Propofol Pretreatment Reduces Ceramide Production andAttenuates Intestinal Mucosal Apoptosis Induced byIntestinal Ischemia/Reperfusion in Rats

Ke-Xuan Liu, PhD, MD*

Shu-Qing Chen, PhD, MD†

Wen-Qi Huang, MD*

Yun-Sheng Li, MD*

Michael G. Irwin, MD‡

Zhengyuan Xia, PhD, MD‡

BACKGROUND: Apoptosis has been shown to be a major mode of intestinal epithelialcell death caused by intestinal ischemia/reperfusion (II/R), a condition that isassociated with increased oxidative stress. Ceramide has been proposed as amessenger of apoptosis. We investigated if pretreatment with propofol, an anes-thetic with antioxidant properties, could reduce ceramide production, and conse-quently, mucosal epithelial apoptosis induced by II/R in rats.METHODS: Rat II/R injury was produced by clamping the superior mesenteric arteryfor 1 h followed by 3 h of reperfusion. Thirty rats were randomly allocated intocontrol, injury (II/R) and propofol (pretreatment) groups (n � 10 per group). In thepropofol group, propofol 50 mg/kg, a dose that has been shown to cause the lossof reflex responses to a painful stimulus while remaining sensitive to skin incisionin rats, was administered intraperitoneally 30 min before inducing intestinalischemia, while animals in control and untreated injury groups received an equalvolume of intralipid. Intestinal mucosal epithelial apoptosis was detected viaelectron microscopy and TUNEL analysis. Lipid oxidation product malondialde-hyde and the activities of superoxide dismutase were assessed by colorimetricanalyses. Ceramide generation and sphingomyelinase mRNA expression in intes-tinal mucosa were determined by high performance thin layer chromatographyand reverse transcriptase polymerase chain reaction, respectively.RESULTS: II/R caused intestinal mucosal epithelial apoptosis and over-production ofceramide accompanied by up-regulation of sphingomyelinase mRNA expressionand increases in lipid oxidation (all P � 0.01 versus control). Propofol pretreatmentsignificantly attenuated these changes (all P � 0.01, propofol versus injury).CONCLUSION: The findings indicate that propofol pretreatment attenuates II/R-induced intestinal epithelial apoptosis, which might be attributable to its antioxi-dant property modulating the ceramide pathway.(Anesth Analg 2008;107:1884–91)

Intestinal ischemia/reperfusion (II/R) injury is apotentially serious consequence of acute mesentericischemia, hemorrhagic, traumatic or septic shock, se-vere burns or some surgical procedures, includingsmall bowel transplantation and abdominal aortic sur-gery.1 II/R leads not only to injury of the intestine itself,but also involves severe destruction of distant tissue dueto disruption of the intestinal mucosal barrier which

causes a systemic inflammatory reaction. II/R can evenresult in multiple organ dysfunction.2,3

Intestinal mucosal epithelial cells are the maincomponent of the intestinal mucosal barrier. Apopto-sis is a major mode of cell death in the destruction ofrat small intestinal epithelial cells induced by ischemiaand I/R injury.4–6 Therefore, prophylactic antiapop-totic treatment could be an effective therapeutic strat-egy for the prevention of II/R injury, which has beendemonstrated by various studies.7–9

Ceramide, a novel second messenger, plays animportant role in a variety of physiologic and patho-logic events, including apoptosis and injuries.10

Modulation of ceramide levels may be regarded as anovel therapeutic approach.11 The intracellular con-centration of ceramide can be influenced by manyinducers, including reactive oxygen species (ROS) andIR injury.12 Recently, we demonstrated that ceramidecontributes to the intestinal mucosal cell apoptosisinduced by II/R.9

Propofol, an IV anesthetic with antioxidant proper-ties,13,14 is commonly used for the induction and

From the Departments of *Anesthesiology, †Gynecology andObstetrics, The First Affiliated Hospital, Sun Yat-sen University,Guangzhou, China; and ‡Department of Anesthesiology, Universityof Hong Kong, Hong Kong SAR, China.

Accepted for publication May 7, 2008.Supported, in part, by a grant from National Natural Science

Foundation of China (No: 30672021, to K.X.L.).The authors K.X.L. and Z.X. share senior authorship.Address correspondence and reprint requests to Dr. Ke-Xuan

Liu, Department of Anesthesiology, The First Affiliated Hospital,Sun Yat-sen University, No.58, Zhongshan 2th Rd., Guangzhou,China, 510080. Address e-mail to liukexuan807@yahoo.com.cn.

