Journal of Medical Sciences

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 42  |  Issue : 6  |  Page : 274--281

Benefit of broccoli extract-sulforaphane prophylaxis in ventilator-induced lung injury


Chen-Liang Tsai1, Chih-Ying Changchien2, Chi-Huei Chiang3, Shan-Yueh Chang1, Ying-Chieh Chen1, Chih-Feng Chian1,  
1 Division of Pulmonary and Critical Care, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
2 Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
3 Department of Chest, Taipei Veterans General Hospital, Taipei, Taiwan

Correspondence Address:
Dr. Chih-Feng Chian
Division of Pulmonary and Critical Care, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, 114 No. 325, Chengong Road, Sec. 2, Neihu District, Taipei City
Taiwan

Abstract

Background: Owig to the extensive use of mechanical ventilation, risks of acute lung injury are significant in the intensive care unit. Broccoli extract-sulforaphane (SFN) has been investigated as bioactive polyphenol in chronic lung diseases. Aim: The present study aimed to evaluate the preventive effect of SFN in a rat model of ventilator-induced lung injury. Methods: SFN supplement was administrated 30 min before intubation with the dosage of 3 mg/kg. Then, rats were assigned to receive ventilation with a high tidal volume of 40 mL/kg for 6 h, and low ventilation of 6 mL/kg served as controls. Results: The severity of pulmonary edema was mitigated in the SFN-pretreated group with decreased weight ratios of wet to dry lung and total lung to the body, respectively. From bronchoalveolar lavage, SFN treatment suppressed both leukocytes counts and cytokines production. Following ventilator-exerted oxidative burst with the rescue of glutathione level was identified in SFN-pretreated group. Besides, SFN-reduced cell apoptosis was confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and cleavage of caspase-3. Western blotting from lung tissues revealed the upregulation of hemeoxygenase-1 with decreased nuclear factor κB and p38 phosphorylation in SFN-treated group. Conclusion: Our results elucidated the prophylaxis of broccoli extract-SFN could attenuate ventilator-induced oxidative stress, inflammation reaction, and pulmonary edema.



How to cite this article:
Tsai CL, Changchien CY, Chiang CH, Chang SY, Chen YC, Chian CF. Benefit of broccoli extract-sulforaphane prophylaxis in ventilator-induced lung injury.J Med Sci 2022;42:274-281


How to cite this URL:
Tsai CL, Changchien CY, Chiang CH, Chang SY, Chen YC, Chian CF. Benefit of broccoli extract-sulforaphane prophylaxis in ventilator-induced lung injury. J Med Sci [serial online] 2022 [cited 2022 Dec 8 ];42:274-281
Available from: https://www.jmedscindmc.com/text.asp?2022/42/6/274/335767


Full Text



 Introduction



Micronutrient supplementation comprising vitamins and trace elements has rapidly gained interest in the critical care field, particularly in alleviating oxidative stress.[1] In ventilated patients through enteral/parenteral feeding solutions, micronutrients such as polyphenols are frequently unavailable than generally acknowledged.[2],[3],[4] Broccoli is recognized as a super-food due to the high content of polyphenols and sulforaphane (SFN), a major bioactive compound with excellent anti-inflammation properties.[5],[6],[7] In the field of chronic lung diseases, few publications had discussed the benefit of SFN in asthma and chronic obstructive pulmonary disease.[8] Furthermore, SFN pretreatment suppressed leukocyte infiltration in human subjects exposed to diesel exhaust particles.[9] The therapeutic efficacy of SFN had been explored in several models of lung injury. The administration of SFN was found to alleviate inflammation and exert oxidative stress in mice of oxidant-induced lung injury, rabbits with acute respiratory distress syndrome, and lipopolysaccharide (LPS)-induced acute lung injury (ALI) through nuclear factor erythroid 2 (Nrf-2) signaling.[10],[11],[12] However, the potential of SFN as pharmaco-nutrition has not been explored in ALI induced by mechanical ventilation.

