Tryptamine-gallic acid hybrid prevents non-steroidal anti-inflammatory drug-induced gastropathy: Correction of mitochondrial dysfunction and inhibition of apoptosis in gastric mucosal cells

Article abstract of DOI:10.1074/jbc.M111.307199

We have investigated the gastroprotective effect of SEGA(3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O₂^.-) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin- induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled (⁹⁹mTc- labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.

Full text of DOI:10.1074/jbc.M111.307199

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 5, pp. 3495–3509, January 27, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Tryptamine-Gallic Acid Hybrid Prevents Non-steroidal
Anti-inflammatory Drug-induced Gastropathy
CORRECTION OF MITOCHONDRIAL DYSFUNCTION AND INHIBITION OF APOPTOSIS IN
□
S
GASTRIC MUCOSAL CELLS*
Received for publication, September 23, 2011, and in revised form, November 15, 2011 Published, JBC Papers in Press, December 7, 2011, DOI 10.1074/jbc.M111.307199
Chinmay Pal‡, Samik Bindu‡, Sumanta Dey‡, Athar Alam‡, Manish Goyal‡, Mohd. Shameel Iqbal‡, Souvik Sarkar‡,
Rahul Kumar‡, Kamal Krishna Halder§, Mita Chatterjee Debnath§, Susanta Adhikari¶, and Uday Bandyopadhyay‡1
From the ‡Division of Infectious Diseases and Immunology and the §Nuclear Medicine Division, Indian Institute of Chemical
Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India and the ¶Department of Chemistry, University of
Calcutta, 92 A.P.C. Road, Kolkata-700009, West Bengal, India
Background: Non-steroidal anti-inflammatory drugs (NSAIDs) induce gastropathy by promoting mitochondrial pathol-
ogy, oxidative stress, and apoptosis in gastric mucosal cells.
Results: We have synthesized SEGA (3a), a tryptamine-gallic acid hybrid, which prevents NSAID-induced gastropathy by
preventing mitochondrial oxidative stress, dysfunction, and apoptosis.
Conclusion: SEGA (3a) bears an immense therapeutic potential against NSAID-induced gastropathy.
Significance: This novel molecule is a significant addition in the discovery of gastroprotective drugs.
NSAIDs2 are the most popular drugs commonly used
throughout the world for the treatment of pain, inflammation,
rheumatic disorders, and osteoarthritis (1, 2). Approximately
30 million patients consume NSAIDs on a daily basis (1). How-
ever, NSAIDs have limitations; they induces gastropathy, and
ϳ107,000 patients are hospitalized every year due to NSAID-
related gastrointestinal complications (3). Extensive studies
have established that besides acid secretion, there are other
important factors, such as gastric mucosal blood flow, mucus-
bicarbonate secretion, antioxidant level, reactive oxygen spe-
cies (ROS), mitochondrial oxidative stress (MOS), apoptosis,
and mucosal cell renewal, involved in the pathogenesis of gas-
troduodenal injury (4–11). It is well established that the major
cause of NSAID-induced gastropathy is the development of
oxidative stress in gastric mucosa due to the excess generation
of ROS. It has also been documented that ROS induces gas-
tropathy through the induction of gastric mucosal cell apopto-
sis (4–6, 12). NSAID acts as an inhibitory uncoupler in human
mitochondria (13). Indomethacin (NSAID) interacts with the
complex I of electron transport chain and results in the leakage
of electrons, which in turn leads to the formation of superoxide
We have investigated the gastroprotective effect of SEGA (3a),
a newly synthesized tryptamine-gallic acid hybrid molecule
against non-steroidal anti-inflammatory drug (NSAID)-in-
duced gastropathy with mechanistic details. SEGA (3a) prevents
indomethacin (NSAID)-induced mitochondrial oxidative stress
(MOS) and dysfunctions in gastric mucosal cells, which play a
pathogenic role in inducing gastropathy. SEGA (3a) offers this
mitoprotective effect by scavenging of mitochondrial super-
.
oxide anion (O2) and intramitochondrial free iron released as
a result of MOS. SEGA (3a) in vivo blocks indomethacin-me-
diated MOS, as is evident from the inhibition of indometha-
cin-induced mitochondrial protein carbonyl formation, lipid
peroxidation, and thiol depletion. SEGA (3a) corrects indo-
methacin-mediated mitochondrial dysfunction in vivo by
restoring defective electron transport chain function, collapse
of transmembrane potential, and loss of dehydrogenase activity.
SEGA (3a) not only corrects mitochondrial dysfunction but also
inhibits the activation of the mitochondrial pathway of apopto-
sis by indomethacin. SEGA (3a) inhibits indomethacin-induced
down-regulation of bcl-2 and up-regulation of bax genes in gas-
tric mucosa. SEGA (3a) also inhibits indometacin-induced acti-
vation of caspase-9 and caspase-3 in gastric mucosa. Besides the
gastroprotective effect against NSAID, SEGA (3a) also expedites
the healing of already damaged gastric mucosa. Radiolabeled
.
.
anion radical (O2) (14). The dismutation of mitochondrial O2
by superoxide dismutase leads to the formation of hydrogen
.
peroxide (H2O2) (15, 16), and H2O2 further reacting with O2
(
99mTc-labeled SEGA (3a)) tracer studies confirm that SEGA
ꢀ
generates highly reactive hydroxyl radical ( OH) through the
.
(3a) enters into mitochondria of gastric mucosal cell in vivo, and
it is quite stable in serum. Thus, SEGA (3a) bears an immense
potential to be a novel gastroprotective agent against NSAID-
induced gastropathy.
Haber-Weiss reaction (6). The excess O2, if not dismutated,
2 The abbreviations used are: NSAID, non-steroidal anti-inflammatory drugs;
ROS, reactive oxygen species; MOS, mitochondrial oxidative stress; RCR,
respiratorycontrolratio;JC-1, 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylben-
zimidazolcarbocyanine iodide; EDC, N-(3-dimethylaminopropyl)-NЈ-ethyl-
carbodiimide hydrochloride; DPPH, 2,2-diphenyl-1-picrylhydrazyl; TPTZ,
2,4,6-tris(2-pyridyl)-s-triazine; FRAP, ferric reducing antioxidant power;
b.w., body weight; HBSS, Hanks’ balanced salt solution; MTT, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PI, propidium
iodide; DTPA, diethylene triamine pentaacetic acid; 5HT, 5Ј-hy-
droxytryptamine; GA, gallic acid.
* This work was supported by the Council of Scientific and Industrial
Research, New Delhi, and the University Grants Commission, New Delhi.
□S
This article contains supplemental Schemes S1–S4 and detailed method-
ology for chemical synthesis, analytical data, and NMR spectra.
1 To whom correspondence should be addressed. Fax: 91-33-24730284;
E-mail: ubandyo_1964@yahoo.com.
JANUARY 27, 2012•VOLUME 287•NUMBER 5
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
offers toxic insult by oxidatively damaging and inactivating purchased from Cell Signaling Technology. All other reagents
mitochondrial aconitase, resulting in the release of iron from its were of analytical grade purity.
General EDC Coupling Procedure for Formation of Esters or
Amide—To a solution of 3,4,5-tris(benzyloxy)benzoic acid/4-
hydroxycinnamic acid/indole-3-acetic acid (1 eq, 12 mmol),
amines (hydrochloride)/alcohol (1.2 eq, 14.4 mmol), 1-hy-
droxybenzotriazole (1 eq, 12 mmol, for amines), and Et3N (6 eq,
72 mmol, for amines)/DMAP (1 eq, 12 mmol, for alcohols) in
N,N-dimethylform amide, EDC hydrochloride (1.2 eq, 14.4
mmol) was added at 0 °C. Then the reaction mixture was stirred
at room temperature overnight until completion of the reac-
tion, monitored by thin layer chromatography (TLC). After that
reaction, the mixture was quenched by the addition of ice-cold
H2O and extracted with ethyl acetate. The combined organic
phase was washed with brine and dried over Na2SO4. The
organic phase was then reduced in vacuo; the concentrated
ethyl acetate extracts were chromatographed over a silica gel
column.
