Inhibitor of nuclear factor-kB, SN50, attenuates lipopolysaccharide-induced lung injury in an isolated and perfused rat lung model CHIH-FENG CHIAN, CHI-HUEI CHIANG, CHIAO-HUI CHUANG, and SHIOU-LING LIU TAIPEI, TAIWAN
NF-kB cell permeable inhibitory peptide (SN50) inhibits translocation of nuclear factor-kB (NF-kB) and production of inflammatory cytokines that are implicated in lipopolysaccharide (LPS)-induced lung injury (LPSLI). However, the protective effect of SN50 in LPSLI is unclear. We explored the cellular and molecular mechanisms of SN50 treatment in LPSLI. LPSLI was induced by intratracheal instillation of 10 mg/kg LPS using an isolated and perfused rat lung model. SN50 was administered in the perfusate 15 minutes before LPS was administered. Hemodynamics, lung histologic change, inflammatory responses, and activation of apoptotic pathways were evaluated. After LPSLI, increased pulmonary vascular permeability and lung weight gain was observed. The levels of interleukin (IL)-1b, tumor necrosis factor (TNF)-a, myeloperoxidase, and macrophage inflammatory protein-2 increased in bronchoalveolar lavage fluids. Lung-tissue expression of TNF-a, IL1b, mitogen-activated protein kinases (MAPKs), caspase-3, p-AKT (serine-threonine kinase, also known as protein kinase B), and plasminogen activator inhibitor-1 (PAI-1) was greater in the LPS group compared with controls. Upregulation and activation of NF-kB was associated with increased lung injury in LPSLI. SN50 attenuated the inflammatory responses, including expression of IL-1b, TNFa, myeloperoxidase, MAPKs, PAI-1, and NF-kB; downregulation of apoptosis indicated by caspase-3 and p-AKT expression was also observed. In addition, SN50 mitigated the increase in the lung weight, pulmonary vascular permeability, and lung injury. In conclusion, LPSLI is associated with inflammatory responses, apoptosis, and coagulation. NF-kB is an important therapeutic target in the treatment of LPSLI. SN50 inhibits translocation of NF-kB and attenuates LPSLI. (Translational Research 2014;163:211–220) Abbreviations: ALI ¼ acute lung injury; DWt/Dt ¼ rate of weight change; ERK ¼ extracellular signal-regulated kinase; IL ¼ interleukin; Kfc ¼ pulmonary capillary filtration coefficient; LPS ¼ lipopolysaccharide; LPSLI ¼ lipopolysaccharide-induced lung injury; MAPKs ¼ mitogen-activated protein kinases; MIP ¼ macrophage inflammatory protein; MPO ¼ myeloperoxidase; NF-kB ¼ nuclear factor-kB; PAI-1 ¼ plasminogen activator inhibitor type 1; Ppa ¼ pulmonary artery pressure; Ppc ¼ pulmonary capillary pressure; Ppv ¼ pulmonary venous pressure; Ra ¼ pulmonary arterial resistance; Rv ¼ pulmonary venous resistance; SN50 ¼ NF-kB cell permeable inhibitory peptide; TLR4 ¼ Toll-like receptor 4; TNF ¼ tumor necrosis factor
From the Division of Pulmonary and Critical Care Medicine, Internal Medicine Department, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Division of Pulmonary Immunology and Infectious Diseases, Chest Department, Taipei Veterans General Hospital, Taipei, Taiwan; Institute of Emergency and Critical Care Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. Submitted for publication June 14, 2013; revision submitted October 3, 2013; accepted for publication October 7, 2013.
Reprint requests: Chi-Huei Chiang, MD, Division of Pulmonary Immunology and Infectious Diseases, Chest Department, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei, Taiwan; e-mail: [email protected] or [email protected]. tw. 1931-5244/$ - see front matter Ó 2014 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2013.10.002
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AT A GLANCE COMMENTARY Chian C-F, et al. Background
Pulmonary infection by bacteria or virus is the leading cause of acute lung injury and acute respiratory distress syndrome, which results in patient death. Nuclear factor-kappaB (NF-kB) appears to be a key in the regulation of the inflammatory process during sepsis. The NF-kB cell permeable inhibitory peptide (SN50) inhibits translocation of NF-kB in 30 to 60 minutes. However, the role of SN50 in the treatment of septic lung injury remains unknown. Translational Significance
Our results demonstrated SN50 attenuated the inflammatory response, reduced cytokines release, and mitigated hypercoagulation in septic lung injury. Specific agents targeting NF-kB may offer novel treatment options for this group of patients.
Sepsis is a significant public health problem and is the tenth leading cause of death in the United States.1 Acute lung injury (ALI) and acute respiratory distress syndrome commonly complicate sepsis and often result in the prolonged use of mechanical ventilator support and a mortality of approximately 30%–50%.2 ALI caused by sepsis is associated with a severe systemic inflammatory response to bacterial infection characterized by the accumulation of inflammatory cells and platelets, disruption of vascular endothelial and epithelial barriers, and increased permeability of the alveolus.3 Toll-like receptor 4 recognizes gram negative bacteria and bacterial components (lipopolysaccharide) and thereby activates an innate immune response.4 Understanding the complex mechanisms responsible for the pathogenesis of ALI will provide insight toward developing novel treatments in the future.5 Nuclear factor-kappa B (NF-kB) is a ubiquitous transcriptional factor that plays a pivotal role in immune and inflammatory responses.6 NF-kB regulates the transcription of various genes that encode inflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, (IL-6), chemokines, plasminogen-activator inhibitor type 1 (PAI-1), adhesion molecules, and inducible nitric oxide synthase.7-9 NF-kB conjugated with IkB proteins are sequestered in the cytosol in an inactive form. Upon activation, degradation of IkB unmasks the nuclear localization sequence of NF-kB. The nuclear
localization sequence allows the NF-kB dimer to translocate into the nucleus. NF-kB binds to the promoter regions of NF-kB regulated genes resulting in genes transcription. Previous transfection studies have shown that masking the nuclear localization sequence of the NF-kB dimer prevents its translocation to the nucleus.10,11 The pathogenesis of sepsis appears to involve the activation of NF-kB, a critical pathway of septic lung injury. As such, NF-kB would be an important therapeutic target in the treatment of septic lung injury. NF-kB cell permeable inhibitory peptide (SN50) was first synthesized by Lin et al and was comprised of the hydrophilic region of the signal peptide of Kaposi fibroblast growth factor as a membrane translocating motif and a nuclear localization sequence derived from the p50 subunit of NF-kB.12 SN50 has been used as an inhibitor of NF-kB translocation. SN50 has been shown to attenuate GATA-3 expression and IL 5 and IL 13 production in developing TH2 cells.13 Liu et al has demonstrated SN50 reduced levels of TNF-a and PAI-1 in bronchoalveolar lavage fluid and inhibited NF-kB activation and deoxyribonucleic acid binding in a mouse model of augmented hyperoxia and ventilator-induced lung injury14; however, the mechanism and protective effect associated with the introduction of SN50 in lipopolysaccharideinduced lung injury (LPSLI). LPSLI remains unclear. We currently hypothesize that SN50 attenuates septic lung injury by inhibiting translocation of NF-kB, resulting in the downregulation of inflammation and apoptosis and coagulation in LPSLI. METHODS Animal preparation. The study was performed in accordance with animal ethics guidelines of the National Institutes of Health. The study protocol was approved by the Institutional Review Board of the Taipei Veterans General Hospital for animal care and use. The in situ isolated-perfused lung model has been previously described.15 Briefly, male Sprague-Dawley rats weighing 250–350 g were anesthetized with intraperitoneal injection of sodium pentobarbital. A tracheotomy was performed and mechanical ventilation was applied (Rodent ventilator Model 683; Harvard Apparatus, South Natick, Mass) at a tidal volume of 6 mL/kg and a positive end-expiratory pressure of 2 cm H2O. After a sternotomy, heparin (1 unit/g) was injected into the right ventricle. The right ventricle was also used to catheterize the pulmonary artery, which had a tight ligature placed around its main trunk. Another catheter was inserted into the left atrium through the left ventricle and mitral valve and fixed by a ligature at the
