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Exogenous Hydrogen Sulfide Protects Against Traumatic Hemorrhagic Shock Attenuation Oxidative Stress

Exogenous Hydrogen Sul?de Protects Against Traumatic Hemorrhagic Shock Via Attenuation of Oxidative Stress

Wei Chai,M.D.,*,2Yan Wang,M.D.,?,2Jia-Yan Lin,M.D.,*Xu-De Sun,Ph.D.,*Li-Nong Yao,Ph.D.,* Yong-Hui Yang,M.B.,*Hui Zhao,Ph.D.,*Wei Jiang,M.B.,*Chang-Jun Gao,Ph.D.,*,1and Qian Ding,Ph.D.*,1 *Department of Anesthesiology,Tangdu Hospital,Fourth Military Medical University,Xi’an,China;and?Department of Intensive Care

Unit,Shaanxi Provincial People’s Hospital,Xi’an,China

Originally submitted February22,2011;accepted for publication July8,2011

Objective.This study was designed to investigate the protective effects of exogenous hydrogen sul?de (H2S)on trauma-hemorrhagic shock(T-H).

Materials and Methods.Forty-eight male Sprague-Dawley rats were anesthetized,while32were sub-jected to both midline laparotomy and hemorrhagic shock(35–40mmHg for90min)by bleeding them from the femoral artery.One hour later,resuscitation was initiated with Ringer lactate.NaHS(28m mol/kg) or vehicle alone was administered intraperitoneally at the onset of resuscitation.Two hours later,eight an-imals from each group were re-anesthetized to deter-mine cardiac function,blood gas concentrations,and hepatic and renal function.Superoxide dismutase ac-tivity(SOD),malondialdehyde concentrations(MDA), and the activity of myeloperoxidase(MPO)in the se-rum were measured and pulmonary wet/dry(W/D)ra-tio and histopathologic evaluations performed. Results.NaHS resulted in an increase in mean arterial blood pressure,left ventricular pressure and positive(D dP/dt max)and negative(–dP/dt max)?rst de-rivatives of pressure as compared with the vehicle only group.The pH,PaO2and base excess(BE)were in-creased in the NaHS-treated group compared with the vehicle-treated group.Aspartate aminotransferase,al-anine aminotransferase,blood urea nitrogen,and se-rum creatinine were reduced in the NaHS-treated group.NaHS also signi?cantly reduced the high mor-tality rate at24h otherwise caused by T-H.The NaHS-treated group showed a remarkable decrease in MDA and MPO concentrations in plasma and an in-crease in SOD as compared with the vehicle-treated group.Histopathologic analysis indicated less edema, congestion,in?ammatory cell in?ltration and necrosis in heart,lung,liver and kidney tissue in NaHS-treated group.

Conclusions.The present study demonstrates that exogenous H2S administered at an appropriate dose confers protective effects after T-H and resuscitation, by preventing a decrease in the antioxidant defense system.ó2011Elsevier Inc.All rights reserved.

Key Words:antioxidant;hydrogen sul?de;trauma-hemorrhagic shock.

INTRODUCTION

Trauma hemorrhage(T-H)due to severe traf?c acci-dents,battle wounds,or surgery is the main cause of death below44y of age world-wide[1].T-H causes se-vere hypotension and hypoxemia initially,inducing is-chemic damage to organs and tissues[2].Despite?uid resuscitation,it can still be associated with ischemia-reperfusion(I/R)injuries,a systematic in?ammatory response,organ dysfunction,and the‘no-re?ow’phe-nomenon[2–5].Severe and developing T-H and resusci-tation injuries ultimately result in the systematic in?ammatory response syndrome or multiple organ failure.There is experimental evidence that oxidative stress is implicated in the high mortality and morbidity of T-H and resuscitation.Studies have indicated that treatment with anti-oxidative stress agents can im-prove the outcome after T-H and resuscitation[5–7]. Since its biological activity was discovered in1996 [8],hydrogen sul?de(H2S)has been shown to

1To whom correspondence and reprint requests should be ad-dressed at Department of Anesthesiology,Tangdu Hospital,Fourth Military Medical University,Xi’an,China.E-mail:gaocj74@https://www.wendangku.net/doc/174755877.html, or dingqian@https://www.wendangku.net/doc/174755877.html,.

2These authors contributed equally to this

work.

0022-4804/$36.00

ó2011Elsevier Inc.All rights reserved.