Fe-S cluster (6, 17). Again, the released iron in presence of H2O2
ꢀ
generates OH through the Fenton reaction (17). Heme oxyge-
nase 1 may also generate free iron by catabolizing excess free
heme. Heme oxygenase 1 translocates to mitochondria and
decreases intramitochondrial free heme accumulated during
gastric injury by NSAID. Excess free heme and overactivity of
heme oxygenase 1 inside mitochondria may favor the accumu-
lation of free iron in excess to ferritin sequestration (18). Free
iron overload in cells has been shown to be associated with the
development and progression of several pathological condi-
tions (19–21). Intramitochondrial free iron and ROS lead to
MOS and consequent dysfunction (19–25). MOS disrupts cel-
lular integrity and promotes cell death (5, 26, 27), which ulti-
mately leads to organ damage.
The overproduction of ROS develops mitochondrial pathol-
ogy (22, 24, 28, 29), as indicated by the defect in electron trans-
port chain and ATP synthesis, opening of mitochondrial per-
meability transition pore (MPTP), fall in transmembrane
potential (⌬⌿m), oxidative damage of mitochondrial DNA,
proteins, and phospholipids (30), and finally the activation of
the mitochondrial pathway of apoptosis (6, 31). Thus, mito-
chondrial dysfunction triggers the mitochondrial pathway of
apoptosis (6, 32–38). Mitochondrial dysfunction and concur-
rent apoptosis play an important role in NSAID-induced gas-
tropathy (4, 6–7, 12). Hence, it is clear that the molecule that
will prevent MOS and consequent mitochondrial dysfunction
will be effective against NSAID-induced gastropathy.
General Procedure of Debenzylation (Hydrogenolysis)—A
mixture of compound and 10% palladium on carbon (catalytic)
in a methanol/chloroform mixture (5:1) (10 ml) was hydroge-
nated at 40 p.s.i. for 2 h and was filtered through Celite, after
removal of catalyst. The filtrate was evaporated in vacuo to
dryness, to give the product. The details of the materials and
methods for synthesis are described in the supplemental
material.
Determination of in Vitro Antioxidant Property by Following
Ferric Reducing Antioxidant Power (FRAP)—The assay was
performed in a 96-well microplate as described earlier (4).
FRAP reagent was prepared by mixing of 10 ml of acetate buffer
(200 mM, pH 3.6), 1 ml of TPTZ solution (10 mM in 40 mM HCl),
and 1 ml of ferric chloride solution (20 mM) in distilled water.
The solution was kept for 1 h in a water bath at 37 °C. In a
96-well microplate, 25 ␮l of the compounds under investiga-
tion dissolved (in methanol or water) at different concentra-
tions in the range 1–100 ␮M were placed in triplicate, and
freshly prepared FRAP solution (175 ␮l) was added to this sam-
ple. Absorbance was monitored at 595 nm at different time
intervals up to 150 min in a microplate reader. Absorbance of
175 ␮l of FRAP solution and 25 ␮l of methanol or water mixture
was taken, which was subtracted from the absorbance of the
samples at each time interval to calculate the absorbance
change (⌬A). The FRAP value at time interval t (FRAP valuet)
was calculated according to the formula,
The aim of the present study is to design a small molecule
that will correct NSAID-induced mitochondrial pathology,
apoptosis, and gastropathy. Here, we report the designing of a
tryptamine-gallic acid hybrid molecule, SEGA (3a), which pre-
vented NSAID-induced mitochondrial pathology, apoptosis,
and gastropathy by blocking MOS, chelating intramitochon-
drial free iron, and correcting mitochondrial pathology enter-
ing into mitochondria.
EXPERIMENTAL PROCEDURES
Indomethacin, thiobarbituric acid, 5,5Ј-dithiobis(nitroben-
zoic acid), 2,2-diphenyl-1-picrylhydrazyl (DPPH), DMSO,
albumin, collagenase type I, hyaluronidase, paraformaldehyde,
the caspase-3 assay kit, and 4-hydroxycinnamic acid were
obtained from Sigma. Serotonin was purchased from Alfa
Aesar. 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) was obtained
from Acros Organics (Geel, Belgium). Gallic acid and indole-3
acetic acid were procured from SRL (New Delhi, India). Fetal
bovine serum was obtained from Invitrogen. 5,5Ј,6,6Ј-Tetra-
chloro-1,1Ј,3,3Ј-tetraethylbenzimidazolcarbocyanine iodide
(JC-1) was procured from Molecular Probes (Eugene, OR), and
the caspase-9 assay kit was bought from Biovision (Mountain
View, CA). MitoSOX, Mitotracker Red, and Phen Green SK
were purchased from Invitrogen. The mitochondria isolation
kit was purchased from the Biochain Institute (Hayward, CA).
The Dead-End colorimetric TUNEL assay kit was purchased
FRAP valuet͑M͒ ϭ ͑⌬atT/⌬atFe2ϩ͒ ϫ 10Ϫ5
(Eq. 1)
where ⌬atT is the absorbance change after the time interval t (6
min) relative to the tested tryptamine derivatives at a concen-
tration of 10 ␮M, and ⌬atFe2ϩ is the absorbance change at the
same time interval relative to ferrous sulfate at the same con-
centration (4).
Free Radical-scavenging Activity by Following DPPH Radical
Assay—DPPH is a stable free radical and shows absorbance at
517 nm. Antioxidant molecules scavenge the DPPH radical by
donating hydrogen, as visualized by discoloration of the DPPH
from Promega, and the APO-BrdUTM TUNEL assay kit was radical from purple to yellow (4, 39). The assay system con-
purchased from Invitrogen. Anti-active caspase-3 antibody was tained 1 ml of compounds under investigation dissolved (in
3496 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 5•JANUARY 27, 2012

Tryptamine-GA Hybrid against NSAID-induced Gastropathy
methanol or water) at different concentrations in the range acin, served as the autohealing group. Starting from 0 h, the
10–100 ␮M and 4 ml of DPPH (0.15 mM) in methanol (80% (v/v) stomach was dissected out from all groups at intervals of 2, 4, 8,
in water) and mixed well. It was allowed to stand for 30 min at and 24 h, respectively, for measuring injury index as described
room temperature away from light. Ascorbic acid and gallic (6, 12) and histological studies.
acid were used as positive control. The absorbance of the solu-
tion was measured at 517 nm.
Histological Study—Stomach tissue from control and exper-
imental groups was washed a number of times with phosphate-
Iron Chelating Activity in Vitro—The assay system has a buffered saline (PBS, pH 7.4) and fixed in 10% buffered formalin
total volume of 1 ml containing Fe(II) (10 ␮M) in 20 mM for 12 h at 25 °C. The fixed tissues were then dehydrated and
phosphate buffer, pH 7.4. SEGA (3a) at different concentra- embedded in paraffin for preparing semithin sections (4). A
tions (500 nM to 100 ␮M) and TPTZ (20 ␮M solution) were microtome was used to prepare the semithin sections, which
added to the Fe(II) solutions in small volumes to the sample were then taken over poly-L-lysine-coated glass slides for hema-
cuvette with the concomitant addition of the same volume of toxylin-eosin staining. The stained sections were observed
DMSO to the reference cuvette (SEGA (3a) was dissolved in under a microscope (Leica DM-2500, Leica Microsystems
DMSO). For the control group, the assay system is the same GmbH, Wetzlar, Germany) and were documented by a high
without SEGA (3a). Desferrioxamine, a well known iron chela- resolution digital camera.
tor was used as a positive control. Iron chelating ability of SEGA
Soret Spectroscopy to Detect Hemoglobin Released in Stomach
(3a) at different concentrations was monitored by recording during Mucosal Injury—After opening the stomach, gastric
the absorbance of the Fe(II)-TPTZ complex immediately after mucosal tissues from control and experimental groups were
each addition in the quartz cells of the 1-cm light path in a washed with PBS (pH 7.4). This PBS solution was collected and
PerkinElmer Life Sciences Lamda 15 UV-visible spectropho- clarified by centrifugation at 12,000 ϫ g for 20 min. The clari-
tometer at 25 Ϯ 1 °C. The contents were mixed well before the fied PBS solution was monitored to detect hemoglobin by
spectrum was recorded.
