COMBINATION THERAPY WITH MOLECULAR HYDROGEN AND HYPEROXIA ...kelieng.com/uploads/file/150726/07-26162046_6804.pdf · Oxygen is often used in critically ill patients. Early goal-directed

COMBINATION THERAPY WITH MOLECULAR HYDROGEN ANDHYPEROXIA IN A MURINE MODEL OF POLYMICROBIAL SEPSIS

Keliang Xie,* Wenzheng Fu,† Weibin Xing,‡ Ailin Li,§ Hongguang Chen,*Huanzhi Han,* Yonghao Yu,* and Guolin Wang*

*Department of Anesthesiology, General Hospital of Tianjin Medical University; †Department of Colorectaland Anal Surgery, Tianjin Union Medical Center, Tianjin People’s Hospital, Tianjin; ‡Department of

Dermatology, Cangzhou People’s Hospital, Hebei Province, Hebei; and §Department of Neurosurgery,Tianjin Huanhu Hospital, Tianjin, People’s Republic of China

Received 19 Jul 2012; first review completed 7 Aug 2012; accepted in final form 21 Sep 2012

ABSTRACT—Sepsis is the most common cause of death in intensive care units. Some studies have found that hyperoxiamay be beneficial to sepsis. However, the clinical use of hyperoxia is hindered by concerns that it could exacerbate organinjury by increasing free radical formation. Recently, it has been suggested that molecular hydrogen (H2) at low con-centration can exert a therapeutic antioxidant activity and effectively protect against sepsis by reducing oxidative stress.Therefore, we hypothesized that combination therapy with H2 and hyperoxia might afford more potent therapeutic strategiesfor sepsis. In the present study, we found that inhalation of H2 (2%) or hyperoxia (98%) alone improved the 14-day survivalrate of septic mice with moderate cecal ligation and puncture (CLP) from 40% to 80% or 70%, respectively. However,combination therapy with H2 and hyperoxia could increase the 14-day survival rate of moderate CLP mice to 100% andimprove the 7-day survival rate of severe CLP mice from 0% to 70%. Moreover, moderate CLP mice showed significantorgan damage characterized by the increases in lung myeloperoxidase activity, lung wet-to-dry weight ratio, proteinconcentration in bronchoalveolar lavage, serum biochemical parameters (alanine aminotransferase, aspartate amino-transferase, creatinine, and blood urea nitrogen), and organ histopathological scores (lung, liver, and kidney), as well as thedecrease in PaO2/FIO2 ratio at 24 h, which was attenuated by either H2 or hyperoxia alone. However, combination therapywith H2 and hyperoxia had a more beneficial effect against lung, liver, and kidney damage of moderate or severe CLP mice.Furthermore, we found that the beneficial effect of this combination therapy was associated with the decreased levelsof oxidative product (8-iso-prostaglandin F2!), increased activities of antioxidant enzymes (superoxide dismutase andcatalase) and anti-inflammatory cytokine (interleukin 10), and reduced levels of proinflammatory cytokines (high-mobilitygroup box 1 and tumor necrosis factor !) in serum and tissues. Therefore, combination therapy with H2 and hyperoxiaprovides enhanced therapeutic efficacy via both antioxidant and anti-inflammatory mechanisms and might be potentiallya clinically feasible approach for sepsis.

KEYWORDS—Sepsis, organ damage, molecular hydrogen, hyperoxia, antioxidant enzyme, high-mobility group box 1

ABBREVIATIONS—BALVbronchoalveolar lavage; CATVcatalase; CLPVcecal ligation and puncture; H2Vmolecularhydrogen; HMGB1Vhigh-mobility group box 1; 8-iso-PGF2!V8-iso-prostaglandin F2!; MPOVmyeloperoxidase; O2Voxygen;ROSVreactive oxygen species; SODVsuperoxide dismutase; W/DVwet-to-dry

INTRODUCTION

Despite substantial advances in antibiotic therapy and inten-

sive care, sepsis remains the most common cause of death in

intensive care units, with a mortality of 30% to 50%, which is

often accompanied by multiple organ dysfunction (1, 2). In the

United States, there are 751,000 cases of severe sepsis each

year, with a total annual cost of $16.7 billion and a progressive

increase in its incidence over time of 8.7% (1, 2). It is exceed-

ingly difficult to develop effective therapeutic interventions that

reduce so high mortality because the factors responsible for

sepsis are not fully understood (3). Therefore, there is consid-

erable interest in exploring an effective therapy for patients

with sepsis.

