Banhasasim-Tang Treatment Reduces the Severity of...

Research ArticleBanhasasim-Tang Treatment Reduces the Severity of EsophagealMucosal Ulcer on Chronic Acid Reflux Esophagitis in Rats

Mi-Rae Shin12 Bu-Il Seo2 Chang Gue Son3 Seong-Soo Roh2 and Hyo-Jin An1

1Department of Pharmacology College of Korean Medicine Sangji University Wonju-si Gangwon-do 26339 Republic of Korea2Department of Herbology College of Korean Medicine Daegu Haany University 136 Shinchendong-ro Suseong-guDaegu 42158 Republic of Korea3Liver and Immunology Research Center Daejeon Oriental Hospital Oriental Medical College Daejeon University176-9 Daeheung-ro Jung-gu Daejeon 34929 Republic of Korea

Correspondence should be addressed to Seong-Soo Roh ddededhuackr and Hyo-Jin An hjansangjiackr

Received 6 December 2016 Revised 6 February 2017 Accepted 13 February 2017 Published 2 March 2017

Academic Editor Tadayuki Oshima

Copyright copy 2017 Mi-Rae Shin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study was conducted to evaluate both antioxidant and anti-inflammatory activity of Banhasasim-tang (BHSST) onchronic acid reflux esophagitis (CRE)model Rat CREmodel was established operatively and then treated with BHSST (1 gkg bodyweight per day) for 15 days Esophageal pathological changes were analyzed using macroscopic examination and hematoxylineosinstaining The antioxidant and inflammatory protein levels were determined using Western blotting The administration of BHSSTsignificantly reduced both the overexpression of serum reactive oxygen species (ROS) and an excessive formation of thiobarbituricacid-reactive substances (TBARS) in esophagus tissue Thus the severity of esophageal ulcer was lower in BHSST treated rats thancontrol rats on the gross and histological evaluation Nuclear factor-erythroid 2-related factor 2 (Nrf2) led to the upregulationof antioxidant enzyme including SOD GPx-12 and HO-1 by binding to antioxidant response element (ARE) Moreover BHSSTadministration markedly reduced the expression of inflammatory proteins through mitogen-activated protein kinase- (MAPK-)related signaling pathways and decreased significantly the protein expressions of inflammatory mediators and cytokines byinhibition of nuclear factor-kappa B (NF-120581B) activation Taken together these results support the fact that BHSST administrationcan suppress the development of esophageal mucosal ulcer via regulating inflammation through the activation of the antioxidantpathway

1 Introduction

Gastroesophageal reflux disease (GERD) was defined as acondition that develops when the reflux of gastric contentsinto the esophagus causes troublesome symptoms such asacid regurgitation heartburn and dysphagia [1 2] Protonpump inhibitors (PPIs) for acid suppression are the mainstaytherapeutic strategy for GERD and irreplaceable drugs inthe management of acid-related disorders [3] Despite theirexcellent efficacy such agents have adverse effects associatedwith a long-term inappropriate use namely about one-thirdof patients with suspected GERD caused by weakly acidicreflux and duodenogastrooesophageal reflux experiencedfailure of PPIs [4ndash6] Accordingly the recent researches are

focused on a new and rational approach about the effectiveand safe replacement therapy

The cellular biochemical process for maintenance ofhomeostasis is regulated by redox balance The redox imbal-ance may result in excessive production of reactive oxygenspecies (ROS) which may lead to oxidative stress Redoximbalanceoxidative stress deteriorate when there is aug-mented production and inefficient scavenging of ROS [7 8]A continuous reflux of gastric contents causes inflammationulceration and destruction of the normal squamous epithe-lium of esophagus The damaged squamous epithelium ofthe esophagus is replaced with an intestine-like columnarepithelium which has strong resistance about acid attack andit is called Barrettrsquos esophagus (BE) BE is commonly noted

HindawiBioMed Research InternationalVolume 2017 Article ID 7157212 10 pageshttpsdoiorg10115520177157212

2 BioMed Research International

Table 1 Composition of Banhasasim-tang (BHSST) used in thisstudy

Herbs Amounts (g)Pinelliae Rhizoma 167Scutellariae Radix 100Zingiberis Rhizoma Siccus 083Ginseng Radix 100Glycyrrhizae Radix 100Jujubae Fructus 100Coptidis Rhizoma 033Banhasasimtang Ex Granule (15 g) is contained in 1 pack (35 g)Banhasasimtang Ex Granule was purchased from Hankook Shinyak Corp(Nonsan-si Chungcheongnam-do Republic of Korea)

in humans with chronic reflux esophagitis and increases arisk for development of adenocarcinoma of the esophagusand gastric cardia [9 10] Accordingly the suppression of thegastroesophageal reflux is the most important in treatment ofGERD

Banhasasim-tang (BHSST Hange-shashin-to in JapaneseTraditional Kampo Medicine Banxia-xiexin-tang in Tra-ditional Chinese Medicine) has been used as an herbalprescription to improve dyspepsia gastric ulcerative disor-ders laryngopharyngitis colitis and diarrhea [11ndash14] Thisformula is composed of Pinelliae Rhizoma ScutellariaeRadix Zingiberis Rhizoma Siccus Ginseng Radix AlbaGlycyrrhizae Radix Zizyphi Fructus and Coptidis Rhizoma(Table 1) BHSST described in Shang-Han Lun (Treatise onCold Damage and Miscellaneous Diseases) written by ZhangZhong-jing (150ndash219 AD) is widely accepted by Chineseherbal doctors and has been applied for treating the symptomassociated to GERD in Korea [15 16] Despite previousvarious reports related to the improvement of GERD theprotective mechanisms of BHSST treatment in esophagealulcer by chronic reflux are not fully understood Thereforewe investigated the effects of BHSST on rats with chronic acidreflux esophagitis (CRE) to examine its ameliorating effectagainst oxidative stress-related inflammation

2 Materials and Methods

21 Materials The protease inhibitor mixture solutionand ethylenediaminetetraacetic acid (EDTA) were pur-chased from Wako Pure Chemical Industries Ltd (OsakaJapan) Phenylmethylsulfonyl fluoride (PMSF) was pur-chased from Sigma-Aldrich (St Louis MO USA) 2101584071015840-Dichlorofluorescein diacetate (DCF-DA) was obtained fromMolecular Probes (Eugene OR USA) The pierce bicin-choninic acid (BCA) protein assay kit was obtained fromThermoFisher Scientific (WalthamMAUSA) ECLWesternBlotting Detection Reagents and pure nitrocellulose mem-branes were supplied by GE Healthcare (Chicago IL USA)Rabbit polyclonal antibodies against nuclear factor-kappa Bp65 (NF-120581Bp65 1 1000 SC-372) nuclear factor-erythroid 2-related factor 2 (Nrf2 1 1000 SC-7228) heme oxygenase-1 (HO-1 1 1000 SC-10789) superoxide dismutase (SOD1 1000 SC-11407) glutathione peroxidase-12 (GPx-12

1 1000 SC-30147) goat polyclonal antibodies against Kelch-like ECH-associated protein 1 (Keap1 1 1000 SC-15246)tumor necrosis factor-120572 (TNF-120572 1 1000 SC-1351) andinterleukin-6 (IL-6 1 1000 SC-1266) and mouse mon-oclonal antibodies against phosphor-c-Jun NH