recording Soret absorbance immediately in quartz cells of 1-cm
Animals and Indomethacin (NSAID)-induced Gastric Dam- light path in a PerkinElmer Lamda 15 UV-visible spectropho-
age (Gastropathy)—All of the in vivo studies were done in tometer at 25 Ϯ 1 °C (41, 42).
accordance with the institutional animal ethical committee
Gastric Mucosal Cell Culture—Gastric mucosal cells were
guidelines. Sprague-Dawley rats (180–220 g) were used for this isolated and cultured as described earlier (4). Mucosa from the
study. Each group (control or experimental) of animals was rat stomach was scraped in Hanks’ balanced salt solution
maintained at 24 Ϯ 2 °C with a 12-h light and dark cycle. The (HBSS) (pH 7.4) containing 100 units/ml penicillin, 100
animals were fasted for 24 h before the start of experiments to units/ml streptomycin, and 10 ␮g/ml gentamycin. The mucosa
avoid food-induced increased acid secretion and its effect on was then minced and suspended in HBSS (pH 7.4), containing
gastric lesions. The rats were provided with water ad libitum. A 0.05% hyaluronidase and 0.1% collagenase type I. The suspen-
gastric mucosal lesion was induced by indomethacin as sion was incubated for 30 min at 37 °C in a 5% CO2 environ-
described (5). Briefly, all of the animals were divided into con- ment with shaking and then filtered through a sterile nylon
trol, indomethacin-treated, and drug-pretreated indometha- mesh. The filtrate was centrifuged at 600 ϫ g for 5 min, and the
cin-treated groups. Oral administration of indomethacin at a cell pellet was washed with HBSS (pH 7.4) and further centri-
Ϫ1
dose of 48 mgꢀkg b.w. was given to the fasted animals to fuged. The pellet was incubated in 5 ml of Ham’s F-12 medium
induce gastric injury. In the drug-pretreated groups, the ani- in a T25 flask supplemented with 10% fetal bovine serum (FBS)
mals were given SEGA (3a) (50, 20, 10, 5, 3, and 1 mgꢀkgϪ1 b.w.), and 100 units/ml penicillin, 100 units/ml streptomycin, and 10
intraperitoneally 30 min prior indomethacin treatment. The ␮g/ml gentamycin. Cells were cultured at 37 °C with 5% CO2
control group received vehicle only. After 4 h of indomethacin and grown to ϳ90% confluence before treatment. 90% of the
treatment, the animals were sacrificed under proper euthana- cells obtained following this protocol possessed epithelial char-
sia, and stomachs were collected. The severity of mucosal acteristics. For all in vitro experiments, cultured cells were first
lesions was scored as the injury index (40) according to the divided into three groups: control, indomethacin-treated, and
following scale: 0, no pathology; 1, a small injury (1–2 mm); 2, a SEGA (3a)-pretreated indomethacin-treated in 12-well plates
medium injury (3–4 mm); 4, a large injury (5–6 mm); 8, a larger with 106 cells/well. Each well of the “indomethacin group” was
injury (Ͼ6 mm). The sum of the total scores in each group of treated with indomethacin, each well of the “indomethacin plus
rats divided by the number of the animals was expressed as the SEGA (3a) group” was treated with SEGA (3a) 30 min prior to
mean injury index (4–6).
indomethacin treatment, and the control was treated with only
For the healing study, gastric mucosal injury was first in- vehicle.
.
Ϫ1
duced with indomethacin treatment at a dose of 48 mgꢀkg
Measurement of Intramitochondrial Superoxide Anion (O2)
.
b.w. 4 h after the induction of mucosal injury, some of the ani- in Gastric Mucosal Cells—Mitochondrial O2 was detected by
mals were divided into two different groups (n ϭ 6) (i.e. auto- fluorescence microscopy using the specific dye MitoSOX.
healing and SEGA (3a)-induced healing). This time point was Equal numbers of gastric mucosal primary cultured cells from
referred as 0 h of healing. At this point of time, in the SEGA control and experimental groups were used for the detection of
.
(3a)-induced healing group, SEGA (3a) was administered intramitochondrial O2 using MitoSOX, a superoxide-sensitive
(intraperitoneally) at a dose of 50 mgꢀkgϪ1 b.w. (this dose was fluorescence probe, following the protocol as described in the
selected from the dose-response curve). The animals, which product catalogue (6, 43). Cells were stained with the fluores-
were not treated with SEGA (3a) and received only indometh- cent probe in HBSS (pH 7.4) and incubated for 15 min at 37 °C
JANUARY 27, 2012•VOLUME 287•NUMBER 5
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
in the dark. After the incubation, cells were washed with HBSS a liquid phase oxygen measurement system (Oxygraph, Han-
three times and used for fluorescence microscopy (Leica satech, Norfolk, UK) (46). Complex I (state 3)-mediated oxygen
DM-2500). Staining of MitoSOX was visualized using a red consumption was initiated by the incorporation of glutamate
filter.
and malate (5 mM each) to 1 ml of respiratory medium (250 mM
Measurement of Intramitochondrial Free Iron in Gastric sucrose, 5 mM KH2PO4, 5 mM MgCl2, 0.1 mM EDTA, and 0.1%
Mucosal Cells—Equal numbers of gastric mucosal primary cul- BSA in 20 mM HEPES, pH 7.2). Basal respiration (state 2) was
tured cells from control and experimental groups were used for measured following the addition of mitochondrial suspension.
free iron localization using Phen Green SK, an iron-sensitive The addition of ADP (1 mM) marks the initiation of state 3
fluorescence probe, following the protocol as described in the respiration. State 4 respiration was recorded in the absence of
product catalogue. Cells were first incubated with Phen Green ADP. The respiratory control ratio (RCR) was obtained from
SK (20 ␮M) for 15 min at 37 °C in the dark. After the incubation, the ratio of state 3 respiration (nmol of O2 consumed) and state
cells were washed with HBSS and used for fluorescence micros- 4 respiration (nmol of O2 consumed) (18, 47).
copy (Leica DM-2500). The fluorescence of Phen Green SK was
visualized using a green filter (6).
Measurement of ⌬⌿m—Isolated mitochondria from stomach
tissue of rats were used to detect ⌬⌿m. Equal amounts of mito-
Isolation of Mitochondria—Mitochondria were isolated and chondria (25 ␮g) from control and experimental groups were
purified by following the protocol reported earlier (18, 44). In taken in 100 ␮l of JC-1 assay buffer (100 mM MOPS, pH 7.5,
brief, the scraped gastric mucosa from the control, indometh- containing 550 mM KCl, 50 mM ATP, 50 mM MgCl2, 50 mM
acin-treated (48 mgꢀkgϪ1 b.w.), and SEGA (3a)-pretreated (50 sodium succinate, 5 mM EGTA) and were incubated in the dark
mgꢀkgϪ1 b.w.) indomethacin-treated groups were minced and with JC-1 (300 nM) for 15 min at 25 °C. The fluorescence of each
homogenized in isolation buffer, followed by centrifugation at sample was measured in a Hitachi F-7000 fluorescence
600 ϫ g for 10 min to remove the cell debris and nuclear pellet. spectrofluorimeter (excitation 490 nm, emission 530 nm for
This was further centrifuged at 12,000 ϫ g for 15 min to obtain JC-1 monomer; emission 590 nm for JC-1 aggregates). ⌬⌿m
the crude mitochondrial pellet. A 25–50% Percoll density gra- was expressed as fluorescence ratio of 590 nm/530 nm (4, 43).
dient was prepared. Over the 25% Percoll, the crude mitochon-
Measurement of Mitochondrial Dehydrogenase Activity—
drial pellet (resuspended in cold 15% Percoll solution) was lay- Mitochondrial metabolic function was studied by observing the
ered. It was further centrifuged at 30,000 ϫ g at 4 °C for 30 min ability of mitochondrial dehydrogenases to reduce 3-(4,5-dime-
to obtain pure mitochondria at the interface between the Per- thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
coll (25–50%) layers. The mitochondria were isolated from the into formazan dye (4). Equal numbers of gastric mucosal pri-
interface. Further, they were washed with isolation buffer and mary cultured cells were taken (106 cells) in each well of a
centrifuged at 16,700 ϫ g at 4 °C for 10 min. The supernatant 12-well plate and were divided into control and experimental
was discarded, and 10 mgꢀmlϪ1 fatty acid-free BSA was added groups. After incubating for 16 h, cells were dissociated and
and mixed. Afterward, the mixture was centrifuged at 6,900 ϫ g centrifuged at 500 ϫ g for 5 min, and the supernatant was then
at 4 °C for 10 min. The purified mitochondria (pellet) were discarded. Cell pellet was reconstituted in fresh cell culture
resuspended in storage buffer. Mitochondrial protein content medium. Equal numbers of cells (105 cells) in a final volume of
was determined by using the method of Lowry (45).