Acute respiratory failure is one of the most common causes for intensive care unit admission. In contrast, indispensable mechanical ventilation may damage lung parenchyma through physical stress, so-termed “ventilator-induced lung injury” (VILI).[13] In the early 1970s, scientists have observed the development of perivascular and alveolar edema within 35 min in ventilated rats.[14] Other physiological abnormalities included elevated wet-to-dry ratio of the isolated lung, composite alterations in bronchoalveolar lavage fluid (BALF), and increased thickness of hyaline membranes.[15] At the cellular level, VILI is characterized by the release of cytokines, including interleukin-1 (IL-1) β and tumor necrosis factor-α (TNF-α), recruitment of leukocytes, predominately neutrophils, and massive apoptosis of pneumocytes.[16],[17] Clinical practice has mainly focused on ventilation strategies, whereas pharmacological interventions remain uncertain areas, especially inflammation-targeted agents.[18] Taken advantage of high bioavailability and less systemic toxicity, natural extracts have emerged as a promising drug class in ALI.

Despite better understanding of pathophysiology in ALI, clinicians still lack consensus for its systemic pharmacotherapy. These experimental therapies encompass prostaglandin E1 (PGE1) as a vasodilator, glucocorticoid as an anti-inflammatory agent, and N-acetylcysteine (NAC) as an anti-oxidant drug.[19] PGE1 has the potency to modulate neutrophils activation, whereas its nonselective vasodilatory effects exert severe hypotension.[20],[21] Although steroids have been experimented in the prevention and treatment of acute respiratory distress syndrome,[22] its inhibition of host defense is highly concerned. As NAC alleviates oxidative stress, the evidence to support its clinical use is not adequate.[23] Therefore, the development of the new drug in ALI is in urgent need. The present study aimed to assess the prevention use of broccoli concentrate-SFN in the rat VILI model through mitigating leukocyte infiltration, oxidative burst, and lung edema.

 Materials and Methods



Animals

Taipei Veterans General Hospital Research Animal Care Subcommittee approved the study (IACUC-2012-201). Sprague − Dawley virus-free rats weighing between 250 and 300 g were obtained from National Laboratory Animal Center, Taiwan.

Experimental protocols

Male Sprague − Dawley rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (20–25 mg) while breathing room air. PE 240 tubing was inserted into the trachea and connected to a Harvard apparatus ventilator, model 55-7058 (Holliston, MA, USA). We used our established ventilator protocol in the rat model of VILI as previously described.[24] The rats were then randomly assigned into four groups (N = 7 for each group) and ventilated for 6 h. These groups consisted of: (a) a control group using a tidal volume (VT) of 6 ml/kg, (b) a vehicle control group using dimethyl sulfoxide (DMSO), (c) a VILI group using a high VT of 40 ml/kg, and (d) a VILI + SFN treatment group. R, S-SFN, purchased from LKT Laboratories, Inc. (Minnesota, USA) was prepared with DMSO and given 3 mg/kg 30 min before starting the mechanical ventilator. A positive end-expiratory pressure of 2 cm H2O was applied in all groups. End-tidal CO2 was monitored intermittently by a microcapnograph (Columbus Instruments, Columbus, OH, USA) and was kept between 35 and 45 torrs by adjusting the respiratory rate of the ventilator. The femoral artery and vein were cannulated. Tracheal airway pressure and arterial blood pressure were monitored with a polygraph (Gould Instruments, Cleveland, OH, USA). During the period of ventilator use, intra-peritoneal sodium pentobarbital (0.05 mg/g) was administered every 30 min, and the intraperitoneally administered anesthetic fluid was sufficient to correct for hypovolemia. The chest was opened by incision of the left border of the sternum 6 h after the experiment.

