research article gastroprotective activity of violacein

Research ArticleGastroprotective Activity of Violacein Isolated fromChromobacterium violaceum on Indomethacin-Induced GastricLesions in Rats Investigation of Potential Mechanisms of Action

Paulrayer Antonisamy1 Ponnusamy Kannan2 Adithan Aravinthan1

Veeramuthu Duraipandiyan3 Mariadhas Valan Arasu3 Savarimuthu Ignacimuthu2

Naif Abdullah Al-Dhabi3 and Jong-Hoon Kim1

1 Biosafety Research Institute College of Veterinary Medicine Chonbuk National University 664-14 1GA Duck Jin-DongDeokjin-gu Jeonju City Jeollabuk-do 561-756 Republic of Korea

2Division of Ethnopharmacology Entomology Research Institute Loyola College Chennai Tamil Nadu 600 034 India3 Department of Botany and Microbiology Addiriyah Chair for Environmental Studies College of Science King Saud UniversityPO Box 2455 Riyadh 11451 Saudi Arabia

Correspondence should be addressed to Jong-Hoon Kim jhkim1jbnuackr

Received 17 April 2014 Revised 18 June 2014 Accepted 26 June 2014 Published 5 August 2014

Academic Editor Fulvio DrsquoAcquisto

Copyright copy 2014 Paulrayer Antonisamy et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chromobacterium violaceum Gram-negative bacteria species found in tropical regions of the world produces a distinct deep violet-colored pigment called violacein In the present study we investigated whether violacein can promote a gastroprotective effectand verified the possible mechanisms involved in this action For this study an indomethacin-induced gastric ulcer rat modelwas used The roles of biomolecules such as MPO PGE

2 pro- and anti-inflammatory cytokines growth factors caspase-3 NO

K+ATP channels and 1205722-receptors were investigated Violacein exhibited significant gastroprotective effect against indomethacin-

induced lesions while pretreatment with L-NAME and glibenclamide (but not with NEM or yohimbine) was able to reverse thisaction Pretreatment with violacein also restored cNOS level to normal and led to attenuation of enhanced apoptosis and gastricmicrovascular permeability Our results suggest that violacein provides a significant gastroprotective effect in an indomethacin-induced ulcermodel through themaintenance of some vital proteinmolecules and this effect appears to bemediated at least in partby endogenous prostaglandins NOS K+ATP channel opening and inhibition of apoptosis and gastric microvascular permeability

1 Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) remain thefirst line therapy for rheumatoid arthritis and osteoarthritisUnfortunately their therapeutic effects are problematic dueto gastrointestinal toxicity [1] primarily via inhibition ofprostaglandin synthesis [2] neutrophil infiltration [3] nitricoxide imbalance [4] induction of apoptosis [5] and produc-tion of free radicals [6] These radicals play a prominent rolein microvascular injury [7] and cell death [5]

In the pharmaceutical industry microorganisms havebecome an important source of natural products Nearly

63 of commercially available drugs are directly or indi-rectly derived from microorganisms plants or animals[8] Chromobacterium violaceum is a Gram-negative 120573-proteobacterium facultative anaerobic saprophyte free-living soil- and water-associated microorganism found inwater body soils in tropical and subtropical regions of theworld Violacein a purple pigment is a main characteristicof this bacterium and exhibits significant activity againstessential tropical pathogens such as Mycobacterium tubercu-losis Trypanosoma cruzi Leishmania sp and Plasmodiumfalciparum It is reported to have antifungal antioxidantantitumor bactericidal cytotoxic and antiviral activities [9]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 616432 10 pageshttpdxdoiorg1011552014616432

2 The Scientific World Journal

Several studies have shown that violacein is also capable ofinducing apoptosis in a variety of cancer cell lines includingleukemia lineages suggesting a promising clinical applicationin cancer treatment The therapeutic application of violaceinto cancer chemoprevention has been the focus of recentstudies [10 11] Violacein has been shown to induce apoptosisin HL60 leukemic cells but is ineffective in normal humanlymphocytes and monocytes [12] In our previous studies weshowed that violacein possesses immunomodulatory anal-gesic antipyretic antidiarrheal and antiulcerogenic activities[13 14]

In this study we investigated the gastroprotective effect ofviolacein in an indomethacin-induced ulcer model in Wisterrats to determine its mechanisms of action

2 Materials and Methods

21 Animals Adult Wistar albino rats (200ndash220 g) of bothsexes were used for experiments Animals were housed witha 12 h lightdark cycle at 25 plusmn 1∘C at a relative moisture of60ndash70 they had access to diet and water ad libitum andunderwent at least twoweeks of adaptation before starting theexperiments All studies were carried out using six animalsin each group All animal experiments were conductedaccording to the ethical norms approved by the Ministry ofSocial Justice and Empowerment Government of India andthe procedures of the Institutional Animal Ethics Committee

22 Chemicals and Drugs Indomethacin omeprazole cele-coxib SC560 N-G-nitro-L-argininemethyl ester (L-NAME)N-ethylmaleimide (NEM) yohimbine and glibenclamidewere obtained from Sigma-Aldrich (Sigma Chemicals CoSt Louis MO USA) Carboxymethyl cellulose (CMC) wasobtained from Himedia (Mumbai India) The PGE

2EIA

kit vascular endothelial growth factor (VEGF) hepatocytegrowth factor (HGF) epidermal growth factor (EGF) andenzyme-linked immunosorbent assay (ELISA) kits were pur-chased from Cayman Chemical (Ann Arbor MI USA)Tumor necrosis factor (TNF)-120572 interleukin (IL)-1120573 IL-4IL-6 and IL-10 ELISA kits were purchased from PierceBiotechnology (Rockford IL USA) The apoptosis assay kitwas acquired from Boehringer Mannheim and caspase-3activity assays were conducted using the Quanti Zyme assaysystem from Biomol Research Laboratories Inc (USA) Allother chemicals usedwere of analytical reagent gradeThe testcompound violacein was isolated from C violaceum ESBV4400 which was isolated from forest water body soil samplesfrom Kolli Hills of Tamil Nadu India at latitude 11∘ 101015840 to11∘301015840N and longitude 78∘ 151015840 to 78∘ 301015840 E (Figure 1) and

reported in our previous study [14]

23 Determination of Doses To determine the lowest effec-tive dose of violacein gastric ulcers were induced byindomethacin followed by treatment with violacein Afterfasting for 24 h rats were distributed into eight groups (119899 =6group) Animals orally received vehicle alone (05mL of05 CMC) for sham and indomethacin treated controlgroup omeprazole as the positive control (40mgkg) or

OH

HNH

N

HN

O

O

Figure 1 Structure of violacein

violacein (10 20 40 80 or 160mgkg) After 30min theanimals orally received 20mgkg of indomethacin exceptsham treated group Six hours later the animals were sacri-ficed under ether anesthesia and the stomach was surgicallyremoved immersed in 5 formalin for 30min and openedalong the greater curvature to macroscopically examinelesions according to the ulcer score described by previousmethod [15]

