Novel model for NSAID induced gastroenteropathy in rats

A novel model for NSAID induced gastroenteropathy in rats☆
Devendra Pratap Singh a,b
, Swapnil P. Borse a,b
, Manish Nivsarkar a,
⁎
a
Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Ahmedabad, Gujarat, 388054, India
b
Institute of Pharmacy, NIRMAUniversity, Sarkhej-Gandhinagar Highway, Ahmedabad, Gujarat 382481, India
a b s t r a c ta r t i c l e i n f o
Article history:
Received 3 September 2015
Received in revised form 30 November 2015
Accepted 30 November 2015
Available online 2 December 2015
Introduction: Progress in management of Nonsteroidal anti-inflammatory drug (NSAID) induced gastrointestinal
toxicity requires the availability of appropriate experim ental anim al m odels that are as close to humans as
feasible. Our objective was to develop a rat model for NSAID-induced gastroenteropathy and also to simulate
the common clinical scenario of co-administration of NSAID and proton pump inhibitor (PPI) to explore if PPI
contribute to exacerbation of NSAID-enteropathy.
Methods: Rats were treated twice daily with pantoprazole sodium (PTZ; 10 mg/kg peroral) or vehicle for a total of
10 days. In some experiments, Diclofenac sodium (DCF; 9 mg/kg) or vehicle was administered orally twice daily
for the final 5 days of PTZ/vehicle administration. After the last dose on 9th day, rats in all the groups were fasted
but water was provided ad libitum.12 h after the last dose on 10th day, rats in all the groups were euthanized and
their gastrointestinal tracts were assessed for haemorrhagic lesions, lipid peroxidation, intestinal permeability
and gastrointestinal luminal pH alterations. Changes in haemoglobin, haematocrit and serum levels of albumin,
total protein, ALT and bilirubin were calculated.
Results: The macroscopic and histological evidence suggested that administration of DCF resulted in significant
gastroenteropathic damage and co-administration of PTZ resulted in significant exacerbation of NSAID enterop-
athy, while attenuation of NSAID induced gastropathy was observed. Our results were further supported by the
significant decrease in haemoglobin and haematocrit levels and serum levels of albumin and total proteins, an
increase in oxidative stress and intestinal permeability with the use of DCF either alone or in combination with
PTZ.
Conclusions: This m odel was developed to simulate the hum an clinical situation during NSAID therapy and
indeed the present DCFregimen caused both gastric and small bowel alterations, such as multiple erosive lesions,
together with a decrease in haemoglobin, haematocrit, serum albumin, serum total protein levels and IP
alteration, known to occur in patients receiving NSAIDs. Additionally, this paper provides yet another evidence
for PPI induced exacerbation of NSAID enteropathy.
© 2015 Elsevier Inc. All rights reserved.
Keywords:
NSAIDs
Gastroenteropathy
Methods
PPIs
Rats
Enteropathy
Haemorrhagic lesions
1. Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) which are among
the most prescribed drugs worldw ide (Brune & Patrignani, 2015;
Conaghan, 2012) are often preferred because of their low abuse poten-
tial, long history of clinical use and robust efficacy in alleviating pain and
inflammation (Atchison, Herndon, &Rusie, 2013). However, their use is
associated with adverse events in the upper gastrointestinal (GI) tract of
experimental anim als and humans, and now it is being increasingly
appreciated that these drugs can also exert detrimental effects on the
lower GI tract, with potential serious outcomes like; ulceration, perfora-
tion, overt bleeding and diaphragm-like strictures (Fornai et al., 2014;
Scarpignato & Hunt, 2010; Zeino, Sisson, & Bjarnason, 2010). The
frequency and severity of lower GI toxicity of NSAIDs such as Diclofenac
sodium (DCF) is frequently underestimated (Scarpignato &Hunt, 2010;
Zeino et al., 2010). The fact that there are no proven-effective treat-
ments for NSAID-enteropathy likely contributes to the lack of recogni-
tion of this serious condition (Wallace, 2012). This is a major clinical
concern as NSAID induced enteropathic damage is more difficult to
detect, more expensive to treat, and is associated with longer hospital
stays and higher mortality rates, com pared to gastropathic damage
(Lanas et al., 2009; Wallace, 2013).