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apex of the heart. Pulmonary venous outflow was diverted into a reservoir. To prevent backflow into the ventricles, an additional ligation was performed above the atrioventricular junction. The lungs were perfused with 10 cc blood mix with 30 cc 0.9% normal saline (Minipulse 2; Gilson Medical Electronic, Middleton, Wisc) at a constant flow (30 mL.min21.g21 body weight). Pulmonary artery pressure (Ppa), pulmonary venous pressure (Ppv), circulating perfusate pH, and peak airway pressure were monitored. The weight of each rat was reflective of lung weight in the in situ system and was recorded continuously as the lung weight gain. Pulmonary arterial resistance (Ra) and venous resistance (Rv) were calculated using the following equations: Ra 5 (Ppa - Ppc)/Q, and Rv 5 (Ppc - Ppv)/Q, where Q is perfusate flow. Determination of (Ppc). The Ppc was
pulmonary
capillary
pressure
estimated by using the doubleocclusion method.16 Briefly, arterial inflow and venous outflow lines were occluded simultaneously, and the equilibrium Ppa and Ppv were measured. Measurement of microvascular permeability. The pulmonary capillary filtration coefficient (Kfc) was used as an index of microvascular permeability to water. The method for Kfc measurement has been previously described.16 Briefly, after an isogravimetric period, Ppv was rapidly elevated to 6–8 cm H2O for 15 minutes. The increase in lung weight was recorded, and a characteristic rapid weight gain (vascular filling) was followed by a slower rate of weight gain. The rate of weight change (DWt/Dt) during the 6- to 14-minute interval was analyzed using linear regression of the log10-transformed rates of weight changes per minute. The initial rate of weight gain was calculated using an extrapolation of DWt/Dt to time 0. Kfc was calculated by dividing DWt/Dt at time 0 by the changes in Ppc that occurred after venous outflow pressure was increased. The resulting value was then normalized using the baseline wet-lung weight, and was expressed as milliliters per minute per centimeter H2O per 100 g of lung tissue. Experimental protocols. Isolated lung preparations were separated into 3 experimental groups: (1) sham (control), (2) intratracheal LPS (LPS), and (3) intratracheal LPS plus SN50 (LPS 1 SN50) and ventilated with tidal volume settings at 6 mL/kg. Sham controls were ventilated with a tidal volume setting at 6 mL/kg and neither exposed to LPS nor SN50. The protocol for LPS-induced injury was performed as follows: 10 mg/kg LPS was administered intratracheally to the LPS-induced lung injury group. SN50 (Enzo Life Sciences, Farmingdale, NY) was administered in the perfusate at 15 minutes before LPS initiation. SN50
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was administered in total volume 40 cc circulating perfusate (30 cc normal saline 110 cc blood) with a final concentration of 1 mM. Hemodynamics were continuously monitored for 6 hours in all groups. Vascular permeability was measured using the pulmonary capillary filtration coefficient (Kfc) as previously described.17 Measurement of white cell count in bronchoalveolar lavage fluid. All experiments were terminated after
6 hours of closed extracorporeal perfusion, and the lungs were removed and wet weights were measured. The lungs were lavaged twice with saline (2.5 mL/ lavage). Lavage samples were centrifuged at 1500 g at room temperature for 10 minutes. The white cell count was determined as previously described.15,18 Myeloperoxidase assay. The concentration of myeloperoxidase (MPO), an index of neutrophil sequestration in the lungs, was measured as previously described in right middle lung tissue.18 Cytokines assays. IL-1b, TNF-a, and macrophage inflammatory protein (MIP)-2 were measured in lavage fluids using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Oxon, UK). The absorbance was read at 450 nm (SpectraMax M5; Molecular Devices). Western blotting analysis. Lung tissues were homogenized using lysis buffer containing protease inhibitor cocktail (Roche, Pleasanton, CA) and phosphatase inhibitor cocktail (Roche). The total protein concentration in the extract was determined with a bicinchoninic acid protein assay (Pierce, Rockford, Ill): 80 mg protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel, and electro-transferred onto polyvinylidene fluoride membrane (Millipore, Billerica, Mass). The membrane was blocked with 5% nonfat dry milk in trisbuffered saline (TBS) containing: 0.1% Tween 20 for 1 hour. Antibodies against phospho-p44/42 mitogenactivated protein kinase (MAPK) (extracellular signalregulated kinase [ERK] 1/2), phospho-stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), phospho-p38 MAPK, anti-p44/42 MAPK (ERK1/2), anti- SAPK/JNK, and anti-p38 MAPK [1:1,000; Cell Signaling Technology, Beverly, Mass] were used. Antibodies against GADPH (1:10,000; Lab Frontier, Abfrontier, Seoul, Korea), c-Jun N-terminal kinase 1 (JNK1) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3 (1:2000; Cell Signaling Technology, p-AKT (1:1000; Cell Signaling Technology), AKT (1:1000; Cell Signaling Technology), PAI-1 (1:1,000; Cell Signaling Technology) were used. The appropriate secondary antibodies (1:10,000 horseradish peroxidase anti-rabbit [Jackson Immuno Research Laboratories, West Grove, Pa]) were also used. Visualization was performed by enhanced chemiluminescence (Visual
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Table I. Hemodynamics Group
Baseline Control LPS LPS 1 SN50 After 6 hours Control LPS LPS 1 SN50
N
PPa
PPv
PPc
Ra
Rv
7 7 7
8.4 6 3.6 8.2 6 1.4 8.4 6 1.1
4.7 6 0.4 3.0 6 0 3.3 6 0.3
6.3 6 1.7 5.3 6 0.6 5.5 6 0.5
0.04 6 0.04 0.06 6 0.02 0.06 6 0.01
0.03 6 0.03 0.05 6 0.01 0.05 6 0.01
7 7 7
9.9 6 3.9 7.1 6 1.4 8.7 6 1.2
4.6 6 0.4 3.0 6 0 3.3 6 0.3
6.9 6 1.9 4.8 6 0.3 5.7 6 0.5
0.06 6 0.04 0.05 6 0.01 0.06 6 0.02
0.05 6 0.03 0.04 6 0.01 0.05 6 0.01
Abbreviations: LPS, lipopolysaccharides; N, number; Ppa (mm Hg), pulmonary arterial pressure; Ppv (mm Hg), pulmonary venous pressure; Ppc (mm Hg), pulmonary capillary pressure; Ra (cm Hg$min21 mL21), pulmonary arterial resistance; Rv (cm Hg$min21 mL21), pulmonary venous resistance; SN50, NF-kB cell permeable inhibitory peptide. Values are mean 6 SD.
Protein Biotechnology Crop, New Taipei City, Taiwan). The protein bands were quantified using Kodak 1D Image Analysis (Eastman Kodak Company, Rochester, NY). Lung histopathology. After the termination of each experiment, the right lower lobe of each lung was dissected and fixed immediately in 10% neutral buffered formalin. After fixation, the lung tissue was dehydrated using a graded series of alcohol, cleared in xylene, and embedded in paraffin. All sections were cut to 5 mm and stained with hematoxylin/eosin. Immunohistochemistry. Lung slides coated with polyL-lysine (Sigma, St. Louis, MO) were deparaffinized and rehydrated using xylene and ethanol, and placed in 3% H2O2 for 15 minutes. The slides were incubated with 1:60 dilution of monoclonal nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (Cell Signaling Technology), incubated at 4 C overnight, and stained with diaminobenzidine (Dako, Carpinteria, CA) and Mayer’s hematoxylin (Dako). Analysis was performed under Eclipse 80i microscope (Nikon, Tokyo, Japan) using Image Pro Plus 5.0 (Media Cybernetics, Rockville, MD). Cells with positive nuclear NF-kB staining were counted out of a total of 100 cells in each slide from 7 animals in each group of control, LPS or LPS 1 SN50, respectively. Cell counting was performed by a pathologist who was blinded to the experimental conditions. Statistical analysis. Systat10.0 (Systat Software Inc, San Jose, CA) was used for statistical analyses. Comparisons among all groups were conducted using an analysis of variance followed by Dunnett’s post-hoc analysis. Comparison between baseline and post-I/R values within group was conducted using Student paired t-test. Values are expressed as mean 6 standard deviation. P , .05 was considered statistically significant.
