Biochemical and Biophysical Research Communications...chial epithelial cells in 5 random, high-power ﬁelds per section 137 were counted with the samples’ identities masked. 138

1

2

3

4

5

6789

1011

1 3

141516

1718192021222324

2 5

45

46

47

48

49

50

51

52

53

54

Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx

YBBRC 26553 No. of Pages 6, Model 5G

10 April 2011

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate /ybbrc

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lunginjury via a mechanism involving nuclear factor-kappa B activation

Chien-Sheng Huang a,b, Tomohiro Kawamura a, Ximei Peng a, Naofumi Tochigi c, Norihisa Shigemura a,Timothy R. Billiar d, Atsunori Nakao a,d,⇑, Yoshiya Toyoda a

a Department of Cardiothoracic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, United Statesb Division of Thoracic Surgery, Department of Surgery, Taipei-Veterans General Hospital and National Yang-Ming University School of Medicine, Taipei, Taiwanc Department of Pathology, University of Pittsburgh Medical Center, PA, United Statesd Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, United States

a r t i c l e i n f o a b s t r a c t

2627282930313233343536

Article history:Received 30 March 2011Available online xxxx

Keywords:HydrogenNuclear factor jBVentilator-induced lung injuryApoptosisBcl-2Inhalation

373839404142

0006-291X/$ - see front matter � 2011 Published bydoi:10.1016/j.bbrc.2011.04.008

Abbreviations: Bax, B-cell lymphoma-2 (Bcl-2)-B-cell lymphoma-2; BSA, bovine serum albumin;traacetic acid; ELISA, enzyme-linked immuno sorbentmobility shift assay; MV, mechanical ventilation; NVILI, ventilator induced lung injury.⇑ Corresponding author at: Department of Cardioth

Pittsburgh Medical Center, E1551 Biomedical SciencPittsburgh, PA 15213, United States. Fax: +1 412 624

E-mail address: [email protected] (A. Nakao).

Please cite this article in press as: C.-S. Huang einvolving nuclear factor-kappa B activation, Bio

We recently demonstrated the inhalation of hydrogen gas, a novel medical therapeutic gas, amelioratesventilator-induced lung injury (VILI); however, the molecular mechanisms by which hydrogen amelio-rates VILI remain unclear. Therefore, we investigated whether inhaled hydrogen gas modulates thenuclear factor-kappa B (NFjB) signaling pathway. VILI was generated in male C57BL6 mice by performinga tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg or 10 ml/kgwithout positive end-expiratory pressure). The ventilator delivered either 2% nitrogen or 2% hydrogenin balanced air. NFjB activation, as indicated by NFjB DNA binding, was detected by electrophoreticmobility shift assays and enzyme-linked immunosorbent assay. Hydrogen gas inhalation increased NFjBDNA binding after 1 h of ventilation and decreased NFjB DNA binding after 2 h of ventilation, as com-pared with controls. The early activation of NFjB during hydrogen treatment was correlated with ele-vated levels of the antiapoptotic protein Bcl-2 and decreased levels of Bax. Hydrogen inhalationincreased oxygen tension, decreased lung edema, and decreased the expression of proinflammatorymediators. Chemical inhibition of early NFjB activation using SN50 reversed these protective effects.NFjB activation and an associated increase in the expression of Bcl-2 may contribute, in part, to the cyto-protective effects of hydrogen against apoptotic and inflammatory signaling pathway activation duringVILI.

� 2011 Published by Elsevier Inc.

43

44

55

56

57

58

59

60

61

62

63

64

1. Introduction

Recent basic and clinical research studies have revealed thathydrogen is an important physiological regulatory molecule withantioxidant, anti-inflammatory and antiapoptotic protective effectson cells and organs [1]. Although the therapeutic efficacies of hydro-gen have been extensively studied, there is very limited informationon the pathways and processes regulated in vivo by the hydrogenmolecule. In the body, hydrogen may serve as a modulator of signaltransduction, like other gaseous signaling molecules (e.g., nitric

65

66

67

68

69

70

71

72

73

Elsevier Inc.

associated X-protein; Bcl-2,EDTA, ethylene diaminete

assay; EMSA, electrophoreticFjB, nuclear factor-kappa B;

oracic Surgery, University ofe Tower, 200 Lothrop Street,6666.

t al., Hydrogen inhalation reducchem. Biophys. Res. Commun.

oxide, carbon monoxide and hydrogen sulfide) [2–5], and hydrogenhas been proposed as ‘‘the fourth signaling gaseous molecule’’ [6].

