Preventive Effect of Probiotics and a-Tocopherol on Ethanol-InducedGastric Mucosal Injury in Rats

Altug Senol,1 Mehmet Isler,1 Aynur G. Karahan,2 Gulden B. Kilic,3 Hakan Kuleasan,2 Selcuk Kaya,4

Muharrem Keskin,1 Ibrahim Goren,1 Ulku Saritas,1 Buket C. Aridogan,4 and Namik Delibas5

Departments of 1Gastroenterology, 4Microbiology, and 5Biochemistry, Faculty of Medicine, and 2Department of FoodEngineering, Faculty of Engineering, Suleyman Demirel University, Isparta; and 3Burdur Vocational School of Higher Education,

Department of Dairy Products, Mehmet Akif Ersoy University, Burdur, Turkey

ABSTRACT The protective effect of a probiotic mixture of 13 different bacteria and a-tocopherol on 98% ethanol-induced

gastric mucosal injury was evaluated. Levels of gastric mucosal pro- and anti-inflammatory cytokines, malondialdehyde, and

secretory immunglobulin A were measured. Rats were allocated into four groups: control, ethanol, probiotic, and a-

tocopherol. The control and ethanol groups received skim milk for 14 days. Probiotic and a-tocopherol groups were ad-

ministered probiotic mixture suspended in skim milk and 100 mg=kg a-tocopherol, respectively, by daily gavage for 14 days.

On Day 15, gastric lesions were induced by administration of ethanol 98% (1 mL) to all rats except those in the control group.

Probiotic, but not a-tocopherol, seemed to inhibit ethanol-induced gastric mucosal tumor necrosis factor-a, interferon-g, and

interleukin-2 production (P> .05). Ethanol caused the elevation of mucosal interleukin-4 level (compared to the control,

P< .05). Probiotic pretreatment significantly suppressed the ethanol-induced increase of gastric mucosal interleukin-4 levels.

Pretreatment with either probiotic or a-tocopherol inhibited the ethanol-induced increase of mucosal malondialdehyde con-

centration (P< .01 and P< .05, respectively). Probiotic pretreatment enhanced the gastric mucosal secretory immunoglobulin

A concentration (P< .001). In conclusion, probiotic mixture and a-tocopherol reduced ethanol-induced gastric mucosal lipid

peroxidation, suggesting that they may be beneficial for gastric lesions induced by lower ethanol concentration.

KEY WORDS: � ethanol � gastric injury � probiotic � rat � a-tocopherol

INTRODUCTION

Ethanol has long been recognized as the causativeagent of acute hemorrhagic gastric lesions in experi-

mental and clinical studies. Ethanol-induced gastric mucosaldamage is associated with the depletion of gastric mucuscontent, decreased mucosal blood flow, and mucosal orgastric cell injury.1,2 Additionally, administration of abso-lute ethanol has been shown to significantly increase tumornecrosis factor (TNF)-a and other pro-inflammatory cyto-kines.1,3,4 Pentoxifylline, having anti-TNF properties, hasbeen documented to prevent alcohol-induced gastric muco-sal injury in rats.5 TNF-a augments neutrophil-derived su-peroxide generation,6 leading to oxygen radical-mediatedtissue damage. The radical-removing agents have beenshown to stimulate the healing of ethanol-induced acutegastric mucosal injury in rats.7 Enzymatic antioxidants suchas superoxide dismutase and catalase are also effective inulcers induced by ethanol.8–10

Vitamin E is a major lipid-soluble antioxidant in thecell antioxidant defense system at low concentrations. Inischemia-reperfusion (I=R)-induced gastric mucosal injuryin rats, pretreatment with a water-soluble vitamin E deriv-ative significantly inhibited the increase of pro-inflammatorycytokines and TNF-b.11 It has been reported that vitamin Escavenges chain-carrying peroxyl radicals, singlet oxygen,12

and superoxide anion radical.13,14 Some studies have shownthat vitamin E has a protective effect, with decreasing lipidperoxidation, against gastric mucosal injury induced byI=R,15–17 by nonsteroidal anti-inflammatory drugs,18,22 andby ethanol.2

