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Effect of Curculigo orchioides on Reux Esophagitis by Suppressing Proinammatory Cytokines Sae-Kang Ku, * Jae-Soo Kim, Young-Bae Seo, ** Yong-Ung Kim, Seung-Lark Hwang, || Young-Chul Lee, †† Chae-Ha Yang, § Hui-Young Kim, § Bu-Il Seo, || Ji-Ha Park, || You-Hong Min and Seong-Soo Roh || * Department of Histology and Anatomy, College of Oriental Medicine Department of Acupuncture and Moxibustion, College of Oriental Medicine Department of Herbal Biotechnology, College of Herbal Bio Industry § Department of Physiology, College of Oriental Medicine Department of Herbal Skin Care, College of Herbal Bio Industry || Department of Herbology, College of Oriental Medicine Daegu Haany University, Gyeongsan 712-715, Republic of Korea ** Department of Herbology, College of Oriental Medicine Daejeon University, Daejeon 300-716, Republic of Korea †† Department of Herbology, College of Oriental Medicine Sangji University, Wonju 220-702, Republic of Korea Abstract: This study was performed to investigate effects of Curculigo orchioides rhizome (curculiginis rhizome) on acute reux esophigitis (RE) in rats that are induced by pylorus and forestomach ligation operation. Proinammatory cytokine, as well as tumor necrosis factor (TNF)-, interleukin (IL)-1β and IL-6 were all assayed and the expression of TNF- and COX2 analyzed by RT-PCR. The esophagic tissue damage of reux esophagitis rat was increased compared to that of normal intact group. However, the esophagic damage per- centage from the extract of curculiginis rhizoma (ECR) 600 mg/kg and ECR 300 mg/kg were signicantly lower than that of the RE control group. Administration of -tocopherol (30 mg/kg) and ECR (600 mg/kg, 300 mg/kg, and 150 mg/kg) had a signicant effect on the gastric acid pH in rats with induced reux esophagitis (p < 0:05). The treatment with ECR signicantly reduced the production of cytokines TNF-, IL-1β and IL-6 levels compared to Correspondence to: Prof. Seong-Soo Roh, Department of Herbology, College of Oriental Medicine, Daegu Haany University, Yougok-dong, Gyeongsan-si 712-715, Republic of Korea. Tel: (þ82) 53-770-2296, Fax: (þ82) 53- 768-6340, E-mail: [email protected] or Prof. You-Hong Min, Department of Herbal Skin Care, College of Herbal Bio Industry, Daegu Haany University, Gyeongsan 712-715, Republic of Korea. Tel: (þ82) 53-819-1587, Fax: (þ82) 53-819-1157, E-mail: [email protected] The American Journal of Chinese Medicine, Vol. 40, No. 6, 12411255 © 2012 World Scientic Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X12500929 1241 Am. J. Chin. Med. 2012.40:1241-1255. Downloaded from www.worldscientific.com by STATE UNIVERSITY OF NEW YORK on 05/12/13. For personal use only.

Effect of Curculigo orchioides on Reflux Esophagitis by Suppressing Proinflammatory Cytokines

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Page 1: Effect of               Curculigo orchioides               on Reflux Esophagitis by Suppressing Proinflammatory Cytokines

Effect of Curculigo orchioideson Reflux Esophagitis by Suppressing

Proinflammatory Cytokines

Sae-Kang Ku,* Jae-Soo Kim,† Young-Bae Seo,** Yong-Ung Kim,‡ Seung-Lark Hwang,||

Young-Chul Lee,†† Chae-Ha Yang,§ Hui-Young Kim,§ Bu-Il Seo,|| Ji-Ha Park,||

You-Hong Min¶ and Seong-Soo Roh||

*Department of Histology and Anatomy, College of Oriental Medicine

†Department of Acupuncture and Moxibustion, College of Oriental Medicine

‡Department of Herbal Biotechnology, College of Herbal Bio Industry

§Department of Physiology, College of Oriental Medicine¶Department of Herbal Skin Care, College of Herbal Bio Industry