We recently demonstrated that hydrogen inhalation amelio-rates ventilator-induced lung injury (VILI) through its antiapopto-tic and anti-inflammatory properties [7]. Lung cell apoptosis occursduring VILI and is one of the most detrimental processes duringVILI, initiating inflammation and subsequent tissue injury [8]. Inour previous study, we determined that the antiapoptotic abilitiesof hydrogen were at least partially explained by upregulation ofthe antiapoptotic gene, B-cell lymphoma-2 (Bcl-2) [5,7]. Nuclearfactor-jB (NFjB), a multisubunit transcription factor, plays a piv-otal role in protecting cultured cells and lung epithelial cellsin vivo against apoptotic cell death by induction of antiapoptoticgenes, such as Bcl-2 family members (e.g., Bcl-2, Bcl-xl) [9–16].Accordingly, we hypothesized that the antiapoptotic effects ofhydrogen inhalation during mechanical ventilation may be medi-ated by an activation of NFjB. The results of this study may definea novel mechanism underlying the antiapoptotic/anti-inflamma-tory effects of hydrogen during VILI.

ed epithelial apoptosis in ventilator-induced lung injury via a mechanism(2011), doi:10.1016/j.bbrc.2011.04.008

http://dx.doi.org/10.1016/j.bbrc.2011.04.008

mailto:[email protected]


http://www.sciencedirect.com/science/journal/0006291X

http://www.elsevier.com/locate/ybbrc


74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

2 C.-S. Huang et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx


10 April 2011

2. Methods

2.1. Animals

Male wild type C57BL6 mice (10–12 weeks old) were purchasedfrom the Jackson Laboratory (Bar Harbor, ME). All animals weremaintained in laminar flow cages in a specific-pathogen-free facil-ity at the University of Pittsburgh. The experimental protocol wasapproved by the Institutional Animal Care and Use Committee ofthe University of Pittsburgh and all experiments were performedin adherence to the National Institute of Health guidelines for theuse of laboratory animals.

2.2. VILI model

VILI was generated in mice using high tidal volume mechani-cal ventilation (MV) as previous described [7]. Briefly, mice wereanesthetized by intraperitoneal injection of 85 mg/kg ketamineand 15 mg/kg xylazine. Then, under the sterile conditions, a tra-cheostomy was performed with a 20-gauge angiocatheter, whichwas then sutured in place. Mice were placed supine on a warmingdevice and connected to a ventilator (Harvard Apparatus Co.,

Fig. 1. (A) EMSA for NFjB, representative of three independent experiments. (B) HistograELISA-based measurement of p65 DNA binding using nuclear protein isolated from lung

Please cite this article in press as: C.-S. Huang et al., Hydrogen inhalation reducinvolving nuclear factor-kappa B activation, Biochem. Biophys. Res. Commun.

Holliston, MA, USA) on volume-control mode at a constant inspi-ratory flow. MV was initiated with a tidal volume of 30 ml/kgwithout an end-expiratory pressure at a respiratory rate of120 breaths/min. Mean arterial blood pressure was continuouslymonitored using a blood pressure monitor (Cardiomax-III, Colum-bus, OH) via catheterization of a femoral artery. Mice receivedintravenous injection of 0.05 ml/h saline, as well as intraperito-neal ketamine and xylazine to maintain the blood pressure at75–80 mm Hg. At the end of the experiment, the mice wereeuthanized with 150 mg/kg ketamine intraperitoneally and tissuewas collected for analysis.

2.3. Chemical inhibition of NFjB

Chemical blockade of NFjB was performed by intraperitonealinjection of SN50 (2 lg/g, Calbiochem, San Diego, CA) 30 min priorto ventilation [17].

2.4. Experimental design

The mice were randomly assigned to 1 of 5 experimentalgroups. The animals in sham group (Group 1, n = 6) received 2%

m of band intensity calculated using NIH image (n = 3 for each group, ⁄p < 0.05). (C)tissue after 1 h of MV (n = 3–4 for each group, ⁄p < 0.05).



