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ANESTHETIC PHARMACOLOGY

Desflurane Preconditioning Inhibits Endothelial Nuclear Factor--B Activation by Targeting the Proximal End of Tumor Necrosis Factor- Signaling

Yuan Li, MD^*, Xiaonan Zhang, MSc, Biao Zhu, MD^*, and Zhanggang Xue, MD^*

From the *Department of Anesthesiology, Zhongshan Hospital, Fudan University, Shanghai, China; and Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.

Address correspondence and reprint requests to Zhanggang Xue, MD, Department of Anesthesiology, Zhongshan Hospital, Fudan University, Fenglin Road 180, Shanghai 200032, China. Address e-mail to xue.zhanggang{at}zs-hospital.sh.cn.

	Abstract

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
BACKGROUND: Volatile anesthetics interfere with inflammatory cytokine production and expression of adhesion molecules which are critical for ischemia reperfusion induced injury. Nuclear factor (NF)-B has been reported to be suppressed in this process, but the detailed molecular mechanism is still unclear.

METHODS: In this study, ECV304 (a human umbilical vein endothelial cell line) was preconditioned with 30 min desflurane (1 minimal alveolar concentration), after 15 min washout, 30 min anoxia, and 60 min reoxygenation was performed. ECV304 was finally stimulated with tumor necrosis factor (TNF)- (10 ng/mL). Control groups, which were not preconditioned and/or not stimulated, were also included in the protocol. IB-, phospho-IB-, phospho-IB kinase (IKK)/IKKβ, and phopho-p38 were detected by Western blotting. The nuclear NF-B p65 subunit was measured by subcellular fractionation and Western blotting. The surface expression of TNF-R1 was measured by flow cytometry. Receptor-associated signaling adaptors, e.g., TNF receptor-associated factor 2 (TRAF2) and IKK-, were evaluated by immunoprecipitation by TNF-R1 antibody and subsequent Western blotting.

RESULTS: Desflurane preconditioning inhibits IB- phosphorylation, degradation, and p65 nuclear localization. Desflurane also affects p38 phosphorylation, which is needed for optimal inflammatory response. The phosphorylation of IKK/IKKβ was suppressed by preconditioning while the surface abundance of TNF-R1 was not affected. The association of TRAF2 and IKK- with TNF-R1 was compromised by desflurane.

CONCLUSIONS: Our results suggest that the molecular target of desflurane in the NF-B pathway is upstream of IKK activation. The abundance of TNF-R1 on the cell membrane is not affected by anesthetic preconditioning. We suggest that desflurane preconditioning targets the proximal end of TNF- signaling.

	Introduction

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
Volatile anesthetic preconditioning, especially with desflurane, halothane, isoflurane, and sevoflurane, can protect against ischemia-reperfusion (IR) and anoxia-reoxygenation (A/R) injury both in vivo and in vitro.1–5 Extensive analyses have indicated that secretion of inflammatory cytokines are involved in IR-induced damage.6–8 Nuclear factor NF-B is a pivotal transcription factor in oxidative stress and inflammatory response and is activated during IR.6 Activation of NF-B induces various genes, of which cytokines (e.g., tumor necrosis factor [TNF]-, interleukin [IL]-6), chemokines, and adhesion molecules are implemented in IR injury.9 Moreover, NF-B is also a central transcription factor in TNF- or endotoxin-mediated responses.10 Indeed, in an animal model of acute myocardial IR injury, attenuation of NF-B activation and subsequent down-regulation of NF-B-dependent inflammatory cytokines have been reported by volatile anesthetic preconditioning (APC).6,11

According to the present understanding (Fig. 1), TNF- initiates a cellular response by binding to the extracellular domain of TNF-R trimer, which leads to the recruitment of the TNF-R1-associated death domain protein and subsequent recruitment of FAS-associated death domain protein, TNF receptor-associated factor 2 (TRAF2), and receptor-interacting protein kinase. Binding of TRAF2 and RIP to TNF-R1-associated death domain activates the IB kinase (IKK) complex. The IKK complex is composed of two catalytic subunits, IKK and IKKβ, and an essential regulatory subunit, IKK. This complex phosphorylates IB and targets it for ubiquitin-proteasome degradation,12,13 this directly triggers the release and nuclear localization of NF-B and subsequent transactivation programs.