100 ␮l of cell culture medium from each group were then taken
Measurement of MOS—Isolated mitochondria from stomach in a 96-well plate in triplicates. MTT (0.1% final concentration)
tissue of control, indomethacin-treated (48 mgꢀkgϪ1 b.w.), and solution was added to each well of both control and experimen-
Ϫ1
SEGA (3a)-pretreated (50 mgꢀkg b.w.) and indomethacin- tal groups, mixed well, and incubated for 3 h at 37 °C in a CO2
treated (48 mgꢀkgϪ1 b.w.) rats were used to detect MOS. MOS incubator. After the incubation, 100 ␮l of MTT solubilization
was measured as described earlier through the quantification of solution containing 10% Triton X-100 plus 0.1 N HCl in anhy-
total thiol, lipid peroxidation products, and protein carbonyl in drous isopropyl alcohol was added to solubilize the insoluble
mitochondria. In brief, thiol content was measured by its reac- formazan crystals at the bottom of the well. The MTT reduc-
tion with 5,5Ј-dithiobis(nitrobenzoic acid) to yield the yellow tion (absorbance of formazan dye) was measured at 570 nm.
chromophore of thionitrobenzoic acid, which was measured at
Assay of Caspase-9 and Caspase-3 Activities—Caspase-9 and
412 nm. Mitochondrial lipid peroxidation was assayed by add- caspase-3 activities were measured using a commercially avail-
ing 1 ml of the mitochondrial fraction in 0.9% normal saline to able kit (Biovision and Sigma, respectively). In brief, stomach
Ϫ1
2 ml of thiobarbituric acid/TCA mixture (0.375% (w/v) and 15% tissue from control, indomethacin-treated (48 mgꢀkg b.w.),
Ϫ1
(w/v), respectively) in 0.25 N HCl and was mixed and boiled for and SEGA (3a)-pretreated (5, 10, and 50 mgꢀkg b.w.) indo-
15 min. The samples were then cooled, and after centrifugation, methacin-treated rats were minced and homogenized in
the absorbance of the supernatant was read at 535 nm. Tetra- caspase lysis buffer (provided with the respective kits). The
ethoxypropane was used as a standard. Protein carbonyl was homogenate was centrifuged at 16,000 ϫ g for 15 min. For the
measured by following the standard colorimetric method that caspase-9 assay, the supernatant was collected, containing an
measures the binding of dinitrophenylhydrazine to the car- equal amount of protein for each sample, and mixed with 50 ␮l
bonyl group and was quantified by taking the absorbance at 362 of 2ϫ reaction buffer (provided with the kit). This was followed
nm (4, 43).
by the addition of the substrate, LEHD-p-nitroanilide (200 ␮M
Assessment of Mitochondrial Respiratory Function by Follow- final concentration) for caspase-9. In case of caspase-3, 5 ␮l of
ing Mitochondrial Oxygen Consumption—Mitochondrial oxy- the supernatant was taken together with 1ϫ reaction buffer and
gen consumption was measured using a Clark-type electrode in 10 ␮l of substrate (provided with the kit), Ac-DEDV-p-nitroa-
3498 JOURNAL OF BIOLOGICAL CHEMISTRY
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
nilide (200 ␮M final concentration). The mixture was incubated
Radiolabeling of SEGA (3a)—SEGA (3a) was labeled with
at 37 °C for 1 h, and absorbance was taken at 405 nm (4, 43).
99mTc by a standard stannous reduction method (48) as per
RT-PCR for Proapoptotic and Antiapoptotic Genes—Equal Reaction 1. Nitrogen-purged water was used for the prepara-
Ϫ
amounts of stomach tissue (30 mg) from control, indometha- tion of aqueous 99mTcO4 solution and stannous chloride solu-
Ϫ1
Ϫ
cin-treated (48 mgꢀkg b.w.), and SEGA (3a)-pretreated (50 tion. Briefly, aqueous 99mTcO4 (2 mCi/ml) was mixed with 0.03
Ϫ1
Ϫ1
mgꢀkg b.w.) indomethacin-treated (48 mgꢀkg b.w.) rats ml of freshly prepared stannous chloride solution (1 mg/ml) at
were used for total RNA isolation using a commercially avail- pH 3.2 and further mixed with 1 ml of SEGA (3a) solution (3
able kit (RNeasy kit, Qiagen). RNA (2 ␮g) was used to prepare mg/ml). The mixture was incubated separately for 20 min at
cDNA using oligo(dT)18. Equal amounts of cDNA were used for room temperature (30 °C).
PCR amplification using specific forward and reverse primers
Reduction
of bcl-2, bax, and actin. The PCR-amplified products were
SEGA ͑3a͒ ϩ 99mTcO4Ϫ
¡
99mTc-labeled SEGA ͑3a͒
resolved in 2% agarose gel and documented in a gel documen-
tation system (Alpha Infotech). The intensity of each band was
quantified with densitometric software (Lab Image beta ver-
sion, Kapelan GmbH, Germany). The intensity of each band
was normalized with that of actin (6).
͑Sn2ϩ͒
REACTION 1
The effect of stannous chloride on the labeling efficiency at
Terminal Deoxynucleotidyltransferase dUTP nick end label- different concentrations was studied to find the optimum con-
ing (TUNEL) Assay in Vitro in Cultured Gastric Mucosal Cells centration needed for maximum labeling. After adding SEGA
Ϫ
and in Vivo in Rat Gastric Mucosa—In vitro apoptosis was (3a) to the mixture of 99mTcO4 and SnCl2 adjusted at the opti-
detected in the cultured gastric mucosal cells using a commer- mum pH (pH 3.2), the solution was incubated for various time
cially available APO-BrdUTM TUNEL assay kit (Invitrogen). In periods to see the effect of incubation time on the yield of label-
brief, cultured cells were first divided into control, indometha- ing. The extent of labeling of SEGA (3a) was determined by
cin-treated and SEGA (3a)-pretreated indomethacin-treated ascending TLC using 2.5 ϫ 10-cm silica gel strips as the station-
groups. After 16 h of incubation, the cells were washed with PBS ary phase and either acetone, methanol, or brine solution as the
twice and fixed with 1% paraformaldehyde in PBS (pH 7.4), mobile phase. The test sample (incubation mixture) (2–3 ␮l)
followed by treatment with 70% ethanol in ice. The cells were was applied 1 cm from the base of the TLC plate and dried at
then loaded with DNA labeling solution, containing terminal room temperature. The plates were then developed in appro-
deoxynucleotidyltransferase. Cells were then stained with priate solvent systems. Acetone was used for the determination
Alexa Fluorꢁ 488 dye-labeled anti-BrdU antibody. The cells of free pertechnetate, whereas either methanol or brine solu-
were finally stained with propidium iodide (PI) solution con- tion was used for the determination of radiocolloid. After devel-
taining RNase A. The cells were then visualized under a fluo- oping, the plates were dried, and the distribution of radioactiv-
rescence microscope (Leica DM-2500) using appropriate filters ity was determined by cutting the portion of the strips and
for Alexa Fluor 488 and PI (4, 6, 43). counting it in a ␥-scintillation counter (Electronic Corp. of
For the detection of apoptosis in vivo, the gastric mucosa India, model LV4755 (Hyderabad, India)) at 140 keV).