Copyright © 2008 International Anesthesia Research SocietyDOI: 10.1213/ane.0b013e3181884bbf

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maintenance of anesthesia during surgery and forpostoperative sedation.15–17 We have recently demon-strated that pretreatment with propofol at a sedativedose attenuated intestinal mucosal injury induced byII/R in rats.18 It is unknown, however, whether itsprotection of intestinal mucosa is due to its effectagainst intestinal mucosal cell apoptosis.

Based on the above findings, we hypothesized thatmoderate pretreatment doses of propofol would de-crease intestinal mucosal cell apoptosis. The presentstudy was designed to investigate the effect of propo-fol pretreatment on II/R-induced intestinal mucosalepithelial apoptosis as well as its effects on ceramidesignaling.

METHODSAnimal Model

The current study was approved by the AnimalCare Committee of Sun Yat-sen University, Guang-zhou, China and was performed in accordance withNational Institutes of Health guidelines for the use ofexperimental animals. Thirty adult pathogen-freemale Wistar rats weighing between 240 and 305 g,were housed in individual cages in a temperature-controlled room with alternating 12 h light/darkcycles and acclimatized for 1 wk before the study.Food was removed 8 h before the study, but allanimals had free access to water. All animals wereanesthetized with pentobarbital (30 mg/kg bodyweight, intraperitoneally [ip]), and the small intestinewas exteriorized by midline laparotomy. The II/Rinjury was established by occluding the superiormesenteric artery (SMA) with a microvessel clip for1 h followed by 3 h reperfusion as previouslydescribed.9,18 Ischemia was recognized by the devel-opment of pulselessness and pale color of the smallintestine. The return of pulses and the reestablishmentof the pink color were assumed to indicate validreperfusion.

Experimental ProtocolThe rats were randomly allocated into 1 of the 3

groups (n � 10 per group): i) control group (control),in which sham surgical preparation including isola-tion of the SMA without occlusion was performed; ii)injury group (injury), in which II/R was produced byclamping the SMA (ischemia) for 1 h followed bydeclamping the SMA (reperfusion) for 3 h; iii) Propo-fol pretreatment group (propofol), in which propofolwas given 30 min before intestinal ischemia wasinduced. In the treatment groups, propofol (Diprivan,propofol 1%, CG411, AstraZeneca, Caponago, Italy) 50mg/kg was administrated ip. Animals in the controland injury groups received an equal volume of in-tralipid (vehicle solution of propofol) by ip injection.The dose of propofol was chosen based on our previ-ous experiment18 as well as a study from anothergroup that shows that administration of propofol 50

mg/kg ip inhibited rat hippocampal acetylcholinerelease to a lesser degree than propofol 100 mg/kgip19, and produced a sedative response in rats, esti-mated as loss of reflex responses to a painful stimulus,while remaining sensitive to skin incision (i.e., seda-tion rather than anesthesia). By contrast, propofol 60mg/kg ip, as reported by Brasil et al.,20 providedsatisfactory anesthesia in rats. In addition, during ourpreliminary experiments, we found that neither ipintralipid (n � 4) or physiological saline (n � 3)influenced the extent of intestinal mucosal cell apo-ptosis in the injury group. Therefore, only intralipidwas used as a vehicle control in the ensuing studies.

Preparation of SpecimensAfter the completion of the experiments, the rats

were killed with an IV overdose of pentobarbitalsodium. A segment of 0.5–1.0 cm intestine was cutfrom 5 cm to terminal ileum, fixed in 4% formalde-hyde polymerisatum, and embedded in paraffin forpreparation. Another segment of small intestine waswashed with cold saline and the intestinal mucosawas gently scraped off, dried with suction paper, andpreserved at �70°C.

Intestinal Mucosal Epithelial Apoptosis Detection underTransmission Electron Microscopy

The ileal segments were taken from a similar posi-tion in each rat and were fixed with phosphate-buffered glutaraldehyde (Ladd Research Industries,England), postfixed with osmium tetroxide (Prolabo,France) and then dehydrated, processed, sliced andobserved under a Zeiss 902 electron microscope (CarlZeiss, Thornwood, NY).