Bronchoalveolar lavage fluid

The lungs were removed en bloc, and tubing was inserted into the trachea and secured. The right lung was clamped at the bronchus to prevent lavage fluid from entering. The left lung was lavaged by instilling 2.5 mL of normal saline twice. The recovered lavage samples were centrifuged at 1,500 g at the room temperature (RT) for 10 min. The supernatant was stored in a –80 °C refrigerator for the later measurement of cytokines. The white cell count in the BALF was determined by using a hemocytometer. Measurement of the concentration of cytokines, including macrophage inflammatory protein 2 (MIP-2), IL-1 β, and TNF-α, was performed by enzyme-linked immunosorbent assay kits (RandD Systems, Oxon, UK).

Measurement of hydrogen peroxide

BALF was centrifuged at 1,000 g within 30 min, the supernatant was collected, and 50 μl hydrogen peroxide (H2O2) reaction mix containing 46 μl assay buffer, two μl OxiRed™ probe solution, and 2 μl HRP solution (BioVision, USA) was added to the supernatant of the BALF and incubated for 10 min. Absorbance was read at 570 nm (SpectraMax M5; Molecular Devices, USA). The concentration was calculated based on H2O2 standard curves.

Measurement of thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS) are formed as a byproduct of lipid peroxidation diagnostic for tissue damage produced by oxidative stress. The TBARS level in the BALF supernatant was measured using the OxiSelectTM TBARS assay kit (Geneteks Biosciences, Inc.).

Measurement of myeloperoxidase, protein carbonyl, and reduced glutathione levels in lung tissue

The concentration of myeloperoxidase (MPO) in the right middle lung tissue, an index of neutrophil sequestration in the lungs, was measured as previously described. Lung tissue protein carbonyl content was measured by protein carbonyl assays (Geneteks Biosciences, Inc., San-Chong City, Taipei). Measurement of glutathione concentrations was carried out according to the method described by a commercially available kit (Sigma–Aldrich Fine Chemicals, Munich, Germany).

Western blotting analysis

Lung tissues were homogenized using lysis buffer containing a protease inhibitor cocktail (Roche, USA) and a phosphatase inhibitor cocktail (Roche, USA). Total protein extracts were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto polyvinylidine fluoride membrane (Millipore, USA). The membrane was blocked with 5% nonfat dry milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) for 1 h. Antibodies against phospho-p44/42 mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase [ERK] 1/2), phospho-stress-activated protein kinases/Janus kinase/signal transducers and activators of transcription (JNK), and phospho-p38 MAPK (1:1,000; Cell Signaling Technology, Beverly, MA, USA) were used. Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GADPH, 1:10,000; Lab Frontier, Korea), JNK (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-3 (1:2,000; Cell Signaling Technology), p-AKT (1:1,000; Cell Signaling Technology), AKT (1:1,000; Cell Signaling Technology), and heme oxygenase-1 (HO-1, 1:1000, Abcam, UK), were also used. The appropriate secondary antibodies were used (1:10,000 horseradish peroxidase anti-rabbit; Jackson Immuno Research Laboratories, West Grove, PA, USA). Visualization was performed by enhanced chemiluminescence (Visual Protein Biotechnology Corp., Taiwan). Protein bands were quantified using Kodak 1D image analysis (Eastman Kodak Company, Rochester, NY, USA).

Nuclear factor κB analysis of nuclear protein

Lung tissue was homogenized with a Dounce tissue homogenizer in 5-mL of Solution A (0.6% Nonidet P-40, 150 mM NaCl, 10 mM HEPES, pH 7.9, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged for 30 s at 2,000 rpm, and the supernatants were collected and centrifuged for 5 min at 5,000 rpm. The pelleted nuclei were resuspended at 4°C in 300 μL of solution B (25% glycerol, 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 5 μg/mL pepstatin A, 5 μg/mL leupeptin, 5 μg/mL aprotinin) and incubated on ice for 20 min. Samples were centrifuged at 15,000 rpm for 1 min. The total protein concentration in the extract was determined with a bicinchoninic acid protein assay (Pierce). The membrane was blocked for 1 h. Anti-nuclear factor κB (NF-κB) antibody (1:1,000; Cell Signaling Technology) and anti-PCNA antibody (1:1,000; Cell Signaling Technology) were diluted in TBST buffer (TBS/0.1% Tween 20) and incubated at 4°C overnight. The appropriate secondary antibody was used (1:10,000 horseradish peroxidase anti-rabbit Jackson ImmunoResearch Laboratories) at RT for 1 h. Visualization was performed by enhanced chemiluminescence (Visual Protein Biotechnology Corp). The protein bands on the destained gels were quantified with the Kodak 1D Image Analysis version 3.5 software package (Eastman Kodak Company). Anti-PCNA antibody was used as a loading control to correct for the pixel values of NF-κB.