View larger version (18K): [in this window] [in a new window]   Figure 1. Schematic illustration of TNF--induced NF-B signaling and a proposed mechanism for desflurane-mediated suppression of NF-B activation.

Interestingly, our previous work has shown that desflurane could also significantly inhibit the second phase of IR injury, i.e., TNF- induced adhesion molecule (intercellular adhesion molecule-1, ICAM-1, vascular cell adhesion molecule-1, VCAM-1, and E-selectin) expression and subsequent neutrophil attachment on human endothelial cells.14 These results suggest that APC can dampen the inflammatory response by inhibiting neutrophil recruitment through vascular endothelium to inflamed tissues. The present study aimed to elucidate whether desflurane could do so by interfering with NF-B activation and explore the possible molecular mechanism of this phenomenon.

	METHODS

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No human or animal subjects were used for this study. Desflurane was purchased from Baxter (USA). ECV304 cells (obtained from Liver Cancer Institute, Zhongshan Hospital, Fudan University) were maintained in RPMI 1640 medium (Gibco BRL) with 10% fetal calf serum (PAA Austria), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Recombinant TNF- was from Peprotech (Rocky Hill, USA). Antibody to p65 was from Active Motif (USA). TNF-R1, TRAF2, IKK, pERK antibodies, and peroxidase-conjugated secondary antibodies were from Santa Cruz (USA). pIB- and phspho-p38 antibodies were from Cell signaling (USA). Antibody to β-actin and -tubulin were from Sigma(USA). Poly-ADP ribose polymerase (PARP) antibody was from BD Biosciences (USA). Flow cytometric antibody to TNF receptor 1 (TNFR1) and PE-conjugated rat anti-mouse IgG were from BD pharmingen (USA). The chemiluminescence reagent was from Perking Elmer (USA).

ECV304 cells, seeded in 12 well or 6 well plates, were divided into five groups and treated as shown in Figure 2. The MAC value used in this study was 7.0% for desflurane. ECV304 was placed in a standard airtight incubator (BB 16; Heraeus, Hanau, Germany) providing a gas mixture of 5% CO₂ and 95% oxygen and exposed to 1.0 MAC desflurane for 30 min in the same incubator and desflurane was delivered with a standard anesthetic machine (Primus; Dräger, Lueck, Germany), concentrations of all gases at outlet were continuously monitored with a multigas analyzer (Datex, Helsinki, Finland). IR was simulated by replacing 95% O₂ to 5% CO₂ with 95% N₂ to 5% CO₂ in the buffer for 30 min, followed by a 60-min oxygenated recovery period. TNF- (10 ng/mL) was added into the medium in group three and five after A/R.

View larger version (12K): [in this window] [in a new window]   Figure 2. Experimental protocol. ECV304 cells were treated as described in Materials and methods. 30min of desflurane (1.0 MAC) exposure and 15 min washout was applied before anoxia/reoxygenation (A/R) in group four and five. TNF- was added into the medium after A/R for indicated time in various experiments in group three and five.

Cytoplasmic and nuclear fractions were obtained as described by Rahmouni et al.15 A total of 5 x 10⁶ cells were resuspended in ice-cold hypotonic buffer (42 mM KCl, 10 mM HEPES [pH 7.4], 5 mM MgCl₂, 1 mM Na₃VO₄, and EDTA-free protease inhibitor cocktail) and incubated on ice for 15 min. Cells were then sheared by five passes through a 30-gauge needle. The lysates were centrifuged at 500g for 10 min. The supernatant (cytosol) was collected, and the pellet was washed three times in hypotonic buffer and then resuspended in 20 mM HEPES-KOH (pH 7.9), 20 mM NaCl, 10 mM NaF, 0.2% Triton X-100, 1 mM EDTA, 25% glycerol, EDTA-free protease inhibitor cocktail. Lysates were then vortexed, incubated on ice for 15 min, and centrifuged at 13,000g for 15 min, and the supernatant containing the nuclear proteins was collected. Extracts were diluted in Laemmli buffer and analyzed by Western blotting using anti-PARP, anti--tubulin, and anti-p65 antibodies.