||Department of Herbology, College of Oriental Medicine

Daegu Haany University, Gyeongsan 712-715, Republic of Korea

**Department of Herbology, College of Oriental Medicine

Daejeon University, Daejeon 300-716, Republic of Korea

††Department of Herbology, College of Oriental Medicine

Sangji University, Wonju 220-702, Republic of Korea

Abstract: This study was performed to investigate effects of Curculigo orchioides rhizome(curculiginis rhizome) on acute reflux esophigitis (RE) in rats that are induced by pylorus andforestomach ligation operation. Proinflammatory cytokine, as well as tumor necrosis factor(TNF)-�, interleukin (IL)-1β and IL-6 were all assayed and the expression of TNF-� andCOX2 analyzed by RT-PCR. The esophagic tissue damage of reflux esophagitis rat wasincreased compared to that of normal intact group. However, the esophagic damage per-centage from the extract of curculiginis rhizoma (ECR) 600 mg/kg and ECR 300 mg/kgwere significantly lower than that of the RE control group. Administration of �-tocopherol(30 mg/kg) and ECR (600 mg/kg, 300 mg/kg, and 150 mg/kg) had a significant effect on thegastric acid pH in rats with induced reflux esophagitis (p < 0:05). The treatment with ECRsignificantly reduced the production of cytokines TNF-�, IL-1β and IL-6 levels compared to

Correspondence to: Prof. Seong-Soo Roh, Department of Herbology, College of Oriental Medicine, Daegu Haany

University, Yougok-dong, Gyeongsan-si 712-715, Republic of Korea. Tel: (þ82) 53-770-2296, Fax: (þ82) 53-768-6340, E-mail: [email protected] or Prof. You-Hong Min, Department of Herbal Skin Care, College of HerbalBio Industry, Daegu Haany University, Gyeongsan 712-715, Republic of Korea. Tel: (þ82) 53-819-1587, Fax:

(þ82) 53-819-1157, E-mail: [email protected]

The American Journal of Chinese Medicine, Vol. 40, No. 6, 1241–1255© 2012 World Scientific Publishing Company

Institute for Advanced Research in Asian Science and MedicineDOI: 10.1142/S0192415X12500929

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the model group (p < 0:05). The expression of TNF-� and COX2 in the intact esophagealmucosa was low while those of the RE control group were significantly higher due to aninflammatory reaction in the esophagus. Compare to the model group, treatment with�-tocopherol or ECR significantly inhibited the expression levels of COX2 and TNF-� in adose-dependent manner. These results suggest that anti-inflammatory and protective effectsof ECR could attenuate the severity of reflux esophagitis and prevent esophageal mucosaldamage.

Keywords: Curculigo orchioides; Curculiginis Rhizome; Reflux Esophagitis; Anti-inflammation; TNF-�, COX2.

Introduction

The Curculigo orchioides Gaertner (Amaryllidaceae) rhizome, also known as curculiginisrhizome, is an herbal medicine mainly used in Asian countries, especially in China, Indiaand South Korea. In traditional Chinese medicine, the rhizome of C. orchioides was used totreat pile, asthma, jaundice, diarrhea, colic and gonorrhea (Kirtikar and Basu, 1935) andwas reported to be an immunostimulant (Bafna and Mishra, 2006).

The curculiginis rhizoma has been reported to contain different types of compoundssuch as curculigosaponins A�M (Xu and Xu, 1992a,b), curculigoside A�C (Fu et al.,2004), orcinol glycoside (Wu et al., 2005), 2, 6-dimenthoxyl benzoic acid, curculiginesA�C (Chen et al., 1999) and orchiosides A and B (Gupta et al., 2005), tannin, resin andalkaloid (Anon, 2004).

Curculigoside A is a major bioactive compound present in C. orchioides. It stimulatesthe secretion of estradiol on primary cultural granulosa cell and exhibits potent inhibitoryactivity against matrix metalloproteinase (MMP)-1 in cultured human skin fibroblasts(Dong et al., 2006; Lee et al., 2009), attenuates human umbilical vein endothelial cellinjury induced by H2O2 (Wang et al., 2010), and up-regulates VEGF in MC3T3-E1 Cells(Ma et al., 2011). We reported an inhibitory effect of lonicerae flos on acute reflux eso-phagitis throughout its antioxidant activity (Ku et al., 2009).

Gastroesophageal reflux disease (GERD) is one of the most common diseases affectingabout 40% of the world’s population (Ford, 2005; Watanabe et al., 2007). Reflux eso-phagitis is a multifactorial disease because it involves transient lower esophageal sphincter(LES) relaxation, mucosal resistance and other factors, and is often associated with LESpressure (Bell and Hunt, 1992).

Several studies have been published reporting that the presence of reactive oxygenspecies (ROS), pro-inflammatory cytokines (such as tumor necrosis factor (TNF)-�,interleukin (IL)-1 and IL-6), and proinflammatory mediators (such as prostaglandins E2(PGE2) and nitric oxide (NO) generated from the induction of inducible nitric oxidesynthase (iNOS)) may play an important part in the pathophysiology of reflux esophagitis(Holzer, 2010; Oh et al., 2001). Abnormal gastric motility, associated with rapid or delayedgastric emptying, also contributes to gastric ulcer development. Yasunaga et al. reported

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that increased production of IL-1β in tissue and serum may contribute to foveolar hyper-plasia in enlarged fold gastritis (Yasunaga et al., 1996).

