Reactive Oxygen Species by Isoflurane Mediates Inhibition of Nuclear Factor B Activation in Lipopolysaccharide-Induced Acute Inflammation of the Lung

Chung, In Sun MD*; Kim, Jie Ae MD, PhD; Kim, Ju A. MS; Choi, Hyun Sung MD, PhD; Lee, Jeong Jin MD, PhD; Yang, Mikyung MD, PhD; Ahn, Hyun Joo MD, PhD; Lee, Sang Min MD, PhD

doi: 10.1213/ANE.0b013e31827aec06
Anesthetic Pharmacology

BACKGROUND: Although anesthetic-induced inhibition of lipopolysaccharide (LPS)-induced lung injury has been recognized, the underlying mechanism is obscure. Some studies suggest that reactive oxygen species (ROS) by isoflurane play a crucial role for anesthetic-induced protective effects on the brain or the heart; however, it still remains controversial. In this study, we examined the role of isoflurane-derived ROS in isoflurane-induced inhibition of lung injury and nuclear factor κB (NFκB) activation in LPS-challenged rat lungs.

METHODS: Male Sprague-Dawley rats were subjected to inhalation of 1.0 minimum alveolar concentration of isoflurane for 60 minutes, and intratracheal LPS 0.1 mg was administered 60 minutes later. In some cases, ROS scavenger, 2-mercaptopropinyl glycine or N-acetylcysteine was given 30 minutes before isoflurane. ROS generation was measured by fluorometer before LPS challenge and 4 hours after. Isoflurane’s preconditioning effect was assessed by histologic examination, protein content, neutrophil recruitment, and determination of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 levels in bronchoalveolar lavage fluid and lung tissue. Western blotting measured phosphorylation of inhibitory κB α (ser 32/36), NFκB p65, and inducible nitric oxide synthase (iNOS). TNF-α and IL-6 mRNA expression and immunofluorescence staining for iNOS were also assessed.

RESULTS: Isoflurane preconditioning reduced inflammatory lung injury and TNF-α, IL-1β, and IL-6 release in the lung. Isoflurane upregulated ROS generation before LPS but inhibited a ROS burst after LPS challenge. ROS scavenger administration before isoflurane abolished the isoflurane preconditioning effect as well as isoflurane-induced inhibition of phosphorylation of inhibitory κBα, NFκB p65, iNOS activation, and mRNA expression of TNF-α and IL-6 in acute LPS-challenged lungs.

CONCLUSIONS: This study suggests a crucial role of upregulated ROS generation by isoflurane for modification of inflammatory pathways by isoflurane preconditioning in acute inflammation of the lung.

From the *Department of Anesthesiology and Pain Medicine, Asan Medical Center, University of Ulsan College of Medicine, Songpa-gu, Seoul; Department of Anesthesiology and Pain Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; and Samsung Biomedical Research Institute, Seoul, South Korea.

Accepted for publication October 19, 2012.

Supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 20110005905).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Jie Ae Kim, MD, PhD, Department of Anesthesiology and Pain Medicine, Samsung Medical Center, 50 Ilwon-dong, Kangnam-gu, Seoul 135-710, South Korea. Address e-mail to

Article Outline

Several researchers have noted a protective effect of isoflurane on the endotoxin-induced acute inflammation of the lung in both in vitro and in vivo studies.1–6 However, in contrast to the comprehensive delineation of the signaling pathways of the isoflurane-induced cardiac or brain protection in the ischemia/reperfusion model, there has been limited progress in elucidating the mechanism of the volatile anesthetic-induced preconditioning effect on inflammatory lung injury. Alveolar macrophages and epithelial cells cause a massive release of proinflammatory cytokines and nitric oxide in response to an endotoxin challenge to the lung,5,7 resulting in inflammation and pulmonary edema. Endotoxin-challenged macrophages activate signaling kinases such as phosphatidylinositol-3′-kinase and downstream inhibitory κBα (IκBα) and nuclear factor κB (NFκB), thus inducing expression of nitric oxide synthase (iNOS).8 Isoflurane preconditioning reduces proinflammatory cytokines and nitric oxide release and prolongs survival in rats that inhaled lipopolysaccharide (LPS).5 In LPS-challenged monocytes, isoflurane inhibited NFκB translocation,9 but the pathway used before an endotoxin challenge has not been investigated.