RESULTS Hemodynamic and lung weight change. There was no significant difference in hemodynamics among the groups at baseline and at the end of study (Table I). Lung weight gain was higher in the LPS groups than in the control group, but the weight gain was attenuated by the presence of SN50 (Table II). The observed lung weight gain was consistent with increased lung Kfc (Table II) in the LPS group. The leukocyte counts in bronchoalveolar lavage fluid increased in the LPS group, but the increase was mitigated in the presence of SN50 (Table II). Histologic findings. Histologic analysis of the lung tissue further supported the lung weight data and Kfc by showing perivascular edema, interstitial and intraalveolar leukocytic infiltrates (Fig 1, B), intra-alveolar hemorrhage, and proteinaceous intra-alveolar exudates in the LPS group (not shown) compared with the control group (Fig 1, A). Pretreatment with SN50 reduced the histologic alterations associated with LPSLI (Fig 1, C). Inflammatory responses. The concentration of MPO in the LPS group was higher than that in the control group (Table II). SN50 treatment was associated with decreased MPO concentration comparable with that observed in the control group (Table II). The levels of IL-1b, TNF-a, and MIP-2 in lavage fluids were higher in the LPS group compared with the control group. The administration of SN50 attenuated these cytokine responses (Fig 2, A–C ). IL-1b, TNF-a, and NF-kB in lung tissue of the LPS group were higher than that observed in the control group, however, the group treated with SN50 had lower levels of IL-1b, TNF-a, and NF-kB compared with the LPS group (Fig 3, A–C). MAPK signaling pathways and apoptosis. There was an increase in ERKs, JNK, and P-38 activation in response
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Table II. Lung weight gain, pulmonary capillary filtration coefficient, white blood cell count, and myeloperoxidase in various groups Groups
N
LWG (g)
Kfc (cmH2O．min21． mL21)
WBC (3106/L)
MPO (U/mg)
Control LPS LPS 1 SN50
7 7 7
0.10 6 0.12 1.78 6 0.83* 0.65 6 0.58*,†
0.08 6 0.12 0.47 6 0.20* 0.14 6 0.19*,†
118.5 6 44.15 250.94 6 56.19* 120.0 6 50.6†
10.5 6 3.1 17.5 6 3.3* 11.1 6 3.1†
Abbreviations: Kfc, pulmonary capillary filtration coefficient; LPS, lipopolysaccharides; LWG, lung weight gain; N, number; MPO, myeloperoxidase; SN50, NF-kB cell permeable inhibitory peptide; WBC, white blood cell count (in bronchoalveolar lavage fluid). Values are mean 6 SD. *P , 0.05 compared with control. † P , 0.05 compared with LPS.
Fig 1. NF-kB cell permeable inhibitory peptide (SN50) attenuated lung inflammation in lipopolysaccharideinduced lung injury. Acute lung injury was characterized by perivascular edema (white arrow), interstitial, and intra-alveolar leukocyte infiltration (arrow head) in the lipopolysaccharide (LPS) group (B) compared with the control group (A). Treatment with SN 50 improved pathologic changes of lung (C). Lung tissue was stained by hematoxylin/eosin (HE) stain and it showed the picture with amplification (magnification, 3200).
to LPS (Fig 4, A–C). SN50 treatment attenuated ERKs, JNK, and P-38 activation (Fig 4, A–C). Caspase-3 and p-AKT were also elevated in lung tissue from the LPS group compared with controls, however, this increase was attenuated by the administration of SN50 (Fig 5, A and B). Plasminogen activator inhibitor-1. PAI-1 was upregulated in lung tissue from the LPS group compared with controls and decreased with SN50 treatment (Fig 5, C). NF-kB signaling pathway. Immune staining of nuclear NF-kB increased in lung tissue in response to LPS compared with controls (Fig 6, A and B). Administration of SN50 decreased the translocation of NF-
kB after exposing to LPS seen in the LPS 1 SN50 group (Fig 6, C). The quantitative data of cells with positive nuclear NF-kB staining are reported in Figure 6, D. DISCUSSION
We demonstrate that LPS-induced lung injury increased pulmonary vascular permeability, infiltration of inflammatory cells, pulmonary edema, cytokine responses, MAPK activation, NF-kB translocation, expression of apoptotic enzymes (caspase-3 and p-AKT), and upregulation of coagulation (PAI-1). Treatment with
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Fig 2. NF-kB cell permeable inhibitory peptide (SN50) decreased the production of interleukin (IL)-Ib, tumor necrosis factor (TNF)-a, and macrophage inflammatory protein (MIP)-2 in bronchoalveolar lavage fluid. The levels of cytokines are shown in IL-1b (A), TNF-a (B), and MIP-2 (C), respectively.
Fig 3. Interleukin (IL)-1b, tumor necrosis factor (TNF)-a in lung tissue, and nuclear factor-kappa B (NF-kB) in nucleic protein of lung tissue were higher in the lipopolysaccharide (LPS) group than those of control. Treatment of NF-kB cell permeable inhibitory peptide (SN50) mitigated the increased of IL-1b, TNF-a, and NF-kB in lung tissue. The levels of cytokines are shown in IL-1b (A), TNF-a (B), and NF-kB (C), respectively.
the SN50 efficiently suppressed NF-kB expression, reduced proinflammatory cytokine release, inhibited MAPK activation, attenuated apoptosis responses, and decreased PAI-1. However, such changes as lung weight gain, Kfc, inflammatory cytokines, MAPK, apoptotic enzymes, and PAI-1 did not return to baseline in the SN50treated LPS-exposed group. This implies that SN50 only attenuated but not abrogated LPSLI. The causes may come from other gene expressions activated by LPS19 or the dose of SN50 in this experiment was only a partial, and not a complete, blockade of NF-kB activity. Our LPS model has reproduced many features reported in other ALI studies, including inflammatory responses and structural lung damage.20 The effects of pharmacologic intervention to reduce LPSLI have been previously examined, including
some focused on the downregulation of NF-kB translocation by inhibiting the different signal transduction pathways using toll-like receptor signal transduction inhibitor,21 suppressing the generation of reactive oxygen species,22 and peroxisome proliferator-activated receptor-b/d knockout.23 During the course of sepsis many nuclear transcription factors and genes are involved through different signaling transduction pathways.9,24 Although both the attenuation of LPSLI and the downregulation of NF-kB expression have been demonstrated in previous studies, it raises the possibility of nonspecific activation or inhibition of other nuclear transcription factors that are involved in the attenuation of LPSLI. SN50 contains a specific nuclear localization sequence derived from the p50 subunit of NF-kB and conserves the inhibitory
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Fig 4. NF-kB cell permeable inhibitory peptide (SN50) attenuated activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase 1 (JNK1), and P-38 in lung tissue. The relative density of ERK, JNK and P-38 in lung tissue were shown in various groups. ERK, JNK, and P38 MAPK were showed in (A), (B), and (C) respectively.