It was postulated that gastric acid can cause submucosal inflammation, increaseexpression of cytokines such as TNF-� and IL-1β, and promote thickening of the laryngealepithelium. Reflux esophagitis is mediated by oxygen-derived free radicals (Wetscheret al., 1995a,b). Administration of various free radical scavengers has been found toprevent esophageal mucosa damage (Wetscher et al., 1995c).

Curculigoside shows powerful antioxidant capacity and antiosteoporotic activity(Borek, 2001). It was reported that curculigoside played a neuroprotective role through theextinction of high mobility group box protein-1 release and nuclear factor-B activationtriggered by TNF-� and ameliorates middle cerebral artery occlusion (MCAO)-inducedcerebral ischemia/reperfusion injury. There are limitations associated with the investigationof the pathophysiology of GERD in humans. Thus, experiments using animal models arefundamental to this investigation. Because aqueous extract of curculiginis rhizoma (ECR)has an antioxidant effect and may have an inhibition effect on NF-�B activation induced byproinflammatory action in the esophagus, we investigated the anti-inflammatory effect ofECR on acute reflux esophagitis using a rat model.

Materials and Methods

Plant Material

The dry roots of Curculigo orchioides Gaertner (Curculiginis Rhizoma; CR) were pur-chased from Omniherb Co. (Youngcheon, South Korea). Voucher specimens (CR-20110805) were retained for future reference at the herbal laboratory of oriental college,Daegu Haany University, South Korea. 500 g of CR were cut into thick slices and wasboiled in 2 L of water. The extract of curculiginis rhizoma (ECR) was filtered and evap-orated on a rotary evaporator (Buchi, Flawil, Switzerland), dried by in a freeze drier (EyelaFDU-540, Tokyo, Japan). The extract powder was stored at �20�C. The percentage yieldwas about 14% (w/w). The CR aqueus extract powder was dissolved in distilled water(2ml/kg) before it was used for oral administration.

Animals

Six week old male Sprague–Dawley rats (Central Lab. Animal Inc., Seoul, Korea)weighing 180–200 g were housed under normal laboratory conditions at 25� 1�C with acontrolled 12 h light–dark cycle and maintained on standard rodent chow and tap water.The experimental protocols were carried out in accordance with the internationallyaccepted principles for laboratory animal use and care, as stated in the US guidelines.When necessary, the rats were deprived of food 18 h before the experiments but still hadaccess to water. All animals were kept in raised mesh-bottom cages to prevent coprophagy.Six to seven rats were used in each group.

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Acute Reflux Esophagitis Induction

All five groups of rats were deprived of food 18 h before the experiment, but had freeaccess to water. The rats were anaesthetized with an injection of zoletil 0.75mg/kg (VirbacS.A., France). A midline laparotomy was performed to expose the stomach, and then boththe pylorus and the transitional junction between the fore stomach and the corpus were firstexposed and later ligated with a 2-0 silk thread but without a pyloric ring, contrary to theprocedure originally proposed by Omura et al. (1999).

The vagus nerves were left intact. Thirty-six rats with reflux esophagitis were dividedinto five groups of six rats each. In Group I (intact control group) and Group II (refluxesophagitis control group), no further treatment was performed in addition to the abovesurgical procedure. However, Group III was additionally treated with antioxidant (of�-tocopherol) 1 h before surgery; Group IV pretreated with an extract of CR concentrationof 600 mg/kg, Group V pretreated with an extract of CR 300mg/kg, and Group VIpretreated with an extract of CR 150 mg/kg, 1 h before abdominal surgery.

Esophageal Mucosa Injury Evaluation in Acute Reflux Esophagitis

The rat esophagus was removed immediately after sacrifice and cut with scissor in thelongitudinal direction from the gastroesophageal junction to the pharynx. The innermucous was washed away with phosphate-buffered saline (PBS) using a micropipette. Thedissected esophagus was laid out on paper and photographed with an optical digitalcamera, and the diagnosis was analyzed using the i-solution lite software program.