1

Journal of Surgical Research-,1–10(2011) doi:10.1016/j.jss.2011.07.016

participate in multiple physiologic and pathologic processes.Previous studies have demonstrated the sig-ni?cant protective effects of H2S in restoring organ function due to I/R injuries[9,11,12].Administration of physiologic concentrations of NaHS protects neurons against oxidative stress by increasing glutathione con-centrations and scavenging hydrogen peroxide[13, 14].Recently,Chang found that administration of exog-enous H2S protects the myocardium from oxidation in-duced by Hcy[15],which suggests that H2S may be a novel strategy for treatment of oxidative stress.It is well accepted that intracellular Ca2t([Ca2t]i)overload is an immediate precipitant of cardiac injury during ischemia-reperfusion[16].Research has suggested that H2S has activates K ATP and decreases[Ca2t]i.,con-ferring cardio-protection against hypoxic insults[9,17]. Elrod et al.have provided proof that administration of 50m g/kg H2S preserves mitochondrial function and membrane integrity[18].Moreover,it has been re-ported that physiologic concentrations of H2S can slow down metabolism without in?uencing cardiac function and induce‘suspended animation-like states’[19].En-dogenous H2S also contributes to cardio-protection by metabolic inhibition when preconditioned in rat ven-tricular myocytes[20].Therefore,the‘suspended animation-like states’induced by H2S may also confer bene?t by reducing the‘oxygen debt’in T-H and resus-citation.Although H2S has been previously been re-ported[8,10,14]to mediate the redox status of cells and attenuate oxidative stress,the effects of this on T-

H and resuscitation have not yet been clari?ed[8,10,

14].In the present study,we investigated the protective effects of H2S in T-H and resuscitation,and examined the possible role of H2S in oxidative stress.

MATERIALS AND METHODS

Animals and Materials

Male Sprague-Dawley rats(220–250g)were provided by the Ani-mal Department of The Fourth Military Medical University(Xi’an, China).All animals were housed in wire-bottom cages at25 C (room temperature)with a12h-light/dark cycle and fed standard rat chow and water.All animal care and experimental protocols used in this study were approved by the Animal Research Ethics Com-mittee and conducted in accordance with the Guidelines for Animal Experimentation of our institution.A stock solution of NaHS(Sigma, St.Louis,MO)was freshly prepared by dissolving NaHS immediately before use.Other chemicals and reagents were of analytical grade.All reagents used in determinations of superoxide dismutase(SOD),ma-londialdehyde(MDA),and myeloperoxidase(MPO)were purchased from Nanjing Jiancheng Biotechnology(Nanjing,China).

Treatment and Animal Groups

Forty-eight rats were divided randomly into the following three groups:(1)Sham group(n?16);sham-operated animals under-went anesthesia only;(2)Vehicle group(n?16);and(3)NaHS group(n?16).The rats in vehicle group and NaHS group were given the same volume of vehicle or NaHS solution(28m mol/kg) alone intraperitoneally at the beginning of resuscitation.

Trauma Hemorrhage

Protocol

After an overnight fast with free access to water,all animals were anesthetized with1%pentobarbital sodium intraperitoneally(40 mg/kg).T-H was induced and resuscitation carried out on the animals in vehicle group and NaHS group as previously described[21].Brie?y, a5cm midline laparotomy was performed to create soft tissue trauma. The abdominal wound was then closed in two layers with sutures. Polyethylene catheters(PE-50)were placed in both femoral arteries and the right femoral vein.Hemodynamic variables were measured via one of the arteries using a blood pressure analyzer(Powerlab Sys-tem,Chart5;AD Instruments,Bella Vista,NSW,Australia).In the animals in vehicle group and NaHS group,blood was then withdrawn rapidly to a mean arterial pressure(MAP)of35–40mm Hg through the other artery until they could no longer maintain this MAP unless some Ringer lactate solution(RL)was administered;this procedure took about10min.The time taken and volume removed were recorded as maximal bleed out(MBO).After MBO had been achieved,hypoten-sion was maintained between35and40mm Hg by giving small vol-umes of RL until40%of the MBO volume of RL had been returned (about90min from the beginning of bleeding).The volume of blood withdrawn was about60%of the total volume(6%of the body weight). The animals were then resuscitated with four times the volume of MBO of RL over60min through the venous catheter.After resuscita-tion,all catheters were removed and all wounds closed with sutures. All incisions were?ushed with lidocaine to reduce pain.At the end of the whole procedure,each animal was put in a solo cage with food and water ad libitum.Two hours after the end of the experimental or sham operations,eight animals from each group were re-anesthetized to determine cardiac function,blood gas concentrations, and hepatic and renal function.Arterial blood samples were taken to assess changes in blood gas variables,including pH,arterial oxygen tension(PaO2),arterial carbon dioxide tension(PaCO2),and base ex-cess(BE)levels,by using the OMNI Modular System(AVL List