Terminal deoxynucleotidyl transferase dUTP nick end labeling stain for apoptosis

Lung slides coated with poly-L-lysine (Sigma, St. Louis, MO, USA) were deparaffinized and rehydrated using xylene and ethanol. The background was diminished by preincubating samples with 3% bovine serum albumin (BSA) and 20% normal bovine serum in PBS for 30 min at RT. The specimens were then exposed for 1 h at 37°C in a moist chamber to a labeling mix containing 0.135 U/mL calf TdT, 0.0044 nmol/mL digoxigenin-11-2'-deoxy-uridine-5'-triphosphate and 1 mM Co chloride in distilled water. Following washing, the specimens were re-saturated in 3% BSA and 20% normal sheep serum, then treated (1 h at RT) with a 1.25 peroxidase U/mL dilution of peroxidase-labeled anti-digoxigenin sheep Fab fragment, followed by washing with 0.05% 3-3'-diaminobenzidine tetrahydrochloride (Dako, USA) color reaction. Analysis was performed under an Eclipse 80i microscope (Nikon, Japan) using Image Pro Plus 5.0 (Media Cybernetics, USA). The cells with positive terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining in nuclei were counted in groups of 100 cells on 3 slides of immunohistochemical stain for each animal tissue. Two pathologists blinded to the experimental condition carried out the morphological assessments.

Statistical analysis

Systat10.0 (Systat Software Inc., San Jose, CA, USA) was used for the statistical analyses. The comparisons among all groups were conducted using an ANOVA for repeated measurements. The comparisons between baseline and post-VILI values within each group were conducted using the Student's paired t-test. The values are expressed as the means ± standard deviation. P < 0.05 was considered statistically significant.

 Results



Effects of sulforaphane on systolic pressure and heart rate

Rats were randomly assigned as (a) control group ventilated with VT of 6 ml/kg, (b) 0.1% DMSO (vehicle control) group ventilated with VT of 6 ml/kg, (c) VILI group ventilated with high VT of 40 ml/kg, and (d) SFN-pretreated group ventilated with high VT of 40 ml/kg. SFN was prepared with 0.1% DMSO and administrated 30 min before intubation with the dosing of 3 mg/kg. After mechanical ventilation for 6 h, systolic pressure and heart rate were not statistically different by SFN treatment [Table 1]. The therapeutic mechanism of SFN might not be through the regulation of hemodynamic.{Table 1}

Decreased severity of lung edema and inflammation in sulforaphane -pretreated group

The lung wet to dry (W/D) ratio served as an indicator of pulmonary edema, as well as a total lung to body weight ratio. Following 6 h ventilation, the VILI group showed a significant increase in both ratios of lung W/D and total lung to body weight [Table 2]. Compared with the VILI group, SFN pretreatment significantly suppressed the severity of pulmonary edema. The number of white blood cells and cytokines level in BALF reflected pulmonary inflammatory status. High VT ventilation recruited leukocytes infiltration in VILI [Table 2]. However, fewer leukocytes count was identified in the SFN-pretreated group. The lavage fluid analysis revealed a general increase in cytokine levels and oxidative stress after 6 h of high VT ventilation [Figure 1]a. Furthermore, SFN suppressed VILI-induced proinflammatory cytokines and oxidative stress, such as TNF-α, IL-1 β, MIP-2, H2O2, and lipid peroxidation (TBARS). MPO activity has a positive correlation with neutrophils infiltration. Compared with the VILI group, MPO activities were lower in SFN pretreated group [Figure 1]b. Besides, pretreated SFN diminished VILI-induced protein decarbonylation and restored the glutathione level. In conclusion, rats with oral SFN supplement showed a protective effect against ventilator-induced pulmonary edema, inflammation, and oxidative stress.{Figure 1}{Table 2}