Analysis of Gastric Secretions

The rat stomach was removed immediately after sacrifice and washed with 1mL of PBSusing a micropipette. The gastric contents were then collected. The gastric contents werecentrifuged at 3,000 rpm for 5min and their volumes were measured. The pH of collectedgastric juice was also measured using pH paper (MFRS Toshniwal, instrument manu-facturing limited, Ajmer, India). We measured a volume of gastric juice obtained from ratsof each groups and an aliquot was used to determine the concentration of acid. Sampleswere titrated with 0.1 N NaOH to an endpoint of pH 7.0. The amount of mucin in eachsample was measured by using enzyme-linked immunosorbent assay (ELISA). ELISAprocedure was modified and used, based on the methods previously reported (Shimizuet al., 2003; Ishbashi et al., 2004).

Gastric juices were diluted with PBS at a 1:10 ratio and 100�L of each sample wasincubated at room temperature in a 96-well plate for 2 h. Wells were washed three timeswith PBS and blocked with 2% BSA for 1 h at room temperature. Wells were again washedthree times with PBS and then incubated with 100�L of IgG 17Q2, a mouse monoclonalanti-total mucin antibody, which was diluted with PBS containing 0.05% Tween 20(1:1000), and dispensed into each well. After 1 h, wells were washed three times with PBS,and 100�L of horseradish peroxidase-goat antimouse IgG conjugate (1:3000) was

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dispensed into each well. After 1 h, wells were washed three times with PBS. Colorreaction was developed with 3,3 0,5,5 0-tetramethylbenzidin (TMB) peroxide solution andstopped with 1N H2SO4. Absorbance was read at 450 nm. The effect of agents on mucinsecretion was measured as follows: the amount of mucin secreted during the treatmentperiod was divided by the amount of mucin secreted during the pretreatment period and theratio was expressed as a secretory index. Means of secretory indices of each group werecompared and the differences were assessed using statistics.

Effect of ECR on Cytokine Levels in the Plasma

In order to study the effect of ECR on cytokine levels in the plasma, immediately after thetermination of experiment, venous blood samples were drawn from the abdominal vein andplaced into 410 EDTA-containing vials and used for the determination of plasma inter-leukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-�). Bloodwas collected and centrifuged at 3500 rpm for 10min with a constant temperature of 15�C.The plasma were collected with a micropipette and stored in �80�C until the ELISA assay.

The serum levels of proinflammatory cytokines IL-1β, TNF-� and IL-6 were evaluatedwith the Multi-Analyte ELISArray® Kit (Milipore, Rockford, IL, USA) according to themanufacturer’s instructions. The intensity of the color reaction was estimated using theLuminex luminometer (Awareness Technology Inc., Pal City, FL, USA) at 490 nm.

Effect of ECR on TNF-� and COX2 mRNA Transcript Expression Level by ReverseTranscriptasepolymerase Chain Reaction (RT-PCR)

RNA was extracted from esophageal samples using TRIZOL Reagent (Invitrogen LifeTechnologies, Karlsruhe, Germany). cDNA was generated from 5�g of total RNA usingthe RETROscript kit (MACROgene, Seoul, South Korea) following the manufacturer’sinstructions. Genes for COX2, TNF-� and β-actin were amplified with specific primer setsas previously described (Konturek et al., 2003; Wei et al., 2003).

cDNA samples were annealed at 94�C (5min) and amplified for 35 cycles with thefollowing cycling conditions: 94�C for 1min; respective annealing temperature for TNF-�at 70�C, and β Actin at 55�C for 1min; 72�C for 1min followed by a final extension at72�C for 10min and was run on a Bioer XP Cycler. PCR products were electrophoresed ona 1.0% agarose gel using a 100-bp ladder (MACRO gene, Seoul, South Korea) and theintensity was measured using Biovis gel documentation software and expressed as therelative intensity of PCR-product/β-actin ratio. The primers for the rat β-actin, COX2 andTNF-� were purchased in Biometra (MACRO gene, Seoul, South Korea). The TNF-�primer sequences were as follows: up-stream, 5 0TAC TGAACTTCG GGG TGA TTGGTC C; downstream, 5 0CAG CCT TGTCCC TTG AAG AGA ACC-3. The expectedlength product was 296 bp. Amplification of control rat β-actin was performed on the samesamples to verify the integrity of the RNA.

The COX2 primer sequences were as follows: upstream: 5 0-ACA ACA TTC CCT TCCTTC-3 0, downstream: 5 0-CCT TAT TTC CTT TCA CAC C-3 0. The expected length of this

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PCR product was 201 bp. Primer annealing was carried out at 56�C for COX2 and theamplification of the control rat β-actin was performed on the same samples to verify theRNA integrity. The β-actin primer sequences were as follows: upstream, 5 0-TTG TAACCA ACTGGG ACG ATA TGG-3 0, downstream, 5 0-GAT CTT GAT CTTCAT GGTGCT AGG-3 0 and primers annealing temperature was 54�C. The expected length of PCRproduct was 764 bp. PCR products were separated by electrophoresis in 2% agarose gelcontaining 0.5�g/ml ethidium bromide and then visualized under UV light. Location ofpredicted PCR product was confirmed by using O’Gene Ruler 50 bp DNA ladder (Fer-mentas, Life Sciences, San Francisco, USA) as standard marker.