The most com mon approach used clinically to minim ize NSAID
induced gastropathic injury has been the co-administration of a proton
pump inhibitor (PPI) with the NSAID (Wallace, 2012). This has
been show n to significantly attenuate the incidence and severity of
gastro-duodenal damage (Scarpignato & Hunt, 2010; Scheiman et al.,
2006), but recent evidence from anim al studies suggest that these
Journal of Pharmacological and Toxicological Methods 78 (2016) 66–75
Abbreviations: DCF, Diclofenac sodium; EB, Evans Blue; Hb, Haemoglobin; HCT,
Haem atocrit; LPO, Lipid peroxidation; MDA, Malondialdehyde; NSAIDs, Nonsteroidal
anti-inflammatory drugs; PPIs, Proton pump inhibitors; PTZ, Pantoprazole sodium; ROS,
Reactive oxygen species..
☆ Institutional Communication Reference Number: PERD440815.
⁎ Corresponding author at: B. V. Patel Pharmaceutical Education and Research
Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad – 380054, Gujarat, India.
E-mail address: manishnivsarkar@gmail.com (M. Nivsarkar).
http://dx.doi.org/10.1016/j.vascn.2015.11.008
1056-8719/© 2015 Elsevier Inc. All rights reserved.
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gastroprotective drugs rather synergistically exacerbate NSAID-induced
small intestinal injury and bleeding (Satoh, Amagase, &Takeuchi, 2014;
Wallace et al., 2011). There are several clinical studies too that report
high levels of intestinal damage in healthy volunteers taking NSAIDs
plus a PPI (Fujim ori et al., 2010; Goldstein et al., 2005; Maiden,
Thjodleifsson, Theodors, Gonzalez, & Bjarnason, 2005).
Thus, it is imperative to find novel therapeutic agents to prevent the
NSAID-induced gastroenteropathy as well as the PPI induced exacerba-
tion of NSAID enteropathy. In this purview, future studies need to
include a more rigorous evaluation of the therapeutic interventions or
novel NSAIDs in the entire GI tract, rather than focusing almost entirely
on gastro-duodenal region. In addition, we also need to examine the GI
safety of these compounds when administered together with PPIs, as
PPIs have become the mainstay therapy for preventing NSAID-induced
gastro-duodenal damage.
Many studies have been done for investigating the pathogenesis of
GI lesions and for screening drugs for the treatm ent of GI ulcers in
humans, however, in most of those studies or the models thereof, the
focus has been on either NSAID-induced gastric (gastropathy) or small
intestinal (enteropathy) injury (Tajima, 2014; Wallace, 2012). Likewise,
to model NSAID induced gastropathy and/or enteropathy different dose
of NSAIDs, dosing regimen and rats with different nutritional status (fed
and non-fed rats) have been used (Atchison, Balakumaran, et al., 2000;
Cheung, Kim, Park, & Kim, 2014; Fornai et al., 2014; Kim et al., 2005;
Satoh et al., 2014; Saud, Nandi, Ong, Finocchiaro, & Levine, 2005).
However, these approaches have their own limitations and do not
m im ic the clinical scenario of NSAID use in humans and may not
give the com plete idea about the entire GI safety/efficacy/toxicity of
either novel and current NSAIDs or any other therapeutic agent
aim ed at preventing GI toxicity of NSAIDs ( Blackler, Syer, Bolla,
Ongini, & Wallace, 2012), thus increasing the chances of failure or
safety concerns at a later stage. For exam ple, the drugs w hich m ay
prove to be gastroprotective in m odel of NSAID induced gastropathy
m ay not be effective in preventing enteropathic dam age or rather
prove to be detrim ental in nature; like the case w ith PPIs (Wallace
et al., 2011).
This likely necessitates the development of animal models with both
gastric (gastropathy) as well as small intestinal (enteropathy) injury
sim ulatin g the clin ical scen ario of NSAID use, that is; a m odel for
NSAID-induced gastroenteropathy. Indeed, this approach will m ake
the data more predictive of the hum an response, therefore, provid-
ing m ore insight on the potential GI toxicity of current and novel
NSAIDs or efficacy/safety of drugs intended for use as treatm ents of
the GI toxicity of NSAIDs. In the present study, w e have developed
a rat m odel for NSAID-induced gastroenteropathy. In addition,
w e sim ulated the com m on clinical scenario of co-adm inistration
of NSAID and PPI to explore if PPI contribute to exacerbation of
NSAID-enteropathy.
2. Materials and methods
2.1. Chemicals and assay kits
Diclofenac sodium (DCF) and Pantoprazole sodium (PTZ) w ere
purch ased from Yarrow Ch em Products, Mum bai, In dia. Sodium
bicarbonate ( NaHCO3) and carboxym ethyl cellulose ( CMC) w ere
products of Him edia, Mum bai, India. The assay kits for total protein,
album in, alanine transam inase (ALT) and bilirubin (both total and
direct) w ere p roducts of ERBA Diagn ostics Man nh eim Gm bH,
Man nh eim , Germ an y. Heparin w as purch ased from Biological E.