IB-, pIB-, and phosphos-p38 were measured by Western blotting by specific antibodies. After various treatments, ECV304 cells were washed with prechilled phosphate-buffered saline and lysed with Laemmli buffer containing protease inhibitor cocktail (Roche)' sodium vanadate (1 mM) and NaF (10 mM).

TNF-R1 staining was performed essentially as described in the Techniques for immune function analysis Application Handbook (BD Biosciences). Briefly, ECV304 cells were initially digested by 0.05% trypsin; after washing, cells were resuspended in staining buffer and TNF-R1 antibody was added at 4°C for 30 min, followed by washes and PE-conjugated rat anti-mouse secondary antibody staining. Finally, the cells were fixed with 3% formaldehyde in phosphate-buffered saline. The BD FACS Calibur system was used to detect the staining pattern.

Immunoprecipitation was performed as previously described.16 Immunoprecipated protein complex or ECV304 cells were extracted by using SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, and 0.1% w/v bromophenol blue). Equal amounts of protein extracts were loaded on a 7.5 or 10% SDS-PAGE gel. After electrophoresis, protein was transferred onto a 0.2 µm nitrocellulose membrane in transfer buffer. The membranes were incubated with various antibodies, followed by the addition of horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG. Proteins were visualized by an ECL Western blotting system.

The data were analyzed by SPSS 11.5. The quantitative value is expressed as mean ± sd. The one-way ANOVA test was used to compare mean values across multiple treatment groups and Student–Newman–Keuls is used in multiple comparisons.

	RESULTS

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Typically, the hallmark of NF-B activation is the nuclear translocation of p65 and p50 heterodimers. We evaluated the amount of p65 in the nucleus by subcellular fractionation. The quality of the nuclear exact was confirmed by Western blotting of the cytosolic and nuclear markers, PARP and -tubulin (Fig. 3A). A/R markedly induced the amount of nuclear p65 (Fig. 3B, lane 2). Furthermore, much more p65 was observed in the A/R and TNF- group (>3-fold, Fig. 3B). Notably, desflurane preconditioning reduced 88.6% of nuclear p65 compared with Group 3 (Fig. 3B). As, IB phosphorylation and proteasomal degradation is the prerequisite for NF-B release into the nucleus, we also examined the status of IB by specific antibodies. Indeed, A/R and TNF- treatment resulted in IB phosporylation and its degradation, whereas desflurane preconditioning significantly blocked these posttranslational modifications (86.1% decrease in IB phosphorylation, 4.72 fold in IB level compared with Group 3, Fig. 3C). Thus, we conclude that desflurane preconditioning markedly inhibits IB phosphorylation/ degradation and subsequent NF-B activation.

View larger version (20K): [in this window] [in a new window]   Figure 3. Desflurane preconditioning inhibits NF-B activation. ECV304 cells were treated as described in Figure 2, TNF- was treated for 1 h, and nuclear and cytoplasmic fractionation was performed. The quality of the extracts was assessed by Western blotting with PARP and -tubulin (A). The amount of p65 in the nuclear fraction was further measured (B). Densitometric and statistical analysis were performed. *P < 0.001 versus A/R group. , P < 0.001 versus A/R TNF- group. (C) The phosphorylation and degradation of I-B was further evaluated after 1 h TNF- treatment. Densitometric and statistical analysis were performed. *0.001 versus pI-B A/R group. **0.001 versus pI-B A/R TNF- group. , P < 0.001 versus I-B A/R group. , P < 0.001 versus I-B A/R TNF- group.