Determination of Gross and Microscopic Esophageal Mucosa Damage

The rats were deprived of food but were still given water. Six hours later, the rats weresacrificed and the entire esophagus, was removed which we examined for gross mucosainjury. The esophagus was opened, gently rinsed with 0.9% NaCl, and photographs weretaken for specific areas of damage under a dissecting microscope. Esophageal mucosa wasstripped off the muscle layer and stored at �80�C for the following biochemical assaysincluding RT-PCR.

For microscopic evaluation, the opened esophagus was divided at its middle segment.The segments was embedded in paraffin, cut into 2�m sections and stained by hematoxylinand eosin (H&E) for microscopic evaluation. The stained slices were then observed underthe optical microscope, and diagnosis was analyzed by i-solution lite software program(Innerview Co., Sungnam, South Korea). The macroscopic injury damage percentage ofthe squamous epithelium was determined as follows.

Damage percentage (%)¼width of area with mucosa injury on esophagus (mm2Þ/widthof total area on esophagus (mm2) � 100

Statistical Analysis

Results are expressed as means� S.E.M. Statistical analysis was done using analysis ofvariance and two-way ANOVA test with a Tukey post-hoc test where appropriate. Data areexpressed as mean� S.D. of each group. Analysis of student’s t-test was used for statisticalanalysis; p < 0:05, p < 0:01 and p < 0:001 were considered statistically significant.

Results

Effects of Mucosa Damage

The lesion damage of the normal intact group was without mucosa injury such ashyperemia or serosanginous exudates (Figs. 1A and 1B). However, severe longitudinalulcerations with serosanginous exudates were observed in the esophagus of all of the refluxesophagitis-provoked group (RE control Group; Figs. 1C and 1D). The pathological areaon the esophagus of the RE control group was grossly increased compared with that in the

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sham operated group. However, the positive control group treated with �-tocopherol30mg/kg had less damage compared with the RE control group (Figs. 1E and 1F), and thegross appearance of esophagus from ECR vehicle group respectively pretreated withconcentration of ECR 600 mg/kg, ECR 300 mg/kg and ECR 150mg/kg, all showed onlyscattered erosions or mild hemorrhagic spots with whitish exudates scattered along theesophagus (Figs. 1G, 1H, 1I, 1J, 1K, 1L).

Effect of Gastric Volume

The gastric volume of the normal intact group was 1.28� 0.27ml (containing 1ml PBS).That of the reflux esophagitis (RE) control group was significantly increased comparedwith the gastric volume of the normal intact group (2.98� 0.3ml; p < 0:05). And those ofPC (positive control group treated with �-tocopherol 40 mg/kg), ECR-A (RE group treated

Figure 1. Effects of ECR on esophageal mucosal injury in acute reflux esophagitis. (A), (B) intact rat esophagus innormal group; (C), (D) acute reflux esophagitis model group; (E), (F) 30mg/kg �-tocopherol treated group; (G),

(H) ECR 600mg/kg treated group, (I); (J) ECR 300mg/kg; and (K), (L) ECR 150mg/kg. INT: normal intact rat;RE: acute reflux esophagitis model group; PC: positive control treated with �-tocopherol 30mg/kg; and ECRtreated groups: ECR-A (600mg/kg), ECR-B (300mg/kg), and ECR-C (150mg/kg). Values are expressed as themean� standard deviation (S.D.) of six rats; #p < 0:05 compared with the RE control rat group.

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with a concentration of 600mg/kg), ECR-B (RE group treated with a concentration of300mg/kg) and ECR-C (RE group treated with a concentration of 150mg/kg) wasdecreased compared to that of RE control group (Fig. 2). The PC group and ECR-A grouphad significant reduction of gastric volume (p < 0:05).

Effect of Gastric pH, Acidity and Mucin Content in Gastric Juice

The severity and incidence of acute reflux esophagitis appeared when each esophagus wasexamined 6 h later. Thus, we selected 6 h as the time limit for the experiment. Theadministration of �-tocopherol (30 mg/kg) and ECR (600 mg/kg, 300 mg/kg and 150 mg/kg) had a significant effect on gastric acid pH in reflux esophagitis rats (p < 0:05). Thevehicle groups treated with ECR 300 mg/kg and 150 mg/kg significantly decreased thegastric acid pH compared to that of the RE control group (Fig. 3). The administration of�-tocopherol (30 mg/kg) and ECR (600 mg/kg) significantly decreased the acidity ofgastric juice (p < 0:05).