Increasing evidence has suggested a role of reactive oxygen species (ROS) generated by volatile anesthetics for a cardio-protective effect,10–13 and there has been some evidence that ROS act as important mediators in signal transduction pathways by modulating kinases and redox-sensitive proteins in ischemic preconditioning.14 However, details of the interactions between a small amount of ROS by isoflurane and inflammatory pathways remain open to question. We hypothesized that upregulated ROS generation by isoflurane triggers an isoflurane-induced preconditioning effect on LPS-stimulated lung inflammation and modifies endotoxin-derived proinflammatory cytokines and iNOS expression. We evaluated the effects of preisoflurane ROS scavenger administration on the isoflurane-induced preconditioning effect, NFκB activation, and iNOS expression in acute LPS challenge to the rat lung.

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Animals and Experimental Protocols

The animal experiments were reviewed and approved by the Animal Care and Use Committee at Samsung Medical Center. Male Sprague-Dawley rats (270–320 g) were obtained from Orient Bio Inc. (Seongnam, South Korea). They were housed throughout the experiments in laminar flow, temperature-controlled units and maintained on a standard laboratory diet ad libitum. Experimental animals were anesthetized with intraperitoneal thiopental sodium (70 mg·kg−1), and additional bolus doses of thiopental sodium were titrated as required to ensure that the pedal reflex was absent throughout the experiments. The rats were placed supine on a heating blanket to maintain a body temperature of 37°C and were allowed to breathe spontaneously to avoid the risk of ventilator-related lung injury. After stabilization, the rats were randomized to 1 of 5 groups: (1) control; (2) isoflurane + LPS; (3) oxygen free radical scavenger + isoflurane + LPS; (4) oxygen free radical scavenger + LPS; and (5) LPS. The isoflurane groups received a 1.0 minimum alveolar concentration of isoflurane whereas other groups breathed room air for 60 minutes through fitted facial masks. The end-tidal isoflurane concentration was measured continuously with a gas analyzer (Capnomac Ultima; Datex, Helsinki, Finland). Sixty minutes after the discontinuation of isoflurane, 0.1 mg LPS (dissolved in distilled water at a concentration of 5 mg·mL−1; Merck KGaA, Darmstadt, Germany) or saline was injected intratracheally using a 24-gauge angiocatheter. The control and LPS groups were allowed to breathe room air for 60 minutes before saline or LPS administration. In the experiments involving oxygen free radical scavenger, the rats received IV N-acetylcysteine 150 mg·kg−1 (Sigma-Aldrich, St. Louis, MO) for 30 minutes before inhalation of isoflurane. The rats were killed by decapitation with brief anesthesia 4 hours after the LPS injection (Fig. 1).

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Histologic Assessment of Lung Injury

After exsanguination, the lungs were fixed with intratracheal injection of 4% paraformaldehyde, embedded in paraffin, cut in 4-µm sections, and stained with hematoxylin–eosin. Neutrophil and cellular infiltration and septal thickening were evaluated by a pathologist blinded to the treatment condition.

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Bronchoalveolar Lavage Fluid and Phenotyping

A 22-gauge angiocatheter was placed into the trachea, and the lungs were lavaged with 1 mL of cold phosphate-buffered saline (PBS) 5 times. The recovery ratio of the fluid was approximately 85%. Bronchoalveolar lavage (BAL) fluid was centrifuged at 520g for 10 minutes at 4°C. The supernatant was removed and frozen at −80°C for later analyses. BAL fluid protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL). The absorbance was measured at 562 nm, and protein concentration was determined using albumin standard curves. The cell pellet was resuspended in 1 mL of red cell lysis buffer for 5 minutes, and white cells were then repelleted by centrifugation at 520g for 10 minutes at 4°C. The cell pellets were resuspended in 200 µL PBS, and 10-µL aliquots were used for cell counting using a Countess® Automated Cell Counter (Life Technologies Corporation, Grand Island, NY). Trypan Blue dye exclusion was used to assess cell viability. Differential cell counting was performed on Cytospin-prepared slides (Thermo Shandon, Pittsburgh, PA) stained with Diff-Quik (Dade Behring, Newark, DE). After BAL fluid had been obtained, the lungs were perfused free of blood, harvested, frozen in liquid nitrogen, and stored at −80°C for later analysis.