Fig 5. NF-kB cell permeable inhibitory peptide (SN50) reduced activation of caspase-3, AKT, and plasminogen activator inhibitor type 1(PAI-1) in lung tissue. The relative density of caspase -3, AKT, and PAI-1in lung tissue were showed in (A), (B), and (C), respectively.
translocation of NF-kB into the nucleus. To the best of our knowledge, we are the first to report that treatment with the SN50 at the onset of LPS attenuated LPSLI. Activation of NF-kB associated with LPSLI may be a significant therapeutic target in the management of septic lung injury. Mechanisms of activation of NF-kB by activators were either by canonical or noncanonical (or both) pathways. Strategies of blocking NF-kB activation to attenuate LPSLI have included reducing activation of the
IkB kinase complex,25 decreasing degradation of IkB proteins,26 inhibited translocation of NF-kB,27 and reducing deoxyribonucleic acid binding activity of NF-kB.28 Despite the available literature discussing the role of NF-kB in LPSLI, there is a paucity of data regarding the inhibition of NF-kB by more specific single peptides in LPSLI. Inhibition of NF-kB translocation may be an important mechanism underlying the therapeutic effects of SN50 treatment because NF-kB plays a crucial role in initiating the complex cytokine and
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Fig 6. SN50 decreased nuclear factor-kappa B (NF-kB) activation. Nuclear staining of NF-kB in lung tissue and it showed the picture of amplification (magnification, 3 400) in control group (A), lipopolysaccharide (LPS) group (B), LPS 1 SN50 (C), respectively. Arrows indicated nuclear NF-kB staining. Percentage of cells with positive nuclear staining for NF-kB were reported in (D).
inflammatory response in sepsis.8,29 We demonstrated that treatment with SN50 dramatically reduced NF-kB translocation into nuclei and was associated with antiinflammatory responses, including downregulation of cytokines and MAPK activation in LPSLI. Although it is difficult to pinpoint exactly the time course for SN50 import into cells in our in vivo model, it has been demonstrated that within 30–60 minutes SN50 reaches maximal intracellular localization in cultured endothelial cells, thereby inhibiting nuclear translocation of NF-kB induced by LPS.12 Nuclear translocation of NF-kB is essential for the production of proinflammatory cytokines, PAI-1, prolonged MAPK activation, and cell death. It has been shown that secretion of chemokines and cytokines is dependent on the activation of MAPKs and NF-kB.8,30 We have previously demonstrated that NF-kB inhibition produced attenuation of ischemia/reperfusion and ventilator-induced lung injury and downregulation of cytokine production.15 In the current study, we observed NF-kB activation in LPSLI, and SN50 treatment induced downregulation of NF-kB translocation and cytokine production. Taken together, SN50 appears to have anti-inflammatory effects. Furthermore, we have shown for the first time that SN50 has an antiapoptotic effect by inhibiting AKT and caspase-3 and anticoagulation effects by decreasing of PAI-1 content in the lung.
SN50 blockade of NF-kB has been demonstrated to inhibit translocation of NF-kB without causing cellular cytotoxicity.12 In addition, SN50 also blocks trafficking of NFAT, STAT1, and AP-1 into the nucleus in Jurkat T lymphocytes when the concentration of SN50 is greater than 75 mM.31 Liu et al has shown that SN50 peptide decreased production of TNF-a, interferon-g, and IL 2 in Staphylococcal enterotoxin B-stimulated splenocytes.32 Similar findings have been noted using SN50 in human T cells through reduced expression of T-bet.33 Although SN50 has also been shown to improve survival rate in an endotoxemia rat model,34 the role of SN50 in the treatment of septic lung injury has not been elucidated. Our results suggest that SN50 might confer protection by preventing ALI in the acute phase of sepsis, however, further study is needed to determine the onset and duration of NF-kB blockade in SN50 treatment, including possible adverse sequelae. The molecular mechanism of the effect of SN50 treatment on LPSLI has not been well documented. In the current study, we demonstrated SN50 treatment induced a decrease in NF-kB nuclear content in lung tissue, indicative of blocking NF-kB activation by decreasing NF-kB translocation into the nuclei. We also showed SN50 treatment in LPSLI reduced expression of the inflammatory cytokines (IL-1b, MIP-2, and TNF-a). Furthermore, we are the first to report that SN50
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treatment downregulates MPO, MAPK pathway (p38, ERK 1/2, and JNK), apoptosis index (caspase 3 and p-AKT), and hyper-coagulation (PAI-1). There are several limitations to consider when evaluating the current results. First, we used an isolated, perfused lung model to minimize hemodynamic effects on lung injury, which precludes any interactions with other organ systems. Second, as in other previous studies, which used different doses of SN50 pretreatment in different animal models, SN50 was administered prior to LPS as a proof-of-concept study14,34-36; however, our results showed that NF-kB was an important therapeutic target for the treatment of LPSLI. Third, we chose the dose of SN50 that was modulated from our previous study,15 the optimal dose of SN50 to attenuate LPSLI is unknown. However, SN50 has proven to attenuate LPSLI in this study. In summary, our results demonstrate LPS induced acute lung injury is associated with inflammation, apoptosis, and hypercoagulation; furthermore, treatment with SN50 inhibited nuclear translocation of NF-kB and attenuated LPSLI via reduced inflammation, apoptosis, and coagulation. ACKNOWLEDGMENTS
This work was supported by grants from National Science Council (NSC99-2314-B-075-34; NSC96-2314B-075-045) and Taipei Veteran General Hospital (V100-C1-044), Taipei, Taiwan. Dr Chian received support from North East Great Dental Clinic in New Taipei City, Taiwan. Granting agencies and their members did not participate in the design or execution of the study. This work was assisted in part by the Division of Experimental Surgery of the Department of Surgery, Pathology and Statistics, Taipei Veterans General Hospital, Taiwan. The authors have read the journal’s policy on disclosure of potential conflicts of interest, and have not disclosed any potential conflicts of interest. REFERENCES
1. Melamed A, Sorvillo FJ. The burden of sepsis-associated mortality in the United States from 1999 to 2005: an analysis of multiplecause-of-death data. Crit Care 2009;13:R28. 2. Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. 3. Matthay MA, Ware LB, Zimmerman GA. The acute respiratory distress syndrome. J Clin Invest 2012;122:2731–40. 4. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499–511. 5. Komissarov AA, Stankowska D, Krupa A, et al. Novel aspects of urokinase function in the injured lung: role of a2-macroglobulin. Am J Physiol Lung Cell Mol Physiol 2012;303:L1037–45.
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6. Ghosh S, Hayden MS. Celebrating 25 years of NF-kB research. Immunol Rev 2012;246:5–13. 7. Rao P, Hayden MS, Long M, et al. IkBbeta acts to inhibit and activate gene expression during the inflammatory response. Nature 2010;466:1115–9. 8. Liu SF, Malik AB. NF-kB activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol 2006;290:L622–45. 9. Mattick JS, Yang Q, Orman MA, Ierapetritou MG, Berthiaume F, Androulakis IP. Long-term gene expression profile dynamics following cecal ligation and puncture in the rat. J Surg Res 2012;178:431–42. 10. Ganchi PA, Sun SC, Greene WC, Ballard DW. I kB/MAD-3 masks the nuclear localization signal of NF-kB p65 and requires the transactivation domain to inhibit NF-kB p65 DNA binding. Mol Biol Cell 1992;3:1339–52. 11. Beg AA, Ruben SM, Scheinman RI, Haskill S, Rosen CA, Baldwin AS Jr. I kB interacts with the nuclear localization sequences of the subunits of NF-kB: a mechanism for cytoplasmic retention. Genes Dev 1992;6:1899–913. 12. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kB by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem 1995;270: 14255–8. 13. Das J, Chen CH, Yang L, Cohn L, Ray P, Ray A. A critical role for NF-kB in GATA3 expression and TH2 differentiation in allergic airway inflammation. Nat Immunol 2001;2:45–50. 14. Liu YY, Liao SK, Huang CC, Tsai YH, Quinn DA, Li LF. Role for nuclear factor-kB in augmented lung injury because of interaction between hyperoxia and high stretch ventilation. Transl Res 2009; 154:228–40. 15. Chiang CH, Pai HI, Liu SL. Ventilator-induced lung injury (VILI) promotes ischemia/reperfusion lung injury (I/R) and NF-kB antibody attenuates both injuries. Resuscitation 2008;79:147–54. 16. Drake R, Gaar KA, Taylor AE. Estimation of the filtration coefficient of pulmonary exchange vessels. Am J Physiol 1978;234: H266–74. 17. Chiang CH, Chuang CH, Liu SL. Apocynin attenuates ischemiareperfusion lung injury in an isolated and perfused rat lung model. Transl Res 2011;158:17–29. 18. Chiang CH, Hsu K, Yan HC, Harn HJ, Chang DM. PGE1, dexamethasone, U-74389G, or Bt2-cAMP as an additive to promote protection by UW solution in I/R injury. J Appl Physiol 1997; 83:583–90. 19. Croner RS, Hohenberger W, Jeschke MG. Hepatic gene expression during endotoxemia. J Surg Res 2009;154:126–34. 20. Hagiwara S, Iwasaka H, Hasegawa A, et al. Adenosine diphosphate receptor antagonist clopidogrel sulfate attenuates LPS-induced systemic inflammation in a rat model. Shock 2011;35:289–92. 21. Seki H, Tasaka S, Fukunaga K, et al. Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury. Inflamm Res 2010;59:837–45. 22. Chian CF, Chiang CH, Yuan-Jung C, et al. Apocynin attenuates lipopolysaccharide -induced lung injury in an isolated and perfused rat lung model. Shock 2010;38:196–202. 23. Kapoor A, Shintani Y, Collino M, et al. Protective role of peroxisome proliferator -activated receptor-b/d in septic shock. Am J Respir Crit Care Med 2010;182:1506–15. 24. Nguyen TT, Foteinou PT, Calvano SE, Lowry SF, Androulakis IP. Computational identification of transcriptional regulators in human endotoxemia. PLoS One 2011;6:e18889. 25. Ansaldi D, Hod EA, Stellari F, et al. Imaging pulmonary NF-kB activation and therapeutic effects of MLN120B and TDZD-8. PLoS One 2011;6:e25093.