2-terminal

kinase (p-JNK 1 1000 SC-6254) phosphor-extracellularsignal-regulated kinase 12 (p-ERK12 1 1000 SC-7383)phosphor-p38 (p-p38 1 1000 SC-7973) cyclooxygenase-2(COX-2 1 1000 SC-19999) inducible nitric oxide synthase(iNOS 1 1000 SC-7271) histone (1 1000 SC-8030) and 120573-actin (1 1000 SC-4778) were purchased from Santa CruzBiotechnology Inc (Santa Cruz CA USA) Mouse mon-oclonal antibody against activator protein-1 (AP-1) subunitc-Jun (1 1000 2315) was obtained from Cell SignalingTechnology Inc (Cell Signaling MA USA) Rabbit anti-goat (1 3000 SC-2774) goat anti-rabbit (1 3000 SC-2004)and goat anti-mouse (1 3000 SC-2005) immunoglobulin G(IgG) horseradish peroxidase- (HRP-) conjugated secondaryantibodies were acquired from Santa Cruz BiotechnologyInc (Santa Cruz CA USA) All other chemicals and reagentswere purchased from Sigma-Aldrich (St Louis MO USA)

22 Test Material Light brown granules of Banhasasim-tang(Hankook Shinyak Corp Nonsan-si Chungcheongnam-doRepublic of Korea) produced according to Korean GoodManufacturing Practice (GMP)were permitted and regulatedby the Korean Food amp Drug Administration (KFDA SeoulRepublic of Korea) BHSST was dissolved in distilled waterThe Banhasasimtang Ex Granule is included as 15 g inBanhasasim-tang (1 package 35 g) used in this study andillustrated in Table 1

23 Experimental Animals and Treatment Animal experi-ments were carried out according to the ldquoGuidelines forAnimal Experimentationrdquo approved by the Ethics Committeeof the Daegu Haany University on 20102016 with certificatenumber DHU2016-81 Five-week-old male Sprague-Dawleyrats (BW 150ndash155 g) were purchased from Nara Biotec Co(Pyeongtaek Republic of Korea) Rats were maintainedunder a 12 h lightdark cycle housed at controlled tem-perature (24 plusmn 2∘C) and humidity (about 60) and keptin raised mesh-bottom cages to prevent coprophagy Afteradaptation (1 week) the rats were fasted for 18 h prior tosurgical procedures and kept in raised mesh-bottom cages toprevent coprophagy And then rats were anaesthetized withan injection of Zoletil at 075mgkg (Virbac SA France)The CRE model (chronic acid reflux esophagitis model) wasdeveloped by following the methods proposed by Omuraet al [17] A midline laparotomy was performed to exposethe stomach and the transitional region (ie limiting ridge)between the fundus and the glandular portion of the stomachwas ligated with 2ndash0 silk thread in order to restrict thecompliance of the stomach which led to the reflux ofgastric contents into the esophagus Additionally a latex ring(2mm in thickness ID 4mm made from 18-Fr Nelatoncatheter) was placed around the pyloric sphincter so as torestrict the emptying of gastric contents Rats were injectedwith gentamicin sulfate (antibiotic subcutaneous injection)

BioMed Research International 3

and dexamethasone (anti-inflammatory agent subcutaneousinjection) for 3 days to prevent infection After surgery therats fasted for a further 48 h but water was provided 24 hafter surgery All animals had an operation adjustment for7 days after surgery Body weight was recorded during 22days from surgery day (an operation adjustment period 7days + drug treatment period 15 days) and food intakewas recorded during 15 days (drug treatment period) At 22days after surgery rats were sacrificed and the esophagealtissues were obtained for further processing and analysisRats were divided into three groupsThe normal and chronicacid reflux esophagitis control groups were given water whilethe drug group was orally administered with Banhasasim-tang at a dose of 1 gkg body weight daily using a stomachtube for 15 consecutive days (119899 = 7 in each group) Theentire esophaguswas removed immediately and examined forgross mucosal injury The esophageal tissue was immediatelyfrozen in liquid nitrogen and blood samples were collected byvena cava puncture from anesthetized rats Subsequently theesophagus and serum were kept at minus80∘C until analysis

24 Esophageal Ulcer Ratio The rat esophagus was cut withscissors in a longitudinal direction from the gastroesophagealjunction to the pharynx after sacrifice The inner mucouswas washed away with 09 NaCl and the remaining tissuewas laid out on paper Thereafter the dissected esophaguswas photographed with an optical digital camera (SonyTokyo Japan) and analyzed using the i-solution lite softwareprogram The gross mucosal ulcer ratio was calculated asfollows The gross mucosal ulcer ratio () = [width of areawith esophageal mucosal ulcer (mm2)width of total area ofesophagus (mm2)] times 100

25 ROS and TBARS Levels Measurements Serum ROSlevel was measured employing the method of Ali et al[18] Esophageal tissues were homogenized on ice with1mM EDTA-50mM sodium phosphate buffer (pH 74) andthen 25mM DCF-DA was added to homogenates Afterincubation for 30min the changes in fluorescence valueswere determined at an excitation wavelength of 486 nm andemission wavelength of 530 nm The 2-thiobarbituric acid-reactive substance (TBARS) level was estimated according tothe method of Mihara and Uchiyama [19]

26 Preparation of Cytosol and Nuclear Fractions Proteinextraction was performed according to the method ofKomatsu with minor modifications [20] Esophageal tissuesfor cytosol fraction were homogenized with ice-cold lysisbuffer A (250mL) containing 10mMHEPES (pH 78) 10mMKCl 2mM MgCl

2 1 mM DTT 01mM EDTA 01mM

PMSF and 1250 120583L protease inhibitor mixture solution Thehomogenate was incubated at 4∘C for 20min And then10 NP-40 was added and mixed well After centrifugation(13400timesg for 2min at 4∘C) using Eppendorf 5415R (Ham-burgGermany) the supernatant liquid (cytosol fraction)wasseparated in new e-tube The left pellets were washed twiceby buffer A and the supernatant was discarded Next thepellets were suspended with lysis buffer C (20mL) containing

50mM HEPES (pH 78) 50mM KCl 300mM NaCl 1mMDTT 01mM EDTA 01mM PMSF 1 (vv) glycerol and100 120583L protease inhibitor mixture solution suspended andincubated at 4∘C for 30min After centrifugation (13400timesgfor 10min at 4∘C) the nuclear fractionwas prepared to collectthe supernatant Both cytosol and nuclear fractions were keptat minus80∘C before the analysis

