital imaging system UVIDOCMW version 99.03 (Uvtec Ltd, Cambridge, UK).

Analysis of reverse transcriptase-polymerase chain reaction Messenger RNA from macrophages exposed to PPF, or LPS or a combination of PPF and LPS was prepared for analyses of reverse transcriptase-polymerase chain reaction (RT-PCR) of iNOS and of b-actin according to the instruction of the ExpressDirect mRNA Capture and RT System for RT-PCR kit (Pierce). Oligonucleotides for PCR analyses of mouse iNOS and b-actin were designed and synthesized by the Clontech Laboratories, Inc. (Palo Alto, CA, USA). The oligonucleotide sequences of upstream and downstream primers for iNOS mRNA analysis were, respectively, 5¢-CCCTT-CCGAAGTTTCTGGCAGCAGC-3¢ and 5¢-CGACTCCTTTTC-CGCTTCCTGAG-3¢. The oligonucleotide sequences of sense and antisense primers for b-actin mRNA analysis were 5¢-GTGGGC-CGCTCTAGGCACCAA-3¢ and 5¢-CTCTTTGATGTCACGCA-CGATTTC-3¢, respectively. The PCR reaction was carried out using 35 cycles including 94C for 45 s, 60C for 45 s and 72C for

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2 min, and the products were loaded and separated in a 1.8% agarose gel containing 0.1 lg/ml ethidium bromide. The amounts of b-actin mRNA in macrophages were detected as an internal standard. The intensities of DNA bands in the agarose gel were quantiﬁed with the aid of UVIDOCMW version 99.03 digital imaging system as described above.

Statistical analysis

The statistical significance of the difference between control and PPF-treated groups was evaluated by the Student’s t-test. A P-value less than 0.05 was considered as statistically significant. The sta-tistical difference between groups was considered significant when the P-value of the Duncan’s multiple range test was less than 0.05.

Results

The exposure of macrophages to 25, 50 and 75 lM PPF

for 1, 6 and 24 h did not aﬀect the cell viability

(Table 1). However, treatment with 100 lM PPF for 6

and 24 h caused 16 and 32% cell death, respectively.

LPS at 0.5, 1, 1.5 and 2 ng/ml was not cytotoxic to

macrophages (Table 1). Co-treatment with 100 lM PPF

and 1 ng/ml LPS for 6 and 24 h led to signiﬁcant cell

death of 21 and 35%, respectively. Treatment with

50 lM PPF and various concentrations of LPS was not

cytotoxic to macrophages (Table 1).

LPS at 0.5, 1, 1.5 and 2 ng/ml resulted in signiﬁcant

6-, 14-, 18- and 20-fold increases of nitrite in the culture

medium of macropahges, respectively (Fig. 1). Exposure

of macrophages to 50 lM PPF blocked LPS-enhanced

nitrite levels by 40, 53, 56, and 50%, respectively. PPF

did not inﬂuence the amounts of nitrite released from

macrophages (Fig. 2). Following treatment with 25, 50,

75 and 100 lM PPF, the LPS-enhanced nitrite levels

were signiﬁcantly decreased by 18, 33, 52 and 55%,

respectively.

In untreated macrophages, iNOS protein was not

detectable (Fig. 3, top panel, lane 1). Following treatment

with LPS, the levels of iNOS protein were signiﬁcantly

increased (lane 2). PPF per se did not induce the

ex-pression of iNOS protein (lane 3). Exposure of

macro-phages to PPF suppressed LPS-induced iNOS protein

(lane 4). The amounts of b-actin protein in macrophages

were immunodetected and used as an internal standard

(Fig. 3, bottom panel). Quantiﬁcation of immunorelated

protein bands revealed that PPF signiﬁcantly decreased

by 74% the LPS-enhanced iNOS protein (Table 2).

After pretreatment with the drugs, mRNA from

macrophages was prepared for RT-PCR analyses of

iNOS and b-actin (Fig. 4). The molecular size of

RT-PCR products for iNOS and b-actin mRNA was 497

and 540 base pairs, respectively. In untreated

macro-phages, iNOS mRNA was not detectable (Fig. 4, top

panel, lane 2). Following treatment with LPS, iNOS

Table 1 Eﬀects of propofol (PPF),lipopolysaccharide (LPS) and a combination of PPF and LPS on macrophage viability. Macrophages were exposed to PPF, LPS or a combination of PPF and LPS for 1, 6 and 24 h. Cell viability was determined by the MTT assay as described in Materials and methods section. Each value represents mean ±SEM for n=12