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Measurement of Cytokines from BAL Fluid and Lung Tissue

A commercially available enzyme-linked immunoassay kit was used according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) to measure tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels in BAL fluid supernatants. The snap-frozen lungs were thawed, weighed, and transferred to ice-cold tubes containing tissue protein extraction reagent. The lungs were homogenized at 4°C and centrifuged at 10,000g for 20 minutes. Total protein concentrations in lung tissue homogenates were assayed, and IL-6 levels in lung tissue were measured using an enzyme-linked immunoassay kit (R&D Systems).

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ROS Measurement in the Cells Obtained from the BAL Fluid

Intracellular levels of ROS were measured using the redox-sensitive dye, carboxy-2′,7′-dichlorodihydrofluorescein diacetate, in conjunction with a microplate fluorometer. Viable cells (5 × 104 cells in 200 ìL PBS in a 96-well plate) obtained from BAL fluid were reacted with 15 ìM of carboxy-2′,7′-dichlorodihydrofluorescein diacetate (Invitrogen, Carlsbad, CA) and incubated at 37°C for 30 minutes in the dark. The cells were washed with PBS, and cellular fluorescence was determined using a microplate reader at 490 nm/520 nm (GloMax®-Multi Microplate Multimode Reader, Promega, Madison, WI).

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Quantitative Real-Time Polymerase Chain Reaction in Lung Tissue

The mRNA levels of TNF-α and IL-6 in whole lung tissues were assessed by quantitative real-time polymerase chain reaction (PCR). Total RNA was extracted from lungs with Trizol (Invitrogen) according to the manufacturer’s instructions. Primers and probes were purchased from TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA): TNF-α (assay ID, Rn 99999017_m1); glyceraldehyde 3-phosphate dehydrogenase (GAPDH; assay ID, Rn 99999916_s1); and IL-6 (assay ID, Rn 01410330_m1). Assays were performed with the ABI 7000 Taqman system. Gene expression of each sample was normalized to the GAPDH level. Final levels were expressed as n-fold change relative to the control group.

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Immunofluorescence Staining

Immunofluorescence histochemistry for iNOS was performed on the deparaffinized 4 µm thick lung sections. Heat-induced antigen retrieval was performed on the Target Retrieval Solution (S 1699, Dako, Glostrup, Denmark) at 93°C for 7 minutes. The primary antibodies were incubated overnight at 4°C with 1:50 anti-iNOS rabbit polyclonal antibody (AbCam, Cambridge, MA) diluted in a blocking solution, containing 0.5% (v/v) bovine serum albumin in PBS. The following secondary antibodies were incubated at a 1:200 dilution in the blocking solution at room temperature for 2 hours. Alexa Fluor 488 donkey antirabbit (Molecular Probes, Eugene, OR) was used as secondary antibody. The samples were counterstained with 4′,6-diamidino-2-phenylindole (Vecta shield, Vector Laboratories, Burlingame, CA) for nuclear staining. Fluorescence microscopy was performed at 20× magnification (Eclipse 80i, Nikon Inc., Tokyo, Japan) with 4′,6-diamidino-2-phenylindole/fluorescein isothiocyanate filter, and images were achieved using Nis Elements software (Nikon Inc.).