27 Immunoblotting Analyses For the estimation of Nrf2NF-120581Bp65 and histone 136 120583g of protein from each nuclearfraction was electrophoresed through 8ndash10 sodium dode-cylsulfate polyacrylamide gel (SDS-PAGE) Separated pro-teins were transferred to a nitrocellulose membrane blockedwith 5 (wv) skim milk solution for 1 h and then incubatedwith primary antibodies (Nrf2 NF-120581Bp65 and histone)overnight at 4∘C After the blots were washed they were incu-bated with anti-rabbit or anti-mouse IgG HRP-conjugatedsecondary antibody for 1 h at room temperature In addition8 120583g protein of each cytosol fraction of Keap1 SOD GPx-12 HO-1 COX-2 iNOS TNF-120572 IL-6 and 120573-actin waselectrophoresed through 8ndash15 SDS-PAGE Each antigen-antibody complex was visualized using ECLWestern BlottingDetection Reagents and detected by chemiluminescence withSensi-Q 2000 Chemidoc (Lugen Sci Co Ltd Gyeonggi-do Republic of Korea) Band densities were measured usingATTO Densitograph Software (ATTO Corporation TokyoJapan) and quantified as the ratio to histone or 120573-actin Theprotein levels of the groups are expressed relative to those ofthe normal rat (represented as 1)

28 Statistical Analysis Thedata are expressed as the mean plusmnSEM Significance was assessed by one-way analysis of vari-ance (ANOVA) followed by Dunnettrsquos multiple comparisontest using SPSS version 220 software (SPSS Inc Chicago ILUSA) Values of 119875 lt 005 were considered significant

3 Results and Discussions

Traditional herbal formulas have been widely used to pre-vent and treat various inflammatory disorders in East Asiaincluding Korea China and Japan BHSST among theseformulas consists of seven herbs Pinelliae Rhizoma Scutel-lariae Radix Zingiberis Rhizoma Siccus GinsengRadixAlbaGlycyrrhizae Radix Zizyphi Fructus and Coptidis Rhizomain 5 3 3 3 3 3 1 proportions The previous study reportedthat the major thirteen marker components of BHSST usinganultra-performance liquid chromatography coupled to elec-trospray ionization tandemmass spectrometry are homogen-tisic acid (Pinelliae Rhizoma) 34-dihydroxybenzaldehyde(Pinelliae Rhizoma) spinosin (Zizyphi Fructus) liquiritin(Glycyrrhizae Radix) baicalin (Scutellariae Radix) gin-senoside Rg1 (Ginseng Radix Alba) liquiritigenin (Gly-cyrrhizae Radix) wogonoside (Scutellariae Radix) ginseno-side Rb1 (GinsengRadixAlba) baicalein (Scutellariae Radix)glycyrrhizin (Glycyrrhizae Radix) wogonin (ScutellariaeRadix) and 6-gingerol (Zingiberis Rhizoma Siccus) [21] Par-ticularly the highest content is baicalin and thenwogonosideglycyrrhizin liquiritin and 6-gingerolMoreover the existing


1Day

3 5 7 9 11 13 15 17 19 21

Operation adjustment Drug treatment

NormalControlBHSST

050

100150200250300350

Body

wei

ght (

g)

(a)

9 11 13 15 17 19 21Drug treatment

NormalControlBHSST

05

101520253035

Food

inta

ke (g

)

lowastlowast

(b)

Figure 1 Body weight changes in the whole experimental periods (a) and food intake changes in the drug treatment periods (b) in chronicacid reflux esophagitis rats Normal normal rats Control chronic acid reflux esophagitis rats BHSST BHSST 1 gkg body weightday-treatedchronic acid reflux esophagitis rats Data aremean plusmn SEM (119899 = 7) Significance 119875 lt 0001 versus normal rats and lowastlowast119875 lt 001 versus controlrats

studies demonstrated that Pinelliae Rhizoma [22] Scutel-lariae Radix [23] Zingiberis Rhizoma Siccus [24] GinsengRadix Alba [25] Glycyrrhizae Radix [26] Zizyphi Fructus[27] and Coptidis Rhizoma [28] exert the gastroprotectiveeffects through inhibition of inflammatory proteins Based onprevious studies we predicted that BHSST containing thesebioactive herbs as constituents would enhance amelioratingeffects on esophageal ulcer induced chronic acid refluxesophagitisTherefore the present studywas conducted usingthe same chronic acid reflux esophagitis model used in theprevious experimental [17] First of all body weight gainduring the experimental periods and food intake during drugtreatment periods are confirmed As shown in Figure 1(a)normal rats and chronic acid reflux esophagitis (CRE) modelinduced rats are similar to body weight in starting-pointBodyweight decreased 3 days in a row after surgerywhereas itincreased gradually from 4 days Based on the whole experi-mental periods control rats significantly suffered weight loss(119875 lt 0001) compared with normal rats due to the lowfood intake However the significant increase of food intakeduring drug treatment periods by BHSST treatment (lowastlowast119875 lt001) leads to the body weight rise (without a significance)(Figure 1(b)) The results suggested that changes in the foodintake and body weight were caused by surgery assuming acomparative similar aspect [29]

Gross morphological changes such as mucosal swellingand esophageal ulcer ratio as shown in Figure 2 associatedwith the metaplastic process of mucosal epithelial cell wereobserved in chronic reflux esophagitis rats instead of hyper-emia andmultiple erosions showed in acute reflux esophagitis[30] Herein the different severity of esophageal ulcerationwas seen in rats with CRE (Figure 2(c)) while no visibleesophageal mucosa lesions were observed in normal ratsThese tissue injuries were located in the middle or distalesophagus Moreover the normal esophagus exhibited a thinepithelial layer with squamous cells and few inflammatorycells in the submucosal layer while the CRE esophagus exhib-ited basal layer thickening inflammatory cells infiltrationand the desquamated epithelial cells (Figure 3) [31] Howeverthe CRE rats treated with BHSST had less damage than

the CRE control rats These results were consistent with thehistomorphological staining results

Several herbal therapies have been proposed for the treat-ment of GERD [32] however the role of BHSST against CREstill lacks proved experiments In the current study we clearlydemonstrated that supplementation of BHSST significantlyameliorated chronic acid reflux esophagitis- (CRE-) inducedesophageal ulcer The severity of an esophageal ulcer hasbeen correlated with the overproduction of free radicalsOxidative stress (OS) is caused when production of reactiveoxidative species (ROS) exceeds the potential of cellularantioxidant defenses to detoxify these toxicants [33] Andthen esophageal ulcer by the continuous and chronic refluxprogresses gradually and can deteriorate until esophagealstricture or Barretrsquos esophagus Ultimately it may contributeto the development of esophageal cancer [34] OS can bedetermined by measuring the levels of malondialdehyde(MDA) which is a lipid peroxide generated by the reactionamong oxygen free radicals [35] MDA is reactive markerof membrane damage and forms a colour complex withthiobarbituric acid (TBA) which can be measured spec-trophotometricallyHereinwemeasured the content ofMDAusing the method of TBARS assay In present study the levelsof serum ROS and esophageal TBARS were markedly higherthan those of normal rats whereas the elevated levels ofserum ROS were significantly decreased nearly to the levelsof normal rats On the other hand serum TBARS showed atendency to reduction (without significance) (Figure 4)

A major cellular defense mechanism which is sensitiveto oxidative stress is the nuclear factor E2-related factor2Kelch-like ECH-associated protein 1antioxidant responseelements (Nrf2-Keap1-ARE) pathway and activates as adap-tive response to protect cells from injury [36 37] In normalcells Nrf2 is sequestered by the Keap1 to form Nrf2-Keap1complex However during oxidative stress Nrf2 dissociatesfrom Keap1 translocates into the nucleus and binds to AREpromoting the transcription of the target gene [38] As shownin Figure 5(a) the Nrf2 protein expression was decreased incontrol rats comparedwith normal rats whereas BHSST treat-ment significantly upregulated Nrf2 expression Moreover