*P<0.05, values are considered to be statistically diﬀerent from the respective control

Treatment Cell viability (% of control)

Agent Concentration 1 h 6 h 24 h Control 100 100 100 PPF 25 lM 100±5 99±8 103±6 50 lM 102±6 104±6 97±8 75 lM 99±4 97±5 93±7 100 lM 97±7 84±5* 68±8* LPS 0.5 ng/ml 101±7 101±6 104±7 1.0 ng/ml 95±8 98±7 105±6 1.5 ng/ml 104±5 103±6 99±5 2.0 ng/ml 103±4 99±5 101±7 PPF + LPS 25 lM + 1.0 ng/ml 96±5 95±8 91±9 50 lM + 1.0 ng/ml 93±8 102±6 99±3 75 lM + 1.0 ng/ml 101±5 98±5 102±4 100 lM + 1.0 ng/ml 95±8 79±4* 65±8* 50 lM + 0.5 ng/ml 94±9 94±9 96±7 50 lM + 1.0 ng/ ml 100±7 101±7 104±8 50 lM + 1.5 ng/ml 97±6 100±5 95±9 50 lM + 2.0 ng/ml 97±8 95±6 98±3

Fig. 1 Concentration-dependent effects of lipopolysaccharide (LPS) on nitrite production. Macrophages were exposed to 0.5, 1, 1.5 and 2.0 ng/ml LPS and a combination of 50 lM propofol (PPF) and LPS at various concentrations for 24 h. The amounts of nitrite in the culture medium were determined by the Griess reaction method. Data are expressed as means ±SEM for n=12. *P<0.05, values significantly different from the respective control.

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mRNA was apparently induced (lane 3). PPF did not

enhance the expression of iNOS mRNA (lane 4).

Co-treatment with PPF and LPS inhibited the

endo-toxin-induced iNOS mRNA (lane 5). The amounts of

b-actin mRNA were detected and quantiﬁed as an internal

standard (Fig. 4, bottom panel). Quantiﬁcation of

RT-PCR products revealed that PPF signiﬁcantly inhibited

by 68% the LPS-induced iNOS mRNA (Table 2).

Discussion

Our present study demonstrates that PPF could

modu-late the levels of NO production, measured as nitrite, in

LPS-activated macrophages. This study showed that a

therapeutic concentration of PPF, 50 lM, caused

sig-niﬁcant decreases in the amounts of nitrite in

LPS-stimulated macrophages (Figs. 1 and 2). The suppressive

eﬀect of PPF on cellular nitrite response means that this

anesthetic was able to inhibit LPS-enhancement of

cel-lular NO in macrophages. In parallel to the increase of

cellular NO, the present study showed that the induction

of iNOS at the protein and mRNA levels in

macro-phages was responsive to LPS stimulation (Figs. 3 and

4). This result is similar to previous studies, according to

Fig. 3 Immunoblotting analysis of inducible nitric oxide synthase (iNOS) from untreated (C) macrophages, and those treated with lipopolysaccharide (LPS), propofol (PPF) or a combination of PPF and LPS. Macrophages were exposed to 1 ng/ml LPS, 50 lM PPF and a combination of PPF and LPS. Cytosolic proteins were prepared and subjected to protein blot analysis in which mouse monoclonal antibody was used for probe for iNOS protein. The expression of b-actin protein was regarded as an internal standard. The molecular sizes of iNOS and b-actin are 130 and 42 kD, respectively

Fig. 2 Concentration-dependent inhibitory effects of propofol (PPF) on lipopolysaccharide (LPS)-enhanced nitrite production. Macrophages were exposed to 25, 50, 75 and 100 lM PPF and a combination of 1 ng/ml LPS and PPF at various concentrations for 24 h. The amounts of nitrite in the culture medium were determined by the Griess reaction method. Data are expressed as means ±SEM for n=12.P<0.05, values for the combination of PPF and LPS significantly different from those with LPS alone

Fig. 4 RT-PCR analysis of inducible nitric oxide synthase (iNOS) from untreated (C) macrophages, and those treated with lipopoly-saccharide (LPS), propofol (PPF), or a combination of PPF and LPS. Macrophages were exposed to 1 ng/ml LPS, 50 lM PPF and a combination of PPF and LPS. Cellular mRNA was prepared for RT-PCR analysis of iNOS. The expression of b-actin mRNA was regarded as an internal standard

Table 2 Eﬀects of propofol (PPF) on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) protein and mRNA in macrophages. Cytosolic proteins and cellular mRNA from macrophages exposed to LPS, PPF or a combination of PPF and LPS were isolated for immunoblotting and RT-PCR analyses, re-spectively. Intensities of the protein and mRNA bands (in arbitrary units) were obtained from densitometric analyses of the protein blot and RT-PCR using a digital imaging system. Each value represents mean ±SEM for n>3 (n.d. not detectable)

Treatment iNOS, (arbitrary units)

Protein mRNA

Control n.d. n.d.