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Cytosolic and Nuclear Protein Extraction in Lung Tissue Homogenates

Lung parenchyma were mechanically homogenized and lysed in 2 volumes of buffer A containing 10 HEPES pH 7.9, 10 mM potassium chloride, 1.5 mM magnesium chloride, 0.34 M sucrose, 10% glycerol, 0.1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF; Roche, Basel, Switzerland), and protease inhibitor cocktail on ice and centrifuged at 1500g for 10 minutes at 4°C to remove cellular debris. The supernatants were incubated on ice for 10 minutes, transferred to a 1.7 mL ice-cold Eppendorf tube and further centrifuged at 100,000g for 30 minutes at 4°C. The supernatants containing the cytosolic fraction were collected and stored. The pellets were washed twice in buffer A and resuspended in buffer B containing 50 mM Tris-HCl pH 7.8, 420 mM sodium chloride (NaCl), 1 mM EDTA, 0.34 M sucrose, 10% glycerol, 0.5% NP-40, 1 mM PMSF, and protease inhibitor and then pelleted at 1000g for 15 minutes. The pellets were solubilized in a solution containing 50 mM Tris-HCl pH 7.2, 0.3 M sucrose, 150 mM NaCl, 2 mM EDTA, 20% glycerol, 2% Triton X-100, 2 mM PMSF, and protease inhibitor cocktails. The mixtures were kept on ice for 1 hour with gentle stirring and centrifuged at 14,000g for 30 minutes at 4°C. The supernatants were collected as the nuclear fraction. Protein concentrations were determined with the Bradford reagent (Bio-Rad, Hercules, CA) and bovine serum albumin as a standard.

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Western Blotting

Protein samples (25 µg) were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel, separated by electrophoresis at 120 V for 90 minutes, and transferred to a polyvinylidene difluoride membrane at 250 mA for 90 minutes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline Tween 20 (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 hour and then incubated overnight at 4°C with rabbit polyclonal antibody against GAPDH, mouse monoclonal antibody against phosphorylated-IκBα (ser 32/36), or rabbit polyclonal antibody against NFκB p65 and LaminA (Cell Signaling Technology, Inc., Beverly, MA). Rabbit polyclonal anti-iNOS antibody was purchased from AbCam. Antirabbit or antimouse horseradish peroxidase-conjugated goat immunoglobulin G (1:5000; Cell Signaling Technology, Inc.) was used to detect binding of antibodies. Bands were visualized by enhanced chemiluminescence (SuperSignal, Pierce Biotechnology, Rockford, IL) and exposed on an X-ray film. Densitometric analysis gave the relative intensity of each band using Gel-Pro Analyzer (MediaCybernetics Inc., Bethesda, MD).

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We hypothesized that the endotoxin-derived inflammatory response of rat lungs would be altered by ROS generation during isoflurane preconditioning. Our preliminary study showed 50% reduction of TNF-α level in BAL fluid supernatants with isoflurane preconditioning. With a power of 0.8 and a P value of <0.05, a sample size of at least 5 experiments (analysis of variance, 5 groups) was needed to detect a difference in TNF-α level of 130 pg/mL with a standard deviation of 50 pg/mL based on our preliminary data. Normal distributions of the residuals were checked with the Shapiro-Wilk test and homogeneity of variance by the Levene test. Data are expressed as mean (SD) when normal distribution was given (all P > 0.057). Results without normal distribution are expressed with median and interquartile range.

For a comparison of normally distributed variables (BAL fluid cell counts and IL-6 mRNA level of lung tissues), analysis of variance was conducted to examine differences among the groups. If a significant difference among the groups was found, all pairwise comparisons were followed with post hoc Holm-Sidak to isolate the group or groups that differed from the others. The Holm-Sidak method performs stepwise comparisons between all possible pairs. Data without normal distribution were analyzed by Kruskal-Wallis analysis of variance on ranks followed by Tukey test using ranks. This was done with exact P values, considering the small sample size. Statistical analysis was performed with SigmaPlot 9.0 (Systat Software Inc., Richmond, VA). A P value of 0.05 was considered to be statistically significant.

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We found that isoflurane preconditioning inhibited neutrophil infiltration, interstitial edema, and alveolar septal thickening in the LPS-challenged rat lung. This effect was mitigated by administration of ROS scavenger before isoflurane (Fig. 2A).

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Protein Content and Cellular and Neutrophil Recruitments in BAL Fluid

LPS challenge increased the protein content of BAL fluid compared with that of the control, and isoflurane inhibited this increase in protein in the LPS challenge. Administration of ROS scavenger before isoflurane halted the isoflurane-induced inhibition of protein content increase. Isoflurane preconditioning constrained cellular and neutrophil recruitment into BAL fluid upon LPS challenge, and the previous administration of ROS scavenger eliminated this protective effect on LPS challenge (Fig. 2B).