Effect of sulforaphane against apoptosis and nuclear factor κB activation

Ventilation has been observed to trigger cell apoptosis. TUNEL assay was applied to detect apoptosis activity in lung tissue sections. Increased immunostaining was noticed in the VILI group with massive alveolar collapse [Figure 2]a. In comparison, the SFN-pretreated group revealed decreased apoptosis with relatively intact alveolar space. Western blotting showed consistent results that cleavage caspase-3 expression was suppressed under SFN treatment [Figure 2]b. The pathogenic role of Nf-κB has been confirmed in ALI. Following 6 h ventilation, there was an upward trend of Nf-κB phosphorylation [Figure 2]b. Furthermore, SFN treatment could reverse ventilator-induced Nf-κB nuclear translocation. As a result, SFN pretreatment had the potency to inhibit apoptosis signaling and Nf-κB activation in the VILI model.{Figure 2}

Blockade of p38 phosphorylation and elevated hemeoxygenase-1 expression by sulforaphane pretreatment

The continuous physical force under ventilation was associated with marked activation of MAPK and AKT signaling. There was elevated phosphorylation of ERK, JNK, and p38 but not AKT in lung tissue homogenates [Figure 3]a, [Figure 3]b, [Figure 3]c and [Figure 3]e. Among the above signaling, SFN pretreatment exclusively mitigated p38 activation in the VILI group. Upraised HO-1 expression was beneficial in ALI through the anti-oxidant mechanism. Compared with the VILI group, SFN treatment further enhanced the HO-1 protein level under high VT ventilation [Figure 3]d. Conclusively, the therapeutic mechanism of SFN was postulated through inhibiting p38 phosphorylation with protective HO-1 expression.{Figure 3}

 Discussion



Following the trend of nutraceuticals as anti-inflammation agents, the purpose of the current study is to evaluate the efficacy of broccoli extract-SFN against VILI. Rats receiving high VT ventilation resulted in ALI manifested by lung edema, cytokines release, leukocytes infiltration, and pneumocytes apoptosis. In contrast, SFN pretreatment ameliorated the above pathogenic indices with significantly decreased oxidative stress. Our in vivo data supported the notion that SFN is capable of alleviating VILI in a prophylactic manner.

Neutrophil recruitment and activation are regarded as the vital feature in the early phase of VILI.[25] In the present experiment, leukocytes migrated into the lungs in response to high VT ventilation, as manifested by elevation in BALF cell count and tissue MPO activity. Pretreatment with SFN remarkably attenuated inflammatory cells sequestration, as well as cytokines, MIP-2 and Nf-κB elevation. These findings suggest that the protective effect of SFN could be attributed to immune-modulator against high ventilation-induced inflammation.

Apart from neutrophils accumulation, elevated oxidative stress is another feature of VILI. SFN was well-known as potent antioxidant through activation of Nrf-2 and its down-stream target HO-1.[26] Literature reviews have established SFN owned advantage of less systemic toxicity and high bioavailability.[27],[28] Our results are the first to describe the treatment of VILI with SFN, which could suppress reactive oxygen species production and subsequent pneumocytes apoptosis. Moreover, there has been reported that HO-1 upregulation exerted anti-inflammatory and anti-oxidant effects in ALI.[29] Increased HO-1 expression was found under VILI conditions and further augmented in SFN-treated group. Our results suggested that SFN has a role in adjuvant therapy of VILI targeted oxidative damage with HO-1 preconditioning.