Situ Detection of Intestinal Mucosal Epithelial ApoptosisThe ileal fragments were fixed in 4% formaldehyde

polymerisatum and embedded in paraffin. The apopto-sis of intestinal mucosal epithelial cell was detec-ted by the terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling (TUNEL)method. Cell death was assessed using an assay kit(Roche, Indianapolis, IN). Briefly, specimens weredewaxed and immersed in phosphate-buffered salinecontaining 3 g/L hydrogen peroxide for 10 min atroom temperature and then incubated with 20 �g/mLproteinase K for 15 min at room temperature. Equili-bration buffer (75 �L) was applied directly onto thespecimens for 10 min at room temperature, followedby incubation with 55 �L of terminal deoxynucleoti-dyl transferase enzyme at 37°C for 1 h. The reactionwas terminated by transferring the slides to pre-warmed stop/wash buffer for 30 min at 37°C. Thespecimens were covered with a few drops of rabbitserum and incubated for 20 min at room temperatureand then covered with 55 �L of antidigoxigeninperoxidase and incubated for 30 min at room tempera-ture. Specimens were then soaked in Tris buffer con-taining 0.2 g/L diaminobenzidine and 0.2 g/L hydro-gen peroxide for 1 min for color development. Finally,

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the specimens were counterstained by immersion inhematoxylin. The cells with clear nuclear labelingwere defined as TUNEL-positive cells. The apoptosiswas initially evaluated independently by two patholo-gists who were blinded to the study groups. The rateof cell apoptosis (apoptotic index) was calculated aspercentage of TUNEL-positive cells using the follow-ing formula: the number of TUNEL-positive cellnuclei/the number of total cell nuclei) � 100.

Detection of Lipid Peroxidation and SuperoxideDismutase (SOD) Activity in Intestinal Mucosa

Intestinal mucosal tissues were homogenized on icewith normal saline and centrifuged for 15 min at 4000g.Supernatants were transferred into fresh tubes for theevaluation. The lipid peroxidation product malonediade-hyde (MDA) was measured by chemical analysis (Assaykits were supplied by Nanjing Jiancheng Biological Prod-uct, Nanjing, China) as previously described.21 The resultswere expressed as nanomoles per 100 mg tissue. SODactivity was evaluated by inhibition of nitroblue tetrazo-lium reduction by superoxide anion generated by thexanthine/xanthine oxidase system using a commercialassay kit (Nanjing Jiancheng Biological Product, Nan-jing, China) as previously described.21 The resultswere expressed as Units per 100 mg protein.

Detection of Ceramide Level in Intestinal MucosaThe ceramide level was determined by high perfor-

mance thin layer chromatography as described byDasgupta and Hogan.22 Tissue was homogenized withchloroform-methanol-water 2:4:1 (v/v/v; 14 mL/g oftissue), stirred for 1 h and centrifuged. The pellet wasreextracted with the same solvent. A third extractionwas performed with chloroform-methanol 2:1 (v/v).The extracts were pooled, dried, and suspended in aminimum volume of chloroform. The chloroform sus-pension was applied to a silicic acid column (0.5 � 5cm) and washed with 15 column volumes of chloro-form to remove nonpolar lipids (which can be assayedby TLC). The column was then eluted successivelywith chloroform-acetone 9:1 (v/v; 15 column vol-umes), and then dried and stored at 4°C until use.Ceramide, purified by silicic acid chromatography[chloroformacetone 9:1 (v/v)], was dissolved in adefined volume of chloroform (1.0 mL/g tissue) and15 �L was applied to a high performance thin layerchromatography plate, developed with chloroform-methanol-acetic acid 95:4.5:0.5 (v/v/v) and visualizedby benzidine spray. A measured volume (5 �L) of theceramide solution was removed and methanolyzedwith methanol-water-HCl 29:4:3 (v/v/v) at 80°C for18 h, and the recovered base was analyzed by gaschromatography as the trimethylsilyl derivative. Therecovery of the standard ceramide purified throughthe silicic acid column, using chloroform-acetone 9:1(v/v), was more than 95%.

Detection of Neutral- Sphingomyelinase (SMase)mRNA Expression by Semi-QuantitativeReverse-Transcription Polymerase Chain Reaction(RT-PCR) in Intestinal Mucosa

The hydrolysis of sphingomyelin regulated bysphingomyelinase (SMase) is known as the mainregulating mode of endogenous ceramide level.11,23

At least three kinds of SMase, including acid-SMase,neutral-SMase, and alkaline-SMase have been dem-onstrated to be involved in the generation of cer-amide.24,25 It is reported that neutral SMase plays amore important role in the development of apopto-sis relative to that of the acid-SMase and alkaline-SMase.26 –29 Therefore, we only investigated thechange of neutral SMase expression in the currentstudy.