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Cytokine Measurement in BAL Fluid or Lung Homogenates

Proinflammatory cytokines released from alveolar macrophages and epithelial cells are crucial for the recruitment of neutrophils. We measured TNF-α and IL-1β in BAL fluid and IL-6 in lung tissue homogenates. LPS challenge increased levels of TNF-α, IL-1β, and IL-6 compared with the controls except in the isoflurane preconditioning group. ROS scavenger administered before isoflurane halted the isoflurane-induced inhibition of IL-1β and IL-6 release (Fig. 3).

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Measurement of ROS in Cells Obtained from BAL Fluid

To clearly demonstrate that a ROS burst occurs upon LPS challenge, we investigated levels of ROS in isolated BAL cells. LPS challenge resulted in an approximately 5-fold increase in ROS production compared with that in the control, and isoflurane preconditioning clearly attenuated ROS release. ROS scavenger administration before isoflurane eliminated isoflurane-induced inhibition of the ROS burst (Fig. 4A). Isoflurane alone upregulated ROS generation approximately 1.25-fold compared with that of the control (Fig. 4B).

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mRNA Expression in Lung Tissue Homogenates

We used quantitative real-time PCR to explore the effect of isoflurane on the expression of IL-6 and TNF-α at the mRNA level. LPS challenge induced IL-6 and TNF-α mRNA expression in the lung. In addition, isoflurane preconditioning halted LPS-dependent upregulated expression of IL-6 and TNF-α mRNAs. ROS scavenger administration before isoflurane mitigated isoflurane-induced reduction of IL-6 mRNA expression in lung tissue homogenates (Fig. 5A).

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iNOS Staining in Lung Tissue

iNOS staining was stronger in the lung epithelium of the LPS group, although weak staining was also observed in the isoflurane group. ROS scavenger administration before isoflurane attenuated isoflurane-induced inhibition of iNOS staining in the lung (Fig. 5B). This finding was confirmed by the iNOS expression measured with Western analysis (Fig. 6).

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Measurement of Phospho-IκBα (ser 32/36), NFκB p65, and iNOS Expression in Lung Tissue

Nuclear translocation of NFκB and subsequent iNOS expression are important features of LPS-induced inflammatory lung injury and coincide with activation of IκBα. We found that LPS challenge increased expression of phospho-IκBα (ser 32/36), NFκB p65, and iNOS. Isoflurane preconditioning inhibited expression of these proteins, and ROS scavenger administration before isoflurane reversed the isoflurane-induced inhibition (Fig. 6). Isoflurane alone, without LPS challenge, did not affect either phospho-IκBα or NFκB p65 expression compared with controls (data are not shown).

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In this study, we demonstrated an elimination of isoflurane-induced preconditioning effect by ROS scavenger administration before isoflurane in LPS-induced acute lung inflammation, indicating that upregulated ROS generation mediates the isoflurane-induced preconditioning effect on LPS inhalation. This is the first animal study demonstrating a crucial role of upregulated ROS production by isoflurane in the isoflurane-induced protective effect on acute inflammation of the lung.

Our finding of upregulated ROS production by isoflurane is in accord with some previous reports. Isoflurane has complex effects on electron transport channels in mitochondria, altering electron fluxes and enhancing ROS production.11 It has been recently found that isoflurane inhibits electron transport at complex I and provokes ROS generation at complex I and III.12 Acute myocyte exposure to desflurane results in increased ROS generation, coinciding with the anesthetic preconditioning effect in ischemia/reperfusion.15 Sevoflurane-derived ROS generation preceded brain protection against ischemic injury.16 Other reports on the central role of mitochondria-derived ROS in diminishing LPS-induced inflammatory lung injury are interesting. Rotenone (complex I inhibitor)17 or mitochondrial complex III inhibitor18 treatment increased ROS generation in neutrophils and inhibited neutrophil and NFκB activation and severity of LPS-induced lung injury. It is interesting to note that the simultaneous use of rotenone with isoflurane enhanced the inhibitory effect of isoflurane on complex I, resulting in enhanced ROS generation in mitochondria compared with that of isoflurane alone.12 The effect of the combined use of complex I inhibitor and volatile anesthetic on the preconditioning effect should be evaluated. Although the mechanism responsible for the upregulated ROS generation by isoflurane is beyond the scope of this study, our results clearly support an essential role of upregulated ROS by isoflurane in the isoflurane preconditioning effect on LPS-challenged rat lung.