MAPK pathways have been identified as critical regulators to translate mechanical force to gene regulation in VILI development.[30] In addition, MAPKs regulated the phosphorylation of Nrf2 and ARE-mediated HO-1 gene expression.[31] Western blotting analysis showed widespread elevation of phosphorylated MAPKs in high VT, whereas p38 MAPK signaling was exclusively mitigated under SFN pretreatment. The p38 MAPK kinase has been implicated in the mechanotransduction-associated apoptosis.[32] Moreover, increased p-p38 was correlated with MIP-2 production in the study of LPS -induced lung injury.[33] Our results elucidated therapeutic mechanism of SFN treatment through reduced p38 activation and inflammatory cytokine cascades.

 Conclusion



The application of SFN can effectively attenuate VILI through the inhibition of inflammation and reduction of oxidative stress and apoptosis in a high VT-induced lung injury rat model [Figure 4]. Our results hinted that bioactive food compound-SFN might function mechanistically to elevate HO-1, and subsequently proceed through the reduction of P38 and NF-κB activation to exert its protective effects.{Figure 4}

Financial support and sponsorship

This work was financially supported by Tri-Service General Hospital (TSGH C108-109 and TSGH C104-088) in Taiwan.

Conflicts of interest

There are no conflicts of interest.

References

1Wernerman J. Micronutrients against oxidative stress – Time for clinical recommendations? Crit Care 2012;16:124.
2Iacone R, Scanzano C, Santarpia L, D'Isanto A, Contaldo F, Pasanisi F. Micronutrient content in enteral nutrition formulas: Comparison with the dietary reference values for healthy populations. Nutr J 2016;15:30.
3Ojo O, Brooke J. Recent advances in enteral nutrition. Nutrients 2016;8:E709.
4Miyauchi T, Uchida Y, Kadono K, Hirao H, Kawasoe J, Watanabe T, et al. Preventive effect of antioxidative nutrient-rich enteral diet against liver ischemia and reperfusion injury. JPEN J Parenter Enteral Nutr 2019;43:133-44.
5Houghton CA, Fassett RG, Coombes JS. Sulforaphane and other nutrigenomic Nrf2 activators: Can the clinician's expectation be matched by the reality? Oxid Med Cell Longev 2016;2016:7857186.
6Liang J, Jahraus B, Balta E, Ziegler JD, Hübner K, Blank N, et al. Sulforaphane inhibits inflammatory responses of primary human T-cells by increasing ROS and depleting glutathione. Front Immunol 2018;9:2584.
7Mazarakis N, Snibson K, Licciardi PV, Karagiannis TC. The potential use of l-sulforaphane for the treatment of chronic inflammatory diseases: A review of the clinical evidence. Clin Nutr 2020;39:664-75.
8Brown RH, Reynolds C, Brooker A, Talalay P, Fahey JW. Sulforaphane improves the bronchoprotective response in asthmatics through Nrf2-mediated gene pathways. Respir Res 2015;16:106.
9Heber D, Li Z, Garcia-Lloret M, Wong AM, Lee TY, Thames G, et al. Sulforaphane-rich broccoli sprout extract attenuates nasal allergic response to diesel exhaust particles. Food Funct 2014;5:35-41.
10Lv Y, Jiang H, Li S, Han B, Liu Y, Yang D, et al. Sulforaphane prevents chromium-induced lung injury in rats via activation of the Akt/GSK-3β/Fyn pathway. Environ Pollut 2020;259:113812.
11Qi T, Xu F, Yan X, Li S, Li H. Sulforaphane exerts anti-inflammatory effects against lipopolysaccharide-induced acute lung injury in mice through the Nrf2/ARE pathway. Int J Mol Med 2016;37:182-8.
12Sun Z, Niu Z, Wu S, Shan S. Protective mechanism of sulforaphane in Nrf2 and anti-lung injury in ARDS rabbits. Exp Ther Med 2018;15:4911-5.
13Kuchnicka K, Maciejewski D. Ventilator-associated lung injury. Anaesthesiol Intensive Ther 2013;45:164-70.