Total RNA was extracted from intestinal mucosaltissue using Trizol reagent (Gibco BRL, Gercy-Pontoise, France), cDNA was synthesized from 4 �g ofthe total RNA by extension of random primers withMMLV reverse transcriptase (Promega, Madison, WI).Polymerase Chain Reaction (PCR) of the cDNA wasperformed in a final volume of 25 �L containing 2 mMMgCl2, 4 Ues Taq DNA polymerase (Gibco BRL,Gercy-Pontoise, France), and 25 pmol specific primers.�-action was used as a control. The PCR was per-formed in the following conditions: denaturation for 3min at 95°C, annealing for 30 s at 61°C for SMase and62°C for �-actin, extension for 30 s at 72°C and for 35cycles followed by further extension for 7 min at 72°C.Primer sequences were as follows: neutral-SMase, 5�TGA TGC CTT TGT TGA GAC CGA 3� (sense); 5�GGA AGC CAG AGA CTG CCT TGTA 3� (antisense);�-action, 5� AGC CAT GTA CGT AGC CAT CC-3�(sense); 5� GTC CAT GCA GTT CTT GGT CA 3�(antisense). The synthesized PCR products were sepa-rated by electrophoresis on a 20 g/L agarose gel andanalyzed by Gel-Pro analyzer version 3.1 software(Media Cybernetics, GA). The ratio of target genesover �-actin was used for the relative level of mRNAexpression.

Statistical AnalysisResults are expressed as mean � sd. Biochemical

assays for MDA, SOD, and ceramide were performed induplicate or triplicate for each specific sample. Signifi-cance was evaluated using analysis of variance (Graph-Pad Prism 4, San Diego, CA) followed by Tukey’s post test.The correlation relationships were evaluated by the Pear-sons test. P � 0.05 was considered statistically significant.

RESULTSChanges of Intestinal Mucosal Epithelial Apoptosis UnderTransmission Electron Microscopy and Light Microscopy

No animals died in this experiment. Electron mi-croscopy was used to confirm morphological featuresof cell apoptotic alterations. Figure 1A (control group)shows normal epithelial cells. Significant epithelial cell

1886 Propofol Inhibition of Intestinal Apoptosis ANESTHESIA & ANALGESIA

apoptosis in the intestinal mucosa, manifested bymarked cytoplasm shrinkage, nuclear/chromatin con-densation, and formation of apoptotic bodies, wasseen in the injury group (Fig. 1B), This was attenuatedby pretreatment with propofol (Fig. 1C). TUNEL-positive epithelial cells at the villus surface staineddark brown in the nuclei (Fig. 2). In the control group,few TUNEL-positive cells were found (Fig. 2A). Incontrast, the apoptotic index was significantly higherin the injury group (Fig. 2B) than that in the controlgroup (P � 0.01, Fig. 2D). However, pretreatment withpropofol significantly suppressed apoptosis and re-duced the apoptotic index, compared to the injurygroup (P � 0.01) as shown in Figures 2C and D.

Changes of the MDA Level and the Activity of SOD inIntestinal Mucosa

As shown in Figure 3A, the MDA level in the injurygroup was significantly higher than that in the controlgroup (P � 0.01), and this was significantly reducedby pretreatment with propofol (P � 0.01, propofolgroup versus injury group).However, the SOD activityin the injury group was markedly reduced (P � 0.01,injury versus control group), but propofol signifi-cantly increased and restored SOD activity (P � 0.01,propofol versus injury or control, Fig. 3B).

Change of the Ceramide Level in Intestinal MucosaIn the injury group, the ceramide level was signifi-

cantly higher than that in the control group (P � 0.01).It was reduced by propofol pretreatment (P � 0.01,propofol versus injury, Fig. 4).

Change of SMase mRNA Expression in Intestinal MucosaSMase mRNA expression was detectable in the

control group and was significantly increased in theinjury group (P � 0.01, injury versus control, Fig. 5).Compared to the injury group, SMase mRNA expres-sion was markedly down-regulated in the propofolgroup (P � 0.01) to a level comparable to the controlgroup (P � 0.05) (Fig. 5).

Correlation AnalysisOverall (n � 30), the SMase mRNA expression was

positively correlated to the MDA level (r � 0.554, P �0.002, Fig. 6A), and negatively correlated to the SODactivity (r � �0.414, P � 0.023, Fig. 6B). In addition,strong positive correlations between the SMasemRNA expression and the ceramide level (r � 0.676,P � 0.000, Fig. 6C) and between the ceramide leveland the apoptosis index (r � 0.849, P � 0.000, Fig. 6D)were identified.