from control, indomethacin-treated, and SEGA (3a)-pre-
Transchelation with Diethylene Triamine Pentaacetic Acid
treated indomethacin-treated rats were collected. Then these (DTPA)—This study was performed to check the stability and
tissues were fixed in 10% buffered formalin for 12 h at 25 °C and strength of binding of 99mTc with the SEGA (3a). Radiolabeled
processed as described under “Immunohistological Studies.” preparations of 0.5 ml were challenged against three different
The semithin sections (5 ␮m) were used for TUNEL staining concentrations (10, 30, and 50 mM) of DTPA in 0.9% saline by
using a commercially available kit (Promega).
incubating at 37 °C for 2 h. The effect of DTPA on labeling was
Immunohistochemical Studies—The semithin sections (5 measured by TLC on a silica gel plate using normal saline and
␮m) of mucosal tissues from control and experimental groups acetone as the mobile phase, which allowed the separation of
were deparaffinized in xylene and rehydrated in graded ethanol. free pertechnetate and DTPA chelate (Rf ϭ 0.9) from that of the
After antigen retrieval, slides were rinsed in water and washed 99mTc-labeled SEGA (3a), which remained at the point of appli-
twice with Tris-buffered saline (TBS) (pH 7.4) plus Triton cation (Rf ϭ 0).
X-100 (0.025%) with gentle agitation. The sections were then
Determination of Mitochondrial Uptake of SEGA (3a)—Mi-
blocked with 1% BSA in TBS for 2 h at 25 °C. Primary anti-active tochondrial uptake was carried out according to the following
caspase-3 was added at a dilution of 1:500 to the sections and method. All animal experiments were carried out in compli-
kept at 4 °C overnight. The next day, the sections were rinsed ance with the relevant national laws relating to the conduct of
twice for 5 min in TBS plus Triton X-100 (0.025%) with gentle animal experimentation and with the approval of institutional
agitation. To block the endogenous peroxidases, the slides were animal ethics committees. Sprague-Dawley rats (180–220 g)
incubated with 0.3% H2O2. Then the slides were incubated with were used for this study. The animals were fasted for 24 h before
HRP-labeled anti-rabbit IgG secondary anti-rat antibody at a the start of experiments as described above. After 24 h of fast-
dilution of 1:1000 in 1% BSA in TBS for 2 h. Finally, the slides ing, all of the rats were well hydrated by intraperitoneal admin-
were rinsed three times in TBS. Slides were stained with istration of saline (0.9%, 2 ml) for 1 h. After another 1 h, the
diaminobenzidine and counterstained with hematoxylin. The 99mTc-chelate SEGA (3a) in a total volume 0.03 ml (5–8 ␮Ci)
slides were viewed under the 10ϫ objective of a Leica DM-2500 was administered through an intraperitoneal route in each rat.
Ϫ1
microscope.
After 30 min, indomethacin (48 mgꢀkg b.w.) was adminis-
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
FIGURE 1. General scheme for the synthesis of tryptamine derivatives.
trated orally in each rat. The rats were sacrificed at 4 h postin- may be detoxified by GA through toxic group protection. 5HT
jection. Mitochondria of stomach mucosa were isolated as
described above. Mitochondrial protein was estimated, and the
radioactivity of 99mTc-chelate SEGA (3a) was counted in a
␥-scintillation counter against suitably diluted aliquots of the
injected solution as a standard. The data were expressed as
percentage dose/mg of mitochondrial protein (Mean Ϯ S.E).
Stability Studies—The stability of 99mTc-labeled SEGA (3a)
was determined in vitro using 0.9% sodium chloride and serum
(from rat) by ascending TLC. The labeled complex (0.5 ml) was
mixed with 1.5 ml of normal saline or rat serum and incubated
at 37 °C. The samples were withdrawn at regular intervals up to
24 h, monitored by TLC, and analyzed in a ␥-counter.
was conjugated with GA through an amide linkage to synthe-
size SEGA (3a) and through an ester linkage to synthesize
GASE (4d) (Fig. 1) (supplemental Schemes S1 and S2). Both
conjugates were tested first for their antioxidant property in
vitro by following FRAP and DPPH free radical-scavenging
activity. The FRAP assay is based on the measurement of the
ability of a substance to reduce Fe(III) to Fe(II); the greater the
reducing ability, the better the antioxidant property. Antioxi-
dants reduce the colorless Fe(III)-TPTZ to a blue Fe(II)-TPTZ
complex, which results in an increase in the absorbance at 595
nm, giving a FRAP value. A higher FRAP value indicates a
greater reducing (i.e. antioxidant property) ability of the com-
pound. FRAP values at 6 min were calculated from the equation
described above. At 6 min, the absorbance change takes place
abruptly due to reduction of Fe(III) into Fe(II). The results
clearly indicate that SEGA (3a) shows a reducing ability (Fe(III)
to Fe(II)) that is much better than GASE (4d) (Table 1). In the
DPPH assay, the decrease of absorbance is correlated with
the antioxidant potency of the compounds. The greater the
decrease in absorbance, the higher is the DPPH scavenging
potency (i.e. the antioxidant potency of different synthesized
compounds). The results indicate that SEGA (3a) also shows
greater DPPH scavenging potency compared with GASE (4d)
(Table 1). These results indicate that when 5HT is conjugated
with GA through amide linkage, it appears to be more effective
than when conjugated by ester linkage.
Statistical Analysis—All data are presented as mean Ϯ S.E.
The level of significance was determined by unpaired Student’s
t test with one-way analysis of variance as applied. A p value of
Յ0.05 was considered as significant.
RESULTS
Synthesis of Tryptamine-Gallic Acid Hybrid Molecule—A
small molecule having the iron-chelating property and the
capability of preventing MOS from entering into mitochondria
is necessary to protect gastric mucosa against NSAID-induced
gastric mucosal injury or gastropathy. To design such a small
molecule, we began the synthesis using 5Ј-hydroxytryptamine
(5HT), a hydrophobic amine that enters inside mitochondria
(49). However, 5HT is toxic at high concentration (50). In con-
trast, free amine and hydroxyl groups of 5HT offer a scope to
conjugate a powerful antioxidant bearing an iron-chelating
property to make a non-toxic antioxidant hybrid molecule
retaining mitochondrial penetration. Gallic acid (GA), a natural
polyphenol and antioxidant (4), possesses the iron-chelating
property (4, 51), and that is why we selected it to make a hybrid
molecule with 5HT. The strategy might give double benefits
because the conjugation of GA with 5HT is expected to
enhance the bioavailability of GA in body fluid (lack of bioavail-
Our next objective was to synthesize different types of
tryptamine-antioxidant conjugates through amide linkage
using other antioxidants replacing GA and to evaluate their
activities for comparative efficacy. We replaced GA with 4-hy-
droxycinnamic acid and indole-3-acetic acid to synthesize
other tryptamine-antioxidant conjugates, such as 2b and 2c,
respectively (Fig. 1). These compounds were synthesized by
successive condensation of 5HT with 4-hydroxycinnamic acid
ability is a common problem of bioactive polyphenol), or 5HT and indole-3-acetic acid, respectively through amide linkage
3500 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 5•JANUARY 27, 2012

Tryptamine-GA Hybrid against NSAID-induced Gastropathy
TABLE 1
Evaluation of antioxidant property (reducing property) in FRAP assay
and DPPH radical scavenging assay of synthesized compounds
FIGURE 2. Iron-chelating activity of SEGA (3a) in vitro. Shown is spectro-
scopic analysis for SEGA (3a)-Fe(II) interaction at different concentrations of
SEGA (3a) (500 nM (a), 1 ␮M (b), 10 ␮M (c), 50 ␮M (d), and 100 ␮M (e)). Detailed
descriptions are given under “Experimental Procedures.”
different concentrations. Results indicate that SEGA (3a)
shows excellent antioxidant property in the FRAP assay as well
as in the DPPH-scavenging assay in a concentration-dependent
fashion (data not shown).