Oxygen is often used in critically ill patients. Early goal-

directed therapy for sepsis aims to balance tissue oxygen (O2)

delivery and demand. Our and other studies have found that

hyperoxia has a beneficial effect against sepsis and sepsis-

associated multiple organ damage (4Y8). Yet, the clinical use

of hyperoxia is hindered by concerns that it may exacerbate

organ damage by increasing free radical formation. Oxidative

stress plays an essential role in the pathogenesis of sepsis, and

overproduction of reactive oxygen species (ROS) can exacer-

bate organ damage (9, 10). Recently, it has been suggested

that a low concentration of molecular hydrogen (H2) exerts

a therapeutic antioxidant activity by selectively reducing hy-

droxyl radicals (&OH, the most cytotoxic ROS) and peroxyni-

trite (ONOOj) in vitro and also effectively protects against

many diseases in vivo (11Y15). Furthermore, our studies have

656

SHOCK, Vol. 38, No. 6, pp. 656Y663, 2012

Address reprint requests to Guolin Wang, PhD, MD, or Yonghao Yu, MD, PhD,

Department of Anesthesiology, General Hospital of Tianjin Medical University,

Tianjin 300052, People’s Republic of China. E-mail: [email protected];

([email protected]).

K.X., W.F., and W.X. contributed equally to this work.

This work was supported by research grants from the National Natural Science

Foundation of China (81101409 to K.X., 81071533 to Y.Y., and 30972847 to

G.W.), Tianjin Municipal Science and Technology Commission Foundation

(11JCYBJC12900 to Y.Y.), and the Foundation of Tianjin Bureau of Public

Health (2011KZ108 to K.X.).

The authors have declared that no conflict of interest exists.

Supplemental digital content is available for this article. Direct URL citation

appears in the printed text and is provided in the HTML and PDF versions of this

article on the journal_s Web site (www.shockjournal.com).

DOI: 10.1097/SHK.0b013e3182758646

Copyright � 2012 by the Shock Society

Copyright © 2012 by the Shock Society. Unauthorized reproduction of this article is prohibited.

http://www.shockjournal.com

demonstrated that 2% or 4% H2 can alleviate organ injury

and improve survival rate of animals with sepsis via reducing

oxidative stress and inflammation (16, 17).

Therefore, these findings strongly indicate that combina-

tion therapy with H2 and hyperoxia may afford more potent

therapeutic strategies for sepsis. It is well known that cecal

ligation and puncture (CLP) can cause lethal peritonitis and

sepsis, which is accompanied by multiple organ dysfunction

(18). Thus, the present study was designed to investigate

whether combination therapy with H2 and hyperoxia could

produce enhanced efficacy in a murine model of moderate or

severe CLP. In addition, the roles of oxidative stress and

inflammatory cytokines in the protective effect were studied.

Our results may establish a clinically applicable strategy for

the treatment of sepsis and provide a new avenue for the use of

therapeutic gas in patients.

MATERIALS AND METHODS

AnimalsAdult male C57BL/6 mice weighing 20 to 25 g (specific pathogen-free)

were provided by the Laboratory Animal Center of the Military Medical Sci-ence Academy of the PLA. Animals were housed at 20-C to 22-C with a 12-hlight-dark cycle. Standard animal chow and water were freely available. Allexperimental protocols were approved by the Institutional Animal Care andUse Committee of Tianjin Medical University and performed in accordancewith the National Institutes of Health guidelines for the use of experimentalanimals.

CLP modelSepsis was induced by CLP model performed as described previously

(16, 18, 19). Briefly, the animals were anesthetized by intraperitoneal injectionof 50 mg/kg pentobarbital sodium. We exposed the cecum by a 1-cm abdominalmidline incision and subjected it to ligation below the ileocecal valve and asingle through-and-through perforation of the ligated segment. For severe CLP(100% lethality), we ligated the distal three quarters of cecum and made a singlepuncture with a 20-gauge needle; for moderate CLP (30%Y40% survival), weligated the distal one half of cecum and made a single puncture with a 21-gaugeneedle. A small amount of stool was extruded through the puncture site. We thenreplaced the cecum into abdomen and closed the incision using a sterile 6-0 silksuture. Finally, we administered subcutaneously 1 mL of prewarmed sterile saline(pyrogen-free 0.9% NaCl, 37-C) for fluid resuscitation. Animals with shamoperation underwent the same procedure without CLP.