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26. Yunhe F, Bo L, Xiaosheng F, et al. The effect of magnolol on the Toll-like receptor 4/nuclear factor kB signaling pathway in lipopolysaccharide-induced acute lung injury in mice. Eur J Pharmacol 2012;689:255–61. 27. Xu X, Xie Q, Shen Y, et al. Involvement of mannose receptor in the preventive effects of mannose in lipopolysaccharide-induced acute lung injury. Eur J Pharmacol 2010;641:229–37. 28. Liu G, Park YJ, Tsuruta Y, Lorne E, Abraham E. p53 Attenuates lipopolysaccharide -induced NF-kB activation and acute lung injury. J Immunol 2009;182:5063–71. 29. Rahman A, Fazal F. Blocking NF-kB: an inflammatory issue. Proc Am Thorac Soc 2011;8:497–503. 30. Nick JA, Avdi NJ, Young SK, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. J Clin Invest 1999;103:851–8. 31. Torgerson TR, Colosia AD, Donahue JP, Lin YZ, Hawiger J. Regulation of NF-kB, AP-1, NFAT, and STAT1 nuclear import in T lymphocytes by noninvasive delivery of peptide carrying the nuclear localization sequence of NF-kB p50. J Immunol 1998;161:6084–92.
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32. Liu D, Liu XY, Robinson D, et al. Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor. J Biol Chem 2004;279:19239–46. 33. McCracken SA, Hadfield K, Rahimi Z, Gallery ED, Morris JM. NF-kB-regulated suppression of T-bet in T cells represses Th1 immune responses in pregnancy. Eur J Immunol 2007;37: 1386–96. 34. O’Sullivan AW, Wang JH, Redmond HP. NF-kB and p38 MAPK inhibition improve survival in endotoxin shock and in a cecal ligation and puncture model of sepsis in combination with antibiotic therapy. J Surg Res 2009;152:46–53. 35. Duffy JY, McLean KM, Lyons JM, Czaikowski AJ, Wagner CJ, Pearl JM. Modulation of nuclear factor-kB improves cardiac dysfunction associated with cardiopulmonary bypass and deep hypothermic circulatory arrest. Crit Care Med 2009;37: 577–83. 36. Smuder AJ, Hudson MB, Nelson WB, Kavazis AN, Powers SK. Nuclear factor-kB signaling contributes to mechanical ventilation-induced diaphragm weakness. Crit Care Med 2012;40: 927–34.
NF-kB cell permeable inhibitory peptide (SN50) inhibits translocation of nuclear factor-kB (NF-kB) and production of inflammatory cytokines that are implicated in lipopolysaccharide (LPS)-induced lung injury (LPSLI). However, the protective effect of SN50 in LPSLI is unclear. We explored the cellular and molecular mechanisms of SN50 treatment in LPSLI. LPSLI was induced by intratracheal instillation of 10 mg/kg LPS using an isolated and perfused rat lung model. SN50 was administered in the perfusate 15 minutes before LPS was administered. Hemodynamics, lung histologic change, inflammatory responses, and activation of apoptotic pathways were evaluated. After LPSLI, increased pulmonary vascular permeability and lung weight gain was observed. The levels of interleukin (IL)-1b, tumor necrosis factor (TNF)-a, myeloperoxidase, and macrophage inflammatory protein-2 increased in bronchoalveolar lavage fluids. Lung-tissue expression of TNF-a, IL1b, mitogen-activated protein kinases (MAPKs), caspase-3, p-AKT (serine-threonine kinase, also known as protein kinase B), and plasminogen activator inhibitor-1 (PAI-1) was greater in the LPS group compared with controls. Upregulation and activation of NF-kB was associated with increased lung injury in LPSLI. SN50 attenuated the inflammatory responses, including expression of IL-1b, TNFa, myeloperoxidase, MAPKs, PAI-1, and NF-kB; downregulation of apoptosis indicated by caspase-3 and p-AKT expression was also observed. In addition, SN50 mitigated the increase in the lung weight, pulmonary vascular permeability, and lung injury. In conclusion, LPSLI is associated with inflammatory responses, apoptosis, and coagulation. NF-kB is an important therapeutic target in the treatment of LPSLI. SN50 inhibits translocation of NF-kB and attenuates LPSLI. (Translational Research 2014;163:211–220) Abbreviations: ALI ¼ acute lung injury; DWt/Dt ¼ rate of weight change; ERK ¼ extracellular signal-regulated kinase; IL ¼ interleukin; Kfc ¼ pulmonary capillary filtration coefficient; LPS ¼ lipopolysaccharide; LPSLI ¼ lipopolysaccharide-induced lung injury; MAPKs ¼ mitogen-activated protein kinases; MIP ¼ macrophage inflammatory protein; MPO ¼ myeloperoxidase; NF-kB ¼ nuclear factor-kB; PAI-1 ¼ plasminogen activator inhibitor type 1; Ppa ¼ pulmonary artery pressure; Ppc ¼ pulmonary capillary pressure; Ppv ¼ pulmonary venous pressure; Ra ¼ pulmonary arterial resistance; Rv ¼ pulmonary venous resistance; SN50 ¼ NF-kB cell permeable inhibitory peptide; TLR4 ¼ Toll-like receptor 4; TNF ¼ tumor necrosis factor
From the Division of Pulmonary and Critical Care Medicine, Internal Medicine Department, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Division of Pulmonary Immunology and Infectious Diseases, Chest Department, Taipei Veterans General Hospital, Taipei, Taiwan; Institute of Emergency and Critical Care Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. Submitted for publication June 14, 2013; revision submitted October 3, 2013; accepted for publication October 7, 2013.
Reprint requests: Chi-Huei Chiang, MD, Division of Pulmonary Immunology and Infectious Diseases, Chest Department, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei, Taiwan; e-mail: [email protected] or [email protected]. tw. 1931-5244/$ - see front matter Ó 2014 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2013.10.002
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AT A GLANCE COMMENTARY Chian C-F, et al. Background
Pulmonary infection by bacteria or virus is the leading cause of acute lung injury and acute respiratory distress syndrome, which results in patient death. Nuclear factor-kappaB (NF-kB) appears to be a key in the regulation of the inflammatory process during sepsis. The NF-kB cell permeable inhibitory peptide (SN50) inhibits translocation of NF-kB in 30 to 60 minutes. However, the role of SN50 in the treatment of septic lung injury remains unknown. Translational Significance
Our results demonstrated SN50 attenuated the inflammatory response, reduced cytokines release, and mitigated hypercoagulation in septic lung injury. Specific agents targeting NF-kB may offer novel treatment options for this group of patients.