Normal Control BHSST

(a)

BHSSTNormal Control

(b)

Chronic RE ratsNormal Control BHSST

0

5

10

15

20

25

30

35G

ross

esop

hage

al u

lcer

ratio

()

lowast

(c)

Figure 2 Gross esophagus (a) the opened gross esophageal ulcer (b) and esophagealmucosal ulcer ratio (c) in chronic acid reflux esophagitisrats Normal normal rats Control chronic acid reflux esophagitis rats BHSST BHSST 1 gkg body weightday-treated chronic acid refluxesophagitis rats Data are mean plusmn SEM (119899 = 7) Significance 119875 lt 001 versus normal rats and lowast119875 lt 005 versus control rats

Control BHSSTNormal

(a)

Control BHSSTNormal

(b)

Figure 3 HE staining of esophageal ulcer in chronic acid reflux esophagitis rats (a)Magnification times40 and (b) magnification times200 Normalnormal rats Control chronic acid reflux esophagitis rats BHSST BHSST 1 gkg body weightday-treated chronic acid reflux esophagitis rats



0

100

200

300

400

Seru

m R

OS

(fluo

resc

ence

mL)

lowast

(a)Normal Control BHSST

0

2

4

6

8

Seru

m T

BARS

(nM

mL)

(b)


0

02

04

06

08

10

TBA

RS in

esop

hagu

s (nM

mg

prot

ein)

lowastlowastlowast

(c)

Figure 4 ROS and TBARS levels measurements (a) Serum ROS (b) serum TBARS and (c) TBARS of esophageal tissue Normal normalrats Control chronic acid reflux esophagitis rats BHSST BHSST 1 gkg body weightday-treated chronic acid reflux esophagitis rats Dataare mean plusmn SEM (119899 = 6) Significance 119875 lt 001 versus normal rats and lowast119875 lt 005 lowastlowastlowast119875 lt 0001 versus control rats

Histone

Nrf2


Keap1


Nrf2Keap1Normal Control BHSST

lowast훽-Actin

lowast

0

(Fol

d of

N)

06

12

18

(Fol

d of

N) 12

06

18

0

(a)SOD-1

SOD

GPx-12

HO-1

0Normal Control BHSST

Normal Control BHSST Normal Control BHSST

GPx-12

HO-1


lowast

lowast

lowast훽-Actin

0

05

10

15

(Fol

d of

N)

0

05

10

15

(Fol

d of

N)

06

12

18

(Fol

d of

N)

(b)

Figure 5 Effect of BHSST on antioxidant proteins in chronic acid reflux esophagitis rats (a) Nrf2 and Keap1 protein expressions and (b)SOD GPx-12 and HO-1 protein expressions Normal normal rats Control chronic acid reflux esophagitis rats BHSST BHSST 1 gkg bodyweightday-treated chronic acid reflux esophagitis rats Data are mean plusmn SEM (119899 = 6) Significance 119875 lt 001 119875 lt 0001 versus normalrats and lowast119875 lt 005 versus control rats


Normal Control BHSSTNormal Control BHSST

lowastlowast lowastlowast

p-ERK

p-JNK

p-p38Normal Control BHSST

훽-Actin

0051015202530

(Fol

d of

N)


lowast

0051015202530

(Fol

d of

N)

0051015202530

(Fol

d of

N)

(a)

Histone

c-JUNNormal Control BHSST

NF-휅Bp65



lowastlowast lowast

0

09

18

27

36(F

old

of N

)

0

09

18

27

36

(Fol

d of

N)

(b)

COX-2

iNOS

IL-6


훽-Actin

TNF-훼

COX-2

IL-6

iNOS



lowast

lowastlowastlowast

TNF-훼

0

05

10

15

20

(Fol

d of

N)

0

05

10

15

20(F

old

of N

)

0

05

10

15

20

(Fol

d of

N)

0

10

20

30

40

(Fol

d of

N)

(c)

Figure 6 Effect of BHSST on proinflammatory proteins in chronic acid reflux esophagitis rats (a) p-p38 p-ERK12 and p-JNK proteinexpressions (b) c-JUN and NF-120581Bp65 protein expressions (c) COX-2 iNOS TNF-120572 and IL-6 protein expressions Normal normal ratsControl chronic acid reflux esophagitis rats BHSST BHSST 1 gkg body weightday-treated chronic acid reflux esophagitis rats Data aremean plusmn SEM (119899 = 6) Significance 119875 lt 005 119875 lt 001 and

119875 lt 0001 versus normal rats and lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001versus control rats

Keap1 level of control rats was higher than that of the normalrats but it significantly decreased by BHSST administrationParticularly ARE characterizes its unique responsiveness tooxidative stress Thus change of the cellular redox status dueto ROS overexpression andor a reduced antioxidant capacityappears to be an important signal for triggering the transcrip-tional response [39] Activation of Nrf2 signaling inducesthe transcriptional regulation of ARE-dependent expres-sion of antioxidant defense enzymes (such as SOD GPxand HO-1) In our results chronic acid reflux esophagitis

rats showed decreased expressions of Nrf2 SOD GPx-12and HO-1 in esophageal tissues compared with normal ratshowever BHSST administration effectively upregulated Nrf2and alleviated oxidative stress Besides antioxidant enzymeincluding SOD GPx-12 and HO-1 significantly increasedcompared with control rats (Figure 5(b))

A low level of OS induces Nrf2 a transcription factorrelated to the transactivation of gene coding for antioxi-dant enzymes An intermediate amount of OS triggers aninflammatory response through the activation of NF-120581B


Cytoplasm ROS

Chronic reflux esophagitis

Esophageal mucosal ulcer

p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼

Figure 7 Possible mechanism of BHSST in the esophagus of chronic acid reflux-induced esophageal ulcer rats

whereas a high level of OS results in apoptosis or necrosis[40] Thereby Nrf2 and NF-120581B pathways can be functionallyantagonistic to control the transcription or function of theirdownstream targets Namely Nrf2 encodes for antioxidantand general cytoprotection genes while NF-120581B regulatesthe expression of proinflammatory genes So the activationof Nrf2-mediated antioxidant signaling attenuates NF-120581B-mediated inflammatory response As the result activationof NF-120581B pathway impairs esophageal barrier function andactivation of Nrf2 pathway may play a protective role

Mitogen-activated protein kinase (MAPK) signal trans-duction pathways are among the most widespread mecha-nisms that can be activated by a variety of stimuli includingoxidative stress The 3 principal MAPK components arethe extracellular signal-regulated kinase (ERK) c-Jun N-terminal kinase (JNK) and p38 MAPK [41] MAPK path-ways activated by acid exposure found in the metaplasticesophageal mucosa of patients with Barrettrsquos esophagus[42] Moreover ERK12 and p38 MAPK can intensify theexpression of these inflammatory factors such as COX-2and prostaglandin (PGE) 2 [43] JNK which is activatedby environmental stress phosphorylates and regulates theactivity of transcription factors including c-Jun MoreoverJNK is also activated by proinflammatory cytokines suchas TNF and IL-1 [44] In our study MAPK-related proteinexpressions were significantly augmented in the esophagusof control rats compared to the normal rats but the oraladministration of BHSSTmarkedly lowers the expressions ofp-p38 p-ERK12 and p-JNK as shown in Figure 6(a)