24 Gastric Damage Induced by Indomethacin Rats (119899 =6) were treated with sham (05mL of 05 CMC) vehicle+ Indo (05mL of 05 CMC) violacein (40mgkg po)omeprazole (40mgkg po) SC560 + violacein (5mgkgpo + 40mgkg po) celecoxib + violacein (35mgkg po+ 40mgkg po) L-NAME + violacein (50mgkg ip +40mgkg po) NEM + violacein (10mgkg sc + 40mgkgpo) yohimbine + violacein (2mgkg ip + 40mgkg po)or glibenclamide + violacein (5mgkg po + 40mgkg po)All drugs were administered using 05 CMC as the vehiclesolution After 30min each group of animals except the shamtreated group received a 20mgkg oral dose of indomethacinSelective COX-1 inhibitor (SC560) COX-2 inhibitor (cele-coxib) nonselective nitric oxide synthase (NOS) inhibitor(L-NAME) endogenous sulfhydryl antagonist (NEM) 120572

2-

receptors antagonist (yohimbine) and K+ATP channelsantagonist (glibenclamide) were administered to rats 30minbefore violacein treatment and 1 h prior to ulcer induction byindomethacin Six hours later animals were sacrificed underether anesthesia and the stomach was surgically removedimmersed in 5 formalin for 30min and opened alongthe greater curvature to macroscopically examine lesionsaccording to the ulcer score described by previous method[15]

0 = no damage1 = blood at the lumen2 = pin-point erosions3 = one to five small erosions lt2mm4 = more than five small erosions lt2mm5 = one to three large erosions gt2mm6 = more than three large erosions gt2mm

The Scientific World Journal 3

The inhibition percentage was calculated using the follow-ing formula from Demirbilek et al [(UI nontreated minus UItreated)UI nontreated] times 100 [16]

25 Determination of MPO Activity Level MPO activity inthe gastric mucosa was determined according to the methoddescribed previously [17] MPO activity in gastric tissues wasexpressed as 120583molminmg tissue

26 Determination of Inflammatory Mediators and TissueGrowth Factors Levels PGE

2 TNF-120572 IL-1120573 IL-4 IL-6 IL-

10 VEGF EGF and HGF were quantified in the stomachhomogenate using enzyme-linked immunosorbent assay kitsaccording to the manufacturerrsquos specifications The resultswere expressed as pggm tissue or nggm tissue

27 Determination of NOS Activity Gastric mucosal NOSactivity was measured spectrophotometrically with the oxi-dation of oxyhemoglobin to methemoglobin by NO aspreviously described [18 19] The absorption deferencebetween 401 and 421 nm was constantly observed with a dualwavelength recording spectrophotometer at 37∘C InducedNOS (iNOS) activity was calculated by subtraction of cNOSactivity from total NOS activity

28 Determination of Apoptosis and Caspase-3 Activity LevelApoptosis and caspase-3 levels have been assayed spec-trophotometrically by previous methods [20 21] Apoptosisand caspase-3 levels were expressed as unitmg protein andas pmolmg protein respectively

29 Determination of Microvascular Permeability Rats weredivided into six groups each containing six animals Animalswere fasted for 24 h prior to experiments and allowed freeaccess to waterThe first group of rats received 05mL of 05CMC and served as sham treated group The second groupwas subjected to gastric injury by intragastric installationof indomethacin at a dose of 20mgkg and was used asthe ulcer-induced group The remaining four groups weregiven violacein (40mgkg) sucralfate (400mgkg) SC560 +violacein (30mgkg + 40mgkg) or celecoxib + violacein(30mgkg + 40mgkg) by intragastric administration at 1 hrbefore ulcer induction using indomethacin All drugs includ-ing indomethacin violacein sucralfate SC560 and celecoxibwere suspended in 05 CMC Gastric microvascular per-meability was evaluated 4 h after indomethacin treatment bymeasuring the extravasated amount of Evanrsquos blue dye in themucosa according to the previously mentioned method [22]In each animal 1mL of 1 (wv) Evanrsquos blue in sterile salinewas injected intravenously 30min before sacrifice Underether anesthesia animals were sacrificed by bleeding fromthe descending aorta the stomachs were removed and thegastric mucosa was scraped off and immersed in distilledwater The dye was extracted with formamide and quantifiedspectrophotometrically at 620 nm and results are expressedas 120583gmg protein

210 Statistical Analysis Data were statistically analyzedusing analysis of variance (ANOVA) followed by Studentrsquost-test A probability level lower than 005 was consideredstatistically significant

3 Results and Discussion

Because of their anti-inflammatory analgesic and antipyreticeffects NSAIDs are commonly used to treat rheumatoidarthritis pyrexia bone pain headache migraine acute goutand many other conditions [23] High dosage inoppor-tune consumption or sustained use of NSAIDs occasion-ally causes severe intestinal ulcer and gastroduodenal sick-nesses [23] The gastroprotective effect of violacein againstNSAID-induced ulcer has not been reported until now Inthe present study we evaluated the gastroprotective effectsof orally administered violacein on indomethacin-inducedgastric damage in rats Our macroscopic analyses exposedthat administration of indomethacin (20mgkg) producednoticeable mucosal injury in the abdomen Violacein pre-treated group (Figure 2(c)) or omeprazole group (Figure 2(d))considerably reduced gastric lesion compared to the ulcercontrol group where indomethacin induced intense gastricmucosal damage in the form of elongated band of hemor-rhages (Figure 2(b)) Normal group shows intact stomachwithout any incisions (Figure 2(a)) Regarding effective doseevaluation the vehicle + Indo group and the 10 and 20mgkggroups and the 40mgkg dose showed a significant ulcerprotective effect (119875 lt 005) The 80 and 160mgkg doses ofviolacein produced the same effect as the 40mgkg dose so40mgkg was selected as the upper limit for further experi-ments Rats receiving only vehicle (sham treated) showed nogastric mucosal lesions while indomethacin administrationproduced mucosal lesions in rat stomachs Compared withrats in untreated group the indomethacin damage scores inviolacein (40mgkg)mdashand omeprazolemdashtreated groupswerereduced by 8639 and 8830 correspondingly (Figure 3)

MPO activity is known to increase in ulcerated sit-uations and to be reduced through the curing processMPO activity level is regularly used as a threat indicatorand investigative device for evaluating the harshness of anintestinal ulcer [24] In this study we found that gastricMPOactivity was significantly increased in the indomethacingroup from 360 120583molminmg tissue (sham treated) to1072 120583molminmg tissue The effect of violacein (40mgkg)against MPO level (372 120583molminmg tissue) was greaterthan that of omeprazole (391 120583molminmg tissue) butthe difference was not statistically significant (Figure 4)NSAIDs exert their therapeutic action by inhibiting theCOXsisoenzymes (COX-1 and COX-2) and reducing the levels ofcirculating PGs However the reduced levels of PGs in theintestinal mucosa are known to cause gastric ulceration andto exacerbate preexisting gastric ulcers in both rodents andhumans [25] PGs stimulatemucus and bicarbonate secretionas well as mucosal blood flow and encourage angiogenesis[24] All of these elements contribute to reducing healing timeand repairing ulcers In this study mucosal PGE