Sepsis is a significant public health problem and is the tenth leading cause of death in the United States.1 Acute lung injury (ALI) and acute respiratory distress syndrome commonly complicate sepsis and often result in the prolonged use of mechanical ventilator support and a mortality of approximately 30%–50%.2 ALI caused by sepsis is associated with a severe systemic inflammatory response to bacterial infection characterized by the accumulation of inflammatory cells and platelets, disruption of vascular endothelial and epithelial barriers, and increased permeability of the alveolus.3 Toll-like receptor 4 recognizes gram negative bacteria and bacterial components (lipopolysaccharide) and thereby activates an innate immune response.4 Understanding the complex mechanisms responsible for the pathogenesis of ALI will provide insight toward developing novel treatments in the future.5 Nuclear factor-kappa B (NF-kB) is a ubiquitous transcriptional factor that plays a pivotal role in immune and inflammatory responses.6 NF-kB regulates the transcription of various genes that encode inflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, (IL-6), chemokines, plasminogen-activator inhibitor type 1 (PAI-1), adhesion molecules, and inducible nitric oxide synthase.7-9 NF-kB conjugated with IkB proteins are sequestered in the cytosol in an inactive form. Upon activation, degradation of IkB unmasks the nuclear localization sequence of NF-kB. The nuclear
localization sequence allows the NF-kB dimer to translocate into the nucleus. NF-kB binds to the promoter regions of NF-kB regulated genes resulting in genes transcription. Previous transfection studies have shown that masking the nuclear localization sequence of the NF-kB dimer prevents its translocation to the nucleus.10,11 The pathogenesis of sepsis appears to involve the activation of NF-kB, a critical pathway of septic lung injury. As such, NF-kB would be an important therapeutic target in the treatment of septic lung injury. NF-kB cell permeable inhibitory peptide (SN50) was first synthesized by Lin et al and was comprised of the hydrophilic region of the signal peptide of Kaposi fibroblast growth factor as a membrane translocating motif and a nuclear localization sequence derived from the p50 subunit of NF-kB.12 SN50 has been used as an inhibitor of NF-kB translocation. SN50 has been shown to attenuate GATA-3 expression and IL 5 and IL 13 production in developing TH2 cells.13 Liu et al has demonstrated SN50 reduced levels of TNF-a and PAI-1 in bronchoalveolar lavage fluid and inhibited NF-kB activation and deoxyribonucleic acid binding in a mouse model of augmented hyperoxia and ventilator-induced lung injury14; however, the mechanism and protective effect associated with the introduction of SN50 in lipopolysaccharideinduced lung injury (LPSLI). LPSLI remains unclear. We currently hypothesize that SN50 attenuates septic lung injury by inhibiting translocation of NF-kB, resulting in the downregulation of inflammation and apoptosis and coagulation in LPSLI. METHODS Animal preparation. The study was performed in accordance with animal ethics guidelines of the National Institutes of Health. The study protocol was approved by the Institutional Review Board of the Taipei Veterans General Hospital for animal care and use. The in situ isolated-perfused lung model has been previously described.15 Briefly, male Sprague-Dawley rats weighing 250–350 g were anesthetized with intraperitoneal injection of sodium pentobarbital. A tracheotomy was performed and mechanical ventilation was applied (Rodent ventilator Model 683; Harvard Apparatus, South Natick, Mass) at a tidal volume of 6 mL/kg and a positive end-expiratory pressure of 2 cm H2O. After a sternotomy, heparin (1 unit/g) was injected into the right ventricle. The right ventricle was also used to catheterize the pulmonary artery, which had a tight ligature placed around its main trunk. Another catheter was inserted into the left atrium through the left ventricle and mitral valve and fixed by a ligature at the
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apex of the heart. Pulmonary venous outflow was diverted into a reservoir. To prevent backflow into the ventricles, an additional ligation was performed above the atrioventricular junction. The lungs were perfused with 10 cc blood mix with 30 cc 0.9% normal saline (Minipulse 2; Gilson Medical Electronic, Middleton, Wisc) at a constant flow (30 mL.min21.g21 body weight). Pulmonary artery pressure (Ppa), pulmonary venous pressure (Ppv), circulating perfusate pH, and peak airway pressure were monitored. The weight of each rat was reflective of lung weight in the in situ system and was recorded continuously as the lung weight gain. Pulmonary arterial resistance (Ra) and venous resistance (Rv) were calculated using the following equations: Ra 5 (Ppa - Ppc)/Q, and Rv 5 (Ppc - Ppv)/Q, where Q is perfusate flow. Determination of (Ppc). The Ppc was
pulmonary
capillary
pressure
estimated by using the doubleocclusion method.16 Briefly, arterial inflow and venous outflow lines were occluded simultaneously, and the equilibrium Ppa and Ppv were measured. Measurement of microvascular permeability. The pulmonary capillary filtration coefficient (Kfc) was used as an index of microvascular permeability to water. The method for Kfc measurement has been previously described.16 Briefly, after an isogravimetric period, Ppv was rapidly elevated to 6–8 cm H2O for 15 minutes. The increase in lung weight was recorded, and a characteristic rapid weight gain (vascular filling) was followed by a slower rate of weight gain. The rate of weight change (DWt/Dt) during the 6- to 14-minute interval was analyzed using linear regression of the log10-transformed rates of weight changes per minute. The initial rate of weight gain was calculated using an extrapolation of DWt/Dt to time 0. Kfc was calculated by dividing DWt/Dt at time 0 by the changes in Ppc that occurred after venous outflow pressure was increased. The resulting value was then normalized using the baseline wet-lung weight, and was expressed as milliliters per minute per centimeter H2O per 100 g of lung tissue. Experimental protocols. Isolated lung preparations were separated into 3 experimental groups: (1) sham (control), (2) intratracheal LPS (LPS), and (3) intratracheal LPS plus SN50 (LPS 1 SN50) and ventilated with tidal volume settings at 6 mL/kg. Sham controls were ventilated with a tidal volume setting at 6 mL/kg and neither exposed to LPS nor SN50. The protocol for LPS-induced injury was performed as follows: 10 mg/kg LPS was administered intratracheally to the LPS-induced lung injury group. SN50 (Enzo Life Sciences, Farmingdale, NY) was administered in the perfusate at 15 minutes before LPS initiation. SN50
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was administered in total volume 40 cc circulating perfusate (30 cc normal saline 110 cc blood) with a final concentration of 1 mM. Hemodynamics were continuously monitored for 6 hours in all groups. Vascular permeability was measured using the pulmonary capillary filtration coefficient (Kfc) as previously described.17 Measurement of white cell count in bronchoalveolar lavage fluid. All experiments were terminated after
6 hours of closed extracorporeal perfusion, and the lungs were removed and wet weights were measured. The lungs were lavaged twice with saline (2.5 mL/ lavage). Lavage samples were centrifuged at 1500 g at room temperature for 10 minutes. The white cell count was determined as previously described.15,18 Myeloperoxidase assay. The concentration of myeloperoxidase (MPO), an index of neutrophil sequestration in the lungs, was measured as previously described in right middle lung tissue.18 Cytokines assays. IL-1b, TNF-a, and macrophage inflammatory protein (MIP)-2 were measured in lavage fluids using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Oxon, UK). The absorbance was read at 450 nm (SpectraMax M5; Molecular Devices). Western blotting analysis. Lung tissues were homogenized using lysis buffer containing protease inhibitor cocktail (Roche, Pleasanton, CA) and phosphatase inhibitor cocktail (Roche). The total protein concentration in the extract was determined with a bicinchoninic acid protein assay (Pierce, Rockford, Ill): 80 mg protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel, and electro-transferred onto polyvinylidene fluoride membrane (Millipore, Billerica, Mass). The membrane was blocked with 5% nonfat dry milk in trisbuffered saline (TBS) containing: 0.1% Tween 20 for 1 hour. Antibodies against phospho-p44/42 mitogenactivated protein kinase (MAPK) (extracellular signalregulated kinase [ERK] 1/2), phospho-stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), phospho-p38 MAPK, anti-p44/42 MAPK (ERK1/2), anti- SAPK/JNK, and anti-p38 MAPK [1:1,000; Cell Signaling Technology, Beverly, Mass] were used. Antibodies against GADPH (1:10,000; Lab Frontier, Abfrontier, Seoul, Korea), c-Jun N-terminal kinase 1 (JNK1) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3 (1:2000; Cell Signaling Technology, p-AKT (1:1000; Cell Signaling Technology), AKT (1:1000; Cell Signaling Technology), PAI-1 (1:1,000; Cell Signaling Technology) were used. The appropriate secondary antibodies (1:10,000 horseradish peroxidase anti-rabbit [Jackson Immuno Research Laboratories, West Grove, Pa]) were also used. Visualization was performed by enhanced chemiluminescence (Visual
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Table I. Hemodynamics Group
Baseline Control LPS LPS 1 SN50 After 6 hours Control LPS LPS 1 SN50
N
PPa
PPv
PPc
Ra
Rv
7 7 7
8.4 6 3.6 8.2 6 1.4 8.4 6 1.1
4.7 6 0.4 3.0 6 0 3.3 6 0.3
6.3 6 1.7 5.3 6 0.6 5.5 6 0.5
0.04 6 0.04 0.06 6 0.02 0.06 6 0.01
0.03 6 0.03 0.05 6 0.01 0.05 6 0.01
7 7 7
9.9 6 3.9 7.1 6 1.4 8.7 6 1.2
4.6 6 0.4 3.0 6 0 3.3 6 0.3
6.9 6 1.9 4.8 6 0.3 5.7 6 0.5
0.06 6 0.04 0.05 6 0.01 0.06 6 0.02
0.05 6 0.03 0.04 6 0.01 0.05 6 0.01
Abbreviations: LPS, lipopolysaccharides; N, number; Ppa (mm Hg), pulmonary arterial pressure; Ppv (mm Hg), pulmonary venous pressure; Ppc (mm Hg), pulmonary capillary pressure; Ra (cm Hg$min21 mL21), pulmonary arterial resistance; Rv (cm Hg$min21 mL21), pulmonary venous resistance; SN50, NF-kB cell permeable inhibitory peptide. Values are mean 6 SD.