One of the well-studied transcription factors down-stream MAPKs signaling is the nuclear factor NF-120581B Thephosphorylation of p38 and ERK12 MAPK leads to NF-120581B translocation and NF-120581B promotes the transcriptionof target genes such as TNF-120572 and IL-6 and regulatesthe expression of inducible enzymes such as COX-2 andiNOS [45] The protein expressions of c-Jun and NF-120581B incontrol rats were significantly induced to a greater extentcompared with normal rats The administration of BHSSTled to significant downregulation of these two transcriptionprotein expressions (Figure 6(b)) The results of the presentstudy show that BHSST promotes the suppression of NF-120581Bactivation in esophageal tissue As the result upregulation ofNF-120581B-related inflammatory mediators (iNOS) significantlydecreased In addition the elevated TNF-120572 level was signif-icantly lowered by the administration of BHSST otherwiseCOX-2 and IL-6 showed a tendency to decrease (withoutsignificance) in the esophagus (Figure 6(c))

A recent study showed that ROS are one of the mostimportant factors in the pathogenesis of esophageal mucosalinjury mediated by oxidative stress in an experimentalmodel of reflux esophagitis In the present study theadministration of BHSST reduced the oxidative stressvia Nrf2Keap1ARE pathway Furthermore the anti-inflammatory effect of BHSST suggested that the blockingMAPK such as p38 ERK12 and JNK signaling pathwaysand NF-120581B inactivation led to an inhibition of the release ofproinflammatory cytokines and mediators That is BHSSTameliorated esophageal ulcer caused by experimental reflux


esophagitis in rats as shown in Figure 7 Nevertheless theaction mechanism of BHSST is still ambiguous and furtherprofound researches are required

4 Conclusions

In conclusion BHSST was associated with downregulationof proinflammatory proteins and appeared to contribute tothe antioxidant defense Based on these BHSST treatmentshowed the amelioration of esophageal mucosal ulcer whichis one of chronic GERD symptomsTherefore these datamayprovide a scientific basis for BHSST to expand the indicationin the management of GERD field

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] N Vakil S V Van Zanten P Kahrilas et al ldquoThe Montrealdefinition and classification of gastroesophageal reflux diseasea global evidence-based consensusrdquo The American Journal ofGastroenterology vol 101 no 8 pp 1900ndash1943 2006

[2] K Nakahara Y Fujiwara T Tsukahara et al ldquoAcid refluxdirectly causes sleep disturbances in rat with chronic esophagi-tisrdquo PLoS ONE vol 9 no 9 Article ID e106969 2014

[3] C Scarpignato L Gatta A Zullo and C Blandizzi ldquoEffec-tive and safe proton pump inhibitor therapy in acid-relateddiseasesmdasha position paper addressing benefits and potentialharms of acid suppressionrdquo BMCMedicine vol 14 no 1 2016

[4] R Fass and D Sifrim ldquoManagement of heartburn not respond-ing to proton pump inhibitorsrdquo Gut vol 58 no 2 pp 295ndash3092009

[5] P W Ament D B Dicola and M E James ldquoReducing adverseeffects of proton pump inhibitorsrdquo American Family Physicianvol 86 no 1 pp 66ndash70 2012

[6] D Ang C H How and T L Ang ldquoPersistent gastro-oesophageal reflux symptoms despite proton pump inhibitortherapyrdquo Singapore Medical Journal vol 57 no 10 pp 546ndash5512016

[7] V J Thannickal and B L Fanburg ldquoReactive oxygen speciesin cell signalingrdquo American Journal of Physiology Lung CellularandMolecular Physiology vol 279 no 6 pp L1005ndashL1028 2000

[8] J Zhou L Ge C Jia et al ldquoROS-mediated different homeostasisof murine corneal epithelial progenitor cell line under oxidativestressrdquo Scientific Reports vol 6 Article ID 36481 2016

[9] R C Fitzgerald andG Triadafilopoulos ldquoRecentDevelopmentsin themolecular characterization of Barrettrsquos EsophagusrdquoDiges-tive Diseases vol 16 no 2 pp 63ndash80 1998

[10] M J Cossentino and R K H Wong ldquoBarrettrsquos esophagus andrisk of esophageal adenocarcinomardquo Seminars in Gastrointesti-nal Disease vol 14 no 3 pp 128ndash135 2003

[11] K Kawashima A Nomura T Makino K-I Saito and YKano ldquoPharmacological properties of traditional medicine(XXIX) effect of hange-shashin-to and the combinations of itsherbal constituents on rat experimental colitisrdquo Biological andPharmaceutical Bulletin vol 27 no 10 pp 1599ndash1603 2004

[12] G Xu ldquoTreatment of reflux laryngopharyngitis with modifiedBanxia Xiexin Tang (Pinellia Decoction for Draining theHeart)mdasha report of 40 casesrdquo Journal of Traditional ChineseMedicine vol 26 no 2 pp 127ndash131 2006

[13] K G Lee X Cui and J P Lim ldquoEffect of the concurrentadministration of Banhasasim-tang with cimetidine on gastriculcer in ratsrdquoKorean Journal of Oriental PhysiologyampPathologyvol 16 no 3 pp 572ndash576 2002

[14] Y Kase K Saitoh B Makino K Hashimoto A Ishige andY Komatsu ldquoRelationship between the antidiarrhoeal effectsof Hange-Shashin-To and its active componentsrdquo PhytotherapyResearch vol 13 no 6 pp 468ndash473 1999

[15] F-P Chen F-J ChenM-S Jong H-L Tsai J-RWang and S-J Hwang ldquoModern use of Chinese herbal formulae fromShang-Han Lunrdquo Chinese Medical Journal vol 122 no 16 pp 1889ndash1894 2009

[16] B H Ryu K W Ryu J S Kim and S H Yoon ldquoEvaluation fortherapeutic effectiveness of Banwhasashim-tang in functionaldyspepsiardquo The Journal of Korean Oriental Internal Medicinevol 24 no 2 pp 329ndash336 2003

[17] N Omura H Kashiwagi G Chen Y Suzuki F Yano and TAoki ldquoEstablishment of surgically induced chronic acid refluxesophagitis in ratsrdquo Scandinavian Journal of Gastroenterologyvol 34 no 10 pp 948ndash953 1999

[18] S F Ali C P LeBel and S C Bondy ldquoReactive oxygen speciesformation as a biomarker of methylmercury and trimethyltinneurotoxicityrdquoNeuroToxicology vol 13 no 3 pp 637ndash648 1992

[19] M Uchiyama and M Mihara ldquoDetermination of malonalde-hyde precursor in tissues by thiobarbituric acid testrdquo AnalyticalBiochemistry vol 86 no 1 pp 271ndash278 1978

[20] S Komatsu ldquoExtraction of nuclear proteinsrdquoMethods in Molec-ular Biology vol 355 pp 73ndash77 2007