2levels were

markedly suppressed (341-fold) in indomethacin-induced


(a) (b)

(c) (d)

Figure 2 Gastroprotective activity of violacein (40mgkg) on indomethacin-induced gastric injury in rats (a) Sham treated rats (b) vehicle+ indomethacin treated rats (c) violacein (40mgkg) pretreated rats and (d) omeprazole (40mgkg) pretreated rats Note that indomethacininduced sever injuries to the gastric mucosa that appear as elongated bands of hemorrhage (blue arrow)

rats (119875 lt 005) comparedwith sham treated groupOral treat-mentwith violacein and omeprazole upregulated themucosalPGE2level by 307- and 324-fold respectively (Figure 5)

Pretreatment with SC560 resulted in a significant reductionin PGE

2level in violacein-pretreated ulcerated rats Thus it

is promising that violacein exerts its gastroprotective effect bystimulating synthesis of COX-1-derived PGE

2 On the other

hand celecoxib (COX-2 inhibitor) is unable to prevent thegastroprotective effect of violacein which indicates that theCOX-2mediated prostaglandin synthesis was not involved inviolacein activity The present study also confirmed that theselective COX-1 inhibitor SC560 did not induce any injuryon stomachs of normal rats but decreases the PGE

2level

significantly (data not shown) similar to indomethacin thisis consistent with previous observations [26 27] Howeverpretreatment of SC560 completely inhibit the gastroprotec-tive activity of violacein against indomethacin-induced ulcerthis observation clearly elucidated that the gastroprotectiveactivity of violacein also was facilitated through the COX-1 mediated pathways Previous reports show that PGE

2acts

as a potential inhibitor of TNF-120572 [28 29] apoptosis [30]and activator of K+ATP channels [31] it is possible thatviolacein may be able to stop ulcer induction processes viathe inhibition of TNF-120572 apoptosis and activation of K+ATPchannels through significant production of PGE

2by COX-1

mediated pathways

Indomethacin-induced ulcer shows increased expressionof proinflammatory cytokines [32] which are associatedwith the degree of ulceration Indomethacin administrationelevated proinflammatory Th1 cytokines and reduced anti-inflammatory cytokines [33] ELISA study illustrated thatindomethacin stimulated TNF-120572 (218-fold) IL-1120573 (211-fold)and IL-6 (130-fold) and downregulated IL-4 (259-fold) IL-10 (152-fold) VEGF (279-fold) EGF (226-fold) and HGF(249-fold) levels Violacein at a dose of 40mgkg significantly(119875 lt 005) reduced proinflammatory cytokine (TNF-120572 IL-1120573 and IL-6) level (184-fold 195-fold and 145-fold resp)(Figure 6(a)) and increased anti-inflammatory cytokines (IL-4 and IL-10) level (269-fold and 228-fold resp) (Figure 6(b))and growth factors (VEGF EGF and HGF) level (290-fold 243-fold and 241-fold resp) compared with theindomethacin-induced ulcerated untreated group (Figure 7)Overall above results explicate that the violacein treatmentreducedproinflammatory cytokines (TNF-120572 IL-1120573 and IL-6)and concurrently increased the levels of tissue IL-4 and IL-10all of which may contribute to its healing effect

TNF-120572 seems to be a crucial contributor to many formsof intestinal mucosal injury including that during the appli-cation of NSAIDs NSAIDs have been shown to markedlyelevate the level of TNF-120572 Inhibition of TNF-120572 productionresults in attenuation of the harmful effects of NSAIDs inthe rat intestine [34] Prostaglandins are potent inhibitors

The Scientific World Journal 5U

lcer

inde

x (U

I)

UI

Groups

ns

Sham

Vehi

cle+

Indo

Om

p (40

mg)+

Indo

Vio

(10

mg)+

Indo

Vio

(20

mg)+

Indo

Vio

(40

mg)+

Indo

Vio

(80

mg)+

Indo

Vio

(160

mg)+

Indo

lowastlowast 8830 lowast 8639 lowast 8594lowast 8618

lowast 3534

lowast 6989

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

Figure 3 Effect of violacein (10 20 40 80 and 160mgkg orally)on indomethacin-induced ulcer index in rats Values are mean plusmnSD (119899 = 6) lowast119875 lt 005 compare vehicle + Indo with all the groupsValues in the braces indicate ulcer index inhibition percentage Indoindomethacin Vio violacein UI ulcer index ns nonsignificant

0

2

4

6

8

10

12

14 minus494

MPO

activ

ity le

vel

MPO activity level (120583molminmg tissue)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowast 6529 lowast 6501

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

Figure 4 Effect of COX-1 and COX-2 inhibitors and violacein(40mgkg orally) on MPO level in indomethacin-induced ulcerValues are mean plusmn SD (119899 = 6) lowast119875 lt 005 compare vehicle + Indowith all the groups

119875 lt 005 compare Vio (40mg) + Indo withSC560 (or) Celecoxib + Vio (40mg) + Indo Indo indomethacinVio violacein MPO myeloperoxidase

of TNF-120572 release from both macrophages [29] and mastcells [28] IL-10 has a central role in the downregulation ofthe inflammatory cascade by depressing the production ofa number of proinflammatory cytokines [35] and enhanc-ing the production of anti-inflammatory cytokines [36] Inthis study violacein enhances IL-10 and inhibited TNF-120572significantly which clearly indicates the ulcer curing abilityof violacein on NSAID induced ulcer VEGF is a growthfactor that increases ulcer healing by stimulating angiogenesis

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowastlowast lowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

PGE2

leve

l

PGE2 (pgmg protein)

000

1000

2000

3000

4000

5000

6000

Figure 5 Effect of violacein (40mgkg orally) on mucosal PGE2

level in indomethacin-induced ulcer Values are mean plusmn SD (119899 = 6)lowast119875 lt 005 compare vehicle + Indo with all the groups

119875 lt 005

compare Vio (40mg) + Indo with SC560 (or) Celecoxib + Vio(40mg) + Indo Indo indomethacin Vio violacein

[37] Likewise HGF aids angiogenesis through multiplemechanisms including COX activation and increases EGFexpression that eventually accelerates gastroduodenal ulcerhealing by increasing gastric mucin and diminishing gas-tric acid secretion [38] We determined that indomethacinadministration significantly decreased mucosal VEGF EGFand HGF levels compared with the vehicle-treated con-trol group but violacein treatment considerably enrichedgrowth factors levels After COX-1 inhibitor pretreatmentulcer index (UI) and MPO level were significantly increasedfollowed by reduction of tissue VEGF EGF HGF IL-4 IL-10 and PGE

2levels However the COX-2 inhibitor celecoxib

did not affect the therapeutic activity of violacein at any level(119875 lt 005)