We found that isoflurane preconditioning inhibited the LPS-induced ROS burst and p-IκBα and NFκB activation in the lung. However, isoflurane alone before LPS challenge did not affect IκBα phosphorylation or NFκB activation. This implies that isoflurane-derived ROS affected certain redox-sensitive signaling pathways activated by LPS rather than directly modified redox-sensitive proteins. For instance, Ca2+ influx by LPS activates several protein kinase (PK)Cs, and PKC-β regulates serine phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase, and mitogen-activated/extracellular signal-regulated kinase kinases in turn regulate IκB kinase activation.19 More research is needed to validate how isoflurane-derived ROS affect subsequent inflammatory pathways.

Acute LPS challenge to the lung promotes neutrophil migration from blood into the interstitium and inflammatory cascades. Our study validated the preconditioning effect of isoflurane on in vivo LPS-induced acute lung inflammation. This agrees with previous reports that demonstrated isoflurane-induced attenuation of neutrophil recruitment into the interstitium and alveolar space2 and pulmonary nitrate/nitrite and proinflammatory cytokines release.5 Our study further showed that a ROS burst after LPS challenge was reduced by isoflurane preconditioning. To our knowledge, this is the first demonstration of isoflurane-induced attenuation of a ROS burst in response to LPS inhalation. Considering the biological role of excessive ROS in modifying hyperimmune and inflammatory responses resulting in organ injury, an ability to regulate excessive intracellular ROS is crucial in the acute inflammatory signaling pathway. Numerous reports support an antioxidant defense mechanism in acute inflammation. Antioxidant therapy before endotoxin injection suppressed both NFκB activation in the lung and cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue.20 The ROS scavenging agent amifostine protected the lung against LPS-induced lung vascular leakage and neutrophil recruitment.21

This study demonstrated that isoflurane-derived upregulated ROS generation mediated modulation of NFκB activation and inflammatory lung injury after LPS inhalation; however, there are some limitations. We did not delineate a specific isoflurane-derived ROS-mediated signaling pathway involved in modification of NFκB activation upon LPS challenge. Ongoing investigations in our laboratory are examining the effect of upregulated ROS on the activity of PKCs or toll-like receptor 4 expression upon LPS challenge. In addition, our findings are based on an animal model, limiting the implications for the human lung.

In conclusion, we found that isoflurane preconditioning inhibits a ROS burst, NFκB activation, and proinflammatory cytokines expression in acute LPS-inhaled rat lung, and upregulated ROS generation by isoflurane plays an essential role for the isoflurane-induced preconditioning effect.

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Name: In Sun Chung, MD.

Contribution: This author helped design and conduct the study, analyze the data, and prepare the manuscript.

Attestation: In Sun Chung has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Jie Ae Kim, MD, PhD.

Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.

Attestation: Jie Ae Kim has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Ju A. Kim.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Ju A. Kim has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Hyun Sung Choi, MD, PhD.

Contribution: This author helped analyze the data and prepare the submitted manuscript.

Attestation: Hyun Sung Choi has seen the original study data and approved the final manuscript.

Name: Jeong Jin Lee, MD, PhD.

Contribution: This author helped design the study, analyze the data, and prepare the manuscript.

Attestation: Jeong Jin Lee has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Mikyung Yang, MD, PhD.

Contribution: This author helped collect the data, analyze the data, and prepare the manuscript.

Attestation: Mikyung Yang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Hyun Joo Ahn, MD, PhD.

Contribution: This author helped analyze the data and prepare the manuscript.

Attestation: Hyun Joo Ahn has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Sang Min Lee, MD, PhD.

Contribution: This author helped design the study, analyze the data, and prepare the manuscript.

Attestation: Sang Min Lee has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Marcel E. Durieux, MD, PhD.

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