As shown in Fig. 3, in the �-tocopherol group the concentration of mucin in gastricjuice was significantly higher than that of the RE control group (846.2� 55�g/ml vs552.2� 42.3�g/ml in gastric juice, p < 0:01). We also observed the respectively higherconcentration of mucin contents of rat treated with ECR 600 and 300 compared to that ofthe RE control group (831.9� 51.3�g/ml and 745.6� 58.4�g/ml vs 552.2� 42.3�g/mlin gastric juice, p < 0:01).

Effects of TNF-�, IL-1ß and IL-6 Cytokine Levels in Plasma

To examine the effects of proinflammatory cytokine such as tumor necrosis factor(TNF)-�, interleukin (IL)-1β and interleukin (IL)-6 in surgically induced acute refluxesophagitis, we investigated how ECR had an influence on cytokine production.

Figure 2. Effect of ECR on gastric volume in acute reflux esophagitis rats. INT: normal intact rat; RE: acute reflux

esophagitis model group; PC: positive control treated with �-tocopherol 30 mg/kg; and ECR treated groups: ECR-A (600 mg/kg), ECR-B (300 mg/kg), and ECR-C (150 mg/kg). Values are expressed as mean�S.D. of six rats;#p < 0:05 compared with RE group; *p < 0:05 compared with the INT group.

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Figure 3. Effect of alpha-tocopherol and ECR on gastric acid pH, gastric acidity and mucin release in acute reflux

esophagitis rats. INT: normal intact rat; RE: acute reflux esophagitis model group; PC: positive control treated with�-tocopherol 30 mg/kg; and ECR treated groups: ECR-A (600 mg/kg), ECR-B (300 mg/kg), and ECR-C(150 mg/kg). Values are expressed mean� S.D. of six rats; #p < 0:05, ##p < 0:01 and ###p < 0:001 compared

with RE group; *p < 0:05 and **p < 0:01 compared with INT group.

Figure 4. Effect of alpha-tocopherol and ECR on TNF-�, IL-1beta and IL-6 in plasma of acute reflux esophagitisrats. INT: normal intact rat; RE: acute reflux esophagitis model group; PC: positive control treated with�-tocopherol 30 mg/kg; and ECR treated groups: ECR-A (600 mg/kg), ECR-B (300 mg/kg), and ECR-C(150 mg/kg). Values are expressed mean� S.D. of six rats; #p < 0:05, ##p < 0:01 and ###p < 0:001 compared

with RE group; ***p < 0:001 compared with INT group.

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TNF-� was 14.9� 0.7 pg/ml in intact control rats in plasma. However, reflux eso-phagitis induced in rats resulted in doubling TNF-� production (40� 1.5 pg/ml). Ratstreated with �-tocopherol (36.1� 2.0 pg/ml) and ECR 600 mg/kg (37.2� 1.5 pg/ml) wereinhibited significantly (p < 0:05).

IL-1β was 53.8� 4.6 pg/ml in RE control rats, but the levels of rats treated with�-tocopherol (37.1� 3 pg/ml) and ECR 600 mg/kg (37.9� 5.0 pg/ml), ECR 300 mg/kg(42.0� 5.7 pg/ml) and ECR 150 mg/kg (45.4� 4.9 pg/ml) were significantly decreasedcompared to those of the RE control group (p < 0:01).

IL-6 levels of the normal intact group in plasma were 0.38� 0.21 pg/ml. However,surgically induced acute reflux esophagitis rats resulted in an increase in IL-6 productionon plasma (13.7� 0.5 pg/ml). Rats treated with �-tocophero l30 mg/kg (12.6� 1.4 pg/ml)and ECR 600 mg/kg (12.4� 1.4 pg/ml) significantly decreased compared to those of theRE control group (p < 0:05).

Effects of ECR on COX2 and TNF-� Expression Assayed by RT-PCR

As shown in Fig. 5, the expression of COX2 and TNF-� mRNA showed weak signals inthe intact esophageal mucosa whereas those of the RE control group were significantlyincreased because of an inflammatory reaction in the esophagus. But in �-tocopherol and

Figure 5. Effect of alpha-tocopherol and ECR on COX2 and TNF-� mRNA transcripts in the esophageal mucosain acute reflux esophagitis rats. INT: normal intact rat; RE: acute reflux esophagitis model group; PC: positive

control treated with �-tocopherol 30 mg/kg; and ECR treated groups: ECR-A (600 mg/kg), ECR-B (300 mg/kg),and ECR-C (150 mg/kg).