Molecular hydrogen and/or hyperoxia treatmentThe animals were put in a sealed Plexiglas chamber with inflow and out-

flow outlets (16, 17). Molecular hydrogen, O2, or N2 was supplied through agas flowmeter, respectively, and delivered into the chamber through a tube at arate of 4 L/min. The concentrations of O2 and H2 in the chamber were con-tinuously monitored with a gas analyzer (Medical Gas Analyzer LB-2, Model40 M; Beckman, Fullerton, Calif ) and a commercially available detector(Hy Alerta Handheld Detector Model 500; H2 Scan, Valencia, Calif ), respec-tively. Carbon dioxide was removed from the chamber gases with Baralyme(Chemetron Medical Division, Allied Healthcare Products, Inc., St. Louis,Mo). The mixed gases were maintained at the predetermined level (2% H2,21% O2, and 77% N2; 0% H2, 98% O2, and 2% N2; 2% H2, 98% O2, and0% N2) during the treatment. The animals without H2 or hyperoxia treatmentwere exposed to room air in the chamber. Food and water were available ad libitumduring the treatment.

Experimental designExperiment 1: Effects of H2 and/or hyperoxia treatment on the survival rate

of septic mice with moderate or severe CLP—Effects of H2 and/or hyperoxiatreatment on the survival rate of septic mice with moderate CLP. One hundredfifty animals were randomly divided into five groups (n = 30 per group): sham,moderate CLP, moderate CLP + O2, moderate CLP + H2, and moderate CLP +H2 + O2 groups. The treatment concentrations of hydrogen and hyperoxia weredetermined based on our previous studies and preliminary observations (8, 16).The animals in the moderate CLP + O2 and moderate CLP + H2 groups wereexposed to 98% O2 or 2% H2 for 3 h starting at 1 and 6 h after moderate CLPoperation, respectively. The animals in the moderate CLP + H2 + O2 groupwere exposed to 98% O2 and 2% H2 at the same time points. As a control, the

animals from the sham and moderate CLP groups were given room airtreatment at the same time points. The survival rate was observed on days 1, 2,3, 5, 7, and 14 after CLP or sham operation.

Effects of H2 and hyperoxia treatment on the survival rate of septic micewith severe CLP. Based on the above experiments, 60 animals were randomlydivided into two groups (n = 30 per group): severe CLP and severe CLP + H2 +O2 groups. The animals in both groups were exposed to severe CLP operation.The animals in the severe CLP + H2 + O2 group were exposed to 98% O2 and2% H2 for 3 h starting at 1 and 6 h after CLP operation, respectively. As acontrol, the animals from the severe CLP group were given room air treatmentat the same time points. The survival rate was observed on days 1, 2, 3, 5, and7 after CLP operation.

Experiment 2: Effects of H2 and/or hyperoxia treatment on sepsis-associatedorgan injury in mice with moderate or severe CLP—Additional 42 animalswere used in this experiment and were assigned to seven groups (n = 6 pergroup): sham, moderate CLP, moderate CLP + O2, moderate CLP + H2,moderate CLP + H2 + O2, severe CLP, and severe CLP + H2 + O2 groups.The detailed experimental protocols were the same as described above. Lungmyeloperoxidase (MPO) activity, wet-to-dry (W/D) weight ratio, protein con-centration in bronchoalveolar lavage (BAL) fluid, lung histopathology, andPaO2/FIO2 ratio were observed at 24 h after CLP or sham operation. Moreover,we detected the serum biochemical parameters, as well as liver and kidneyhistopathology at 24 h after CLP or sham operation.

Experiment 3: Effects of H2 and hyperoxia treatment on cytokine as well asoxidant and antioxidant system in mice with moderate or severe CLP—Additional30 animals were used in this experiment and were assigned to five groups(n = 6 per group): sham, moderate CLP, moderate CLP + H2 + O2, severe CLP,and severe CLP + H2 + O2 groups. The detailed experimental protocolswere the same as described above. At 24 h after CLP or sham operation,the proinflammatory cytokines (high-mobility group box 1 [HMGB1] andtumor necrosis factor ! [TNF-!]), anti-inflammatory cytokine (interleukin 10[IL-10]), antioxidant enzymes (superoxide dismutase [SOD] and catalase[CAT]), and oxidative product (8-iso-prostaglandin F2! [8-iso-PGF2!;Cayman Chemical, Ann Arbor, Mich]) in serum, lung, liver, and kidney tissueswere measured.