Now, we tested whether SEGA (3a) could chelate free iron
in vitro. The iron-chelating property was assessed by a TPTZ
assay. Fe(II) solution in the presence of TPTZ gave a broad
peak at 595 nm (Fig. 2). This peak was obtained due to Fe(II)-
TPTZ complex formation. When SEGA (3a) was added to
the Fe(II) solution, no such broad peak was observed after the
addition of TPTZ solution (Fig. 2). Thus, from this experiment,
it is evident that SEGA (3a) chelates free iron in vitro. Now,
SEGA (3a), because of its maximum antioxidant potential and
iron-chelating property, was subjected to further detailed bio-
logical evaluation and mechanistic studies on NSAID-induced
gastropathy.
SEGA (3a) Prevents Indomethacin (NSAID)-induced Gastric
Mucosal Damage—We tested whether SEGA (3a) could pro-
tect indomethacin (an NSAID)- induced MOS-mediated mito-
chondrial pathology and gastropathy in vivo. SEGA (3a) pro-
tected gastric mucosa from indomethacin-induced gastric
(supplemental Scheme S1). Next, we searched to find out injury in a dose-dependent manner (ED50 ϭ 6.9 mgꢀkgϪ1 b.w.),
whether 5HT is the best possible tryptamine for our purpose. as evident from the gastric injury index (Fig. 3A). For rapid
We replaced 5HT with other tryptamines to synthesize several visualization of the protective effect of SEGA (3a), we present
other tryptamine-antioxidant conjugates, such as TRGA (3b), the real morphological data obtained by opening the stomach
MEGA (3c), 2f, 2h, and 2i, by successive condensation of interior. From the morphology, it is very clear that SEGA (3a)
tryptamine, 5-methoxytryptamine with GA, 4-hydroxy cin- protected the injury, and the oozing out of blood (which
namic acid, and indole-3-acetic acid, respectively (Fig. 1) (sup- appeared black due to oxidation of released hemoglobin under
plemental Scheme S3). 5HT itself showed little antioxidant an acidic environment) in indomethacin exposed rat gastric
activity in vitro (Table 1). We were interested in investigating mucosa (Fig. 3B). The gastroprotective effect of SEGA (3a) was
whether in SEGA (3a), 5HT has any individual antioxidant also verified by following the changes in microscopic structure
property. To explore this, we synthesized dimer of 5HT (Fig. 1) of the actual histology of the gastric mucosa. SEGA (3a)
(supplemental Scheme S4). Now antioxidant potencies were restored normal architecture of gastric mucosa from indo-
evaluated of all of the synthesized compounds by FRAP and methacin-induced increased gastric mucosal cell death and cell
DPPH free radical scavenging assays in vitro. For the prelimi- shedding in the superficial mucosa (Fig. 3C). Excessive gastric
nary screening of antioxidant activity, all of the synthesized mucosal injury by NSAID leads to the release of blood in the
compounds were taken at high concentration (100 ␮M). From stomach. In an indomethacin-treated rat, a sharp Soret peak
the above results, it is evident that SEGA (3a) shows antioxidant (417 nm) was observed, indicating the presence of hemoglobin
property in FRAP as well as DPPH assays (Table 1). We were in the stomach due to a mucosal injury. But in the case of SEGA
interested in checking the antioxidant property of SEGA (3a) at (3a) pretreatment, we did not find any Soret peak. The data
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
FIGURE 3. SEGA (3a) prevents indomethacin-induced gastropathy. A, protection of indomethacin-induced gastric mucosal injury by SEGA (3a) at different
doses as measured by the injury index (*, p Ͻ 0.001 versus indomethacin; n ϭ 6). B, morphology of gastric mucosa from control, indomethacin-treated (48
mgꢀkgϪ1 b.w.), and SEGA (3a)-pretreated (50 mgꢀkgϪ1 b.w.) indomethacin-treated rats. The arrow indicates damage in the gastric mucosa. C, hematoxylin-
eosin staining of gastric mucosal sections from indomethacin-treated and indomethacin treated-SEGA (3a)-pretreated rats. The arrow indicates injury in the
gastric mucosa. D, detection of hemoglobin in gastric washing of control, indomethacin-treated, and SEGA (3a)-pretreated indomethacin-treated rats as
measured by Soret spectroscopy. Detailed descriptions are given under “Experimental Procedures.” Error bars, S.E.
.
FIGURE 4. SEGA (3a) scavenges indomethacin-induced intramitochondrial O2and free iron in gastric mucosal cells and prevents MOS. A, SEGA (3a) (50
.
.
␮M) scavenges intramitochondrial O2 generated by indomethacin (5 mM) in gastric mucosal cells. Mitochondrial generation of O2 was detected by MitoSOX
staining (red). B, SEGA (3a) (50 ␮M) chelates intramitochondrial free iron in vitro in cultured gastric mucosal cells. Mitochondrial generation of free iron was
detected by Phen Green SK staining (green). C, SEGA (3a) (50 mgꢀkgϪ1 b.w.) prevents indomethacin-induced formation of protein carbonyl, peroxidation of
lipid, and depletion of thiol content in mitochondria (***, p Ͻ 0.001 versus control; ###, p Ͻ 0.001 versus indomethacin (n ϭ 5)). Detailed descriptions are given
under “Experimental Procedures.” Error bars, S.E.
.
further confirmed the gastroprotective effect of SEGA (3a) (Fig. indomethacin-induced generation of mitochondrial O2 and
.
3D).
free iron was evaluated. The generation of O2 and free iron was
.
SEGA (3a) Scavenges Intramitochondrial O2, Chelates induced in cultured gastric epithelial cells by indomethacin
.
Intramitochondrial Free Iron, and Prevents MOS—Intramito- (Fig. 4, A and B). Mitochondrial O2 was measured by MitoSOX,
chondrial generation of ROS and subsequent oxidative stress a mitochondria-specific fluorescence indicator (52). MitoSOX
play a critical role in NSAID-induced gastric injury. Because is a derivative of hydroethydium, and due to the cationic
SEGA (3a) protects gastric mucosa from NSAID-induced dam- property, this dye accumulates in huge amounts within the
age, we tested the ROS-scavenging activity of SEGA (3a). Mito- mitochondria in response to negative membrane potential.
.
.
chondrial O2 is a precursor of ROS, and mitochondrial free iron O2-derived oxidation product of MitoSOX has a distinct exci-
played an important role in the generation of ROS and the tation wavelength at 396 nm and emission wavelength at 510
development of MOS (6). Thus, the effect of SEGA (3a) on nm (6, 52). Indomethacin stimulated intramitochondrial gen-
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
TABLE 2
Effect of SEGA (3a) on indomethacin-induced mitochondrial dysfunction
Mitochondrial dehydrogenase
activity (MTT reduction,
absorbance 570 nm)
Mitochondrial
respiratory function (RCR)
(mean ؎ S.E.)
⌬⌿m (fluorescence
ratio, 590 nm/530 nm)
(mean ؎ S.E.)
(mean ؎ S.E.)
Control
6.21 Ϯ 0.8
3.77 Ϯ 0.41a
5.35 Ϯ 0.7b
5.79 Ϯ 0.48
2.68 Ϯ 0.32a
4.29 Ϯ 0.37c
0.82 Ϯ 0.09
0.42 Ϯ 0.07a
0.71 Ϯ 0.09b
Indomethacin
Indomethacin ϩ SEGA (3a)
a p Ͻ 0.001 versus control (n ϭ 6–8).
b p Ͻ 0.01 versus indomethacin (n ϭ 6–8).
c p Ͻ 0.001 versus indomethacin (n ϭ 6–8).
.
eration of O2, but pretreatment with SEGA (3a) significantly mitochondria, but pretreatment with SEGA (3a) under similar
.
inhibited the generation of O2 as revealed by the decreased conditions restored the loss of mitochondrial dehydrogenase
.
fluorescence of the O2-derived oxidation product of MitoSOX activity (Table 2).