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ECR pretreated rats the expression of COX2 mRNA and TNF-� mRNA were dose-dependent and significantly inhibited compared to those of the RE control group (Fig. 5).

The Histological Determination of Esophageal Mucosa and Stomach Wall Injury

After being stained with H&E, no microscopic mucosal changes were observed in intactanimals (Fig. 6A). The normal esophagus exhibited a thin epithelial layer with squamouscells and only a few inflammatory cells were found in submucosal layers. In contrast, after6 h of induction of the reflux esophagitis, RE group rats developed large coalescedlongitudinal ulcers in the lower and middle parts of the esophagus. The mucosa damageand hyperemia of epithelial layers and edema in mucosa and submucosa were observed inthe RE control group (Fig. 6B). Also, a marked mucosal thickening, accompanied byfurther exaggeration of the damage, edema and heavy inflammatory cell infiltrationwere observed. Whereas the esophageal damage, edema and neutrophil infiltration andgastric hemorrhage still occurred, they were significantly less severe in rats treated with

Figure 6. Effect of alpha-tocopherol and ECR on esophageal mucous and stomach wall injury in acute refluxesophagitis rats. (A) normal intact rat; (B) control rat with pylorus and forestomach ligation treated with distilled

water; (C) positive control treated with �-tocopherol (30 mg/kg); rat with pylorus and forestomach ligation treatedwith extract of ECR; (D) 600mg/kg; (E) 300 mg/kg; and (F) 150 mg/kg.

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�-tocopherol (Fig. 6C), 600 mg/kg ECR (Fig. 6D), 300 mg/kg ECR (Fig. 6E), or 150 mg/kg ECR (Fig. 6F) than in the RE control group.

The damage and edema in the mucosa and submucosa were significantly reduced inrats pretreated with ECR and then exposed to acid reflux (Table 1) compared with RErats treated with the vehicle alone. The depth of hemorrhage in the stomach was564.6� 18.8�m in the RE control group but lower in the �-tocopherol (124.9� 30.2�m),600 mg/kg ECR (59� 7�m), 300 mg/kg ECR (61.1� 22.2�m), and 150 mg/kg ECR(154.7� 100.8�m) groups (Table 1).

Discussion

The gastric volume and gastric juice pH of the groups treated with �-tocopherol or ECRwere significantly different from those of the RE control group (Fig. 3), indicating that allof the rats in which vehicle rats treated with �-tocopherol and ECR was induced receivedless stimulation from the gastric acid. The gastric juice mucin concentrations of thetreatment groups (30 mg/kg �-tocopherol, 600 mg/kg ECR, and 300mg/kg ECR) weresignificantly greater (846.2� 55�g/ml, 831.9� 51.3�g/ml, and 745.6� 58.4�g/mlrespectively) than that of the RE control group (552.2� 42.3�g/ml). Therefore, ECR has abeneficial effect on the release of mucin into gastric juice but does not affect the secretionof gastric juice itself.

Acute esophageal mucosal inflammation has been reported to be the primary con-sequence of gastric juice reflux, resulting in dramatically increased COX2 mRNA andprotein expression in rats with esophageal reflux. Therefore, clinical trials are underway toassess the efficacy of selective COX2 inhibitors in preventing the progression of acutereflux esophagitis. In addition, these effects were accompanied in these anastomosedanimals by a rise in the plasma levels of the proinflammatory cytokines IL-1β and TNF-�(Katsunobu et al., 2004). This is in agreement with previous studies (Eksteen et al., 2001;Fitzgerald et al., 2002) and suggests that proinflammatory cytokines such as TNF-�, IL-1β,and IL-6 play critical roles in the inflammation associated with acute gastroesophagealreflux and the progressive injury and congestion of the esophageal mucosa, possibly by

Table 1. Effect of Alpha-Tocopherol and ECR on Esophageal and Gastric Histomorphometry.

Group Thickness ofMucosa in

Esophagus (¹M)

Thickness ofSubmucosa in

Esophagus (¹M)

HemorrhageDepth in

Stomach (¹M)

InjuryPercentage ofMucosa (%)

Intact 88.5 � 7.5 47.3 � 9.5 0.2 � 0.03 N.D.