Figure 1. Changes of intestinal muco-sal epithelial apoptosis under trans-mission electron microscopy. Rats ei-ther underwent a sham operation(control), 1 h occlusion of the supe-rior mesenteric artery (SMA) fol-lowed by 3 h of reperfusion (injury),or received propofol pretreatment be-fore 1 h occlusion of the SMA fol-lowed by 3 h of reperfusion (propo-fol). Arrows indicate normalintestinal mucosal epithelial cells inthe control group (A, �6600), typicalapoptotic intestinal mucosal epithe-lial cells in injury (B, �8900) andpropofol (C, �8900) groups.

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DISCUSSIONWe have demonstrated in a rat model that 1-h

occlusion of the SMA followed by 3 h of reperfusioncaused significant intestinal epithelial apoptosis inaccordance with previous reports.4–6,9 The intestinalepithelial apoptosis induced by II/R was accompa-nied by dramatic increases in the intestinal mucosa of

MDA, ceramide and SMase mRNA expression, and adecrease in SOD activity, confirming our recent studyresults conducted in the same model.9,18 The novelfinding of the current study is that pretreatment withpropofol, at a dose that has been shown to producesedation in the rat,19 attenuated intestinal mucosalapoptosis induced by SMA occlusion and reperfusion.Apoptosis has been established as a major mode ofintestinal mucosal cell death caused by II/R injury.4–6

Therefore, the protective effect of propofol on intesti-nal mucosal injury induced by II/R18 could be attrib-utable, at least in part, to prevention of intestinalmucosal cell apoptosis.

It has been shown that ceramide is involved in theprocess of cellular apoptosis10 and the hydrolysis ofsphingomyelin regulated by SMase is known as themain regulating mode of endogenous ceramide lev-els.11,23 The tight positive correlation between theapoptosis index and the ceramide level, as well as

Figure 2. Effect of propofol on intesti-nal mucosal cell apoptosis by lightmicrographs of the ileum stained us-ing TUNEL assay (�200). Groups arethe same as Figure 1. Apoptotic nu-clei are stained dark brown indicatedby arrows. (A) In the control group,few apoptotic epithelial cells werepresent at the villous tips. (B) In theinjury group, quite a few apoptoticepithelial cells were seen. (C) In thepropofol group, some apoptotic epi-thelial cells were seen, but the apo-ptotic index is significantly lowerthan that in the injury group (D).Data are mean � sem *P � 0.01versus control; #P � 0.01 versus in-jury. n � 10 rats per group.

Figure 3. Effects of propofol on the malondialdehyde (MDA)level (A) and the superoxide dismutase (SOD) activity (B) inintestinal mucosa. Groups as in Figure 1.Data are mean �sem *P � 0.01 versus control; #P � 0.01 versus injury. n � 10rats per group.

Figure 4. Effect of propofol on the ceramide level in ratintestinal mucosa. Groups as in Figure 1. Data are mean �sem *P � 0.01 versus control; #P � 0.01 versus injury. n � 10rats per group.

1888 Propofol Inhibition of Intestinal Apoptosis ANESTHESIA & ANALGESIA

between the ceramide level and SMase mRNA expres-sion in the present study, is in line with our recentstudy results in the same model,9 which supports

ceramide as a mediator of apoptotic cell death in thismodel. The present findings showed that propofolpretreatment could down-regulate SMase mRNA ex-pression and reduce ceramide production, which isanother novel finding of the present study. The abovefindings suggest that the antiapoptotic effect of propo-fol is related to the ceramide-SMase pathway, al-though further study is needed to confirm a potentialcausative role that the ceramide-SMase pathway mayhave played in propofol-mediated protection.

In our understanding, the relationship betweenthe SMase-ceramide pathway and propofol’s effectagainst II/R-induced intestinal mucosal cell apoptosismay be explained as follows. First, studies showedthat antioxidants can inhibit apoptosis by down-regulating neutral SMase expression and reducingceramide production.26,27 The tight negative correla-tion between SOD activity and SMase mRNA expres-sion and the significant positive correlation betweenMDA levels and SMase mRNA expression in thepresent study are, in fact, supportive of the conceptthat ROS are involved in mediating SMase expression.Also, in neuronal and vascular cells, ROS were re-ported to be critical in mediating the downstreameffects of ceramide, including mitochondrial iron up-take, cytochrome c release, caspase 3 activation, andapoptosis.30 These findings indicate that ROS appearto interact with cell apoptosis processes mediated bythe SMase-ceramide pathway in various tissues. Sec-ond, propofol is an excellent free radical scavengerthat has been shown to enhance the antioxidativeability of various tissues both in vivo and in vitrostudies.10,11 Of interest, propofol (50 mg/kg ip) in-creased SOD activity to a level higher than that in thecontrol group (Fig. 3B). This result is similar to thatreported by Zhu et al.31 They found that propofol at aclinically relevant low concentration (1 �M), but not at