Apart from the NF-B translocation, other factors like mitogen-activated protein kinases (MAPKs, e.g., p38) have also been documented to regulate the gene expression profile induced by TNF-.17–21 Of note, p38 is critical for up-regulation of ICAM-1 in human umbilical vein endothelial cells (HUVEC) after TNF- or lipopoly saccharide stimulation.22 We evaluated whether desflurane preconditioning could alter the activation status of p38. The phosphor-specific antibody to p38 detected a marked activation in TNF- treated cells, whereas A/R alone had no effect. Indeed, desflurane preconditioning could effectively suppress p38 activity (25.4% decrease compared with Group 3, Fig. 4, P < 0.001).

View larger version (19K): [in this window] [in a new window]   Figure 4. Effects of desflurane on TNF- induced p38 activity. The activity of p38 was measured by phosphor specific antibody after 0.5 h TNF- treatment. Densitometric and statistical analyses were performed. *P < 0.001 versus A/R TNF- group.

To further study the mechanism by which desflurane acts on NF-B signaling, we examined whether the surface expression of the TNFR itself was altered. TNF signals through two distinct cell surface receptors, TNF-R1 and TNF-R212 in which TNF-R1 initiates the majority of biological activities. In our FACS experiments, TNF-R2 expression was undetectable in ECV304 cells (data not shown) whereas a low level of TNF-R1 was detected at the basal level. A/R did not significantly alter receptor expression, but TNF- administration caused down-regulation of TNF-R1, which was consistent with published results(Figure 5A).23 Desflurane conditioning before A/R did not change the abundance of TNF-R1 (Figure 5 middle) compared with A/R alone; this is also the case for A/R, followed by TNF- treatment (Fig. 5A lower). Taken together, these results indicate that desflurane does not inhibit NF-B by influencing TNF-R1 expression on the cell surface.

View larger version (10K): [in this window] [in a new window]   Figure 5. Desflurane does not affect TNF-R1 expression on the cell surface. (A) ECV304 cells were treated and TNF-R1 expression was quantified by FACS analysis. Histograms of TNF-R1 on the cell surface were shown. The various treatment groups are indicated on the graph (B). The percentage of positive cells after various treatments was plotted.

To further explore the molecular target of desflurane, we examined the phosphorylation status of IB kinase /β, which are upstream of IB and induce its subsequent degradation. IKK/β serine phosphorylation was readily detected when ECV304 cells were treated with TNF- (Fig. 6A) after A/R. However, desflurane preconditioning markedly reduced the IKK/β activation, despite the presence of TNF- (79.94% decrease compared with Group 3, Fig. 6A). These results indicate that desflurane exerts its activity upstream of IKK/β.

View larger version (14K): [in this window] [in a new window]   Figure 6. Effects of desflurane on molecules upstream of IB-. (A)The activity of IKK/β was measured by its phosphor specific antibody. Densitometric and statistical analyses were performed. *P < 0.001 versus A/R TNF- group. (B) ECV304 cells were treated as before, the receptor associated signaling proteins were assessed by immunoprecipitation by TNF-R1 antibody and subsequent immunoblot of IKK- and TRAF-2. *P < 0.001 versus IKK- A/R TNF- group. , P < 0.001 versus TRAF-2 A/R group.

The NF-B pathway activated by TNF- is initiated by receptor trimerization upon ligand binding and recruitment of cytoplasmic adaptors for downstream signaling.12 As desflurane does not inhibit TNF-R1 surface expression and acts upstream of IKK, we reasoned that the receptor-associated signaling complex could be targeted. Indeed, it was observed that desflurane preconditioning greatly compromised the amount TNF-R1 associated IKK- (93.2% decrease) and TRAF2 (92.7% decrease) compared with Group 3 (Fig. 6B).