RE 13.2 � 4.3*** 10.5 � 4.7** 564.6 � 18.8*** 85.4 � 15.6�-tocopherol 67.7 � 6.1*,## 42.1 � 10.1## 124.9 � 30.2***,## 59.8 � 17.5##

ECR600 60.8 � 10.2*,## 38.7 � 8.4## 59 � 7***,# 45.6 � 15.0##

ECR300 42.7 � 11.1**,# 35.3 � 5.2# 61.1 � 22.2***,# 68.2 � 8.8#

ECR150 27.8 � 8.3** 31.8 � 6.7* 154.7 � 100.8*** 69.1 � 12.2

Note: Values are expressed mean � S.D. of six rats; *p < 0:05, **p < 0:01 and ***p < 0:001 compared tonormal intact rat group (Intact). #p < 0:05 and ##p < 0:01 compared to RE control group.

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recruiting and activating target cells that may produce additional cytokines. The biologicalproperties of IL-1 in serum are similar to those of TNF-�, including the induction of fever,inflammation, and hemodynamic shock (Akira et al., 1990; Dinarello, 1991).

Surgical induction of acute reflux esophagitis in rats resulted in increased production ofTNF-� (40� 1.5 pg/mL) and IL-1β (53.8� 4.6 pg/mL) (Fig. 4). Treatment of inducedesophagitis in rats with �-tocopherol (30 mg/kg) or ECR (600 mg/kg and 300 mg/kg)inhibited the release of IL-1β and TNF-� into the blood compared with that in untreatedRE rats (#p < 0:05 and ##p < 0:01). Similarly, the plasma level of IL-6 in the normal intactgroup was 0.38� 0.21 pg/mL. However, surgical induction of acute reflux esophagitisincreased the plasma IL-6 level (13.7� 0.5 pg/mL), while this induction was significantlyinhibited by treatment with 30 mg/kg �-tocopherol (12.6� 1.4 pg/mL) or 600 mg/kg ECR(12.4� 1.4 pg/mL) (p < 0:05) (Fig. 4).

These results indicate that ECR strongly suppresses inflammation, ulceration, andimmunocyte infiltration into the esophageal mucosa by inhibiting the production ofcytokines such as TNF-�, IL-1β, and IL-6. These findings correspond with the microscopicgross mucosal damage percentage as shown in Fig. 1 with histomorphometric analysisusing an image-analyzing program of H&E-stained tissue sections (Fig. 6).

Hayakawa et al. demonstrated that mRNA and protein expression of COX2 wasdrastically increased in rat acute reflux esophagitis compared with intact rat esophagitis(Hayakawa et al., 2006). We showed in Fig. 1 that pretreatment of animals with �-toco-pherol or ECR at 600 mg/kg, 300 mg/kg, or 150mg/kg administered orally has beneficialeffects on the esophageal mucosa in reflux esophagitis. The reduction of COX2 mRNAexpression in the treatment groups also protects the esophageal mucosa against theinflammatory actions of neutrophils and other immunocytes.

In this study, we have shown that pretreatment of animals with various concentrations(600 mg/kg, 300 mg/kg, and 150 mg/kg) of ECR administered orally has no effect ongastric volume orgastric juice pH and increases mucin production; the additional mucinsecretion in the ECR group compared with that of the RE controls may protect againstinjury of the esophageal mucosa. Furthermore, because ECR has antioxidant and anti-inflammatory activities, it significantly inhibited the damage, ulceration, and myxoidhemorrhage in the esophagus and stomach wall by reducing the levels of cytokines such asTNF-�, IL-1β, and IL-6 and also by decreasing levels of nitric oxide synthase and growthfactors such as COX2. It was considered that the mechanism of efficacy on RE by ECR wasthe result of three activities.

The first activity is the direct reduction of tissue damage by ECR. ECR increased therelease of mucin in the stomach and this protects esophageal tissue from gastric acid.Inflammatory immune cells were less infiltrated than the RE control group and this effect isattributable to decreases of TNF-�, IL-1β and IL-6. A second activity is an inactivation ofNF-kB due to the creation of less product from proinflammatory cytokines because of thereduction in esophagus injury. The inhibitory effect of NF-kB by ECR induced a sup-pression of COX2 mRNA in esophageal tissue.

A final activity is the presence of a strong antioxidant which can scavenge the super-oxide radical in the cell-free systems. ECR decreases cell damage from oxidative stress by

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inducing activation of p53, caspases-3, and a number of other kinases. Interestingly, theaddition of curculigoside attenuated the up regulation of p53 mRNA and the up regulationof P53 relate with NF-kB activation too. We think that a further study is needed todetermine if curculigoside has a direct efficacy in RE or if component induces this effect.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grantfunded by the Korean government [MEST] (No. 2012-0009400).

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