As with a-tochopherol, pretreatment with some probioticstrains has been reported to reduce the severity of the pan-creatitis,23 colitis,24 and I=R injury of the liver,25 decreasinglipid peroxidation. Escherichia coli Nissle 1917, a probiotic,has been shown to significantly inhibit the expression of in-terleukin (IL)-2, TNF-a, and interferon (IFN)-g, but to in-crease IL-10 production in peripheral blood T cells.26 Cellwall components of Lactobacillus casei have been found tobe anti-inflammatory.27 Treatment of IL-10-deficient micewith VSL#3 DNA, a mixed probiotic preparation, resulted ina reduction in colonic mucosal secretion of TNF-a and IFN-g

Manuscript received 14 February 2010. Accepted 17 June 2010.

Address correspondence to: Altug Senol, Department of Gastroenterology, Faculty ofMedicine, Suleyman Demirel University, Isparta, Turkey, E-mail: senolaltug@gmail.com

JOURNAL OF MEDICINAL FOODJ Med Food 14 (1/2) 2011, 173–179# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089=jmf.2010.0040

173

and improvement in histological disease.28 Two novel pro-teins, p75 and p40, purified from Lactobacillus rhamnosusGG broth culture supernatant have been shown to signifi-cantly reduce TNF-a-induced colon epithelial damage.29

The findings noted above suggest that pretreatment withprobiotics may prevent ethanol-induced gastric injury. Inthis study, we evaluated the protective effect of a probioticmixture, including 13 different bacteria, on 98% ethanol-induced gastric mucosal injury and compared with that of a-tocopherol. Gastric mucosal pro- and anti-inflammatorycytokine levels and malondialdehyde (MDA) concentra-tions, as an index of lipid peroxidation, were studied.

It has been determined that some orally administeredstrains of lactic acid bacteria were able to increase thenumber of secretory immunoglobulin A (sIgA)-producingcells in the small intestines of mice,30,31 and Enterococcusfaecium SF68 increases intestinal sIgA production in dogs.32

On the other hand, in an in vitro study, Diebel et al.33 havedemonstrated that sIgA modulates the increased epithelialapoptotic response and preserves barrier function after asimulated I=R insult. Therefore gastric mucosal sIgA levelswere also measured.

MATERIALS AND METHODS

Animals

Wistar albino male rats (Suleymen Demirel UniversityExperimental Animal Production Center, Isparta, Turkey),with a weight of 180–220 g, were fed rat chow and tap waterad libitum and kept in cages at 22� 28C with a 12-hourdark–light cycle before and during the experiments. Ex-periments were performed in accordance with the NationalGuidelines for The Use and Care of Laboratory Animals andwere approved by the Suleyman Demirel University Schoolof Medicine Ethical Committee.

Probiotic microorganisms

Thirteen bacteria that showed resistance to the low pHand bile salts among the 107 probiotic bacterial strainswere selected from healthy human stool34 (four strains ofLactobacillus fermentum [BB16-75, AK2-8, AK5-22, andAK6-26], three strains of Lactobacillus plantarum [AA17-73, AK7-28, and AK8-31B], and six strains of E. faecium[AB6-21, AB16-68, AK4-120, AK7-31, BK9-40, andBK13-54]). Molecular identification of these strains wasdone with 16S rRNA analysis.35 Each strain was inoculatedin MRS broth medium and incubated at 378C for 24 hoursuntil the cell number reached 109 colony-forming units=mL.The cells were pelleted by centrifugation at 5,000 g for 10minutes at 208C, and the pellets were washed in phosphate-buffered saline solution (pH 7.4) twice. Finally, the pro-biotic mixture was adjusted to 1.3�1010 colony-formingunits=mL in 10% reconstituted sterile skim milk.