110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

C.-S. Huang et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx 3


10 April 2011

nitrogen in air for 2 h and underwent anesthesia only prior to pro-curement of lung tissue. Animals in the sham/N2/SN50 group(Group 2, n = 6) were treated the same as those in the sham/N2

group, but were injected with SN50 2 h before sacrifice. Mice inthe VILI/N2 group (Group 3) were ventilated for 1 or 2 h with 2%nitrogen in air (n = 6 for each time point) and mice in the VILI/H2group (Group 4) were ventilated for 1 or 2 h with 2% hydrogen inair (n = 6 for each time point). In the final experimental group(Group 5), the mice were treated with SN50 and ventilated with2% hydrogen in air for 1 or 2 h (VILI/H2/SN50, n = 6 for each timepoint).

2.5. Histopathological analysis

For histological evaluation, the right middle lobes of the lungswere fixed in 10% formalin, embedded in paraffin, sectioned to6 lm thickness, and stained with hematoxylin and eosin. Theslides were blindly reviewed by one of the authors (NT) withoutknowledge of experimental groups (n = 6 for each group). Acutelung injury was scored according to the following four items: alve-olar congestion, hemorrhage, infiltration or aggregation of neutro-phils in the airspace or the vessel wall, and thickness of thealveolar wall/hyaline membrane formation. Each item was gradedaccording to a 5-point scale: 0, minimal (little) damage; 1, milddamage; 2, moderate damage; 3, severe damage; and 4, maximaldamage. Terminal deoxynucleotidyl transferase-mediateddeoxyuridine triphosphate nick-end labeling (TUNEL) was usedfor identification of bronchiolar cell apoptosis with the ApopTag

Fig. 2. (A) Bcl-2 mRNA levels. (B) Bax mRNA levels. RNA was isolated from the lungs afterBcl-2 and Bax using cytoplamic proteins isolated from the lungs after 2 h of MV. Represe(HPF, 400�) in lung tissue after 2 h of MV (n = 6, ⁄p < 0.05).


Peroxidase Kit (Intergen Co., Purchase, NY). TUNEL-positive bron-chial epithelial cells in 5 random, high-power fields per sectionwere counted with the samples’ identities masked.

2.6. Arterial blood analysis

At the end of the experiment, arterial blood was obtained fromthe abdominal aorta. Blood gas levels and blood lactate concentra-tion were assessed using iSTAT (Abaxis, Union City, CA).

2.7. Wet-to-dry weight ratio

Pulmonary edema was assessed by the wet-to-dry weight ratioof the lung tissue. The right lower lobe was weighed immediatelyafter collection and then placed into a 60 �C oven to dry. After2 days, the dried tissue was weighed to determine the wet-to-drylung weight ratio.

2.8. Realtime RT-PCR

The mRNAs for TNFa, Bcl-2, Bax and b-actin were quantified induplicate using SYBR Green two-step, real-time RT-PCR, as previ-ously described [7].

2.9. Western blot

Western blot analysis was performed on 15 lg of cytoplasmicextracts from lung tissue, as described previously [5]. After block-

2 h of MV and quantitated by real time RT-PCR (n = 6, ⁄p < 0.05). (C) Western blot forntative of 3 independent experiments. (D) TUNEL-positive cells per high power field



156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

Fig. 3. (A) Partial pressure of oxygen (PaO2). (B) Partial pressure of carbon dioxide (PaCO2). (C) Wet/dry ratio of the lungs. (D) TNFa mRNA levels. All assays were performedafter 2 h of MV (n = 6, ⁄p < 0.05).



10 April 2011

ing of nonspecific binding with nonfat dry milk, membranes wereincubated overnight with the primary antibodies. The followingprimary antibodies were used: anti-Bcl-2 (Cell Signaling Technol-ogy, Beverly, MA), anti-Bax (Cell Signaling Technology), and anti-b-actin (Sigma–Aldrich, St. Louis, MO). After incubation withhorseradish-peroxidase conjugated goat anti-rabbit or anti-mousesecondary antibodies (Pierce Chemical, Rockford, IL), the mem-branes were developed with the SuperSignal detection system(Pierce Chemical) and exposed to film.