JOURNAL OF SURGICAL RESEARCH:VOL.-,NO.-,-2011 2

GmbH Medizintechnik,Graz,Austria).Blood was collected from the inferior vena cava,kept on ice for15min and then centrifuged at 14,000g for12min[10].Serum samples were then obtained by collect-ing the supernatant.The serum samples were analyzed for aspartate aminotransferase(AST),alanine aminotransferase(ALT),blood urea nitrogen(BUN),and serum creatinine(SCr)using an automatic bio-chemistry analyzer(Roche Cobas Integra400plus;Basel,Switzer-land).The survival rate immediately after resuscitation,6h later, and24h later were recorded for the other eight rats in each group.

Determination of Cardiac Function

The right carotid artery was cannulated with PE-50tubing,and the catheter advanced into the left ventricle to monitor left ventricular pressure(LVP)and positive and negative?rst derivatives of pressure (tdP/d t max andàdP/d t max).

Biochemical Determinations

Arterial blood samples were taken and centrifuged at4000g for5 min at4 C.The resultant supernatants provided plasma samples which were stored at70 C for biochemical determinations.The enzy-matic activities of SOD and MDA are often used to evaluate oxidative and anti-oxidative reaction.MDA,a product of lipid hydroperoxide, re?ects damage to lipid peroxidation indirectly.SOD can protect cells against oxidative injury by scavenging superoxide free radicals.Both SOD and MDA were measured using a commercially available kit (Nanjing Jiancheng Biological Institute,Nanjing,China).The assay of MDA is based on the principle that hydroperoxides react strongly with thiobarbituric acid,forming a red substance that can be detected spectrophotometrically at535nm.SOD,a superoxide generator,was measured at550nm with a spectrophotometer following reduction of nitrite by a xanthine–xanthine oxidase system.MPO can deoxidize hydrogen dioxide in neutrophils.MPO activity was measured using a commercially available kit(Nanjing Jiancheng Biological Institute) according to the manufacturer’s recommendations.

Assay of H2S Concentration in Plasma

Blood was withdrawn at the end of the experiment from all rats and centrifuged at4000g for5min at4 C to obtain plasma.Plasma con-centrations of H2S were measured by the following method[22]. Brie?y,0.5mL of1%zinc acetate was added directly to0.1mL plasma to trap H2S.Subsequently,0.5mL of20mmol/L.

N,N-dimethyl-p-phenylenediamine sulfate in7.2mol/L HCl was mixed into the solution,followed by0.4mL of30mmol/L FeCl3in 1.2mol/L HCl.The mixture was then incubated at room temperature for20min.One mL of10%trichloroacetic acid was then added to the reaction mixture to stop the reaction and remove protein.Finally,2.5 mL of water was added to the mixture.After centrifuging at4000g for 5min,the supernatant was removed to measure the absorbance of the resulting solution at670nm with a spectrophotometer.The H2S concentration was then calculated from the calibration curve and presented as m M.

Assay of the Pulmonary Wet/Dry Ratio

Brie?y,the rats were killed and the inferior third of their right lungs excised and weighed.These specimens were placed in a drying oven at80 C for48h until the weight was constant.The weights be-fore and after drying were used to calculate the wet/dry(W/D)ratio.

Histopathologic Evaluations and Index of Quantitative

Assessment

Eight rats from each group were killed at the indicated time by re-moving blood from their hearts.Various organs,including the heart,lung,liver,and kidney,were removed and?xed with a buffered10% formalin solution for24h and embedded in paraf?n.Sections of all tis-sues were stained with hematoxylin and eosin(HE),after which they were observed by light microscopy(Olympus BX51;Tokyo,Japan)to characterize the histopathologic changes.The index of quantitative assessment(IQA)was assayed as previously described[23],using HE stained histopathologic sections of the upper lobe of lung.The IQA score was measured by light microscopy as the ratio of injured to total pulmonary alveoli(pulmonary alveoli containing more than two red blood cells or polymorphonuclear neutrophils were considered to be injured)in10visual?elds.