Gastric mucosal cNOS activity was significantlydecreased in the indomethacin-induced ulcer group Incontrast the iNOS activity in the gastric mucosa of ratssubjected to indomethacin induction was significantlyincreased [39] Treatment with violacein significantlyincreased cNOS and decreased iNOS but this activitywas inhibited by L-NAME a nonspecific inhibitor whichconfirms the involvement of NOS in violacein-mediatedgastroprotection on indomethacin-induced ulcers (Figure 8)

Nonprotein endogenous sulfhydryl (NP-SH) compoundsare important for the maintenance of gastric mucosalintegrity [40] These SH groups have the ability to bind tothe free radicals generated by noxious agents thus controllingthe production and nature of mucus [40] However ourresults showed significant attenuations in the gastric ulcerarea after the blockage of NP-SH compounds by NEM ingroups treated with violacein (40mgkg) in comparisonwith the indomethacin-induced ulcer group suggesting thatthe gastroprotective effects of violacein are not involved inmaintenance of NP-SH compounds (Figure 9) The openingof K+ATP channels a class of ligand-gated proteins appearsto be involved with a variety of physiologic functions of


GroupsSham Vehicle + Indo Vio (40mg) + Indo

lowastlowastlowast

lowast

lowast

lowast

TNF-120572IL-1120573IL-6

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

(pgm

gm

tiss

ue)

TNF-120572

IL-1120573

and

IL-6

leve

l

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast

lowast lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

IL-4IL-10

IL-4

and

IL-10

leve

l (pg

mg

m ti

ssue

)

000500010000150002000025000300003500040000450005000055000600006500070000

(b)

Figure 6 (a) Effect of violacein (40mgkg orally) on proinflammatory cytokines (TNF-120572 IL-1120573 and IL-6) level (b) anti-inflammatorycytokines (IL-4 and IL-10) level in indomethacin-induced ulcer Values are mean plusmn SD (119899 = 6) lowast119875 lt 005 compare vehicle + Indo with allthe groups

119875 lt 005 compare Vio (40mg) + Indo with SC560 (or) Celecoxib + Vio (40mg) + Indo Indo indomethacin Vio violacein

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowastlowast

lowastlowast lowast

lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

VEGFEGFHGF

(ng

gm ti

ssue

)

000

500

1000

1500

2000

2500

3000

3500

VEG

F EG

F an

d H

GF

leve

l

Figure 7 Effect of COX-1 and COX-2 inhibitors and violacein (40mgkg orally) on VEGF EGF and HGF levels in indomethacin-induced ulcerated rats Values are mean plusmn SD (n = 6) lowast119875 lt 005compare vehicle + Indo with all the groups

119875 lt 005 compare Vio(40mg) + Indowith SC560 orCelecoxib +Vio (40mg) + Indo Indoindomethacin Vio violacein

the stomach such as gastric blood flow regulation acidsecretion and stomach contractility [41] In fact Peskaret al demonstrated that endogenous prostaglandins actas activators of K+ATP channels and this mechanism atleast in part mediates gastroprotection [42] A previousstudy by Peskar et al suggested the participation of K+ATPchannels in an indomethacin-induced ulcer model in which

cNOSiNOStNOS

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast lowast

(40

mg)+

Indo

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

000

050

100

150

200

250

300

(nm

olm

inm

g tis

sue)

cNO

S iN

OS

and

tNO

S le

vel

L-N

AM

E+

Vio

Figure 8 Effect of L-NAME (NOS inhibitor) and violacein(40mgkg orally) on cNOS tNOS and iNOS levels inindomethacin-induced ulcerated rats Values are mean plusmn SD(n = 6) lowast119875 lt 005 compare vehicle + Indo with all the groups119875 lt 005 compare Vio (40mg) + Indo with L-NAME + Vio

(40mg) + Indo Indo indomethacin Vio violacein L-NAMEN-G- nitro- L-arginine methyl ester

prostaglandins were shown as probable activators of thesechannels [42] In this way our results showed that the gas-troprotection mechanism of violacein was K+ATP-channeldependent since its gastroprotective effects were reverted bypretreatment with glibenclamide a potent antagonist of these


Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast 8621 lowast 8564lowast 8639 lowast 8636lowast

minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio

Figure 9 Effect of L-NAME (NOS inhibitor) NEM (NP-SH antagonist) yohimbine (1205722-receptors antagonist) glibenclamide (K+ATP-

channels antagonist) SC560 (COX-1 inhibitor) and Celecoxib (COX-2 inhibitor) on gastroprotective activity of violacein againstindomethacin-induced ulcer Values are mean plusmn SD (n = 6) lowast119875 lt 005 compare vehicle + Indo with all the groups

119875 lt 005 compare Vio(40mg) + Indowith L-NAMENEMYOGLIB SC560 or Celecoxib +Vio (40mg) + Indo Values in the braces indicate ulcer index inhibitionpercentage Indo indomethacin Vio violacein L-NAME N-G-nitro-L-arginine methyl ester NEM N-ethylmaleimide YO yohimbineGLIB glibenclamide

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l

Apoptosis (unitmg protein)

000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226

Caspase-3 (pmolmg protein)

Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)

Figure 10 (a) Effect of violacein (40mgkg orally) on apoptosis level (b) caspase-3 level in indomethacin-induced ulcerated rats Valuesare mean plusmn SD (n = 6) lowast119875 lt 005 compare Vehicle + Indo with all the groups Values in the braces indicate apoptosis and caspase-3 levelinhibition percentage Indo indomethacin Vio violacein

channels (Figure 9) From these data we suggest participationofK+ATPchannels in the gastroprotective effects of violaceinin which prostaglandins could be involved in the activationof these channels Presynaptic 120572

2-receptors mediate several

responses in the gastrointestinal tract and are involved in theregulation of gastric acid secretion [43] Pretreatment withthe 120572

2-receptor antagonist yohimbine failed to effectively

block the gastroprotective effect of violacein (40mgkg)against indomethacin-induced ulcers (Figure 9) suggestingthat the gastroprotective effect of violacein is not mediated by1205722-receptors L-NAME significantly inhibited the gastropro-

tection produced by violacein (Figure 9) suggesting that NOparticipates in gastroprotection It is well known that NO is

involved in themodulation of gastricmucosal integrity and inthe regulation of acid and alkaline secretionmucus secretionand gastric mucosal blood flow [44]

Enhancement of caspase-3 activation and considerableepithelial cell apoptosis are important pathological eventsduring NSAIDs-induced cytotoxicity [33] The amplificationand propagation of the cell death signaling cascade inducedby TNF-120572 involve the activation of a family of specificcysteine proteases known as caspases which are under theregulatory control of nitric oxide [45] Indomethacin displaysits effects on apoptogenic signal propagation through theinduction of TNF-120572 [46] our present experiments showedthat violacein significantly inhibited TNF-120572 The apoptotic


Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast 7412 lowast 7368 lowast 7236lowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo

Gastric microvascular permeability

Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300

Figure 11 Effect of COX-1 and COX-2 inhibitors and violacein(40mgkg orally) on gastric microvascular permeability inducedby indomethacin in rats Values are mean plusmn SD (n = 6) lowast119875 lt 005compare vehicle + Indo with all the groups


index in the indomethacin-induced group was 1048-foldhigher than that in the normal control group Pretreat-ment with violacein caused a 6527 reduction in DNAfragmentation (Figure 10(a)) The caspase-3 activity in theindomethacin-induced group was 355-fold higher than inthe sham treated group Pretreatment with violacein caused a5226 reduction in caspase-3 activity (Figure 10(b)) Basedon these results it is possible that violacein may able toinhibit these apoptogenic processes by the inhibition of TNF-120572The effects of violacein sucralfate SC560 and celecoxib onthe indomethacin-induced microvascular permeability of ratstomach are depicted in Figure 11 Indomethacin increasedgastric microvascular permeability by 42-fold Both viola-cein and sucralfate ameliorated indomethacin-induced gas-tric microvascular permeability (7368 and 7412 resp)Microvascular permeability was significantly increased afterCOX-1 inhibitor pretreatment along with violacein howeverthe COX-2 inhibitor celecoxib did not affect the therapeuticactivity of violacein (40mgkg)The above result indicates theinvolvement of COX-1 in the process of inhibition of vascularpermeability These results are consistent with a previousreport [27]

The results of our present study demonstrate that vio-lacein presented significant gastroprotective effects in anindomethacin-induced gastric ulcermodel and that appear tobe mediated at least in part by endogenous prostaglandinsnitric oxide K+ATP channel opening upregulation of thelevels of mucosal growth factors maintenance of the bal-ance of pro- and anti-inflammatory cytokines antiapoptoticfunction and attenuation of enhanced gastric microvascularpermeability These findings indicate that violacein may be auseful natural gastroprotective tool However further studiesare required to evaluate the exact mechanism involved in

its action as well as to better investigate the safety profile ofviolacein use

Conflict of Interests

The authors declare no competing financial interests

Acknowledgment

The authors thank Addiriyah Chair for Environmental Stud-ies Department of Botany and Microbiology College ofScience King Saud University PO Box 2455 Riyadh SaudiArabia for the support

References

[1] M R Griffin and J M Scheiman ldquoProspects for changing theburden of nonsteroidal anti-inflammatory drug toxicityrdquo TheAmerican Journal of Medicine vol 110 no 1 pp 33Sndash37S 2001

[2] U S Akarca ldquoGastrointestinal effects of selective and non-selective non-steroidal anti-inflammatory drugsrdquoCurrent Phar-maceutical Design vol 11 no 14 pp 1779ndash1793 2005

[3] J L Wallace C M Keenan and D N Granger ldquoGastriculceration induced by nonsteroidal anti-inflammatory drugsis a neutrophil-dependent processrdquo The American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 259 no3 pp G462ndashG467 1990

[4] B L Slomiany J Piotrowski and A Slomiany ldquoRole ofendothelin-1 and constitutive nitric oxide synthase in gastricmucosal resistance to indomethacin injury effect of antiulceragentsrdquo Scandinavian Journal of Gastroenterology vol 34 no 5pp 459ndash464 1999

[5] H Kusuhara H Komatsu H Sumichika and K SugaharaldquoReactive oxygen species are involved in the apoptosis inducedby nonsteroidal anti-inflammatory drugs in cultured gastriccellsrdquoEuropean Journal of Pharmacology vol 383 no 3 pp 331ndash337 1999

[6] H Utsumi K Yasukawa T Soeda et al ldquoNoninvasive mappingof reactive oxygen species by in vivo electron spin resonancespectroscopy in indomethacin-induced gastric ulcers in ratsrdquoThe Journal of Pharmacology and Experimental Therapeuticsvol 317 no 1 pp 228ndash235 2006

[7] Y Naito and T Yoshikawa ldquoOxidative stress involvement andgene expression in indomethacin-induced gastropathyrdquo RedoxReport vol 11 no 6 pp 243ndash253 2006

[8] D J Newman and G M Cragg ldquoMicrobial antitumor drugsnatural products of microbial origin as anticancer agentsrdquoCurrent Opinion in Investigational Drugs vol 10 no 12 pp1280ndash1296 2009

[9] N Duran and C F M Menck ldquoChromobacterium violaceum areview of pharmacological and industiral perspectivesrdquo CriticalReviews in Microbiology vol 27 no 3 pp 201ndash222 2001

[10] P S Melo G Z Justo M B M de Azevedo N Duran andM Haun ldquoViolacein and its 120573-cyclodextrin complexes induceapoptosis and differentiation inHL60 cellsrdquoToxicology vol 186no 3 pp 217ndash225 2003

[11] V S Saraiva J Marshall J Cools-Lartigue and M N BurnierJr ldquoCytotoxic effects of violacein in human uvealmelanoma celllinesrdquoMelanoma Research vol 14 no 5 pp 421ndash424 2004


[12] C V Ferreira C L Bos H H Versteeg G Z Justo N Duranand M P Peppelenbosch ldquoMolecular mechanism of violacein-mediated human leukemia cell deathrdquo Blood vol 104 no 5 pp1459ndash1464 2004

[13] P Antonisamy and S Ignacimuthu ldquoImmunomodulatory anal-gesic and antipyretic effects of violacein isolated from Chro-mobacterium violaceumrdquo Phytomedicine vol 17 no 3-4 pp300ndash304 2010

[14] P Antonisamy P Kannan and S Ignacimuthu ldquoAnti-diarrhoealand ulcer-protective effects of violacein isolated from Chro-mobacterium violaceum in Wistar ratsrdquo Fundamental and Clin-ical Pharmacology vol 23 no 4 pp 483ndash490 2009

[15] J B Dekanski A Macdonald P Sacra and D V Parke ldquoEffectsof fasting stress and drugs on gastric glycoprotein synthesis inthe ratrdquo British Journal of Pharmacology vol 55 no 3 pp 387ndash392 1975

[16] SDemirbilek I GursesN SezginAKaraman andNGurbuzldquoProtective effect of polyunsaturated phosphatidylcholind pre-treatment on stress ulcer formation in ratsrdquo Journal of PediatricSurgery vol 39 no 1 pp 57ndash62 2004

[17] P P Bradley D A Priebat R D Christensen and G RothsteinldquoMeasurement of cutaneous inflammation estimation of neu-trophil content with an enzyme markerrdquo Journal of InvestigativeDermatology vol 78 no 3 pp 206ndash209 1982

[18] M Feelisch and E A Noack ldquoCorrelation between nitric oxideformation during degradation of organic nitrates and activationof guanylate cyclaserdquo European Journal of Pharmacology vol139 no 1 pp 19ndash30 1987

[19] R G Knowles M Merrett M Salter and S Moncada ldquoDiffer-ential induction of brain lung and liver nitric oxide synthase byendotoxin in the ratrdquo Biochemical Journal vol 270 no 3 pp833ndash836 1990

[20] B L Slomiany and A Slomiany ldquoPlatelet-activating factormediates Helicobacter pylori lipopolysaccharide interferencewith gastric mucin synthesisrdquo IUBMB Life vol 56 no 1 pp 41ndash46 2004