(Fig. 4A). Mitochondrial free iron was measured by Phen Green
SEGA (3a) Prevents Activation of Mitochondrial Pathway of
SK, a specific fluorescent probe used to assay chelatable iron Apoptosis—The activation of the mitochondrial pathway of
(Fig. 4B). Mitochondria of gastric mucosal cells were tagged by apoptosis in gastric mucosal cells is a consequence of indo-
Mitotracker Red, a specific fluorescent probe for mitochondria. methacin-induced MOS. Because SEGA (3a) prevents MOS as
From the experiment, it is evident that indomethacin treatment well as mitochondrial dysfunction, SEGA (3a) should protect
resulted in increased intramitochondrial free iron accumula- the mitochondrial pathway of apoptosis in gastric mucosal cells
tion, but pretreatment with SEGA (3a) significantly inhibited by indomethacin. The data indicate that pretreatment with
indomethacin-mediated free iron accumulation as revealed by SEGA (3a) significantly attenuated indomethacin-induced
decreased fluorescence of Phen Green SK (Fig. 4B). Mitochon- activation of casapse-9 (marker of mitochondrial pathway of
.
drial O2 and free iron are responsible for MOS (6). SEGA (3a), apoptosis) (Fig. 5A) as well as caspase-3 (general marker for
.
by scavenging O2 and free iron, protected mitochondria from apoptosis) (Fig. 5B) in gastric mucosa in a dose-dependent
MOS and restored the mitochondrial functions in gastric manner. Indomethacin stimulated about 2-fold activation of
mucosal cells during indomethacin-induced gastropathy (Fig. caspase-9 in the gastric mucosal cells (Fig. 5A). Pretreatment
4C). SEGA (3a) significantly prevented indomethacin-induced with SEGA (3a) significantly attenuated the activation of casa-
mitochondrial lipid peroxidation, thiol depletion, and protein pse-9 and brought the activity close to the normal level (Fig.
carbonyl formation (Fig. 4C), which are the markers for MOS. 5A). Indomethacin activated caspase-3 by more than 2-fold in
SEGA (3a) Corrects Indomethacin-induced Mitochondrial the gastric mucosal cells (Fig. 5B). Pretreatment with SEGA
Dysfunction—Because SEGA (3a) scavenges intramitochon- (3a) significantly blocked the activation of caspase-3 (Fig. 5B).
.
drial O2, chelates intramitochondrial free iron, and prevents bcl-2 and bax play a critical role in the mitochondrial pathway
MOS, we were interested to find out whether SEGA (3a) could of apoptosis. Indomethacin was found to down-regulate the
prevent mitochondrial pathology or dysfunction. The fall of the expression of antiapoptotic bcl-2 and up-regulate the expres-
mitochondrial RCR, collapse of mitochondrial transmembrane sion of bax compared with control (Fig. 5, C–E). SEGA (3a)
potential (⌬⌿m), and loss of dehydrogenase activity are hall- pretreatment was found to block indomethacin-induced up-
marks or indicators for mitochondrial pathology or dysfunc- regulation of bcl-2 as well as down-regulation of bax (Fig. 5,
tion. The functional integrity of mitochondria in the presence C–E). The antiapoptotic effect of SEGA (3a) was further tested
or absence of SEGA (3a) in gastric mucosal cells after indo- by following DNA fragmentation performing a TUNEL assay in
methacin treatment was investigated. Mitochondria from indo- gastric mucosal cells in vitro (Fig. 6A) and in vivo in the pres-
methacin-treated gastric mucosa showed severe inhibition of ence of indomethacin (Fig. 6B). In control cells, the absence of
complex I-mediated state 3 (in the presence of ADP) respira- green signal (DNA fragmentation was indicated by green fluo-
tion and mild inhibition of state 4 (in the absence of ADP) rescence of Alexa Fluor 488) indicated no apoptotic DNA frag-
respiration. As a consequence, the RCR (the ratio of state 3 and mentation. However, in indomethacin-treated gastric mucosal
state 4 respiration) was significantly decreased, indicating cells, green fluorescence was prominent, which was co-local-
impairment of mitochondrial respiration. Interestingly, admin- ized with PI-stained nuclei, indicating apoptotic DNA frag-
istration of SEGA (3a) restored the altered RCR value (Table 2). mentation. In SEGA (3a)-pretreated cells, the intensity as well
SEGA (3a) prevented indomethacin-induced loss of ⌬⌿m. as the total number of cells showing green fluorescence were
Indomethacin showed an about 40% decrease of ⌬⌿m, as mea- much less compared with indomethacin-treated cells (Fig. 6A).
sured by the ratio of fluorescence values measured at 590 nm SEGA (3a) pretreatment also significantly prevented indo-
(JC-1 aggregate) and 530 nm (JC-1 monomer). SEGA (3a) pre- methacin-induced gastric mucosal apoptosis in vivo in mucosal
treatment restored the indomethacin-induced fall of ⌬⌿m tissue (Fig. 6B). The TUNEL-positive cells (dark brown stain-
almost to the control level (Table 2). SEGA (3a) protected the ing, indicated by an arrow) were abundant in the gastric muco-
mitochondrial dehydrogenase from indomethacin-induced sal tissue in the presence of indomethacin, whereas the
inactivation. Indomethacin significantly inhibited mitochon- TUNEL-positive cells were decreased significantly in the SEGA
drial dehydrogenases activity (as measured by MTT reduction) (3a)-pretreated indomethacin-treated group (Fig. 6B). The
in gastric mucosal cells, indicating functional impairment of antiapoptotic effect of SEGA (3a) was further confirmed by
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
FIGURE 5. SEGA (3a) prevents the activation of mitochondrial pathway of apoptosis. A, dose-dependent inhibition of indomethacin-induced caspase-9 in
gastric mucosa of rat by SEGA (3a). B, dose-dependent inhibition of indomethacin-induced caspase-3 in gastric mucosa of rat by SEGA (3a) (5, 10, and 50
mgꢀkgϪ1 b.w.). Data are presented as mean Ϯ S.E. (error bars) (***, p Ͻ 0.001 versus control; ###, p Ͻ 0.001; **, p Ͻ 0.01 versus indomethacin (n ϭ 6)). C, SEGA
(3a) (50 mgꢀkgϪ1 b.w.) inhibits down-regulation of bcl-2 and up-regulation of bax by indomethacin in gastric mucosa as measured by RT-PCR. Actin was used
as an internal control. D, densitometric analysis of bcl-2 expression. E, densitometric analysis of bax expression. Detailed descriptions are given under “Exper-
imental Procedures.” Data are presented as mean Ϯ S.E. (***, p Ͻ 0.001 versus control; ###, p Ͻ 0.001 versus indomethacin (n ϭ 5)).
immunohistochemistry using anti-active caspase-3 antibody injury index of 52, 28, and 20, respectively, whereas after treat-
(Fig. 6C). Active caspase-3 immunolabeled mucosal cells (dark ment with SEGA (3a), damage of gastric mucosa was gradually
brown staining, indicated by an arrow) were found after indo- repaired, as evident from an injury index of 14, 8, and 0, respec-
methacin treatment. But SEGA (3a) pretreatment significantly tively. At 20 h, SEGA (3a) completely restored normal architec-
decreased the active caspase-3-immunolabeled cells, indicating ture of gastric mucosa, whereas in the case of the indomethacin
the antiapoptotic role of SEGA (3a) (Fig. 6C). Thus, the data group, there was significant injury. The results indicate that
indicate that SEGA (3a) prevents indomethacin-induced gas- mucosa shows a time-dependent autohealing of the indometh-
tric mucosal cell apoptosis.
acin-induced gastric damage in the absence of SEGA (3a).