Experimental design

Thirty-two rats were allocated into four groups with dif-ferent treatments for 14 days, as follows: (1) control group

(n¼ 7) rats received 0.2 mL=day skim milk by daily gavage;(2) ethanol group (n¼ 7) rats received 0.2 mL=day skimmilk by daily gavage; (3) probiotic group (n¼ 10) rats re-ceived 0.2 mL=day probiotic mixture (0.2 mL; 1.3�1010

colony-forming units=mL) suspended in skim milk by dailygavage; and (4) a-tocopherol group (n¼ 8) rats received100 mg=kg in a volume of 0.2 mL=day a-tocopherol by dailygavage.

On Day 15, gastric lesions were induced by administra-tion of 98% ethanol (1 mL) to all rats except those in thecontrol group, which were given saline (1 mL). Coprophagywas avoided. All agents given orally were administered bygavage through an intragastric tube.

Twelve hours before ethanol (or saline) administration,rats were deprived of food, but they had free access to water2 hours before ethanol (or saline) administration.

Macroscopic analysis

The animals were sacrificed by intramuscular injection of100 mL=kg ketamine hydrochloride (Ketalar�, Parke-Davis,Eczacibasi, Istanbul, Turkey) and 25 mg=kg xylazine hydro-chloride (Rompun�, Bayer, Germany) 1 hour after ethanol(or saline) administration, and the stomach was removed.

Stomachs were opened along the greater curvature, andmucosae were rinsed with cold phosphate-buffered salinesolution to remove blood concomitant, if any. Gastric mu-cosal lesions were evaluated by two independent observersblinded to the treatment. A damage score was assigned usinga scale of 0–6 described by Coleman et al.36

Sample collections

Gastric mucosal tissues were removed by scraping with aglass slide and immediately frozen in liquid nitrogen andstored at �808C until determination of gastric TNF-a, IFN-g, IL-2, IL-4, sIgA, and MDA concentrations.

Determination of cytokines and sIgA

Gastric tissues were weighed and homogenized (1:10,wt=vol) (Ultra Turrax T25, IKA-Labortechnik, Staufen,Germany) in 100 mmol=L of phosphate buffer (pH 7.4)containing 0.05% sodium azide in an ice bath. The ho-mogenate was sonicated (Bandelin, Berlin, Germany) for 30seconds and centrifuged (5,000 g for 10 minutes). Thegastric mucosal levels of rat TNF-a, IFN-g, IL-2, and IL-4were measured using a commercial enzyme-linked immu-nosorbent assay kit (Biosource International Inc., Camarillo,CA, USA), according to the manufacturer’s instructionswithout modification. A standard curve was constructedfrom a series of known concentrations of TNF-a, IFN-g, IL-2, and IL-4 solution provided by the kit manufacturer, andthe TNF-a, IFN-g, IL-2, and IL-4 concentrations of theunknown samples were determined by interpolation. Thegastric mucosal levels of sIgA were measured using acommercial enzyme-linked immunosorbent assay kit (Im-mundiagnostik AG, Bensheim, Germany), according to themanufacturer’s instructions without modification.

174 SENOL ET AL.

The levels are expressed as picograms for TNF-a, IFN-g,IL-2 and IL-4, and nanograms for sIgA concentrations permilligram of total protein in the supernatant, where the totalprotein levels are determined by the method of Lowryet al.37

Determination of MDA

Tissue (500 mg) was homogenized at 2,400 rpm withan Ultra Turrax in a volume of 1.15% KCl. For alkalinehydrolysis of protein-bound MDA, 200 mL of 6 M NaOHwas added to 1 mL of homogenate in an Eppendorf cup,and the sample was incubated in a 608C water bath for 45minutes. An aliquot of 1 mL was diluted with an equalvolume of acetonitrile to precipate proteins. The resultingsuspension was then vortex-mixed for 30 seconds andcentrifuged at 15,000 g for 10 minutes. The upper clearsupernatant (0.25 mL) was transferred to a 2-mL Eppen-dorf cup, mixed with 25 mL of 2,4-dinitrophenylhydrazinesolution, and incubated for 10 minutes. After derivatiza-tion, the sample was filtered (pore size, 0.2 mm). Aliquotsof 20 mL were injected into the high-performance liquidchromatography system.