[21] B L Siomiany and A Siomiany ldquoNitric oxide as a modulator ofgastric mucin synthesis role of ERK and p38mitogen-activatedprotein kinase activationrdquo IUBMB Life vol 54 no 5 pp 267ndash273 2002

[22] C L Chander A R Moore F M Desa D Howat and DA Willoughby ldquoThe local modulation of vascular permeabilityby endothelial cell derived productsrdquo Journal of Pharmacy andPharmacology vol 40 no 10 pp 745ndash746 1988

[23] R Simone Australian Medicines Handbook AustralianMedicines Handbook Adelaide Australia 2006

[24] M K Jones H Wang B M Peskar et al ldquoInhibition ofangiogenesis by nonsteroidal anti-inflammatory drugs insightinto mechanisms and implications for cancer growth and ulcerhealingrdquo Nature Medicine vol 5 no 12 pp 1418ndash1423 1999

[25] F Halter A S Tarnawski A Schmassmann and B M PeskarldquoCyclooxygenase 2 implications on maintenance of gastricmucosal integrity and ulcer healing controversial issues andperspectivesrdquo Gut vol 49 no 3 pp 443ndash453 2001

[26] B Gretzer N Maricic M Respondek R Schuligoi and B MPeskar ldquoEffects of specific inhibition of cyclo-oxygenase-1 andcyclo-oxygenase-2 in the rat stomach with normal mucosa andafter acid challengerdquo British Journal of Pharmacology vol 132no 7 pp 1565ndash1573 2001

[27] A Tanaka S Hase T Miyazawa R Ohno and K TakeuchildquoRole of cyclooxygenase (COX)-1 and COX-2 inhibition in

nonsteroidal anti-inflammatory drug-induced intestinal dam-age in rats relation to various pathogenic eventsrdquo The Journalof Pharmacology and Experimental Therapeutics vol 303 no 3pp 1248ndash1254 2002

[28] CM Hogaboam E Y Bissonnette B C Chin A D Befus andJ L Wallace ldquoProstaglandins inhibit inflammatory mediatorrelease from rat mast cellsrdquo Gastroenterology vol 104 no 1 pp122ndash129 1993

[29] S L KunkelM SpenglerM AMay R Spengler J Larrick andD Remick ldquoProstaglandin E

2regulates macrophage-derived

tumor necrosis factor gene expressionrdquo Journal of BiologicalChemistry vol 263 no 11 pp 5380ndash5384 1988

[30] H Sheng J Shao J D Morrow R D Beauchamp and RN DuBois ldquoModulation of apoptosis and Bcl-2 expression byprostaglandin E2 in human colon cancer cellsrdquoCancer Researchvol 58 no 2 pp 362ndash366 1998

[31] A S Gomes L M F Lima C L Santos F Q Cunha RA Ribeiro and M H L P Souza ldquoLPS from Escherichia coliprotects against indomethacin-induced gastropathy in ratsmdashrole of ATP-sensitive potassium channelsrdquo European Journal ofPharmacology vol 547 no 1ndash3 pp 136ndash142 2006

[32] T Brzozowski P C Konturek S J Konturek et al ldquoClassicNSAID and selective cyclooxygenase (COX)-1 and COX-2inhibitors in healing of chronic gastric ulcersrdquo MicroscopyResearch and Technique vol 53 no 5 pp 343ndash353 2001

[33] B L Slomiany J Piotrowski and A Slomiany ldquoRole of caspase-3 and nitric oxide synthase-2 in gastric mucosal injury inducedby indomethacin effect of sucralfaterdquo Journal of Physiology andPharmacology vol 50 no 1 pp 3ndash16 1999

[34] C B Appleyard D McCafferty A W Tigley M G Swainand J L Wallace ldquoTumor necrosis factor mediation of NSAID-induced gastric damage Role of leukocyte adherencerdquo TheAmerican Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 270 no 1 pp G42ndashG48 1996

[35] P Stordeur and M Goldman ldquoInterleukin-10 as a regulatorycytokine induced by cellular stress molecular aspectsrdquo Interna-tional Reviews of Immunology vol 16 no 5-6 pp 501ndash522 1998

[36] M A Cassatella L Meda S Gasperini F Calzetti and SBonora ldquoInterleukin 10 (IL-10) upregulates IL-1 receptor antag-onist production from lipopolysaccharide-stimulated humanpolymorphonuclear leukocytes by delaying mRNA degrada-tionrdquo Journal of ExperimentalMedicine vol 179 no 5 pp 1695ndash1699 1994

[37] A Tarnawski I L Szabo S S Husain and B Soreghan ldquoRegen-eration of gastric mucosa during ulcer healing is triggered bygrowth factors and signal transduction pathwaysrdquo Journal ofPhysiologymdashParis vol 95 no 1ndash6 pp 337ndash344 2001

[38] T Brzozowski P C Konturek S J Konturek et al ldquoInvolvementof cyclooxygenase (COX)-2 products in acceleration of ulcerhealing by gastrin and hepatocyte growth factorrdquo Journal ofPhysiology and Pharmacology vol 51 no 4 pp 751ndash773 2000

[39] H Matsuda Y Li and M Yoshikawa ldquoRoles of capsaicin-sensitive sensory nerves endogenous nitric oxide sulfhydrylsand prostaglandins in gastroprotection by momordin Ican oleanolic acid oligoglycoside on ethanol-induced gastricmucosal lesions in ratsrdquo Life Sciences vol 65 no 2 pp 27ndash321999

[40] M A Andreo K V R Ballesteros C A Hiruma-Lima LR Machado da Rocha A R M Souza Brito and W VilegasldquoEffect of Mouriri pusa extracts on experimentally inducedgastric lesions in rodents role of endogenous sulfhydryls


compounds and nitric oxide in gastroprotectionrdquo Journal ofEthnopharmacology vol 107 no 3 pp 431ndash441 2006

[41] M L Garcia M Hanner H Knaus et al ldquoPharmacology ofpotassium channelsrdquo Advances in Pharmacology vol 39 pp425ndash471 1997

[42] BM Peskar K Ehrlich andBA Peskar ldquoRole ofATP-sensitivepotassium channels in prostaglandin-mediated gastroprotec-tion in the ratrdquo The Journal of Pharmacology and ExperimentalTherapeutics vol 301 no 3 pp 969ndash974 2002

[43] K Gyires K Mullner S Furst and A Z Ronai ldquoAlpha-2 adrenergic and opioid receptor-mediated gastroprotectionrdquoJournal of Physiology Paris vol 94 no 2 pp 117ndash121 2000

[44] P Kubes M Suzuki and D N Granger ldquoNitric oxide anendogenous modulator of leukocyte adhesionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 88 no 11 pp 4651ndash4655 1991

[45] A Parenti L Morbidelli X Cui et al ldquoNitric oxide is anupstream signal of vascular endothelial growth factor-inducedextracellular signal-regulated kinase l2 activation in post capil-lary endotheliumrdquoThe Journal of Biological Chemistry vol 273no 7 pp 4220ndash4226 1998