Statistical Analysis

Results are presented as mean6standard error of the mean.Dif-ferences between data from different groups were analyzed by one-way analysis of variance followed by the Student–Newman–Keuls test.Differences between the groups in survival rates were tested by the Fisher’s exact test.Differences were considered statistically signi?cant when the P value was<0.05.All data were analyzed using SPSS13.0statistical software(SPSS Inc.,Chicago,IL).

RESULTS

Effect of NaHS on Hemodynamic Parameters and Cardiac

Function

Intraperitoneal administration at the beginning of resuscitation of exogenous H2S in the form of donor NaHS at a dose of28m mol/kg attenuated depression of cardiac function after T-H.The differences between the vehicle-treated and NaHS-treated groups in base-line MAP and HR were not statistically signi?cant (P>0.05).(Vehicle versus NaHS MAP:104.6610.4 versus101.569.8;P>0.05).The measured hemody-namic variables were signi?cantly decreased during the shock period(P<0.05).After15min of resuscita-tion,the mean MAP of the vehicle-treated group re-versed up to96.467.0mmHg,then began to decrease at30min.In contrast,the MAP of the NaHS group remained at a high level during the whole60 min.The HR in both groups was increased at15min af-ter resuscitation(P<0.05).There was no statistical dif-ference in HR between the vehicle and NaHS groups after45min(P>0.05).Furthermore,after2h of resus-citation,the NaHS group had signi?cant improvements in LVP,tdP/dt max and–dP/dt max compared with the vehicle group(Fig.1).

Effect of NaHS on Blood Gases

NaHS protected against increasing acidosis(Table1). Both the pH and BE were signi?cantly better in the NaHS-treated than in the vehicle group(P<0.05), whereas there was a statistically signi?cant difference in BE levels only between the sham and NaHS-treated groups(sham versus NaHS:3.0160.46versus6.516 0.48;P<0.05).NaHS also prevented the reduction in PaO2that was seen in the vehicle-treated group

CHAI ET AL.:EXOGENOUS HYDROGEN SULFIDE PROTECTS AGAINST TRAUMATIC HEMORRHAGIC SHOCK3

(NaHS versus vehicle:101.1666.29versus 92.1366.33;P <0.05).NaHS had no effect on PaCO 2(sham versus vehicle versus NaHS:39.7565.37versus 42.1061.88versus 37.2962.93,P ?0.053).

Effect of NaHS on Lung Injury

Although the W/D ratio (an indicator of lung injury)of the vehicle and NaHS-treated groups were both sig-ni?cantly higher than that of the sham-operated group (4.4760.30,P <0.05),this ratio was much lower in the NaHS-treated than in the vehicle-treated group,dem-onstrating that NaHS confers protection against pul-monary edema (NaHS versus vehicle: 4.5260.36versus 5.0860.60;P ?0.021).Another indication of its protective effect against lung injury was that NaHS resulted in a smaller IQA than that seen in the vehicle-treated group (NaHS versus vehicle:25.1663.14versus 47.9164.03;P <0.05)(Fig.2).

Effect of NaHS on Hepatic Injury and Renal Injury

Indicators of hepatic and renal function were as-sessed in the sham,vehicle,and NaHS-treated groups (Fig.3).Speci?cally,the 28m mol/kg dose of NaHS re-duced serum ALT by 56.8%(P <0.05),serum AST by 43.6%(P <0.05),serum BUN by 52%(P <0.05),and serum SCr by 64.9%(P <0.05).

Both vehicle and NaHS-treated rats had signi?cantly higher liver transaminases,BUN and SCr

(attributable

FIG.1.Effect of NaHS on hemodynamic variables at the indicated times and cardiac function at 2h after resuscitation.(A)Mean ar-terial pressure (MAP);(B)heart rate (HR);(C)left ventricular pressure (LVP);(D)positive and negative ?rst derivatives of pressure (tdP/d t max and –dP/d t max ).*P <0.05versus sham group;**P <0.05versus vehicle group;(n ?8in each group).

TABLE 1

Blood Gas Analysis 2h After Resuscitation

pH

PaO 2(mmHg)PaCO 2(mmHg)BE(mmol/L)Sham 7.3660.05106.7963.7339.7565.37–3.0160.46Vehicle 7.2160.06*92.1366.33*42.1061.88–9.8860.88*NaHS

7.3460.04**

101.1666.29**

37.2962.93

–6.5160.48*,**

Blood gas analysis was assayed at 2h after resuscitation.Data are presented as mean 6SEM.*

P <0.05versus sham group.**

P <0.05versus vehicle group;(n ?8,in each group).BE ?base excess;PaO 2?arterial oxygen tension;PaCO 2?arterial carbon dioxide tension.