The samples were analyzed on a Thermo Finnigan(Waltham, MA, USA) series high-performance liquidchromatography apparatus. The analytical column was ODS2 C18 (particle size, 5 mm; 125�4 mm Nucleosil). The mo-bile phase was acetonitrile–distilled water (38:6 vol=vol)containing 0.2% (vol=vol) acetic acid. The high-perfor-mance liquid chromatography apparatus was operated underisocratic conditions at a flow rate of 1 mL=minute, and theultraviolet detector was set at 310 nm. MDA peaks weredetermined according to its retention time and confirmed byspiking with added exogenous standard. Concentrations ofMDA were calculated from a standard curve prepared from1,1,3,3-tetraethoxypropane and expressed nanomoles permilligram of protein for tissue.

Statistical analysis

Results were presented as mean� SEM values. Macro-scopic damage scores were statistically evaluated using non-parametric Kruskal–Wallis and Mann–Whitney U tests.Comparison of all other variables was performed with one-way analysis of variance and least-significant differencemethod. All analyses were performed using the statisticalsoftware SPSS version 11.0 (SPSS, Chicago, IL, USA). AP value of <.05 was considered statistically significant.

RESULTS

Effects of a-tocopherol and probiotics on gastricmucosal lesions

Intragastric administration of 98% ethanol induced mul-tiple hemorrhagic erosions in the rat stomach. In the controlgroup negligible damage in the rat stomach was observed. Inthe ethanol group, the mean gastric mucosal damage scorewas 5.14� 0.26. Probiotic mixture and a-tocopherol pre-treatment minimally reduced ethanol-induced gastric dam-

age to 4.70� 0.26 and 4.13� 0.48, respectively (P> .05).Figure 1 shows the gastric mucosal damage scores.

Effects of a-tocopherol and probiotics on gastricmucosal cytokine levels

Exposure of gastric mucosa to 98% ethanol (1 mL, 1-hour exposure) resulted in the increase of mucosal pro-inflammatory cytokine levels to different degrees as shownin Table 1. However, the differences between the controland ethanol-treated animals were not statistically significant.Probiotic pretreatment, but not a-tocopherol pretreatment,seemed to inhibit ethanol-induced TNF-a, IFN-g, and IL-2production. Comparing the control group with the ethanol-treated animals, the IL-4 level was elevated (P< .05) in theethanol group; probiotic pretreatment, but not a-tocopherol,significantly suppressed the increase of IL-4 in the gastricmucosa.

Effects of a-tocopherol and probiotics on gastricmucosal lipid peroxidation

Ethanol ingestion significantly increased the gastric mu-cosal levels of MDA, as an index of lipid peroxidation,

FIG. 1. Effects of a-tocopherol and probiotics on gastric mucosallesions.

Table 1. Effects of Vitamin E and Probiotics on Gastric

Mucosal Cytokine Levels

Level (pg=mg of protein)

Group TNF-a IFN-g IL-2 IL-4

Control 14.77� 1.59 3.70� 0.75 13.88� 3.72 2.82� 0.94*Ethanol 16.30� 2.83 4.89� 0.91 19.48� 3.92* 6.59� 1.90{

Probiotic 11.29� 1.61* 3.61� 0.48 11.08� 1.65 2.62� 0.65a-Tocopherol 17.07� 2.39 4.33� 0.60 13.65� 2.09 4.94� 0.88

Statistical significance was evaluated by analysis of variance followed by a

post hoc least-significant difference test: P< .05 *for TNF-a compared with

the a-tocopherol group, for IL-2 compared with the probiotic group, and for

IL-4 compared with the ethanol group; {for IL-4 compared with the probiotic

group.

IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

ETHANOL GASTROPATHY: PROBIOTICS=a-TOCOPHEROL 175

compared to the control group (0.502� 0.153 and0.163� 0.020 nmol=mg of protein, P< .01). Pretreatmentwith either probiotic and a-tocopherol inhibited the ethanol-induced increase of mucosal MDA concentration(0.211� 0.009 nmol=mg of protein and 0.221� 0.022nmol=mg of protein, respectively [P< .01 and P< .05,respectively]) (Fig. 2).