Oxygenation index analysisTo evaluate the oxygenation capability of lung, the ratio of O2 tension to

inspired O2 fraction (PaO2/FIO2) was calculated. At 24 h of CLP or shamoperation, animals were anesthetized and given endotracheal intubation witha 20-gauge catheter. They were subjected to mechanical ventilation with pureO2 at 7 mL/kg. The respiratory rate was 120 breaths/min. The animals wereventilated for 15 min before blood gas sampling. The arterial blood wasobtained from carotid artery and measured with a GEM Premier 3000 gasanalyzer (Instrumentation Laboratory, Milan, Italy).

Lung MPO activity assayAt 24 h after the CLP or sham operation, lungs were obtained and perfused

with phosphate-buffered saline (PBS) to remove all blood, then weighed andstored at j80-C for no more than 1 week. The supernatant from lung homo-genate was prepared for detecting the activity of MPO, an indicator of neu-trophil infiltration, which was measured as previously reported (16, 20). Theabsorbance was measured spectrophotometrically at 590 nm by spectropho-tometer (DU 640B; Beckman).

Lung W/D weight ratioTo quantify the magnitude of pulmonary edema, we evaluated lung W/D

weight ratio. The harvested wet lung was weighed and then placed in an ovenfor 24 h at 80-C and weighed.

BAL and total protein assayAnimals were subjected to BAL for collecting BAL fluid by the methods

described previously (16, 21). Two volumes of 0.5 mL of PBS (pH 7.4) wereinstilled, gently aspirated, pooled, and reaspirated. Lavage samples were cen-trifuged at 1,500g for 10 min at 4-C. The supernatant was stored at j20-C.The total protein concentration in BAL was determined by using a standardcommercial kit (Bio-Rad Laboratories, Hercules, Calif ).

Organ histologic examinationOrgan samples were collected at 24 h after CLP or sham operation for

observing morphologic alterations. The samples were fixed with 10% formalinfor 6 h at room temperature, embedded in paraffin, and sectioned at 5-2mthickness. After deparaffinization and rehydration, the sections were stainedwith hematoxylin-eosin. Organ histologic changes were evaluated by twopathologists who were blinded to the treatment regimen. A scoring system tograde the degree of lung damage was used (16, 22). In addition, according to

SHOCK DECEMBER 2012 H2 AND O2 COMBINATION THERAPY IN SEPSIS 657


the scoring standard in our recent articles (8, 16), the degree of liver and kidneyinjury was also graded.

Antioxidant enzymatic activity assayBlood and organ specimens (lung, liver, and kidney) were collected at 24 h

after CLP or sham operation. The serum was separated by centrifugation at3,000g for 15 min at 4-C, aliquoted, and stored at j80-C. The tissue homo-genates were prepared in chilled PBS (0.1 M, pH 7.4) and centrifuged at10,000g for 10 min at 4-C. The supernatants were collected, aliquoted, andstored at j80-C until the following analysis. The activities of SOD and CATwere measured using commercial kits purchased from Cayman ChemicalCompany (Ann Arbor, Mich). All spectrophotometric readings were performedusing a spectrophotometer (DU 640B; Beckman). The tissue protein concen-tration was determined by a standard commercial kit (Bio-Rad Laboratories,Hercules, Calif).

Detection of 8-iso-PGF2!The serum and tissue homogenates (lung, liver, and kidney) obtained above

were also used for detecting the 8-iso-PGF2! level. Measurement of 8-iso-PGF2!,free radicalYcatalyzed products of arachidonic acid, can offer a reliableapproach for quantitative measurement of oxidative stress status in vivo (23).The levels of serum and tissue 8-iso-PGF2! were detected by specific enzyme-linked immunosorbent assay kits (8-iso-PGF2!; Ann Arbor, Mich) usinga microplate reader (CA 94089; Molecular Devices, Sunnyvale, Calif ).

Detection of inflammatory cytokinesThe serum and tissue homogenates (lung, liver, and kidney) obtained

above were also used for detecting the levels of proinflammatory cytokines(HMGB1 and TNF-!) and anti-inflammatory cytokine (IL-10). The levels ofinflammatory cytokines were detected by specific enzyme-linked immuno-sorbent assay kits (TNF-! and IL-10 from R&D Systems, Minneapolis,Minn; HMGB1 from IBL, Hamburg, Germany) with a microplate reader(CA 94089; Molecular Devices).