Protein Biotechnology Crop, New Taipei City, Taiwan). The protein bands were quantified using Kodak 1D Image Analysis (Eastman Kodak Company, Rochester, NY). Lung histopathology. After the termination of each experiment, the right lower lobe of each lung was dissected and fixed immediately in 10% neutral buffered formalin. After fixation, the lung tissue was dehydrated using a graded series of alcohol, cleared in xylene, and embedded in paraffin. All sections were cut to 5 mm and stained with hematoxylin/eosin. Immunohistochemistry. Lung slides coated with polyL-lysine (Sigma, St. Louis, MO) were deparaffinized and rehydrated using xylene and ethanol, and placed in 3% H2O2 for 15 minutes. The slides were incubated with 1:60 dilution of monoclonal nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (Cell Signaling Technology), incubated at 4 C overnight, and stained with diaminobenzidine (Dako, Carpinteria, CA) and Mayer’s hematoxylin (Dako). Analysis was performed under Eclipse 80i microscope (Nikon, Tokyo, Japan) using Image Pro Plus 5.0 (Media Cybernetics, Rockville, MD). Cells with positive nuclear NF-kB staining were counted out of a total of 100 cells in each slide from 7 animals in each group of control, LPS or LPS 1 SN50, respectively. Cell counting was performed by a pathologist who was blinded to the experimental conditions. Statistical analysis. Systat10.0 (Systat Software Inc, San Jose, CA) was used for statistical analyses. Comparisons among all groups were conducted using an analysis of variance followed by Dunnett’s post-hoc analysis. Comparison between baseline and post-I/R values within group was conducted using Student paired t-test. Values are expressed as mean 6 standard deviation. P , .05 was considered statistically significant.
RESULTS Hemodynamic and lung weight change. There was no significant difference in hemodynamics among the groups at baseline and at the end of study (Table I). Lung weight gain was higher in the LPS groups than in the control group, but the weight gain was attenuated by the presence of SN50 (Table II). The observed lung weight gain was consistent with increased lung Kfc (Table II) in the LPS group. The leukocyte counts in bronchoalveolar lavage fluid increased in the LPS group, but the increase was mitigated in the presence of SN50 (Table II). Histologic findings. Histologic analysis of the lung tissue further supported the lung weight data and Kfc by showing perivascular edema, interstitial and intraalveolar leukocytic infiltrates (Fig 1, B), intra-alveolar hemorrhage, and proteinaceous intra-alveolar exudates in the LPS group (not shown) compared with the control group (Fig 1, A). Pretreatment with SN50 reduced the histologic alterations associated with LPSLI (Fig 1, C). Inflammatory responses. The concentration of MPO in the LPS group was higher than that in the control group (Table II). SN50 treatment was associated with decreased MPO concentration comparable with that observed in the control group (Table II). The levels of IL-1b, TNF-a, and MIP-2 in lavage fluids were higher in the LPS group compared with the control group. The administration of SN50 attenuated these cytokine responses (Fig 2, A–C ). IL-1b, TNF-a, and NF-kB in lung tissue of the LPS group were higher than that observed in the control group, however, the group treated with SN50 had lower levels of IL-1b, TNF-a, and NF-kB compared with the LPS group (Fig 3, A–C). MAPK signaling pathways and apoptosis. There was an increase in ERKs, JNK, and P-38 activation in response
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Table II. Lung weight gain, pulmonary capillary filtration coefficient, white blood cell count, and myeloperoxidase in various groups Groups
N
LWG (g)
Kfc (cmH2O．min21． mL21)
WBC (3106/L)
MPO (U/mg)
Control LPS LPS 1 SN50
7 7 7
0.10 6 0.12 1.78 6 0.83* 0.65 6 0.58*,†
0.08 6 0.12 0.47 6 0.20* 0.14 6 0.19*,†
118.5 6 44.15 250.94 6 56.19* 120.0 6 50.6†
10.5 6 3.1 17.5 6 3.3* 11.1 6 3.1†
Abbreviations: Kfc, pulmonary capillary filtration coefficient; LPS, lipopolysaccharides; LWG, lung weight gain; N, number; MPO, myeloperoxidase; SN50, NF-kB cell permeable inhibitory peptide; WBC, white blood cell count (in bronchoalveolar lavage fluid). Values are mean 6 SD. *P , 0.05 compared with control. † P , 0.05 compared with LPS.
Fig 1. NF-kB cell permeable inhibitory peptide (SN50) attenuated lung inflammation in lipopolysaccharideinduced lung injury. Acute lung injury was characterized by perivascular edema (white arrow), interstitial, and intra-alveolar leukocyte infiltration (arrow head) in the lipopolysaccharide (LPS) group (B) compared with the control group (A). Treatment with SN 50 improved pathologic changes of lung (C). Lung tissue was stained by hematoxylin/eosin (HE) stain and it showed the picture with amplification (magnification, 3200).
to LPS (Fig 4, A–C). SN50 treatment attenuated ERKs, JNK, and P-38 activation (Fig 4, A–C). Caspase-3 and p-AKT were also elevated in lung tissue from the LPS group compared with controls, however, this increase was attenuated by the administration of SN50 (Fig 5, A and B). Plasminogen activator inhibitor-1. PAI-1 was upregulated in lung tissue from the LPS group compared with controls and decreased with SN50 treatment (Fig 5, C). NF-kB signaling pathway. Immune staining of nuclear NF-kB increased in lung tissue in response to LPS compared with controls (Fig 6, A and B). Administration of SN50 decreased the translocation of NF-
kB after exposing to LPS seen in the LPS 1 SN50 group (Fig 6, C). The quantitative data of cells with positive nuclear NF-kB staining are reported in Figure 6, D. DISCUSSION
We demonstrate that LPS-induced lung injury increased pulmonary vascular permeability, infiltration of inflammatory cells, pulmonary edema, cytokine responses, MAPK activation, NF-kB translocation, expression of apoptotic enzymes (caspase-3 and p-AKT), and upregulation of coagulation (PAI-1). Treatment with
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Fig 2. NF-kB cell permeable inhibitory peptide (SN50) decreased the production of interleukin (IL)-Ib, tumor necrosis factor (TNF)-a, and macrophage inflammatory protein (MIP)-2 in bronchoalveolar lavage fluid. The levels of cytokines are shown in IL-1b (A), TNF-a (B), and MIP-2 (C), respectively.