Figure 5. Effect of propofol on the sphingomyelinase (SMase)mRNA expression in rat intestinal mucosa. Groups as inFigure 1. The SMase mRNA expression was analyzed byRT-PCR. The ratio of target genes over �-action was used forthe relative level of mRNA expression (Top). In the injurygroup, SMase mRNA expression was significantly up-regulated (P � 0.01), but was markedly down-regulated inthe propofol and control groups, as demonstrated in the bargraph (Bottom). The internal control gene (�-action) expres-sion was almost the same level in the all samples. Data aremean � sem *P � 0.01 versus control; #P � 0.01 versusinjury. n � 10 rats per group.

Figure 6. The scatter plots of correlativerelationships between different param-eters. (A) The malondialdehyde leveland the sphingomyelinase (SMase)mRNA expression (r � 0.554, P �0.002). (B) The superoxide dismutase(SOD) activity and the SMase mRNAexpression (r � �0.414, P � 0.023). (C)The SMase mRNA expression and theceramide level (r � 0.676, P � 0.0001).D. The ceramide level and the apopto-sis index (r � 0.849, P � 0.0001).

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higher concentrations (�10 �M) increased SOD activ-ity to a level higher than that in the control group inthe primary cultured newborn rat hippocampus.31

The mechanism for this propofol effect is not clear, butit could be attributable to propofol’s preconditioning-like effect as we described previously.15 Thus, thesuppressive effect of propofol on ceramide productioncould be mainly attributable to this antioxidativeproperty. Third, the effect of propofol on the SMase-ceramide pathway could be relevant to its effects oninflammatory cytokines. II/R can damage the intesti-nal barrier, which in turn causes the release of someproinflammatory cytokines such as tumor necrosisfactor and interleukin-6.3,32 These proinflammatorymediators can stimulate the hydrolysis of sphingomy-elin regulated by SMase, and thereby, increase thegeneration of ceramide.18 Thus, proinflammatory cy-tokines could serve as a link between II/R and theceramide pathway. Studies have shown that propofolattenuates cytokine responses (tumor necrosis factor�, interleukin-6) to endotoxemia in vivo33–35 and invitro.36 Taken together, the effect of propofol on theceramide pathway may be related to its antioxidantproperty and inhibition of cytokine responses, al-though further study is needed to confirm this.

Our findings suggest that propofol is a promisingdrug for the prevention of II/R injury. However, itshould be noted that our study may have severalpossible limitations. First, although propofol at 50mg/kg ip, a sedative or subanesthetic dose, may notsignificantly affect cardiac functions, hemodynamicvariables such as arterial blood pressure and cardiacoutput were not monitored. Therefore, we do notknow whether propofol, at the dose used, could affectintestinal blood perfusion during postischemic intes-tinal reperfusion. Second, simultaneous detection ofthe expression of apoptosis-related genes and pro-teins, such as bcl-2, bax and caspase, shouldstrengthen the mechanistic study and confirmation ofapoptosis. However, the TUNEL method in combina-tion with electron microscopy should be sufficient toconfirm the existence of apoptosis, given that intesti-nal mucosal epithelial apoptosis induced by II/R inthis model has been well-established by previousstudies.4–6 Third, the ceramide signal pathway is verycomplex, but we only investigated its upstreamevents, such as oxidative stress and mRNA expressionof netural-SMase. Therefore, whether downstreamevents in the ceramide pathway relevant to II/R-induced intestinal mucosal cell apoptosis are involvedin propofol’s antiapoptotic effect needs to be furtherinvestigated. Also, further studies should be con-ducted to investigate the propofol dose-response rela-tionship with apoptosis on this model, which willprovide more convincing evidence of a cause-effectrelationship.

In conclusion, the present results indicate that pre-treatment with a sedative dose of propofol attenuates

intestinal epithelial apoptosis and this may be attrib-utable to its antioxidant property in mediating theSMase-ceramide pathway. This finding is worthy offurther clinical study in situations where propofol isan option for sedation and anesthesia in patients atrisk of II/R.

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