189

190

191

192

193

194

195

196

197

198

199

200

2.10. Nuclear factor jB (NFjB) DNA binding activity

NFjB DNA binding activity was measured by electrophoreticmobility shirt assay (EMSA) using nuclear extracts from lung tissueobtained 1 or 2 h after starting MV and an NFjB oligonucleotide(Promega, Madison, WI) based on the NFjB sequence in the immu-noglobulin light-chain enhancer. DNA probes were prepared byend labeling with [c-32P]dATP (DuPont, Merck Pharmaceuticals)and T4 polynucleotide kinase (Boehringer–Mannheim BiomedicalProducts, Mannheim, Germany) and purified in Tris–EDTA buffercontaining NaCl (100 mM) using G-50 resin columns (Whatman,Newton, MA). Typically, 10 lg of nuclear extract was incubatedwith 100,000 counts/min of 32P-labeled oligonucleotide (0.5 ng)for 1–2 h at room temperature in a buffer containing 10 mM Tris(pH 7.6), 10% glycerol, 1 mM EDTA, 1 mg/ml BSA, and 0.2% NonidetP-40. Protein–DNA complexes were resolved on 4% nondenaturing


polyacrylamide gels in 0.4� running buffer containing 450 mMTris–borate and 1 mM EDTA (pH 8.0). Gels were dried after electro-phoresis and subjected to autoradiography. The band intensitieswere quantified by NIH image analysis software [18]. NFjB DNAbinding activity in nuclear extracts from lung tissue was also mea-sured with an enzyme-linked immunosorbent assay (ELISA)-basedassay using a commercially available, colorimetric kit (TransAM™NFjB p65 Transcription Factor Assay Kit; ACTIVE MOTIF, Carlsbad,CA).

2.11. Statistical analysis

Results are presented as mean ± standard deviation (SD). TheEZAnalyze add-in for Microsoft Excel was used to perform ANOVAwith an F test and Bonferroni posthoc group comparisons. A prob-ability level of p < 0.05 was considered statistically significant.

3. Results

3.1. Hydrogen inhalation resulted in early NFjB activation

To examine the effect of VILI and hydrogen inhalation on NFjBactivity, EMSA was performed to measure NFjB-DNA bindingactivity in the nuclear extracts of lung tissues after 1 or 2 h ofMV. Compared with the basal levels of NFjB-DNA binding activityin the lung tissue of sham mice, NFjB DNA-binding activity was



201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

C.-S. Huang et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx 5


10 April 2011

increased in the lung tissue after 1 h of MV and was maintained athigh levels after 2 h of MV (Fig. 1A and B). Interestingly, the DNA-binding activity of NFjB was significantly stronger in the lungstreated with 2% hydrogen after 1 h of MV, as comparing with lungstreated with 2% nitrogen, but was significantly less than that seenin the nitrogen-treated lungs after 2 h of MV. ELISA of DNA-boundNFjB p65 using nuclear protein isolated from the lungs after 1 h ofMV further confirmed significantly higher activation of NFjB in themice treated with hydrogen as compared with nitrogen-treatedmice (Fig. 1C). Treatment with SN50, a chemical inhibitor of NFjB,inhibited early activation of NFjB DNA binding in EMSA and ELISAafter 1 h of MV. After both 1 and 2 h of MV, concomitant treatmentwith 2% hydrogen and SN50 resulted in NFjB DNA binding thatwas not significantly different from that of the control group trea-ted with 2% nitrogen (Fig. 1A–C).

3.2. The effects of hydrogen on expression of antiapoptic molecules inVILI

Because NFjB is a key transcription factor in the regulation ofapoptosis, we investigated the effects of hydrogen treatment dur-ing MV on apoptosis and the expression of apoptotic regulatoryproteins. Real-time RT-PCR and Western blot analyses demon-strated that hydrogen inhalation increased expression of the anti-apoptotic protein Bcl-2 and reduced VILI-induced expression of thepro-apoptotic protein Bax after 2 h of MV (Fig. 2A and C). More-over, MV with 2% hydrogen for 2 h significantly reduced TUNEL-positive epithelial cells as compared with ventilation with 2%nitrogen (Fig. 2D). These effects of hydrogen were completely abro-gated by NFjB inhibition with SN50. These results suggest that the

Fig. 4. (A) Hematoxylin and eosin staining of lung tissue after 2 h of MV. The representscores after 2 h of MV (n = 6, ⁄p < 0.05).


ability of hydrogen to prevent VILI is, at least in part, mediated byinduction of Bcl-2 by NFjB.