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to hepatic and renal injury)than did the sham group (Fig.3).

Effect of NaHS on Survival Rate

All the sham-operated animals survived at the end of experiments.Five of the eight rats in the vehicle-treated group were still alive immediately after resusci-tation and three of them 6h later.In contrast,seven of the eight rats in the NaHS-treated group were still alive immediately after resuscitation and six of them 6h later.No statistically signi?cant differences were ob-served between the two groups immediately after and 6h after resuscitation (immediately:P ?0.57;6h:P ?0.315).NaHS increased the survival rate at 24h

after resuscitation by 50%(two of eight rats survived 24h in the vehicle-treated group compared with six of eight in the NaHS-treated group P <0.05)(Table 2).

Effect of NaHS on H 2S Concentrations and Oxidative Stress

Assays of SOD activity and MDA concentration dem-onstrated the extent of anti-and oxidative stress in-jury.MPO activity was assessed as an indicator of neutrophil in?ltration.NaHS protected against oxida-tive stress injury after resuscitation for T-H (Fig.4).T-H and resuscitation induced a decrease in SOD activ-ity in the vehicle-treated group (sham versus vehicle:150.0069.96versus 124.9967.06,P <0.05).NaHS-treated rats showed evidence of protection

compared

FIG.2.Effects of NaHS administration on lung injury.The W/D ratio and IQA were assayed at 2h after resuscitation.(A)Wet/dry ratio (W/D);(B)index of quantitative assessment score (IQA).*P <0.05versus sham group,**P <0.05versus vehicle group;(n ?8in each

group).

FIG.3.Effects of NaHS on hepatic and renal injury.(A)Aspartate aminotransferase (AST);(B)alanine aminotransferase (ALT);(C)blood urea nitrogen (BUN);(D)serum creatinine (SCr).*P <0.05versus sham group,**P <0.05versus vehicle group;(n ?8in each group).

CHAI ET AL.:EXOGENOUS HYDROGEN SULFIDE PROTECTS AGAINST TRAUMATIC HEMORRHAGIC SHOCK

5

with the vehicle-treated group (NaHS versus vehicle:146.4367.17versus 124.9967.06;P <0.05),the results in the latter being similar to those of the sham-treated group (NaHS versus sham:150.0069.96versus 146.4367.17;P ?0.39).Both vehicle and NaHS groups had signi?cantly higher concentrations of MDA and MPO due to injury after T-H and resusci-tation than did the sham-operated group (P <0.05).MDA and MPO were both reduced in NaHS group com-pared with the vehicle group,indicating that NaHS had a bene?cial therapeutic effect (P <0.05).After re-suscitation,the H 2S concentration in plasma was less than in the sham-operated group (vehicle versus sham:23.8064.88versus 37.6463.29;P <0.05).After intraperitoneal administration of exogenous hydrogen

sul?de donor NaHS,plasma H 2S concentration was much higher than in the vehicle group,but similar to that of the sham group (NaHS versus sham:39.6362.51versus 37.6463.29;P ?0.29).

DISCUSSION

This experiment examined the effects of exogenous H 2S on T-H and resuscitation and the potential mech-anism of this protection for the ?rst time.Our data indicate that intraperitoneal administration of a low dose of exogenous H 2S in the form of donor NaHS (28m mol/kg)results in organ-conservation and an im-proved survival rate.The NaHS-treated group showed a signi?cant decrease in oxidative stress,indicating that an anti-oxidative effect is the mechanism for its bene?cial effects.

T-H and resuscitation has multiple pathologic and physiologic effects,which lead to multi-organ damage [6,7].Several studies have reported that oxygen free radicals (OFRs)such as hydroxyl radical and superox-ide play toxic roles in T-H and resuscitation [3].OFRs are originally generated during the ischemia of the ini-tial shock and accumulate during reperfusion.OFRs attack the cellular and mitochondrial membranes,resulting in lipid peroxidation.Dysfunction of these membrane blocks intracellular energy metabolism

TABLE 2

Effects of NaHS on trauma hemorrhage-induced

mortality at the indicated times

0h after resuscitation (alive/8)

6h after resuscitation (alive/8)

24h after resuscitation (alive/8)

Vehicle 5/83/82/8NaHS

7/8

6/8

6/8*

*

P <0.05versus vehicle group;(n ?8in each

group).