Effects of probiotics on gastric mucosal sIgA levels

The gastric mucosal sIgA levels were measured in onlythe control, ethanol, and probiotic groups (n¼ 4–5 rats ineach groups). Ethanol ingestion did not affect the gastricmucosal sIgA level (313.75� 19.75 ng=mg of protein in thenormal control group and 502.74� 280.66 ng=mg of proteinin the ethanol group, P> .05). Probiotic pretreatment en-hanced the gastric mucosal sIgA concentration as shown inFigure 3 (4,006.74� 608 ng=mg of protein; P< .001 com-pared with the control and ethanol groups).

DISCUSSION

Probiotics are defined as live microorganisms that, whenconsumed in adequate numbers, confer a health benefit onthe host.38 There is growing evidence that probiotic treat-ment may be helpful in the treatment of various gastroin-testinal inflammatory disorders, including Crohn’s disease,39

ulcerative colitis,40,41 and pouchitis.42

Probiotics may exert beneficial effects on ethanol-induced gastric damage in several ways. There are manydata indicating that some selected probiotic bacteria couldenhance tight junctional integrity and intestinal barrierfunction against noxious agents.43–45 Selective probioticshave also been reported to induce mucin gene expression incolonic epithelial cell lines.46–48 In an in vivo study, L.rhamnosus GG enhanced gastric ulcer healing via the at-tenuation of cell apoptosis to cell proliferation ratio and anincrease in angiogenesis in acetic acid-induced gastriculcers in rats.

In contrast, in a recent study, Bifidobacterium lactis Bb12did not reduce indomethacin-induced ulceration in the smallintestine of Sprague–Dawley rats, but also L. rhamnosus GG

actually increased some indicators of injury.49 In addition,myeloperoxidase activity induced by indomethacin in thesmall intestine of rats has been shown to be increased by L.rhamnosus treatment.49

Lam et al.50 reported that pretreatment of rats with L.rhamnosus GG at 109 colony-forming units=mL twice dailyfor three consecutive days markedly reduced the 60% ethanol-induced mucosal lesion area, increasing the basal mucosalprostaglandin E2 level, attenuating the suppressive actions ofethanol on the mucus-secreting layer and transmucosal resis-tance, and reducing cellular apoptosis in the gastric mucosa ofrats.

In the present study we tested the pretreatment withprobiotic bacteria mixture and a-tocopherol on the 98%ethanol-induced gastric damage, focusing on their anti-inflammatory and antioxidative actions. Many studies provideevidence for multistrain probiotics being more effectivethan monostrain probiotics.51,52 Moreover, the use of mul-tispecies preparations, containing multiple strains of morethan one genus, could even be more effective than that ofmultistrain probiotics.51 Therefore we prefer the use of amultispecies–multistrain probiotic combination. Some spe-cies of lactobacilli or bifidobacteria produce antioxidantsubstances that could have a protective effect against in-testinal mucosal barrier damage. It has been shown thatBifidobacterium infantis DSM 15159 significantly de-creased MDA concentrations in rats with dextransulfatesodium-induced colitis, compared to the colitis control.24

Xing et al.25 reported that in rats subjected to I=R injury,pretreatment with Bifidobacterium catenulatum ZYB0401and L. fermentum ZYL0401 led to reduced liver MDA(P< .05) and increased liver superoxide dismutase (P< .05)compared to the I=R group. In patients with nonalcoholicfatty liver disease and alcoholic liver chirrhosis, probioticVSL#3 treatment significantly improved plasma levels ofMDA.53

Administration of absolute ethanol has been shown tosignificantly increase plasma levels of pro-inflammatorycytokines, including IL-1b and TNF-a,1 and release of IL-1band IFN-g in gastric mucosa compared to controls.3 Kon-turek et al.4 demonstrated that exposure of gastric mucosa to75% ethanol resulted in numerous mucosal lesions and in

FIG. 2. Effects of vitamin E and probiotics on gastric mucosalmalondialdehyde levels. By analysis of variance followed by a posthoc least-significant difference test, *P< .01 compared with theethanol group, {P< .01 compared with the probiotic group, #P< .05compared with the a-tocopherol group.