Statistical analysisThe survival rates are expressed as percentage. The measurement data are

expressed as mean T SD. The analysis of survival rates was tested by Fisherexact probability method. The intergroup differences of the rest data weretested by one-way analysis of variance followed by least significant difference(LSD)Yt test for multiple comparisons. The statistical analysis was performedwith SPSS 16.0 software (SPSS Inc., Chicago, Ill). In all tests, P G 0.05 wasconsidered statistically significant.

RESULTS

Changes of arterial blood gas during the treatment

In the present study, we investigated the levels of arterial pH,

PaO2, and PaCO2 in mice with or without CLP operation during

the treatment. The arterial blood gas was conducted at 1.5h

after onset of the first treatment (2.5 h after CLP or sham

operation) using a GEM Premier 3000 gas analyzer (Instru-

mentation Laboratory). The levels of PaO2 in the moderate

CLP + O2, moderate CLP + H2 + O2, and severe CLP + H2 +

O2 groups were 415.2 T 18.4, 410.7 T 19.3, and 412.4 T23.8 mmHg, respectively. The PaO2 level in the moderate

CLP + H2 group was 97.23 T 8.42 mmHg, whereas the levels of

PaO2 in the sham, moderate CLP, and severe CLP groups were

96.38 T 7.56, 95.87 T 8.34, and 95.76 T 10.31 mmHg, respec-

tively. There were no differences in the levels of arterial pH and

PaCO2 among all groups (data not shown). The results demon-

strate that hyperoxia exposure can significantly increase the

PaO2 level, and 2% H2 has no marked effects on the PaO2 level

in moderate or severe CLP mice during the treatment.

Combination therapy with H2 and hyperoxia improved thesurvival rate of moderate or severe CLP mice

In this study, we investigated the effects of H2 and/or

hyperoxia treatment on the survival rates of septic mice with

moderate or severe CLP. The 14-day survival rate of moderate

CLP mice was 40% (P G 0.05 vs. sham group, n = 30 per

group; Fig. 1A). We found that either 98% O2 or 2% H2

exposure for 3 h starting at 1 and 6 h after CLP operation,

respectively, improved the 14-day survival rate of moderate

CLP mice to 70% or 80% (P G 0.05 vs. moderate CLP group,

n = 30 per group; Fig. 1A). Furthermore, combination therapy

with 2% H2 and 98% O2 increased the 14-day survival rate

of moderate CLP mice to 100% (P G 0.05 vs. moderate CLP

group, n = 30 per group; Fig. 1A). In addition, combination

therapy with 2% H2 and 98% O2 improved the 7-day survival

rate of severe CLP mice from 0% to 70% (P G 0.05, n = 30

per group; Fig. 1B). The above data suggest that combination

therapy with H2 and hyperoxia can improve the survival rate

of septic mice with moderate or severe CLP in a synergistic

manner.

Combination therapy with H2 and hyperoxia attenuatedacute organ injury in moderate or severe CLP mice

Moderate and severe CLP mice appeared to have profound

acute lung injury, which was assessed by MPO activity, W/D

ratio, protein concentration in BAL, histopathology, and PaO2/FIO2

ratio. Moderate and severe CLP mice showed the significant

increase in lung MPO activity, lung W/D ratio, protein con-

centration in BAL, and lung histologic scores (P G 0.05 vs.

sham group, n = 6 per group; Figs. 2 and 4), as well as the

decrease in PaO2/FIO2 ratio (P G 0.05 vs. sham group, n = 6 per

FIG. 1. Effects of H2 and/or hyperoxia on the survival rate of septic mice with moderate or severe CLP. The values are expressed as survivalpercentage (n = 30 per group). A, Effects of 2% H2 and/or 98% O2 on the survival rate of septic mice with moderate CLP. *P G 0.05 vs. sham group, †P G 0.05 vs.moderate CLP group, ‡P G 0.05 vs. moderate CLP + O2 group,

§P G 0.05 vs. moderate CLP + H2 group. B, Effects of combination therapy with 2% H2 and 98%O2 on the survival rate of septic mice with severe CLP. †P G 0.05 vs. severe CLP group.

658 SHOCK VOL. 38, NO. 6 XIE ET AL.


group; Fig. 2). These abnormal changes in the moderate CLP

mice were attenuated by 98% O2 or 2% H2 treatment alone

(P G 0.05 vs. moderate CLP, n = 6 per group; Figs. 2 and 4).