14Webb HH, Tierney DF. Experimental pulmonary edema due to intermittent positive pressure ventilation with high inflation pressures. Protection by positive end-expiratory pressure. Am Rev Respir Dis 1974;110:556-65.
15Oeckler RA, Hubmayr RD. Ventilator-associated lung injury: A search for better therapeutic targets. Eur Respir J 2007;30:1216-26.
16Ranieri VM, Suter PM, Tortorella C, De Tullio R, Dayer JM, Brienza A, et al. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: A randomized controlled trial. JAMA 1999;282:54-61.
17Imai Y, Parodo J, Kajikawa O, de Perrot M, Fischer S, Edwards V, et al. Injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome. JAMA 2003;289:2104-12.
18Uhlig S, Uhlig U. Pharmacological interventions in ventilator-induced lung injury. Trends Pharmacol Sci 2004;25:592-600.
19Raghavendran K, Pryhuber GS, Chess PR, Davidson BA, Knight PR, Notter RH. Pharmacotherapy of acute lung injury and acute respiratory distress syndrome. Curr Med Chem 2008;15:1911-24.
20Putensen C, Hörmann C, Kleinsasser A, Putensen-Himmer G. Cardiopulmonary effects of aerosolized prostaglandin E1 and nitric oxide inhalation in patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 1998;157:1743-7.
21Vincent JL, Brase R, Santman F, Suter PM, McLuckie A, Dhainaut JF, et al. A multi-centre, double-blind, placebo-controlled study of liposomal prostaglandin E1 (TLC C-53) in patients with acute respiratory distress syndrome. Intensive Care Med 2001;27:1578-83.
22Bernard GR, Luce JM, Sprung CL, Rinaldo JE, Tate RM, Sibbald WJ, et al. High-dose corticosteroids in patients with the adult respiratory distress syndrome. N Engl J Med 1987;317:1565-70.
23Bernard GR, Wheeler AP, Arons MM, Morris PE, Paz HL, Russell JA, et al. A trial of antioxidants N-acetylcysteine and procysteine in ARDS. The Antioxidant in ARDS Study Group. Chest 1997;112:164-72.
24Liu YY, Chiang CH, Chuang CH, Liu SL, Jheng YH, Ryu JH. Spillover of cytokines and reactive oxygen species in ventilator-induced lung injury associated with inflammation and apoptosis in distal organs. Respir Care 2014;59:1422-32.
25Imanaka H, Shimaoka M, Matsuura N, Nishimura M, Ohta N, Kiyono H. Ventilator-induced lung injury is associated with neutrophil infiltration, macrophage activation, and TGF-beta 1 mRNA upregulation in rat lungs. Anesth Analg 2001;92:428-36.
26Morimitsu Y, Nakagawa Y, Hayashi K, Fujii H, Kumagai T, Nakamura Y, et al. A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway. J Biol Chem 2002;277:3456-63.
27Doss JF, Jonassaint JC, Garrett ME, Ashley-Koch AE, Telen MJ, Chi JT. Phase 1 study of a sulforaphane-containing broccoli sprout homogenate for sickle cell disease. PLoS One 2016;11:e0152895.
28Holst B, Williamson G. A critical review of the bioavailability of glucosinolates and related compounds. Nat Prod Rep 2004;21:425-47.
29Zhao WJ, Hu WW. A study of the role of heme oxygenase-1 expression in ventilator induced lung injury and its mechanism in rats. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 2010;22:410-3.
30Uhlig U, Haitsma JJ, Goldmann T, Poelma DL, Lachmann B, Uhlig S. Ventilation-induced activation of the mitogen-activated protein kinase pathway. Eur Respir J 2002;20:946-56.
31Martin D, Rojo AI, Salinas M, Diaz R, Gallardo G, Alam J, et al. Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Biol Chem 2004;279:8919-29.
32Abdulnour RE, Peng X, Finigan JH, Han EJ, Hasan EJ, Birukov KG, et al. Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways. Am J Physiol Lung Cell Mol Physiol 2006;291:L345-53.
33Nick JA, Young SK, Brown KK, Avdi NJ, Arndt PG, Suratt BT, et al. Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J Immunol 2000;164:2151-9.