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
IR triggers a series of events, including production of reactive oxygen species, altered ion distribution, induction of apoptosis and an inflammatory response in various organs.24,25 Inflammatory responses play an important role in the pathophysiology of IR injury. Notably, the cytokine release (TNF-, IL-1, and IL-8) and induction of adhesion molecules (ICAM-1 and VCAM-1) are initiated and regulated by NF-B,26–28 which leads to adhesion and accumulation of neutrophils and finally massive tissue injury.24,29 Volatile APC inhibits myocardial NF-B activation, which results in limited secretion of inflammatory cytokines and expression of adhesion molecules in a rat model.6 Furthermore, a smaller increase in TNF- and shorter stay in the intensive care unit after coronary artery bypass surgery were also documented with sevoflurane preconditioning compared with midazolam-sufentanil anesthesia.8

The above observations reveal the importance of the NF-B pathway in the initial phase of the inflammatory response. Based on our previous findings, which suggest that desflurane could also inhibit the second phase of the inflammatory response, we used ECV304 to study the molecular mechanism by which desflurane interferes with the cellular transcriptional response initiated by TNF-. ECV304 is a spontaneous transformed HUVEC line30 suitable for study of the endothelial inflammatory response.31,32 Rescue of A/R and inflammatory cytokine-induced apoptosis were confirmed in our preliminary results (unpublished results). In addition, exogenous TNF- was administrated to simulate the secondary inflammation in vitro which obviated the complex cross-talks and feedback mechanisms in animal models.

In this study, we proved that desflurane preconditioning could also limit NF-B activation induced by TNF-. We suggest that desflurane may act on the proximal end of TNF- signaling (Fig. 1) but not the abundance of TNF-R1 itself based on these observations: first, desflurane preconditioning can inhibit NF-B nuclear translocation, IB phosphorylation and degradation; second, desflurane also quenched IKK activation; third, the TNFR-associated adaptors upon stimulation are greatly affected by desflurane; fourth, the expression of TNFR1 on the cell surface was not affected by desflurane regardless of TNF- stimulation.

Apart from this mainstream cascade, it should be noted that NF-B alone is not sufficient for optimal gene induction upon inflammatory cytokines. For example, selective inhibition of p38 MAPK dose-dependently reduces TNF- induced ICAM-1 expression in cultured HUVEC cells.22 Blocking of p38 signaling in bone marrow stromal cells partially inhibited IL-1 and TNF- induced receptor activator for nuclear factor B expression and osteoclastogenesis in vitro.20 In another study, TNF- induced matrix metalloproteinase-9 mRNA and protein expression was down-regulated by p38 MAPK inhibitors. Furthermore, inhibition of p38 also increased IB- level, which correlates with decreased NF-B activation.18 These reports suggest that the p38 pathway is a pivotal promoter for TNF- induced transcription program. In our study, we also observed a marked reduction of p38 activity when cells were preconditioned by desflurane, which could also account for the reduced adhesion molecule expression in endothelial cells (Fig. 1).

The volatile anesthetics have been used for a long time, but the molecular pharmacology of these drugs is still not well defined. Based on the current theories, their small surface area, hydrophobicity, and generally unreactive nature predict the weak and nonstringent binding to multiple sites, including ion channels, membrane receptors, and intracellular enzyme systems, which possess hydrophobic cavities.33 Indeed, it is estimated that about 15% of the high-resolution protein structures in the protein data bank have internal cavities large enough to accommodate typical inhaled anesthetics.34 But whether these targets contribute functionally should be further tested. In our study, the data imply that desflurane could target the proximal end of TNF- signaling. Although TNF-R1 expression is not affected by APC, we could not exclude the possibility that receptor trimerization was suppressed. Further investigation is under way to test this hypothesis.

Taken together, our results show that desflurane effectively inhibits TNF- induced NF-B and p38 activity, which is critical for the induction of adhesion molecules and inflammatory cytokines. Detailed analysis of pathway components suggests that desflurane acts upstream of IKK activation. We conclude that desflurane may affect cytoplasmic membrane bound signaling complex, which is recruited and activated in the early events of the TNF- signaling cascade.

	Footnotes

 
Accepted for publication December 28, 2007.

Supported by the Foundation of Scientific Research for Youth of Fudan University, 2006.

The first two authors contributed equally to this work.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Li, Y. Articles by Xue, Z. Search for Related Content PubMed PubMed Citation Articles by Li, Y. Articles by Xue, Z. Related Collections Mechanisms Preclinical Pharmacology Pharmacology