[21] C-S Seo and H-K Shin ldquoQuantitative determination of thethirteen marker components in banhasasim-tang decoctionusing an ultra-performance liquid chromatography coupledto electrospray ionization tandem mass spectrometryrdquo KoreanJournal of Pharmacognosy vol 47 no 1 pp 62ndash72 2016

[22] J H Chen G Y Cui J Y Liu and R X Tan ldquoPinellosidean antimicrobial cerebroside from Pinellia ternatardquo Phytochem-istry vol 64 no 4 pp 903ndash906 2003

[23] S Xing M Wang Y Peng D Chen and X Li ldquoSimulatedgastrointestinal tract metabolism and pharmacological activi-ties of water extract of Scutellaria baicalensis rootsrdquo Journal ofEthnopharmacology vol 152 no 1 pp 183ndash189 2014

[24] D Tiran ldquoGinger to reduce nausea and vomiting duringpregnancy evidence of effectiveness is not the same as proofof safetyrdquo Complementary Therapies in Clinical Practice vol 18no 1 pp 22ndash25 2012

[25] M Yeo D-K Kim S W Cho and H D Hong ldquoGinseng theroot of Panax ginseng CA Meyer protects ethanol-inducedgastric damages in rat through the induction of cytoprotectiveheat-shock protein 27rdquo Digestive Diseases and Sciences vol 53no 3 pp 606ndash613 2008

[26] X Shi M Zou J He H Xie and X Li ldquoStudies on the identifi-cation of constituents in ethanol extract of radix glycyrrhizaeand their anticancer activityrdquo African Journal of TraditionalComplementary and Alternative Medicines vol 11 no 2 pp334ndash338 2014

[27] R Goyal P L Sharma and M Singh ldquoPossible attenuation ofnitric oxide expression in anti-inflammatory effect of Ziziphusjujuba in ratrdquo Journal of Natural Medicines vol 65 no 3-4 pp514ndash518 2011


[28] H Hirano E Osawa Y Yamaoka and T Yokoi ldquoGastric-mucous membrane protection activity of coptisine derivativesrdquoBiological and Pharmaceutical Bulletin vol 24 no 11 pp 1277ndash1281 2001

[29] J-J Wei D-P Tang J-J Xie L-Y Yang and Z-H ZhuangldquoDecreased n-6n-3 polyunsaturated fatty acid ratio reduceschronic reflux esophagitis in ratsrdquo Prostaglandins Leukotrienesand Essential Fatty Acids vol 112 pp 37ndash43 2016

[30] O J Kwon M Y Kim S H Shin et al ldquoAntioxidant andanti-inflammatory effects of rhei rhizoma and coptidis rhizomamixture on reflux esophagitis in ratsrdquo Evidence-Based Comple-mentary and AlternativeMedicine vol 2016 Article ID 205218013 pages 2016

[31] L Zhang G Liu X Han et al ldquoInhibition of p38MAPK activa-tion attenuates esophageal mucosal damage in a chronic modelof reflux esophagitisrdquo Neurogastroenterology and Motility vol27 no 11 pp 1648ndash1656 2015

[32] C DMeletis andN Zabriskie ldquoNatural approaches for gastroe-sophageal reflux disease and related disordersrdquo Alternative ampComplementary Therapies vol 13 no 2 pp 64ndash70 2007

[33] J Limon-Pacheco and M E Gonsebatt ldquoThe role ofantioxidants and antioxidant-related enzymes in protectiveresponses to environmentally induced oxidative stressrdquoMutation ResearchmdashGenetic Toxicology and EnvironmentalMutagenesis vol 674 no 1-2 pp 137ndash147 2009

[34] P Singh N Singh S Sengupta and G Palit ldquoAmeliorativeeffects of Panax quinquefolium on experimentally inducedreflux oesophagitis in ratsrdquo Indian Journal of Medical Researchvol 135 no 3 pp 407ndash413 2012

[35] Y Xu J Zhu X Hu et al ldquoCLIC1 inhibition attenuates vascularinflammation oxidative stress and endothelial injuryrdquo PLOSONE vol 11 no 11 2016

[36] P Muralidharan D Hayes S M Black and H M MansourldquoMicroparticulatenanoparticulate powders of a novel Nrf2activator and an aerosol performance enhancer for pulmonarydelivery targeting the lung Nrf2Keap-1 pathwayrdquo MolecularSystems Design amp Engineering vol 1 no 1 pp 48ndash65 2016

[37] J-M Lee and J A Johnson ldquoAn important role of Nrf2-ARE pathway in the cellular defense mechanismrdquo Journal ofBiochemistry and Molecular Biology vol 37 no 2 pp 139ndash1432004

[38] C-M Ha S Park Y-K Choi et al ldquoActivation of Nrf2 bydimethyl fumarate improves vascular calcificationrdquo VascularPharmacology vol 63 no 1 pp 29ndash36 2014

[39] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo Journal of Biological Chemistry vol 284 no20 pp 13291ndash13295 2009

[40] Y Chtourou B Aouey M Kebieche and H Fetoui ldquoPro-tective role of naringin against cisplatin induced oxidativestress inflammatory response and apoptosis in rat striatum viasuppressingROS-mediatedNF-120581BandP53 signaling pathwaysrdquoChemico-Biological Interactions vol 239 pp 76ndash86 2015

[41] S-Y Cho S-J Park M-J Kwon et al ldquoQuercetin sup-presses proinflammatory cytokines production through MAPkinases and NF-120581B pathway in lipopolysaccharide-stimulatedmacrophagerdquoMolecular and Cellular Biochemistry vol 243 no1-2 pp 153ndash160 2003

[42] R F Souza K Shewmake L S Terada and S J SpechlerldquoAcid exposure activates the mitogen-activated protein kinasepathways in Barrettrsquos esophagusrdquo Gastroenterology vol 122 no2 pp 299ndash307 2002

[43] J G Woo S Y Park J C Lim M-J Joo H R Kim and U DSohn ldquoAcid-induced COX-2 expression and prostaglandin E2production via activation of ERK12 and p38MAPK in culturedfeline esophageal smooth muscle cellsrdquo Archives of PharmacalResearch vol 34 no 12 pp 2131ndash2140 2011

[44] J M Kyriakis and J Avruch ldquoMammalian mitogen-activatedprotein kinase signal transduction pathways activated by stressand inflammationrdquo Physiological Reviews vol 81 no 2 pp 807ndash869 2001

[45] J H Seo J W Lim and H Kim ldquoDifferential role of ERK andp38 on NF-120581B activation in helicobacter pylori-infected gastricepithelial cellsrdquo Journal of Cancer Prevention vol 18 no 4 pp346ndash350 2013

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Composition of Banhasasim-tang (BHSST) used in thisstudy

Herbs Amounts (g)Pinelliae Rhizoma 167Scutellariae Radix 100Zingiberis Rhizoma Siccus 083Ginseng Radix 100Glycyrrhizae Radix 100Jujubae Fructus 100Coptidis Rhizoma 033Banhasasimtang Ex Granule (15 g) is contained in 1 pack (35 g)Banhasasimtang Ex Granule was purchased from Hankook Shinyak Corp(Nonsan-si Chungcheongnam-do Republic of Korea)

in humans with chronic reflux esophagitis and increases arisk for development of adenocarcinoma of the esophagusand gastric cardia [9 10] Accordingly the suppression of thegastroesophageal reflux is the most important in treatment ofGERD