3.3. Inhibition of NFjB activation abrogated the protective effects ofhydrogen in the VILI model

Arterial blood gas levels, wet/dry ratio of the lungs and TNFamRNA levels were measured after 2 h of MV. As in our previousstudy, ventilation with 2% hydrogen in air protected the lungs,improving oxygenation of the arterial blood, reducing lung edema,and attenuating TNF-a mRNA upregulation (Fig. 3A–D). Chemicalinhibition of NFjB using SN50 completely abrogated the protectiveeffects of hydrogen after 2 h of MV, including hydrogen’s ability tofacilitate gas exchange (Fig. 3A and B), prevent lung edema (Fig. 3C)and block upregulation of TNFa mRNA (Fig. 3D). These resultsstrongly suggest that the ability of hydrogen to prevent VILI is, atleast in part, mediated by an early activation of NFjB.

3.4. Hydrogen improved the lung injury scores

MV led to pulmonary inflammation and injury, as indicated bythickening of the alveolar septum and infiltration of inflammatorycells, evident in histopathological examination of the lung samplestaken after 2 h of MV. In the presence of hydrogen, both edema andinflammatory cell infiltration were reduced. When early activationof NFjB was inhibited by SN50 treatment, the protective effects ofhydrogen against edema and inflammatory cell infiltration werenot evident (Fig. 4A and B). SN50 did not cause histopathologicalchanges in sham animals (Fig. 4B).

ative images are shown (n = 6 per treatment). Magnification 400�. (B) Lung injury



254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311312313314315316

317318319320321322323324325326327Q1328329330331332333334335336337338339340341342343344345346347348349350351352353354355356357358359360361362363364365366367368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397398399400



10 April 2011

4. Discussion

In this study, we demonstrated that activation of an NFjB/Bcl-2signaling pathway may contribute to the cytoprotective effects ofhydrogen against apoptotic and inflammatory cascades duringVILI. Our results suggest that activation of NFjB, indirectly or indi-rectly, by hydrogen inhalation may drive the expression of protec-tive genes or cytoprotective enzymes, such as Bcl-2, which thenfurther inhibit apoptotic and inflammatory damage initiated byVILI. The abrogation of the protective effects of hydrogen by chem-ical inhibition of NFjB strongly supports an important role of NFjBactivation in protection against VILI in our model.

The beneficial effects of inhaled hydrogen are undoubtedly due,in part, to its antioxidant properties, although the direct, free-rad-ical-scavenging activities of hydrogen require further investigation[19–21]. However, there is very limited information on the path-ways and processes regulated in vivo by the hydrogen molecule.Scavenging properties are unlikely to be the only explanation,and undefined biological mechanisms of hydrogen as a signalingmolecule may be involved.

The function of NFjB activation during VILI has not been fullyelucidated and conflicting roles for NFjB, protective and injurious,have been proposed. The detrimental effects of NFjB activation arewell-documented. NFjB triggers upregulation of genes involved ininflammation, infection, and stress response to various stimuli andleads to activation of proinflammatory and proapoptotic cascades[17,22–24]. On the other hand, NFjB mediates a cellular survivalmechanism against apoptotic cell death [10,11]. A previous studyshowed that NFjB activation correlated with elevated levels ofBcl-2 and reduced myocardial necrosis/apotosis induced by ische-mia/reperfusion injury [14]. NFjB activation is also involved inthe neuroprotective actions associated with Bcl-2 and Bcl-xL induc-tion [25]. Zhong et al. suggested a protective role of the NFjB/Bcl-2cascade in prevention of lung cell apoptosis induced by tobaccosmoking [16] or hyperoxia lung injury [15,26,27]. In addition, a crit-ical role for NFjB in mediating the proliferation and blocking apop-tosis was seen in mice hepatectomy model [28]. Thus, it is likelythat NFjB may have dual roles and can be pro- or anti-inflamma-tory and pro- or anti-apoptotic depending on the experimentalmodel, cell type, location and timing of stimuli, and a subunit com-position of the NFjB complex.

There are some limitations to our study and several areas re-quire further investigation including which NFjB subunits are in-volved and how hydrogen activates the NFjB pathway. Thetiming of NFjB activation by hydrogen and the underlying mecha-nism and importance of the differences in NFjB DNA binding after1 and 2 h of MV are also areas for future study. Additionally, stud-ies are ongoing in our laboratory to investigate which other geneproducts participate in the preservation and maintenance of tissueintegrity afforded by hydrogen during MV.