FIG.4.Effect of NaHS on MDA,SOD,MPO,and H 2S concentrations in plasma.(A)Hydrogen sul?de concentration in plasma (H 2S);(B)superoxide dismutase activity (SOD);(C)malondialdehyde concentrations (MDA);(D)myeloperoxidase concentrations (MPO)*P <0.05versus sham group,**P <0.05versus vehicle group;(n ?8in each group).

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and depletion of adenosine triphosphate(ATP)results in intracellular edema,necrosis and apoptosis.The OFRs are also major activators of cytokine/chemokine generation and neutrophil adherence.In?ammatory responses are incited by OFRs.Severe impairment of capillary endothelial cell results in generation of chemotactic activity in neutrophils and triggers in?ammatory cytokines.These pro-in?ammatory me-diators evoke an in?ammation cascade,which is characterized by leukocyte in?ltration,as well as gen-eration of reactive oxygen and reactive nitrogen spe-cies.Generation of OFRs is then accentuated and vascular permeability increased,aggravating hyper-tension and edema,which exacerbates‘oxygen de?cit’and acidosis.Impaction of leukocytes within capillary beds hampers oxygen delivery and leads to the condi-tion of‘no-re?ow’[4],even during resuscitation.Oxy-gen consumption and metabolism rates are increased when?uid resuscitation is administered.Meantime, leukocytes-mediated injuries induce a drop in oxygen delivery to cells and tissues.The resultant imbalance between oxygen support and oxygen consumption leads to‘oxygen debt’[5].The inadequate oxygen sup-ply then affects synthesis of ATP in cells,increasing anaerobic rather than aerobic metabolism.All these metabolic changes are responsible for organ damage, which characteristically occurs in lung,liver and kid-ney.A Medline/Cochrane Library literature search identi?ed many cases in which it was concluded that BE could be used to predict the likelihood of trauma patients dying,suggesting that BE can be used to pro-vide an index of the effectiveness of resuscitation in hu-mans as well as in experimental animals[5].We observed evidence of severe acidosis,depression of car-diac function and dysfunction of both liver and kidney after T-H and resuscitation.As evidenced by compari-son with the vehicle-treated group,NaHS can prevent severe acidosis.PaO2was higher in the NaHS-treated than that in the vehicle-treated group(NaHS versus vehicle:101.166 6.29versus92.136 6.33).Data from our experiment also suggests that exogenous H2S results in cardioprotection by conserving cardiac function and promoting hemodynamic stability.The NaHS-treated group showed reversals of decreases in MAP,LVP,tdP/dt max and–dP/dt max.However,HR was not signi?cantly different in the vehicle-treated and NaHS-treated groups,though there was a short period of increased rate(P>0.05).In addition,a28 m mol/kg dose of NaHS reduced serum ALT by56.8%, AST by43.6%,BUN by52%,and SCr by64.9%.The re-storative effects of H2S in relation to hepatic and renal injury after T-H and resuscitation shown in our study are in accordance with the protective effects reported recently for hepatic and renal I/R injury[10,12].Our study also showed that H2S can reduce lung tissue edema and destruction of alveolar walls.Histo-pathologic sections also showed less damage in the NaHS-treated group(Fig.5).The24-h survival rate of the NaHS-treated group was50%higher than that in the vehicle-treated group.