Banhasasim-tang (BHSST Hange-shashin-to in JapaneseTraditional Kampo Medicine Banxia-xiexin-tang in Tra-ditional Chinese Medicine) has been used as an herbalprescription to improve dyspepsia gastric ulcerative disor-ders laryngopharyngitis colitis and diarrhea [11ndash14] Thisformula is composed of Pinelliae Rhizoma ScutellariaeRadix Zingiberis Rhizoma Siccus Ginseng Radix AlbaGlycyrrhizae Radix Zizyphi Fructus and Coptidis Rhizoma(Table 1) BHSST described in Shang-Han Lun (Treatise onCold Damage and Miscellaneous Diseases) written by ZhangZhong-jing (150ndash219 AD) is widely accepted by Chineseherbal doctors and has been applied for treating the symptomassociated to GERD in Korea [15 16] Despite previousvarious reports related to the improvement of GERD theprotective mechanisms of BHSST treatment in esophagealulcer by chronic reflux are not fully understood Thereforewe investigated the effects of BHSST on rats with chronic acidreflux esophagitis (CRE) to examine its ameliorating effectagainst oxidative stress-related inflammation

2 Materials and Methods

21 Materials The protease inhibitor mixture solutionand ethylenediaminetetraacetic acid (EDTA) were pur-chased from Wako Pure Chemical Industries Ltd (OsakaJapan) Phenylmethylsulfonyl fluoride (PMSF) was pur-chased from Sigma-Aldrich (St Louis MO USA) 2101584071015840-Dichlorofluorescein diacetate (DCF-DA) was obtained fromMolecular Probes (Eugene OR USA) The pierce bicin-choninic acid (BCA) protein assay kit was obtained fromThermoFisher Scientific (WalthamMAUSA) ECLWesternBlotting Detection Reagents and pure nitrocellulose mem-branes were supplied by GE Healthcare (Chicago IL USA)Rabbit polyclonal antibodies against nuclear factor-kappa Bp65 (NF-120581Bp65 1 1000 SC-372) nuclear factor-erythroid 2-related factor 2 (Nrf2 1 1000 SC-7228) heme oxygenase-1 (HO-1 1 1000 SC-10789) superoxide dismutase (SOD1 1000 SC-11407) glutathione peroxidase-12 (GPx-12

1 1000 SC-30147) goat polyclonal antibodies against Kelch-like ECH-associated protein 1 (Keap1 1 1000 SC-15246)tumor necrosis factor-120572 (TNF-120572 1 1000 SC-1351) andinterleukin-6 (IL-6 1 1000 SC-1266) and mouse mon-oclonal antibodies against phosphor-c-Jun NH

2-terminal

kinase (p-JNK 1 1000 SC-6254) phosphor-extracellularsignal-regulated kinase 12 (p-ERK12 1 1000 SC-7383)phosphor-p38 (p-p38 1 1000 SC-7973) cyclooxygenase-2(COX-2 1 1000 SC-19999) inducible nitric oxide synthase(iNOS 1 1000 SC-7271) histone (1 1000 SC-8030) and 120573-actin (1 1000 SC-4778) were purchased from Santa CruzBiotechnology Inc (Santa Cruz CA USA) Mouse mon-oclonal antibody against activator protein-1 (AP-1) subunitc-Jun (1 1000 2315) was obtained from Cell SignalingTechnology Inc (Cell Signaling MA USA) Rabbit anti-goat (1 3000 SC-2774) goat anti-rabbit (1 3000 SC-2004)and goat anti-mouse (1 3000 SC-2005) immunoglobulin G(IgG) horseradish peroxidase- (HRP-) conjugated secondaryantibodies were acquired from Santa Cruz BiotechnologyInc (Santa Cruz CA USA) All other chemicals and reagentswere purchased from Sigma-Aldrich (St Louis MO USA)

22 Test Material Light brown granules of Banhasasim-tang(Hankook Shinyak Corp Nonsan-si Chungcheongnam-doRepublic of Korea) produced according to Korean GoodManufacturing Practice (GMP)were permitted and regulatedby the Korean Food amp Drug Administration (KFDA SeoulRepublic of Korea) BHSST was dissolved in distilled waterThe Banhasasimtang Ex Granule is included as 15 g inBanhasasim-tang (1 package 35 g) used in this study andillustrated in Table 1

23 Experimental Animals and Treatment Animal experi-ments were carried out according to the ldquoGuidelines forAnimal Experimentationrdquo approved by the Ethics Committeeof the Daegu Haany University on 20102016 with certificatenumber DHU2016-81 Five-week-old male Sprague-Dawleyrats (BW 150ndash155 g) were purchased from Nara Biotec Co(Pyeongtaek Republic of Korea) Rats were maintainedunder a 12 h lightdark cycle housed at controlled tem-perature (24 plusmn 2∘C) and humidity (about 60) and keptin raised mesh-bottom cages to prevent coprophagy Afteradaptation (1 week) the rats were fasted for 18 h prior tosurgical procedures and kept in raised mesh-bottom cages toprevent coprophagy And then rats were anaesthetized withan injection of Zoletil at 075mgkg (Virbac SA France)The CRE model (chronic acid reflux esophagitis model) wasdeveloped by following the methods proposed by Omuraet al [17] A midline laparotomy was performed to exposethe stomach and the transitional region (ie limiting ridge)between the fundus and the glandular portion of the stomachwas ligated with 2ndash0 silk thread in order to restrict thecompliance of the stomach which led to the reflux ofgastric contents into the esophagus Additionally a latex ring(2mm in thickness ID 4mm made from 18-Fr Nelatoncatheter) was placed around the pyloric sphincter so as torestrict the emptying of gastric contents Rats were injectedwith gentamicin sulfate (antibiotic subcutaneous injection)














1Day

3 5 7 9 11 13 15 17 19 21


NormalControlBHSST

050

100150200250300350

Body

wei

ght (

g)

(a)

9 11 13 15 17 19 21Drug treatment

NormalControlBHSST

05

101520253035

Food

inta

ke (g

)

lowastlowast

(b)









(a)

BHSSTNormal Control

(b)


0

5

10

15

20

25

30

35G

ross

esop

hage

al u

lcer

ratio

()

lowast

(c)


Control BHSSTNormal

(a)

Control BHSSTNormal

(b)




0

100

200

300

400

Seru

m R

OS

(fluo

resc

ence

mL)

lowast


0

2

4

6

8

Seru

m T

BARS

(nM

mL)

(b)


0

02

04

06

08

10

TBA

RS in

esop

hagu

s (nM

mg

prot

ein)

lowastlowastlowast

(c)


Histone

Nrf2


Keap1



lowast훽-Actin

lowast

0

(Fol

d of

N)

06

12

18

(Fol

d of

N) 12

06

18

0

(a)SOD-1

SOD

GPx-12

HO-1



GPx-12

HO-1


lowast

lowast

lowast훽-Actin

0

05

10

15

(Fol

d of

N)

0

05

10

15

(Fol

d of

N)

06

12

18

(Fol

d of

N)