In conclusion we demonstrated that NFjB is activated by hydro-gen inhalation during an early phase of VILI and is correlated withthe elevated levels of the antiapoptotic protein Bcl-2. Furthermore,the protective effects of hydrogen inhalation were reversed by achemical inhibitor of NFjB. We postulate that the NFjB-Bcl-2 path-way might be one of the key mechanisms underlying the protectiveactions of hydrogen in this VILI model.

References

[1] C.S. Huang, T. Kawamura, Y. Toyoda, A. Nakao, Recent advances in hydrogenresearch as a therapeutic medical gas, Free Radic. Res. 44 (2010) 971–982.

[2] A. Nakao, R. Sugimoto, T.R. Billiar, K.R. McCurry, Therapeutic antioxidantmedical gas, J. Clin. Biochem. Nutr. 44 (2009) 1–13.

[3] T. Itoh, Y. Fujita, M. Ito, A. Masuda, K. Ohno, M. Ichihara, T. Kojima, Y. Nozawa,M. Ito, Molecular hydrogen suppresses FcepsilonRI-mediated signal

401


transduction and prevents degranulation of mast cells, Biochem. Biophys.Res. Commun. 389 (2009) 651–656.

[4] N. Kamimura, K. Nishimaki, I. Ohsawa, S. Ohta, Molecular hydrogen improvesobesity and diabetes by inducing hepatic FGF21 and stimulating energymetabolism in db/db mice, Obesity (Silver Spring).

[5] T. Kawamura, C.S. Huang, N. Tochigi, S. Lee, N. Shigemura, T.R. Billiar, M.Okumura, A. Nakao, Y. Toyoda, Inhaled hydrogen gas therapy for prevention oflung transplant-induced ischemia/reperfusion injury in rats, Transplantation(2010).

[6] J.F. George, A. Agarwal, Hydrogen; another gas with therapeutic potential,Kidney Int. 77, 85–87.

[7] C.S. Huang, T. Kawamura, S. Lee, N. Tochigi, N. Shigemura, B.M. Buchholz, J.D.Kloke, T.R. Billiar, Y. Toyoda, A. Nakao, Hydrogen inhalation amelioratesventilator-induced lung injury, Crit. Care 14 (2010) R234.

[8] M.A. Daemen, C. Van’t Veer, G. Denecker, V.H. Heemskerk, T.G. Wolfs, M.Clauss, P. Vandenabeele, W.A. Buurman, Inhibition of apoptosis induced byischemia-reperfusion prevents inflammation, J. Clin. Invest. 104 (1999) 541–549.

[9] C.Y. Wang, D.C. Guttridge, M.W. Mayo, A.S. Baldwin Jr., NF-kappaB inducesexpression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppresschemotherapy-induced apoptosis, Mol. Cell. Biol. 19 (1999) 5923–5929.

[10] A.A. Beg, D. Baltimore, An essential role for NF-kappaB in preventing TNF-alpha-induced cell death, Science 274 (1996) 782–784.

[11] D.J. Van Antwerp, S.J. Martin, T. Kafri, D.R. Green, I.M. Verma, Suppression ofTNF-alpha-induced apoptosis by NF-kappaB, Science 274 (1996) 787–789.

[12] Z.L. Chu, T.A. McKinsey, L. Liu, J.J. Gentry, M.H. Malim, D.W. Ballard,Suppression of tumor necrosis factor-induced cell death by inhibitor ofapoptosis c-IAP2 is under NF-kappaB control, Proc. Natl. Acad. Sci. USA 94(1997) 10057–10062.

[13] W.R. Franek, S. Horowitz, L. Stansberry, J.A. Kazzaz, H.C. Koo, Y. Li, Y. Arita, J.M.Davis, A.S. Mantell, W. Scott, L.L. Mantell, Hyperoxia inhibits oxidant-inducedapoptosis in lung epithelial cells, J. Biol. Chem. 276 (2001) 569–575.

[14] H. Choi, S.H. Kim, Y.S. Chun, Y.S. Cho, J.W. Park, M.S. Kim, In vivo hyperoxicpreconditioning prevents myocardial infarction by expressing bcl-2, Exp. Biol.Med. (Maywood) 231 (2006) 463–472.