Furthermore, these abnormal changes in the moderate and severe

CLP mice were more significantly ameliorated by combination

therapy with 98% O2 and 2% H2 (Figs. 2 and 4).

Moreover, moderate and severe CLP mice appeared to have

significant liver and kidney damage at 24 h, which was assessed

by serum biochemical parameters (alanine aminotransferase,

aspartate aminotransferase, creatinine, and blood urea nitrogen)

and histopathology. Moderate and severe CLP mice showed

a significant increase in the levels of serum alanine amino-

transferase, aspartate aminotransferase, creatinine, and blood

urea nitrogen, as well as liver and kidney histologic scores

(P G 0.05 vs. sham group, n = 6 per group; Figs. 3 and 4).

These abnormal changes in the moderate CLP mice were

attenuated by either 98% O2 or 2% H2 treatment alone

(P G 0.05 vs. moderate CLP, n = 6 per group; Figs. 3 and 4).

Furthermore, these abnormal changes in the moderate and

severe CLP mice were more significantly ameliorated by

combination therapy with 98% O2 and 2% H2 (Figs. 3 and 4).

In addition, the histopathological changes of lung, liver, and

kidney are shown in the Figure, Supplemental Digital Content 1,

at http://links.lww.com/SHK/A145, which shows the effects

of H2 and/or O2 on organ histopathological changes in septic

mice with moderate or severe CLP. The lung, liver, and kidney

were stained with hematoxylin-eosin at 24 h after CLP or sham

operation (original magnification �20). These results demon-

strate that combination therapy with H2 and hyperoxia has an

enhanced efficacy against multiple organ damage in moderate

and severe sepsis.

Combination therapy with H2 and hyperoxia prevented theabnormal changes of antioxidant enzymatic activities,oxidative product, and inflammatory cytokines inmoderate or severe CLP mice

The activities of antioxidant enzymes (SOD and CAT), the

levels of oxidative product 8-iso-PGF2!, and the levels of

proinflammatory cytokines (HMGB1 and TNF-!) and anti-

inflammatory cytokine IL-10 in serum and tissues (lung, liver,

and kidney) of all animals were observed at 24 h after CLP or

sham operation. Our results showed that the decreased activ-

ities of antioxidant enzymes (SOD and CAT) as well as the

increased levels of oxidative product (8-iso-PGF2!) and

inflammatory cytokines (HMGB1, TNF-!, and IL-10) in serum

and tissues (lung, liver, and kidney) occurred to mice with

moderate or severe CLP (P G 0.05 vs. sham group, n = 6

per group; Figs. 5Y8). Combination therapy with 2% H2 and

98% O2 significantly increased the SOD and CAT activities

and decreased the 8-iso-PGF2! level in serum and tissues

(lung, liver, and kidney) of septic mice with moderate or

severe CLP (P G 0.05, n = 6 per group; Figs. 5Y8). In addition,

Combination therapy with H2 and O2 significantly decreased

the HMGB1 and TNF-! levels as well as increased the IL-10

level in serum and tissues (lung, liver, and kidney) of septic

mice with moderate or severe CLP (P G 0.05, n = 6 per group;

Figs. 5Y8). These data suggest that combination therapy with

H2 and hyperoxia provides more beneficial effects on sepsis

FIG. 3. Effects of H2 and/or O2 on liver and kidney function in septic mice with moderate or severe CLP. These indicators were measured at 24 h afterCLP or sham operation. The values are expressed as mean T SD (n = 6 per group). *P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs.moderate CLP + O2 group, §P G 0.05 vs. severe CLP group. ALT indicates alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood ureanitrogen; Cr, creatinine.

FIG. 2. Effects of H2 and/or O2 on lung injury in septic mice with moderate or severe CLP. A, Lung MPO activity, (B) lung BAL total protein, (C) lung W/Dweight ratio, (D) PaO2/FIO2 ratio. These indicators were measured at 24 h after CLP or sham operation. The values are expressed as mean T SD (n = 6 per group).*P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs. moderate CLP + O2 group,

§P G 0.05 vs. severe CLP group.



http://links.lww.com/SHK/A145

and sepsis-associated organ damage, which are associated with

the decreased levels of oxidative product and proinflammatory

cytokines and the increased levels of antioxidant enzymes and

anti-inflammatory cytokine in serum and tissues.