[46] D Wallach ldquoCell death induction by TNF a matter of selfcontrolrdquo Trends in Biochemical Sciences vol 22 no 4 pp 107ndash109 1997

Submit your manuscripts athttpwwwhindawicom

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


Several studies have shown that violacein is also capable ofinducing apoptosis in a variety of cancer cell lines includingleukemia lineages suggesting a promising clinical applicationin cancer treatment The therapeutic application of violaceinto cancer chemoprevention has been the focus of recentstudies [10 11] Violacein has been shown to induce apoptosisin HL60 leukemic cells but is ineffective in normal humanlymphocytes and monocytes [12] In our previous studies weshowed that violacein possesses immunomodulatory anal-gesic antipyretic antidiarrheal and antiulcerogenic activities[13 14]

In this study we investigated the gastroprotective effect ofviolacein in an indomethacin-induced ulcer model in Wisterrats to determine its mechanisms of action

2 Materials and Methods

21 Animals Adult Wistar albino rats (200ndash220 g) of bothsexes were used for experiments Animals were housed witha 12 h lightdark cycle at 25 plusmn 1∘C at a relative moisture of60ndash70 they had access to diet and water ad libitum andunderwent at least twoweeks of adaptation before starting theexperiments All studies were carried out using six animalsin each group All animal experiments were conductedaccording to the ethical norms approved by the Ministry ofSocial Justice and Empowerment Government of India andthe procedures of the Institutional Animal Ethics Committee

22 Chemicals and Drugs Indomethacin omeprazole cele-coxib SC560 N-G-nitro-L-argininemethyl ester (L-NAME)N-ethylmaleimide (NEM) yohimbine and glibenclamidewere obtained from Sigma-Aldrich (Sigma Chemicals CoSt Louis MO USA) Carboxymethyl cellulose (CMC) wasobtained from Himedia (Mumbai India) The PGE

2EIA

kit vascular endothelial growth factor (VEGF) hepatocytegrowth factor (HGF) epidermal growth factor (EGF) andenzyme-linked immunosorbent assay (ELISA) kits were pur-chased from Cayman Chemical (Ann Arbor MI USA)Tumor necrosis factor (TNF)-120572 interleukin (IL)-1120573 IL-4IL-6 and IL-10 ELISA kits were purchased from PierceBiotechnology (Rockford IL USA) The apoptosis assay kitwas acquired from Boehringer Mannheim and caspase-3activity assays were conducted using the Quanti Zyme assaysystem from Biomol Research Laboratories Inc (USA) Allother chemicals usedwere of analytical reagent gradeThe testcompound violacein was isolated from C violaceum ESBV4400 which was isolated from forest water body soil samplesfrom Kolli Hills of Tamil Nadu India at latitude 11∘ 101015840 to11∘301015840N and longitude 78∘ 151015840 to 78∘ 301015840 E (Figure 1) and

reported in our previous study [14]

23 Determination of Doses To determine the lowest effec-tive dose of violacein gastric ulcers were induced byindomethacin followed by treatment with violacein Afterfasting for 24 h rats were distributed into eight groups (119899 =6group) Animals orally received vehicle alone (05mL of05 CMC) for sham and indomethacin treated controlgroup omeprazole as the positive control (40mgkg) or

OH

HNH

N

HN

O

O

Figure 1 Structure of violacein

violacein (10 20 40 80 or 160mgkg) After 30min theanimals orally received 20mgkg of indomethacin exceptsham treated group Six hours later the animals were sacri-ficed under ether anesthesia and the stomach was surgicallyremoved immersed in 5 formalin for 30min and openedalong the greater curvature to macroscopically examinelesions according to the ulcer score described by previousmethod [15]

24 Gastric Damage Induced by Indomethacin Rats (119899 =6) were treated with sham (05mL of 05 CMC) vehicle+ Indo (05mL of 05 CMC) violacein (40mgkg po)omeprazole (40mgkg po) SC560 + violacein (5mgkgpo + 40mgkg po) celecoxib + violacein (35mgkg po+ 40mgkg po) L-NAME + violacein (50mgkg ip +40mgkg po) NEM + violacein (10mgkg sc + 40mgkgpo) yohimbine + violacein (2mgkg ip + 40mgkg po)or glibenclamide + violacein (5mgkg po + 40mgkg po)All drugs were administered using 05 CMC as the vehiclesolution After 30min each group of animals except the shamtreated group received a 20mgkg oral dose of indomethacinSelective COX-1 inhibitor (SC560) COX-2 inhibitor (cele-coxib) nonselective nitric oxide synthase (NOS) inhibitor(L-NAME) endogenous sulfhydryl antagonist (NEM) 120572

2-

receptors antagonist (yohimbine) and K+ATP channelsantagonist (glibenclamide) were administered to rats 30minbefore violacein treatment and 1 h prior to ulcer induction byindomethacin Six hours later animals were sacrificed underether anesthesia and the stomach was surgically removedimmersed in 5 formalin for 30min and opened alongthe greater curvature to macroscopically examine lesionsaccording to the ulcer score described by previous method[15]

0 = no damage1 = blood at the lumen2 = pin-point erosions3 = one to five small erosions lt2mm4 = more than five small erosions lt2mm5 = one to three large erosions gt2mm6 = more than three large erosions gt2mm





2 TNF-120572 IL-1120573 IL-4 IL-6 IL-









2levels were



(a) (b)

(c) (d)






2 On the other


2level


2acts


2by COX-1

mediated pathways




lcer

inde

x (U

I)

UI

Groups

ns

Sham

Vehi

cle+

Indo

Om

p (40

mg)+

Indo

Vio

(10

mg)+

Indo

Vio

(20

mg)+

Indo

Vio

(40

mg)+

Indo

Vio

(80

mg)+

Indo

Vio

(160

mg)+

Indo


lowast 3534

lowast 6989

000

1000

2000

3000

4000

5000

6000

7000

8000

9000


0

2

4

6

8

10

12

14 minus494

MPO

activ

ity le

vel


Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowastlowast lowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

PGE2

leve

l

PGE2 (pgmg protein)

000

1000

2000

3000

4000

5000

6000



119875 lt 005









lowastlowastlowast

lowast

lowast

lowast

TNF-120572IL-1120573IL-6

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

(pgm

gm

tiss

ue)

TNF-120572

IL-1120573

and

IL-6

leve

l

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast

lowast lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

IL-4IL-10

IL-4

and

IL-10

leve

l (pg

mg

m ti

ssue

)

000500010000150002000025000300003500040000450005000055000600006500070000

(b)



Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowastlowast

lowastlowast lowast

lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

VEGFEGFHGF

(ng

gm ti

ssue

)

000

500

1000

1500

2000

2500

3000

3500

VEG

F EG

F an

d H

GF

leve

l




cNOSiNOStNOS

lowast

lowast

lowast

lowast

lowast

lowast


(40

mg)+

Indo

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

000

050

100

150

200

250

300

(nm

olm

inm

g tis

sue)

cNO

S iN

OS

and

tNO

S le

vel

L-N

AM

E+

Vio





Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l


000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226


Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)











Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2 TNF-120572 IL-1120573 IL-4 IL-6 IL-