(b)





p-ERK

p-JNK


훽-Actin

0051015202530

(Fol

d of

N)


lowast

0051015202530

(Fol

d of

N)

0051015202530

(Fol

d of

N)

(a)

Histone


NF-휅Bp65



lowastlowast lowast

0

09

18

27

36(F

old

of N

)

0

09

18

27

36

(Fol

d of

N)

(b)

COX-2

iNOS

IL-6


훽-Actin

TNF-훼

COX-2

IL-6

iNOS



lowast

lowastlowastlowast

TNF-훼

0

05

10

15

20

(Fol

d of

N)

0

05

10

15

20(F

old

of N

)

0

05

10

15

20

(Fol

d of

N)

0

10

20

30

40

(Fol

d of

N)

(c)







Cytoplasm ROS



p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼








4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























1Day

3 5 7 9 11 13 15 17 19 21


NormalControlBHSST

050

100150200250300350

Body

wei

ght (

g)

(a)

9 11 13 15 17 19 21Drug treatment

NormalControlBHSST

05

101520253035

Food

inta

ke (g

)

lowastlowast

(b)









(a)

BHSSTNormal Control

(b)


0

5

10

15

20

25

30

35G

ross

esop

hage

al u

lcer

ratio

()

lowast

(c)


Control BHSSTNormal

(a)

Control BHSSTNormal

(b)




0

100

200

300

400

Seru

m R

OS

(fluo

resc

ence

mL)

lowast


0

2

4

6

8

Seru

m T

BARS

(nM

mL)

(b)


0

02

04

06

08

10

TBA

RS in

esop

hagu

s (nM

mg

prot

ein)

lowastlowastlowast

(c)


Histone

Nrf2


Keap1



lowast훽-Actin

lowast

0

(Fol

d of

N)

06

12

18

(Fol

d of

N) 12

06

18

0

(a)SOD-1

SOD

GPx-12

HO-1



GPx-12

HO-1


lowast

lowast

lowast훽-Actin

0

05

10

15

(Fol

d of

N)

0

05

10

15

(Fol

d of

N)

06

12

18

(Fol

d of

N)

(b)





p-ERK

p-JNK


훽-Actin

0051015202530

(Fol

d of

N)


lowast

0051015202530

(Fol

d of

N)

0051015202530

(Fol

d of

N)

(a)

Histone


NF-휅Bp65



lowastlowast lowast

0

09

18

27

36(F

old

of N

)

0

09

18

27

36

(Fol

d of

N)

(b)

COX-2

iNOS

IL-6


훽-Actin

TNF-훼

COX-2

IL-6

iNOS



lowast

lowastlowastlowast

TNF-훼

0

05

10

15

20

(Fol

d of

N)

0

05

10

15

20(F

old

of N

)

0

05

10

15

20

(Fol

d of

N)

0

10

20

30

40

(Fol

d of

N)

(c)







Cytoplasm ROS



p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼








4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

BHSSTNormal Control

(b)


0

5

10

15

20

25

30

35G

ross

esop

hage

al u

lcer

ratio

()

lowast

(c)


Control BHSSTNormal

(a)

Control BHSSTNormal

(b)




0

100

200

300

400

Seru

m R

OS

(fluo

resc

ence

mL)

lowast


0

2

4

6

8

Seru

m T

BARS

(nM

mL)

(b)


0

02

04

06

08

10

TBA

RS in

esop

hagu

s (nM

mg

prot

ein)

lowastlowastlowast

(c)


Histone

Nrf2


Keap1



lowast훽-Actin

lowast

0

(Fol

d of

N)

06

12

18

(Fol

d of

N) 12

06

18

0

(a)SOD-1

SOD

GPx-12

HO-1



GPx-12

HO-1


lowast

lowast

lowast훽-Actin

0

05

10

15

(Fol

d of

N)

0

05

10

15

(Fol

d of

N)

06

12

18

(Fol

d of

N)

(b)





p-ERK

p-JNK


훽-Actin

0051015202530

(Fol

d of

N)


lowast

0051015202530

(Fol

d of

N)

0051015202530

(Fol

d of

N)

(a)

Histone


NF-휅Bp65



lowastlowast lowast

0

09

18

27

36(F

old

of N

)

0

09

18

27

36

(Fol

d of

N)

(b)

COX-2

iNOS

IL-6


훽-Actin

TNF-훼

COX-2

IL-6

iNOS



lowast

lowastlowastlowast

TNF-훼

0

05

10

15

20

(Fol

d of

N)

0

05

10

15

20(F

old

of N

)

0

05

10

15

20

(Fol

d of

N)

0

10

20

30

40

(Fol

d of

N)

(c)







Cytoplasm ROS



p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼








4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

100

200

300

400

Seru

m R

OS

(fluo

resc

ence

mL)

lowast


0

2

4

6

8

Seru

m T

BARS

(nM

mL)

(b)


0

02

04

06

08

10

TBA

RS in

esop

hagu

s (nM

mg

prot

ein)

lowastlowastlowast

(c)


Histone

Nrf2


Keap1



lowast훽-Actin

lowast

0

(Fol

d of

N)

06

12

18

(Fol

d of

N) 12

06

18

0

(a)SOD-1

SOD

GPx-12

HO-1



GPx-12

HO-1


lowast

lowast

lowast훽-Actin

0

05

10

15

(Fol

d of

N)

0

05

10

15

(Fol

d of

N)

06

12

18

(Fol

d of

N)

(b)





p-ERK

p-JNK


훽-Actin

0051015202530

(Fol

d of

N)


lowast

0051015202530

(Fol

d of

N)

0051015202530

(Fol

d of

N)

(a)

Histone


NF-휅Bp65



lowastlowast lowast

0

09

18

27

36(F

old

of N

)

0

09

18

27

36

(Fol

d of

N)

(b)

COX-2

iNOS

IL-6


훽-Actin

TNF-훼

COX-2

IL-6

iNOS



lowast

lowastlowastlowast

TNF-훼

0

05

10

15

20

(Fol

d of

N)

0

05

10

15

20(F

old

of N

)

0

05

10

15

20

(Fol

d of

N)

0

10

20

30

40

(Fol

d of

N)

(c)







Cytoplasm ROS



p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼








4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















p-ERK

p-JNK


훽-Actin

0051015202530

(Fol

d of

N)


lowast

0051015202530

(Fol

d of

N)

0051015202530

(Fol

d of

N)

(a)

Histone


NF-휅Bp65



lowastlowast lowast

0

09

18

27

36(F

old

of N

)

0

09

18

27

36

(Fol

d of

N)

(b)

COX-2

iNOS

IL-6


훽-Actin

TNF-훼

COX-2

IL-6

iNOS



lowast

lowastlowastlowast

TNF-훼

0

05

10

15

20

(Fol

d of

N)

0

05

10

15

20(F

old

of N

)

0

05

10

15

20

(Fol

d of

N)

0

10

20

30

40

(Fol

d of

N)

(c)







Cytoplasm ROS



p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼








4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cytoplasm ROS



p38P ERK12P

Nrf2

COX-2 iNOS IL-6

JNKP

GPx12

HO-1

SOD

c-JUN

Nucleus

Keap1 Nrf2

ARE NF-휅B

TNF-훼








4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Conclusions


Competing Interests


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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