Hydrogen sul?de used to be recognized as a gas that is toxic to the central nervous and respiratory systems and smells like rotten eggs.Since endogenous H2S has been found in mammals,it has been described as‘third gas signaling’.Recently,Lu reported that exogenous H2S protects cells against H2O2àinduced oxidative damage and decreases the cell death cascade[13].Fur-ther research by Kimura revealed that H2S protects neurons from oxytosis by enhancing the activity of g-glutamylcysteine synthetase and up-regulating cys-teine transport,by which eventually increasing the pro-duction of the antioxidant glutathione[14].Rats were injected with isoproterenol and observed the effects of exogenous H2S on myocardial injury observed.It was found that exogenous H2S ameliorates myocytes and contractile activity by directly scavenging oxygen free radicals and reducing the lipid peroxidations[24].Zhu observed that pretreatment with14m mol/kg of sodium hydrosul?de(NaHS)would decrease the infarct size and mortality after myocardial ischemia induced by iso-proterenol.Additional animal experiments have indi-cated that administration of a new type of exogenous H2S donor(IK1001)may also attenuate hepatic I/R in-jury via antioxidant and reducing lipid peroxidation pathways.Jha found the hepatoprotection of H2S in a murine model in hepatic ischemia-reperfusion(I/R) injury.Administered with H2S donor(IK1001)could at-tenuate the elevation in serum alanine aminotransfer-ase(ALT),aspartate aminotransferase(AST),and the formation of lipid hydroperoxides.By measuring HSP-90,mitochondrial Bcl-2levels and GSH-to-GSSG ratio, H2S was found to protect the murine liver against I/R injury through an up-regulation of intracellular antiox-idant and anti-apoptotic signaling pathways[10].I/ R injuries represent complicated processes involving oxidative damage,activation of the in?ammatory cas-cade,and apoptosis.I/R injuries are associated with development of diseases in diverse clinical settings,in-cluding transplantation,lobectomy,congestive heart failure,shock states,and resuscitation.Therefore,at-tenuation of I/R injury could increase the number of patients that may survive in fatal diseases.Trauma-hemorrhage shares some similarities with I/R,which is a multifactorial injury that includes hypoxemia and reoxygenation injury,ischemic-reperfusion injury, leading to multiple organ dysfunction and failure.Evi-dences of cardiac contractile dysfunction,lung edema, and hepatic damage have been obtained after trauma-hemorrhagic shock and resuscitation.In?ammation, cellular oxidative stress,and apoptosis have been

CHAI ET AL.:EXOGENOUS HYDROGEN SULFIDE PROTECTS AGAINST TRAUMATIC HEMORRHAGIC SHOCK7

hypothesized to contribute to organ injuries after hem-orrhage and resuscitation [6,20].Our experiments found that NaHS resulted in an improved 24-h survival rate after resuscitation,though no statistically signi?-cant differences were observed immediately after and 6h after resuscitation.Pan found that pretreatment with NaHS caused a concentration related increase in cell viability and the ratio of rod-shaped cells exposed to severe metabolic inhibition in 2time windows (w 1h and 16–28h ),showing a delayed cardioprotection [20].Studies observed that H 2S preserved the GSH levels and Trx-1expression much better at 24h after hepatic I/R injury than that at 1or 5h,suggesting that H 2S maintains the intracellular antioxidant capac-ity of hepatocytes [10].Our experiment also showed higher 24-h survival rate in NaHS-treated group.How-ever,it remains unclear whether the salutary effects are sustained for longer period.Although the present experiments are animal studies,H 2S is found to play a part in organ I/R injuries by protecting against oxida-tive stress,reducing leukocyte-endothelial cell interac-tions,and inhibiting the progression of apoptosis in vivo .However,none of the studies have used varying concentrations of H 2S in vivo model and evaluated the different contributions of varying concentrations H 2S in I/R injuries,further studies should be done before the applications in clinical experiments.

In our experiment,data strongly suggest the antiox-idative roles of H 2S play a part in T-H and resuscita-tion.Oxidative stress has well characterized effects on the pathogenesis of organ injury induced by T-H and resuscitation.It has previously been established that therapeutic treatment with an antioxidant can in-duce reversal of organ damage after T-H and resuscita-tion [3,6,7].In this study,we found that the activity of SOD decreased in the vehicle-treated group,accompa-nied by an increase in the MDA concentration.Mean-while,the H 2S plasma concentration was much lower than that in the NaHS-treated group.Supplying exog-enous H 2S resulted in increased activity of SOD and decreased MDA concentrations.Kimura has reported that H 2S can scavenge H 2O 2and ONOO directly,in-ducing antioxidative injuries [14].Yan revealed that the antioxidative ability of H 2S is induced by promot-ing glutathione [25].The dose of NaHS that we chose was based on preliminary experiments.Administra-tion of 28m mol/kg NaHS intraperitoneally did not affect the MAP,HR or plasma concentrations

of

FIG.5.Morphological expression of changes in tissues.(A)Heart tissue;(B)lung tissue;(C)liver tissue;(D)kidney tissue.1:Sham group,no laparotomy,T-H or resuscitation;2:Vehicle-treated rats that received physiologic saline at the beginning of resuscitation;3:NaHS-treated rats that received NaHS solution (28m mol/kg)at the beginning of resuscitation.(A2)There is marked in?ltration by neutrophils into the edem-atous interstitial spaces with fragmentation of myocardium in the vehicle-treated heart.(B2)Vehicle-treated rats showed edema and severe in?ammatory cell in?ltration of the alveolar walls and congestion in the alveolar spaces,with collapse of some alveolar walls.(C2)The hepatic sinusoids are congested and fractured,and there are edematous hepatocytes and necrosis in the vehicle-treated liver.(D2)Vehicle-treated rats showed edema and red cell casts in the renal tubules.(A3,B3,C3,D3)Rats in the NaHS-treated group showed less evidence of tissue injury (HE,original magni?cation 3400,bar ?20m m).