Fig 3. Interleukin (IL)-1b, tumor necrosis factor (TNF)-a in lung tissue, and nuclear factor-kappa B (NF-kB) in nucleic protein of lung tissue were higher in the lipopolysaccharide (LPS) group than those of control. Treatment of NF-kB cell permeable inhibitory peptide (SN50) mitigated the increased of IL-1b, TNF-a, and NF-kB in lung tissue. The levels of cytokines are shown in IL-1b (A), TNF-a (B), and NF-kB (C), respectively.
the SN50 efficiently suppressed NF-kB expression, reduced proinflammatory cytokine release, inhibited MAPK activation, attenuated apoptosis responses, and decreased PAI-1. However, such changes as lung weight gain, Kfc, inflammatory cytokines, MAPK, apoptotic enzymes, and PAI-1 did not return to baseline in the SN50treated LPS-exposed group. This implies that SN50 only attenuated but not abrogated LPSLI. The causes may come from other gene expressions activated by LPS19 or the dose of SN50 in this experiment was only a partial, and not a complete, blockade of NF-kB activity. Our LPS model has reproduced many features reported in other ALI studies, including inflammatory responses and structural lung damage.20 The effects of pharmacologic intervention to reduce LPSLI have been previously examined, including
some focused on the downregulation of NF-kB translocation by inhibiting the different signal transduction pathways using toll-like receptor signal transduction inhibitor,21 suppressing the generation of reactive oxygen species,22 and peroxisome proliferator-activated receptor-b/d knockout.23 During the course of sepsis many nuclear transcription factors and genes are involved through different signaling transduction pathways.9,24 Although both the attenuation of LPSLI and the downregulation of NF-kB expression have been demonstrated in previous studies, it raises the possibility of nonspecific activation or inhibition of other nuclear transcription factors that are involved in the attenuation of LPSLI. SN50 contains a specific nuclear localization sequence derived from the p50 subunit of NF-kB and conserves the inhibitory
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Fig 4. NF-kB cell permeable inhibitory peptide (SN50) attenuated activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase 1 (JNK1), and P-38 in lung tissue. The relative density of ERK, JNK and P-38 in lung tissue were shown in various groups. ERK, JNK, and P38 MAPK were showed in (A), (B), and (C) respectively.
Fig 5. NF-kB cell permeable inhibitory peptide (SN50) reduced activation of caspase-3, AKT, and plasminogen activator inhibitor type 1(PAI-1) in lung tissue. The relative density of caspase -3, AKT, and PAI-1in lung tissue were showed in (A), (B), and (C), respectively.
translocation of NF-kB into the nucleus. To the best of our knowledge, we are the first to report that treatment with the SN50 at the onset of LPS attenuated LPSLI. Activation of NF-kB associated with LPSLI may be a significant therapeutic target in the management of septic lung injury. Mechanisms of activation of NF-kB by activators were either by canonical or noncanonical (or both) pathways. Strategies of blocking NF-kB activation to attenuate LPSLI have included reducing activation of the
IkB kinase complex,25 decreasing degradation of IkB proteins,26 inhibited translocation of NF-kB,27 and reducing deoxyribonucleic acid binding activity of NF-kB.28 Despite the available literature discussing the role of NF-kB in LPSLI, there is a paucity of data regarding the inhibition of NF-kB by more specific single peptides in LPSLI. Inhibition of NF-kB translocation may be an important mechanism underlying the therapeutic effects of SN50 treatment because NF-kB plays a crucial role in initiating the complex cytokine and
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Fig 6. SN50 decreased nuclear factor-kappa B (NF-kB) activation. Nuclear staining of NF-kB in lung tissue and it showed the picture of amplification (magnification, 3 400) in control group (A), lipopolysaccharide (LPS) group (B), LPS 1 SN50 (C), respectively. Arrows indicated nuclear NF-kB staining. Percentage of cells with positive nuclear staining for NF-kB were reported in (D).
inflammatory response in sepsis.8,29 We demonstrated that treatment with SN50 dramatically reduced NF-kB translocation into nuclei and was associated with antiinflammatory responses, including downregulation of cytokines and MAPK activation in LPSLI. Although it is difficult to pinpoint exactly the time course for SN50 import into cells in our in vivo model, it has been demonstrated that within 30–60 minutes SN50 reaches maximal intracellular localization in cultured endothelial cells, thereby inhibiting nuclear translocation of NF-kB induced by LPS.12 Nuclear translocation of NF-kB is essential for the production of proinflammatory cytokines, PAI-1, prolonged MAPK activation, and cell death. It has been shown that secretion of chemokines and cytokines is dependent on the activation of MAPKs and NF-kB.8,30 We have previously demonstrated that NF-kB inhibition produced attenuation of ischemia/reperfusion and ventilator-induced lung injury and downregulation of cytokine production.15 In the current study, we observed NF-kB activation in LPSLI, and SN50 treatment induced downregulation of NF-kB translocation and cytokine production. Taken together, SN50 appears to have anti-inflammatory effects. Furthermore, we have shown for the first time that SN50 has an antiapoptotic effect by inhibiting AKT and caspase-3 and anticoagulation effects by decreasing of PAI-1 content in the lung.
SN50 blockade of NF-kB has been demonstrated to inhibit translocation of NF-kB without causing cellular cytotoxicity.12 In addition, SN50 also blocks trafficking of NFAT, STAT1, and AP-1 into the nucleus in Jurkat T lymphocytes when the concentration of SN50 is greater than 75 mM.31 Liu et al has shown that SN50 peptide decreased production of TNF-a, interferon-g, and IL 2 in Staphylococcal enterotoxin B-stimulated splenocytes.32 Similar findings have been noted using SN50 in human T cells through reduced expression of T-bet.33 Although SN50 has also been shown to improve survival rate in an endotoxemia rat model,34 the role of SN50 in the treatment of septic lung injury has not been elucidated. Our results suggest that SN50 might confer protection by preventing ALI in the acute phase of sepsis, however, further study is needed to determine the onset and duration of NF-kB blockade in SN50 treatment, including possible adverse sequelae. The molecular mechanism of the effect of SN50 treatment on LPSLI has not been well documented. In the current study, we demonstrated SN50 treatment induced a decrease in NF-kB nuclear content in lung tissue, indicative of blocking NF-kB activation by decreasing NF-kB translocation into the nuclei. We also showed SN50 treatment in LPSLI reduced expression of the inflammatory cytokines (IL-1b, MIP-2, and TNF-a). Furthermore, we are the first to report that SN50
Translational Research Volume 163, Number 3
treatment downregulates MPO, MAPK pathway (p38, ERK 1/2, and JNK), apoptosis index (caspase 3 and p-AKT), and hyper-coagulation (PAI-1). There are several limitations to consider when evaluating the current results. First, we used an isolated, perfused lung model to minimize hemodynamic effects on lung injury, which precludes any interactions with other organ systems. Second, as in other previous studies, which used different doses of SN50 pretreatment in different animal models, SN50 was administered prior to LPS as a proof-of-concept study14,34-36; however, our results showed that NF-kB was an important therapeutic target for the treatment of LPSLI. Third, we chose the dose of SN50 that was modulated from our previous study,15 the optimal dose of SN50 to attenuate LPSLI is unknown. However, SN50 has proven to attenuate LPSLI in this study. In summary, our results demonstrate LPS induced acute lung injury is associated with inflammation, apoptosis, and hypercoagulation; furthermore, treatment with SN50 inhibited nuclear translocation of NF-kB and attenuated LPSLI via reduced inflammation, apoptosis, and coagulation. ACKNOWLEDGMENTS
This work was supported by grants from National Science Council (NSC99-2314-B-075-34; NSC96-2314B-075-045) and Taipei Veteran General Hospital (V100-C1-044), Taipei, Taiwan. Dr Chian received support from North East Great Dental Clinic in New Taipei City, Taiwan. Granting agencies and their members did not participate in the design or execution of the study. This work was assisted in part by the Division of Experimental Surgery of the Department of Surgery, Pathology and Statistics, Taipei Veterans General Hospital, Taiwan. The authors have read the journal’s policy on disclosure of potential conflicts of interest, and have not disclosed any potential conflicts of interest. REFERENCES
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