2levels were



(a) (b)

(c) (d)






2 On the other


2level


2acts


2by COX-1

mediated pathways




lcer

inde

x (U

I)

UI

Groups

ns

Sham

Vehi

cle+

Indo

Om

p (40

mg)+

Indo

Vio

(10

mg)+

Indo

Vio

(20

mg)+

Indo

Vio

(40

mg)+

Indo

Vio

(80

mg)+

Indo

Vio

(160

mg)+

Indo


lowast 3534

lowast 6989

000

1000

2000

3000

4000

5000

6000

7000

8000

9000


0

2

4

6

8

10

12

14 minus494

MPO

activ

ity le

vel


Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowastlowast lowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

PGE2

leve

l

PGE2 (pgmg protein)

000

1000

2000

3000

4000

5000

6000



119875 lt 005









lowastlowastlowast

lowast

lowast

lowast

TNF-120572IL-1120573IL-6

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

(pgm

gm

tiss

ue)

TNF-120572

IL-1120573

and

IL-6

leve

l

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast

lowast lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

IL-4IL-10

IL-4

and

IL-10

leve

l (pg

mg

m ti

ssue

)

000500010000150002000025000300003500040000450005000055000600006500070000

(b)



Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowastlowast

lowastlowast lowast

lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

VEGFEGFHGF

(ng

gm ti

ssue

)

000

500

1000

1500

2000

2500

3000

3500

VEG

F EG

F an

d H

GF

leve

l




cNOSiNOStNOS

lowast

lowast

lowast

lowast

lowast

lowast


(40

mg)+

Indo

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

000

050

100

150

200

250

300

(nm

olm

inm

g tis

sue)

cNO

S iN

OS

and

tNO

S le

vel

L-N

AM

E+

Vio





Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l


000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226


Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)











Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)






2 On the other


2level


2acts


2by COX-1

mediated pathways




lcer

inde

x (U

I)

UI

Groups

ns

Sham

Vehi

cle+

Indo

Om

p (40

mg)+

Indo

Vio

(10

mg)+

Indo

Vio

(20

mg)+

Indo

Vio

(40

mg)+

Indo

Vio

(80

mg)+

Indo

Vio

(160

mg)+

Indo


lowast 3534

lowast 6989

000

1000

2000

3000

4000

5000

6000

7000

8000

9000


0

2

4

6

8

10

12

14 minus494

MPO

activ

ity le

vel


Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowastlowast lowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

PGE2

leve

l

PGE2 (pgmg protein)

000

1000

2000

3000

4000

5000

6000



119875 lt 005









lowastlowastlowast

lowast

lowast

lowast

TNF-120572IL-1120573IL-6

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

(pgm

gm

tiss

ue)

TNF-120572

IL-1120573

and

IL-6

leve

l

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast

lowast lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

IL-4IL-10

IL-4

and

IL-10

leve

l (pg

mg

m ti

ssue

)

000500010000150002000025000300003500040000450005000055000600006500070000

(b)



Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowastlowast

lowastlowast lowast

lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

VEGFEGFHGF

(ng

gm ti

ssue

)

000

500

1000

1500

2000

2500

3000

3500

VEG

F EG

F an

d H

GF

leve

l




cNOSiNOStNOS

lowast

lowast

lowast

lowast

lowast

lowast


(40

mg)+

Indo

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

000

050

100

150

200

250

300

(nm

olm

inm

g tis

sue)

cNO

S iN

OS

and

tNO

S le

vel

L-N

AM

E+

Vio





Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l


000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226


Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)











Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lcer

inde

x (U

I)

UI

Groups

ns

Sham

Vehi

cle+

Indo

Om

p (40

mg)+

Indo

Vio

(10

mg)+

Indo

Vio

(20

mg)+

Indo

Vio

(40

mg)+

Indo

Vio

(80

mg)+

Indo

Vio

(160

mg)+

Indo


lowast 3534

lowast 6989

000

1000

2000

3000

4000

5000

6000

7000

8000

9000


0

2

4

6

8

10

12

14 minus494

MPO

activ

ity le

vel


Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowastlowast lowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

PGE2

leve

l

PGE2 (pgmg protein)

000

1000

2000

3000

4000

5000

6000



119875 lt 005









lowastlowastlowast

lowast

lowast

lowast

TNF-120572IL-1120573IL-6

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

(pgm

gm

tiss

ue)

TNF-120572

IL-1120573

and

IL-6

leve

l

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast

lowast lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

IL-4IL-10

IL-4

and

IL-10

leve

l (pg

mg

m ti

ssue

)

000500010000150002000025000300003500040000450005000055000600006500070000

(b)



Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowastlowast

lowastlowast lowast

lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

VEGFEGFHGF

(ng

gm ti

ssue

)

000

500

1000

1500

2000

2500

3000

3500

VEG

F EG

F an

d H

GF

leve

l




cNOSiNOStNOS

lowast

lowast

lowast

lowast

lowast

lowast


(40

mg)+

Indo

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

000

050

100

150

200

250

300

(nm

olm

inm

g tis

sue)

cNO

S iN

OS

and

tNO

S le

vel

L-N

AM

E+

Vio





Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l


000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226


Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)











Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastlowastlowast

lowast

lowast

lowast

TNF-120572IL-1120573IL-6

000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

(pgm

gm

tiss

ue)

TNF-120572

IL-1120573

and

IL-6

leve

l

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast

lowast lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

IL-4IL-10

IL-4

and

IL-10

leve

l (pg

mg

m ti

ssue

)

000500010000150002000025000300003500040000450005000055000600006500070000

(b)



Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast lowastlowast

lowastlowast lowast

lowast

lowastlowast

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

VEGFEGFHGF

(ng

gm ti

ssue

)

000

500

1000

1500

2000

2500

3000

3500

VEG

F EG

F an

d H

GF

leve

l




cNOSiNOStNOS

lowast

lowast

lowast

lowast

lowast

lowast


(40

mg)+

Indo

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

000

050

100

150

200

250

300

(nm

olm

inm

g tis

sue)

cNO

S iN

OS

and

tNO

S le

vel

L-N

AM

E+

Vio





Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l


000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226


Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)











Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ulc

er in

dex

(UI)

UI

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


minus432

Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

(40

mg)+

Indo

323 238

NEM

+Vi

o

YO+

Vio

GLI

B+

Vio

000100020003000400050006000700080009000

L-N

AM

E+

Vio




Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 6527

Apop

tosis

leve

l


000

500

1000

1500

2000

2500

3000

(a)

Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo

lowast

lowast 5226


Casp

ase-3

leve

l

000

500

1000

1500

2000

2500

(b)











Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Groups

Sham

Vehi

cle+

Indo

Vio

(40

mg)+

Indo


Cele

coxi

b+

Vio

(40

mg)+

Indo

(40

mg)+

Indo

SC560+

Vio

307

Sucr

alfa

te(400

mg

kg)+

Indo


Vasc

ular

per

mea

bilit

y (120583

gm

g pr

otei

n)

000

050

100

150

200

250

300








Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article gastroprotective activity of violacein

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