DISCUSSION

In the present study, we found that (1) either H2 or hyper-

oxia treatment alone improved the survival rate of septic mice

with moderate CLP, whereas combination therapy with H2 and

hyperoxia could synergistically increase the survival rate of

septic mice with moderate and severe CLP, which was greater

than treatment with either gas alone; (2) either H2 or hyperoxia

treatment alone protected against the lung, liver, and kidney

damage of moderate CLP mice, whereas combination therapy

with H2 and hyperoxia provided cumulative protection against

these organ damage of moderate and severe CLP mice; (3)

the beneficial effects of combination therapy with H2 and

hyperoxia on sepsis and sepsis-associated organ injury were

associated with the decreased levels of oxidative stress and

proinflammatory cytokines as well as the increased activities

of antioxidant enzymes and anti-inflammatory cytokine in

serum and tissues.

Cecal ligation and puncture is considered to be a clinically

relevant model for studying the pathogenesis and treatment of

sepsis (18). It can cause lethal peritonitis and sepsis due to a

polymicrobial infection, which is accompanied by multiple

organ damage. Therefore, the present study was designed to

investigate the therapeutic effects of H2 and/or hyperoxia on

sepsis in mice with moderate or severe CLP. In this study,

FIG. 4. Effects of H2 and/or O2 on organ histologic scores in septic mice with moderate or severe CLP. These indicators were measured at 24 h afterCLP or sham operation. The values are expressed as mean T SD (n = 6 per group). *P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs.moderate CLP + O2 group,

§P G 0.05 vs. severe CLP group.

FIG. 5. Effects of combination therapy with H2 and O2 on the levels of antioxidant enzymes, oxidative product, and inflammatory cytokines inserum of septic mice with moderate or severe CLP. The serum was harvested for measuring these indicators at 24 h after CLP or sham operation. The valuesare expressed as mean T SD (n = 6 per group). *P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs. severe CLP group.



we successfully produced the moderate or severe CLP model.

Moderate CLP caused a 40% survival rate and moderate organ

damage, whereas severe CLP caused 100% mortality and severe

organ damage. In the present investigation, we observed the

increase in lung MPO activity, W/D weight ratio, protein con-

centration in BAL, PaO2/FIO2 ratio, and histopathological injury,

indicating that CLP causes significant acute lung injury. Fur-

thermore, we found that the increase in serum biochemical

parameters and histopathological injury for liver and kidney

occurred to moderate or severe CLP mice, demonstrating that

FIG. 6. Effects of combination therapy with H2 and O2 on the levels of antioxidant enzymes, oxidative product, and inflammatory cytokines in lungsof septic mice with moderate or severe CLP. The lungs were harvested for measuring these indicators at 24 h after CLP or sham operation. The values areexpressed as mean T SD (n = 6 per group). *P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs. severe CLP group.

FIG. 7. Effects of combination therapy with H2 and O2 on the levels of antioxidant enzymes, oxidative product, and inflammatory cytokines in liverof septic mice with moderate or severe CLP. The liver was harvested for measuring these indicators at 24 h after CLP or sham operation. The values areexpressed as mean T SD (n = 6 per group). *P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs. severe CLP group.



CLP leads to significant liver and kidney injury. Thus, it is

critical to develop effective strategies for treatment of organ

damage in septic patients.

Oxygen therapy is widely used in clinical practice as a

mainstay of supportive treatment for patients experiencing

hypoxemia and critical illness. It is well known that early

goal-directed therapy for sepsis or septic shock aims to bal-

ance O2 delivery and demand. In recent years, some animal

studies have shown that hyperoxia exposure can improve

organ function and survival rate in several models of shock or

sepsis (4Y8, 24, 25). In addition, we also find that 100% O2

exposure for 2 and 3 h starting at 4 and 12 h, respectively,

after zymosan injection benefits the outcome of mice with

sterile sepsis (8). It is believed that the improved tissue oxy-

genation and decreased systemic inflammatory response play

an essential role in the protection of hyperoxia treatment (4Y8).

However, hyperoxia treatment can induce the production of

ROS, which is considered to be associated with O2 toxicity

(9, 10). Therefore, the use of hyperoxia is hindered in critically

ill patients by concerns that it may exacerbate organ damage

by increasing free radical formation.