JOURNAL OF SURGICAL RESEARCH:VOL.-,NO.-,-2011

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SOD,MDA,and MPO.Besides,the plasma concentra-tion of H2S was within the physiologic concentration (0w46m M).

Collin has reported that endogenous H2S is a pro-in?ammatory factor that aggravates organ injuries in endoxemia[26].Zhang et al.found that intraperitoneal injection of NaHS(10mg/kg)induced an in?ammatory response in sepsis and aggravated endotoxemia[27]. However,despite the roles of H2S in the regulation of in?ammation still being in dispute,administration of low doses of donor H2S has also been reported to have the effect of inhibiting production of pro-in?ammatory cytokines,which suggests that H2S has effects on the regulation of in?ammation.Li examined the possible role H2S in the pathogenesis of oleic acid(OA)-induced acute lung injury(ALI)and its regulatory effects on the in?ammatory response.In the trials,down-regulation of endogenous H2S during ALI might be involved in the pathogenesis of https://www.wendangku.net/doc/174755877.html,pared with the OA-treated rats(0.1mL/kg,intravenous injection),admin-istration of the H2S donor sodium hydrosul?de(NaHS, 56m mol/L,intraperitoneal injection)increased the par-tial pressure of oxygen in the arterial blood(PaO2) level,reduced the pulmonary wet/dry weight(W/D)ra-tio and in?ltration of polymorphonuclear(PMN)cells, and alleviated the degree of ALI(measured by the index of quantitative assessment(IQA)score).In addition, NaHS decreased IL-6and IL-8levels but increased IL-10levels in the plasma and lung tissues,suggesting that H2S may regulate the in?ammatory response and play a protective role through changing the levels of in-?ammatory and anti-in?ammatory cytokines in plasma and in the lung during ALI[28].In the present study, a drop in MPO concentration was also found in the NaHS-treated group.Since OFRs can activate neutro-phil adherence and evoke the in?ammation cascade,in-?ammatory responses often occur after oxidative stress responses.Therefore,future researches need to be done to study the pro?le of exogenous H2S in the in?amma-tion after T-H and resuscitation.

In the hemorrhagic shock model,inhibition of forma-tion of endogenous H2S has been reported to restore he-modynamic parameters after hemorrhagic shock[29]. Interestingly,GYY4137,a novel hydrogen sul?de-releasing molecule,has been proven to protect against endotoxic shock in rats when50mg/kg is injected intra-peritoneally[30].Morrison et al.utilized a rat model of lethal hemorrhage and observed that inhaled H2S(300 ppm)or intravenous administration of another H2S do-nor(Na2S,1mg/kg)improved the survival rate[31]. These studies display the protective effects of H2S on different kinds of shock.Hopefully,H2S-related phar-macologic research is a rapidly emerging?eld and early stage drug candidates are now in development.IK-1001,an injectable donor of H2S,is at the stage of phase 1clinical development,and is known to induce sus-pended animation and have therapeutic effects on mul-tiple hypoxic/ischemic conditions.

The contradictory conclusions about H2S on shock that can be found in currently available reports are due to the use of dissimilar models and doses.H2S seems to work as a‘double-edged sword’in the pathologic pro-cess of shock.In preliminary experiments,we found that intraperitoneal injection of high doses(e.g.,112 m mol/kg)of NaHS signi?cantly decreased the MAP. The plasma concentration of H2S was also far beyond the physiologic range.Additionally,the concentrations of MDA and MPO were increased,accompanied by a de-crease in SOD.It seems that high concentrations of H2S play a more complicated role in T-H and resuscitation. Therefore,further investigations are warranted to re-veal the ef?cacy of H2S under varying conditions.

In summary,our studies demonstrate that H2S sig-ni?cantly attenuates organ injury and mortality via preservation of intracellular balance and inhibition of oxidative stress during T-H and resuscitation.These results suggest that H2S is a promising therapeutic agent for protection against T-H and resuscitation injury.

ACKNOWLEDGMENT

This study was supported by grant30872444from the Natural Sci-ence Foundation of China.

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