[15] T.E. Zaher, E.J. Miller, D.M. Morrow, M. Javdan, L.L. Mantell, Hyperoxia-inducedsignal transduction pathways in pulmonary epithelial cells, Free Radic. Biol.Med. 42 (2007) 897–908.

[16] C.Y. Zhong, Y.M. Zhou, K.E. Pinkerton, NF-kappaB inhibition is involved intobacco smoke-induced apoptosis in the lungs of rats, Toxicol. Appl.Pharmacol. 230 (2008) 150–158.

[17] Y.Y. Liu, S.K. Liao, C.C. Huang, Y.H. Tsai, D.A. Quinn, L.F. Li, Role for nuclearfactor-kappaB in augmented lung injury because of interaction betweenhyperoxia and high stretch ventilation, Transl. Res. 154 (2009) 228–240.

[18] A. Nakao, J. Schmidt, T. Harada, A. Tsung, B. Stoffels, R.J. Cruz Jr., J. Kohmoto,X. Peng, K. Tomiyama, N. Murase, A.J. Bauer, M.P. Fink, A singleintraperitoneal dose of carbon monoxide-saturated ringer’s lactate solutionameliorates postoperative ileus in mice, J. Pharmacol. Exp. Ther. 319 (2006)1265–1275.

[19] I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata,K. Katsura, Y. Katayama, S. Asoh, S. Ohta, Hydrogen acts as a therapeuticantioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13(2007) 688–694.

[20] K.I. Fukuda, S. Asoh, M. Ishikawa, Y. Yamamoto, I. Ohsawa, S. Ohta, Inhalationof hydrogen gas suppresses hepatic injury caused by ischemia/reperfusionthrough reducing oxidative stress, Biochem. Biophys. Res. Commun. 361(2007) 670–674.

[21] B.M. Buchholz, D.J. Kaczorowski, R. Sugimoto, R. Yang, Y. Wang, T.R. Billiar, K.R.McCurry, A.J. Bauer, A. Nakao, Hydrogen inhalation ameliorates oxidativestress in transplantation induced intestinal graft injury, Am. J. Transplant. 8(2008) 2015–2024.

[22] C.H. Chiang, H.I. Pai, S.L. Liu, Ventilator-induced lung injury (VILI) promotesischemia/reperfusion lung injury (I/R) and NF-kappaB antibody attenuatesboth injuries, Resuscitation 79 (2008) 147–154.

[23] H.D. Held, S. Boettcher, L. Hamann, S. Uhlig, Ventilation-induced chemokineand cytokine release is associated with activation of nuclear factor-kappaB andis blocked by steroids, Am. J. Respir. Crit. Care Med. 163 (2001) 711–716.

[24] T.A. Chatila, J.B. Smith, NF-kappaB and the innate immune response in therespiratory distress syndrome of the newborn: commentary on the article byCheah et al. on page 616, Pediatr. Res. 57 (2005) 613–615.

[25] M. Tamatani, Y.H. Che, H. Matsuzaki, S. Ogawa, H. Okado, S. Miyake, T. Mizuno,M. Tohyama, Tumor necrosis factor induces Bcl-2 and Bcl-x expressionthrough NFkappaB activation in primary hippocampal neurons, J. Biol. Chem.274 (1999) 8531–8538.

[26] W.R. Franek, D.M. Morrow, H. Zhu, I. Vancurova, V. Miskolci, K. Darley-Usmar,H.H. Simms, L.L. Mantell, NF-kappaB protects lung epithelium againsthyperoxia-induced nonapoptotic cell death-oncosis, Free Radic. Biol. Med. 37(2004) 1670–1679.

[27] G. Yang, A. Abate, A.G. George, Y.H. Weng, P.A. Dennery, Maturationaldifferences in lung NF-kappaB activation and their role in tolerance tohyperoxia, J. Clin. Invest. 114 (2004) 669–678.

[28] M.L. Chaisson, J.T. Brooling, W. Ladiges, S. Tsai, N. Fausto, Hepatocyte-specificinhibition of NF-kappaB leads to apoptosis after TNF treatment, but not afterpartial hepatectomy, J. Clin. Invest. 110 (2002) 193–202.



Documents

Biochemical and Biophysical Research Communications...chial epithelial cells in 5 random, high-power ﬁelds per section 137 were counted with the samples’ identities masked. 138