Molecular hydrogen has been used in medical applications

to prevent decompression sickness in deep divers for safety

profiles. Recently, several studies demonstrate that H2 exerts

a therapeutic antioxidant activity by selectively reducing &OH

and ONOOj and effectively protecting against many diseases,

suggesting that H2 has potential as an antioxidant for preven-

tive and therapeutic applications (11Y15). Our recent studies

have shown that H2 treatment has a beneficial effect on sepsis

and sepsis-induced organ injury in a concentration- and time-

dependent manner (16, 17). Because of the selective nature of

&OH and ONOOj inactivation, it is believed that H2 treat-

ment does not eliminate superoxide anions (O2j) or hydrogen

peroxide (11). Therefore, H2 therapy might spare the innate

immune system (macrophages and neutrophils), which requires

these ROS (O2j and H2O2) to kill some types of phagocytosed

bacteria. In addition, H2 is continuously produced in the body

by colonic bacteria and circulates in the blood under normal

conditions (26). In the present study, we indeed found that

combination therapy with H2 and hyperoxia could produce a

synergistic protective effect against sepsis and sepsis-associated

multiple organ damage.

To further investigate the possible mechanism, we studied

the effects of H2 and/or hyperoxia treatment on oxidant and

antioxidant system in moderate and severe CLP mice. In rodent

sepsis induced by CLP, the activities of SOD, CAT, and glu-

tathione peroxidase in serum and tissues are significantly

decreased during the early and late phages, suggesting that

sepsis sets up an environment favorable for oxidative stress

(27). On the other hand, the detection of products of lipid

peroxidation has been widely used to estimate the overall status

of oxidative stress. In this study, we observed the decrease in

SOD and CAT activities as well as the increase in oxidation

product 8-iso-PGF2! in serum, lung, liver, and kidney at 24 h

after moderate or severe CLP operation. We further found

that combination therapy with H2 and hyperoxia significant-

ly improved the CAT and SOD activities and decreased the

8-iso-PGF2! level in serum and tissues. These results suggest that

FIG. 8. Effects of combination therapy with H2 and O2 on the levels of antioxidant enzymes, oxidative product, and inflammatory cytokines inkidneys of septic mice with moderate or severe CLP. The kidneys were harvested for measuring these indicators at 24 h after CLP or sham operation. Thevalues are expressed as mean T SD (n = 6 per group). *P G 0.05 vs. sham group, †P G 0.05 vs. moderate CLP group, ‡P G 0.05 vs. severe CLP group.



the decrease in oxidative damage and the increase in endoge-

nous antioxidant enzymatic activities may attribute to the pro-

tection of H2 and hyperoxia treatment.

It is also believed that the uncontrolled and exaggerated

inflammatory response plays a major role in the pathogenesis

of sepsis (3). The inflammatory cytokines include early inflam-

matory cytokines such as proinflammatory cytokines, TNF-! and

IL-6, and anti-inflammatory cytokine, IL-10, as well as the late

inflammatory cytokine, HMGB1 (16, 28). Many researchers

discover that a ubiquitous protein HMGB1, which is released

by activated macrophages/monocytes, functions as a late medi-

ator of lethal endotoxemia and sepsis (28, 29). Many studies

have found that HMGB1 is a necessary and sufficient mediator

of lethal organ damage in murine CLP sepsis (28, 29). Phar-

macologic agents that reduce circulating HMGB1 levels, such

as ethyl pyruvate, provide significant protection against poly-

microbial sepsis lethality (30). On the other hand, adminis-

tration of recombinant HMGB1 to mice recapitulates many

clinical signs of sepsis, including fever, intestinal barrier dys-

function, and organ injury (29). Here, we found that combina-

tion therapy with H2 and hyperoxia significantly reduced the

HMGB1 and TNF-! levels as well as increased the IL-10 level

in serum and tissues of moderate or severe CLP mice and thereby

protected against the development of lethal organ damage.

Molecular hydrogen is neither explosive nor dangerous at a

low concentration (G4.1% in O2). Mixed gas therapy with H2

and hyperoxia is a novel, safe, and potent approach for pre-

venting organ damage and mortality in sepsis. Combination

therapy with H2 and hyperoxia may be a more effective ther-

apeutic strategy for septic patients (31, 32). This study sup-

ports the clinical application of therapeutic medical gases and

may justify a new avenue of investigation in therapeutic gas

use in critically ill patients.

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COMBINATION THERAPY WITH MOLECULAR HYDROGEN AND HYPEROXIA ...kelieng.com/uploads/file/150726/07-26162046_6804.pdf · Oxygen is often used in critically ill patients. Early goal-directed