LPS 357086±48612 9478±674

PPF n.d. n.d.

PPF+LPS 91587±6475* 3042±414* *P<0.05, values are considered to be statistically diﬀerent from the LPS-treated group

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which the calcium-independent iNOS protein is involved

in the NO production in LPS-activated macrophages

(Raetz et al. 1991; Nathan 1992; West et al. 1994). PPF,

at a clinically relevant concentration, 50 lM,

signiﬁ-cantly decreased LPS-enhanced cellular NO production

(Figs. 1 and 2). Simultaneously, PPF inhibited

LPS-in-duced protein and mRNA of iNOS (Figs. 3 and 4;

Table 2). Thus, PPF could inhibit the LPS-related

in-duction of iNOS and hence suppress the amounts of NO

in macrophages. From the present data, we suggest that

the mechanism by which PPF is involved in NO

sup-pression is at the pretranslational level.

CD-14 and Toll-like receptors are two membrane

proteins that contribute to the regulation of NO

syn-thesis in macrophages in responses to LPS stimulation

(Kirkland et al. 1993; Schuster and Nelson 2000). The

binding eﬃciency of LPS to CD-14 and Toll-like

receptors plays a critical role in determining the

induc-tive strength with regard to iNOS (Kirkland et al. 1993).

Because PPF is highly lipophilic, it may accumulate in

cellular membrane (Sebel and Lowdon 1989). The

ac-cumulation of PPF might disturb the membrane

integ-rity, aﬀect the conformation of CD-14 and Toll-like

receptors, decrease the binding eﬃciency of LPS to these

membrane proteins, and ﬁnally inhibit iNOS expression.

However, other mechanisms are also possibly involved

in the PPF-induced suppression of NO biosynthesis in

LPS-activated macrophages. For example, our

unpub-lished data reveal that PPF can bind to LPS and form a

complex with a new ﬂorescence spectrum. The binding

between PPF and LPS may interfere with LPS and the

membrane protein interaction, and decrease iNOS

induction.

This study provides another aﬀecting mechanism

about the antioxidant and immunosupressive

charac-teristics of PPF. Structurally, PPF is similar to

a-to-copherol and butylated hydroxytoluene, and has been

indicated as having antioxidant potential (Demiryurek

et al. 1998; Cudic and Ducrocq 2000). Previous studies

revealed that PPF could directly scavenge hydroxyl

chloride, superoxide, hydrogen peroxide and hydroxy

radical, and protect varieties of tissues or cells from

injuries caused by these oxidants (Murphy et al. 1992;

Kokita and Hara 1996; Demiryurek et al. 1998). Our

present study further showed that, in LPS-activated

macrophages, PPF could decrease cellular oxidative

stress via the suppression of NO biosynthesis. NO, just

like hydrogen peroxide, is one of important eﬀectors

produced by macrophages to decompose ingested

microorganisms and tumor cells (Nathan 1992; Albina

et al. 1993). The eﬀects of PPF on descending NO

bio-synthesis may further explain the immunosuppressive

characteristics of this intravenous anesthetic agent. NO

per se can increase cellular stress and contributes to the

pathophysiology of sepsis (Le et al. 1995; Lynn and

Cohen 1995). Therefore, the PPF-caused NO

suppres-sion in LPS-activated macrophages may be helpful to

decrease the oxidative damage of tissues and cells during

sepsis.

In conclusion, the present study demonstrates that

PPF has the ability to decrease NO biosynthesis through

inhibition of iNOS at the levels of protein and mRNA in

LPS-stimulated macrophages, and that its mechanism of

suppression involves a pretranslational event. This

pro-tective eﬀect might beneﬁt the critically ill patients in

clinical situations such as sepsis.

Acknowledgements The authors express their gratitude to Ms Sheau-Lan Tzeng and Ms Wan-Ju Lee for their technical support and data collection of the experiment. This study is supported by grants TMU90-Y05-A123 from Taipei Medical University and NSC90-2314-B-038-045 from the National Science Council, Taiwan, ROC.

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