FIG. 3. Effects of probiotics on gastric mucosal secretory immu-noglobulin A (sIgA) levels (n¼ 4–5 rats in each groups). By analysisof variance followed by a post hoc least-significant difference test,*P< .001 compared with the probiotic group.

176 SENOL ET AL.

the increase of mucosal expression of TNF-a, prostaglandinE2, and transforming growth factor-a with concomitant de-crease in gastric blood flow. On the other hand, pentox-ifylline exhibits well-documented anti-TNF properties andhas been shown to prevent alcohol-induced gastric mucosalinjury in rats, and its effect has been found to be not relatedto synthesis of prostaglandins and changes in gastric acidity,but related to nitric oxide-mediated pathways.5 Salim7

documented that the radical-removing agents allopurinoland dimethyl sulfoxide stimulate the healing of ethanol-induced acute gastric mucosal injury in rats. Dimethylsulfoxide and allopurinol scavenge hydroxyl,54,55 and thelatter agent also inhibits xanthine oxidase, which is re-sponsible for the formation of superoxide radicals.54 Enzy-matic antioxidants such as superoxide dismutase andcatalase are also effective in ulcers induced by ethanol.8,9

We found that both probiotic and a-tocopherol pretreatmentminimally, but not significantly, diminished the ethanol-induced macroscopic gastric mucosal damage. We deter-mined that ethanol exposure tended to increase mucosalTNF-a, IFN-g, and IL-2 levels along with elevation of levelsof IL-4, an anti-inflammatory cytokine. It seems that ourprobiotic mixture used in this study exhibited suppressiveaction on both T helper type 1 and type 2 cytokines. a-Tocopherol pretreatment had no influence on the ethanol-induced cytokine alterations. However, pretreatment witheither probiotic and a-tocopherol significantly reduced theethanol-induced increase of MDA concentration in thegastric mucosa.

Jaarin et al.2 demonstrated that feeding of rats with palmvitamin E supplementation for 3 weeks significantly low-ered the mean ulcer index induced by 100% ethanol after 1week of ethanol exposure. However, the authors observedno significant difference in ulcer index in rats killed at 1hour or 24 hours after ethanol exposure. In the present studygastric mucosal lesions were induced by 98% ethanol, andevaluation of the mucosal damage and collection of tissuesamples were performed 1 hour after ethanol exposure. It isknown that ethanol in concentrations that exceed 30%causes deeper, necrotic mucosal lesions, visible with thenaked eye.56–58 Suppressive effects of the probiotic mixtureused on the pro-inflammatory cytokines and lipid perox-idation suggest that it may exert beneficial effects over timeand may be useful at lower ethanol concentrations.

We also found that pretreatment with probiotic mixtureincreases gastric mucosal sIgA production approximatelyeightfold compared to the ethanol group. Diebel et al.33

reported that sIgA modulates the increased enterocyte apo-ptotic response. Thus, modulation of gastric mucosal epi-thelial apoptosis by an increased quantity of sIgA maydirectly serve to maintain the epithelial barrier function anddecrease the inflammatory response caused by ethanol. Apositive effect of enhanced sIgA production did not appearwith high ethanol exposure. It has been shown that highconcentrations of ethanol caused a necrotic cell death ratherthan apoptotic cell death.59 At lower ethanol concentrations,probiotics may serve to protect the mucosal integrity,modulating apoptosis by producing sIgA.

In conclusion, probiotic mixture and a-tocopherol pre-treatment did not decrease macroscopic gastric mucosallesions induced by 98% ethanol significantly. However,both pretreatments reduced ethanol-induced gastric mucosallipid peroxidation and could be worthwhile in the case oflower ethanol concentration-induced gastric injury.

ACKNOWLEDGMENT

This study has been supported by Sandoz� Turkey with‘‘The Project Supporting Award for Young Researchers.’’

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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