SEGA (3a) Accelerates Healing of Indomethacin-induced However, SEGA (3a) treatment significantly expedites healing
Damage of Gastric Mucosa—Prevention of MOS and the mito- with the progress of time, as evident from the restoration of
chondrial pathway of apoptosis expedites the healing process gastric mucosa (Fig. 7). In indomethacin-treated animals, the
(5). Because SEGA (3a) prevents both MOS and apoptosis, we autohealing at 4 h was negligible, as evident from the distorted
were interested in discovering whether SEGA (3a) could accel- mucosal histology, but SEGA (3a) treatment restored healthy
erate the healing of indomethacin-induced already damaged mucosal architecture at 4 h, with almost complete restoration
gastric mucosa. Interestingly, in addition to the gastroprotec- at 20 h (Fig. 7).
tive effect, SEGA (3a) also accelerated healing of already injured
Quantitation of SEGA (3a) Entering Mitochondria—Because
.
mucosa by indomethacin (Fig. 7). Although autohealing takes SEGA (3a) scavenged intramitochondrial O2, chelated intrami-
place in the case of damaged mucosa, SEGA (3a) treatment tochondrial iron, and prevented MOS, we were interested in
accelerates the healing process. SEGA (3a)-induced healing of quantitating how much of the administered SEGA (3a) entered
gastric mucosal injury was checked by histological analysis (Fig. the mitochondria under in vivo conditions. For this purpose,
7). At 4, 8, and 20 h, the mucosa shows gastric injury with an SEGA (3a) was radiolabeled with 99mTc isotope as reported (48)
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
FIGURE 7. Effect of SEGA (3a) on healing of indomethacin-induced gastric
mucosal injury. Hematoxylin and eosin staining of a gastric mucosal section
of control, indomethacin-treated, and indomethacin ϩ SEGA (3a)-treated (50
mgꢀkgϪ1 b.w.) groups at different time points. 0 h, control. II, injury index. The
arrows indicate mucosal injury.
.
of intramitochondrial O2 and free iron and correcting mito-
chondrial dysfunction.
The mitochondria are a potential subcellular therapeutic
drug target against NSAID-induced gastropathy because
.
they produce O2 and free iron, which play an important role
in triggering this pathological condition. A mitochondria-
targeted molecule is required for this purpose. This molecule
must be small and lipophilic and be an ROS scavenger in
nature. Moreover, free iron is known to generate ROS
through the Fenton reaction. Thus, the iron-chelating prop-
FIGURE 6. SEGA (3a) prevents indomethacin-induced apoptosis in vitro
erty would be an additional advantage in controlling oxida-
tive stress. All of these criteria were considered while design-
ing the molecule. Several antioxidants and iron chelators
have been reported, but none of them can satisfy all of the
above criteria. Thus, a new molecule is essential, which will
satisfy all of these criteria in preventing NSAID-induced gas-
tropathy. Keeping this in mind, we have synthesized a series
of tryptamine-antioxidant hybrid molecules. GA, when con-
jugated with 5HT through amide linkage, shows greater
activity both in vitro and in vivo. Thus, all other tryptamine-
antioxidant hybrid molecules were generated through the
amide linkage. For the structure-activity relationship stud-
ies, we synthesized different tryptamine-antioxidant deriva-
tives. Because SEGA (3a) appears to be the most active
among all of the tryptamine-antioxidant conjugates, it is
suggested that the presence of the 5-hydroxy group in the
indole moiety of SEGA (3a) plays an important role for its
gastroprotective activity. When the 5-hydroxy group in the
indole moiety of SEGA (3a) was replaced by hydrogen and
the methoxy group in the molecules TRGA (3b) and MEGA
(3c), the activity was decreased. Indomethacin was selected
and in vivo. A, SEGA (3a) (50 ␮M) inhibits indomethacin (5 mM)-induced
apoptosis in vitro in primary gastric mucosal cells in culture as measured
by a TUNEL assay. The first column shows nuclei stained (red) with PI, the
second column shows apoptotic DNA stained with Alexa Fluor 488 (green
fluorescence), and the third column shows the merged pictures of the first
(PI) and second (Alexa Fluor 488) columns. B, SEGA (3a) (50 mgꢀkgϪ1 b.w.)
inhibits indomethacin-induced apoptosis in vivo in gastric mucosal cells
as measured by a TUNEL assay in mucosal tissue. The TUNEL assay shows
that indomethacin triggers apoptosis (deep brown staining showing apo-
ptotic DNA fragmentation, indicated by arrows) of gastric mucosal cells,
and SEGA (3a) blocks indomethacin-induced gastric mucosal cell apopto-
sis. C, immunohistochemical staining of mucosal tissue with the anti-ac-
tive caspase-3 antibody (deep brown staining showing apoptotic cells indi-
cated by arrows). Detailed descriptions are given under “Experimental
Procedures.”
and administered to rats. The data indicated that 0.05% of the
administered dose of SEGA (3a) entered per mg of mitochon-
dria of gastric mucosal tissue (Fig. 8A). The stability or the
structural integrity of SEGA (3a) in physiological saline as well
as in serum was checked. SEGA (3a) was found to be very stable
at 37 °C (Fig. 8B).
DISCUSSION
The present study describes the designing and synthesis of a as the representative NSAID over others because it is the
small molecule, tryptamine-gallic acid hybrid (SEGA (3a)), most frequently used NSAID in gastrointestinal toxicity
which prevents NSAID-induced mitochondrial pathology, apo- studies in experimental animals (13). The dose of indometh-
Ϫ1
ptosis, and gastropathy by blocking MOS through scavenging acin was selected as 5 mM and 48 mgꢀkg b.w. for in vitro
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
FIGURE 8. Mitochondrial uptake of SEGA (3a). A, uptake of 99mTc-labeled SEGA (3a) into the mitochondria of gastric mucosal cells 4 h after intraperitoneal
injection. MitochondrialuptakeofSEGA(3a)wasexpressedasapercentageofdose/mgofmitochondrialprotein. B, stabilitystudiesof99mTc-labeledSEGA(3a)
in physiological saline and serum in vitro at 37 °C. Detailed descriptions are given under “Experimental Procedures.”
FIGURE 9. Scheme showing the proposed mode of action of SEGA (3a) to inhibit NSAID-mediated MOS-induced gastropathy. Indomethacin (NSAID)
.
interacts with complex I of the electron transport chain and results in the leakage of electron in mitochondria, leading to the formation of O2, which leads to the
generation of ROS. Increased ROS develops MOS by oxidizing protein and lipid, including cardiolipin and protein thiol. Iron (Fe2ϩ) is released from the Fe-S
ꢀ
cluster of aconitase due to ROS-mediated damage and further aggravates oxidative stress by producing hydroxyl radical ( OH). The MOS results in mitochon-
drial dysfunction or pathology and activation of the mitochondrial pathway of apoptosis, which plays a pathogenic role in gastropathy. The tryptamine-gallic
acid hybrid molecule, SEGA (3a), enters the mitochondria and prevents NSAID-induced gastropathy.
and in vivo studies, as reported earlier (4, 6, 7). The role of injury (53, 54). Indomethacin with its acidic carboxyl group
MOS and consequent apoptosis behind NSAID-induced gas- (pKa ϭ 4.5) and lipid solubility has been found to damage
tric mucosal injury is already well established and is consid- both rat and human mitochondria (13). Moreover, indo-
ered to be the major player in the acid-independent (5) and methacin enhances mitochondrial ROS, which disrupts
COX-independent pathway of NSAID-mediated gastric mitochondrial function (6).
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Tryptamine-GA Hybrid against NSAID-induced Gastropathy
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Signal Transduction:
Tryptamine-Gallic Acid Hybrid Prevents
Non-steroidal Anti-inflammatory
Drug-induced Gastropathy:
CORRECTION OF MITOCHONDRIAL
DYSFUNCTION AND INHIBITION OF
APOPTOSIS IN GASTRIC MUCOSAL
CELLS
Chinmay Pal, Samik Bindu, Sumanta Dey,
Athar Alam, Manish Goyal, Mohd. Shameel
Iqbal, Souvik Sarkar, Rahul Kumar, Kamal
Krishna Halder, Mita Chatterjee Debnath,
Susanta Adhikari and Uday Bandyopadhyay
J. Biol. Chem. 2012, 287:3495-3509.
doi: 10.1074/jbc.M111.307199 originally published online December 7, 2011
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