Ltd., Hyderabad, India. Heparinised capillaries w ere purch ased
from Him edia, Mum bai, India. Rodent diet w as obtained from VRK
nutrition, Pune, India. All other reagents used in this study w ere of
analytical grade an d procured from a leading ch em ical house in
India.
2.2. Animals
Male, Wistar rats (n = 24), 4–6 months old, weighing 200–250 g
were obtained from the anim al house of B. V. Patel PERD Centre,
Ahmedabad, India. All anim als were housed in clean, polypropylene
cages (three animals per cage) and were allowed to acclimatize for a
week before any experiment. A 10%air exhaust conditioning, relative
humidity (60 ± 5%), temperature (25 ± 3 °C) and 12:12 h light:dark
cycle was maintained in the animal house facility (Reg. No. 1661/PO/
a/12/CPCSEA) in accordance with Good Laboratory Practise (GLP)
mentioned in Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA) guidelines. Balanced rodent food
pellet and water was provided ad libitum. All experimental protocols
were reviewed and accepted by the Institutional Animal Ethics Commit-
tee prior to initiation of the experiment.
2.3. Experimental design
2.3.1. Preparation of drugs for administration
Doses required for the study were calculated according to the body
weight of the experimental anim als. Required quantity of DCF was
suspended in 1%CMC and PTZ was suspended in 1%CMC containing
1%NaHCO3 for oral adm inistration (Satoh et al., 2014). Drugs were
prepared just before the experiments and administered in a volume of
0.2 ml/100 g body weight. The same volume of a solution containing
1%CMC was used as a vehicle. A small volume of drug vehicle was
employed in order to m inim ize any possible interference with the
effects of test drugs, as a consequence of repeated daily administrations.
2.3.2. Induction of gastrointestinal damage/dosage regimen
Animals were divided into 4 groups: Group 1: Treated with vehicle
for 10 days, referred as NC group henceforth, Group 2: Treated with
PTZ (10 mg/kg body weight peroral, p.o.) tw ice daily for 10 days,
referred as PTZ group henceforth, Group 3: Treated with vehicle for
10 days and DCF (9 m g/kg body weight, p.o) twice daily on the
final 5 days, referred as DCF group henceforth, Group 4: Treated with
PTZ (10 m g/kg body weight p.o.) tw ice daily for 10 days and DCF (9
mg/kg body weight, p.o) tw ice daily on the final 5 days, referred as
DCF + PTZ group henceforth. The dose and duration of DCF treatment
was selected on the basis of previous literature (Reuter, Davies, &
Wallace, 1997; Satoh, Amagase, & Takeuchi, 2012; Satoh et al., 2014;
Wallace et al., 2011) and preliminary studies. In preliminary studies,
DCF at the dose of 9 mg/kg body weight twice daily when administered
for 5 days (6th to 10th day) was effective in producing gastric as well as
intestinal ulcers without any mortality, while, slightly higher dose of
DCF (10 mg/kg twice daily for 5 days) resulted in death of all the rats
on fifth day and lower dose did not produce lesions in all rats (data
not shown). Dose of PPI was taken as per earlier studies where, PPIs at
the dose of 10 mg/kg have been shown to sufficiently inhibit gastric
acid secretion (N99%) in rats (Wallace et al., 2011). In previous clinical
studies, therapeutic dose of NSAIDs has been reported to cause
gastroenteropathic damage in humans and PPIs have been reported to
exacerbate the enteropathic damage (Fujimori et al., 2010; Kuramoto
et al., 2013; Maiden, 2009; Watanabe et al., 2008). Here, our prim e
objective was the development of a novel model for gastroenteropathy
in rats which may be used in assessing the entire GI safety/efficacy/tox-
icity of therapeutic agent aimed at preventing the GI toxicity of NSAIDs,
thus reducing the chances of failure or safety concerns at a later stage.
PTZ was administered 30 min (min) prior to DCF administration. After
the last dose on 9th day, rats in all the groups were fasted but water
was provided ad libitum. Tw elve hours after the last dose of the test
drugs on 10th day, rats were anaesthetised with isoflurane, blood
samples were collected from each animal and later euthanized by CO2
asphyxiation.
67D.P. Singh et al. / Journal of Pharmacological and Toxicological Methods 78 (2016) 66–75

2.4. Changes in body weight and food intake
During the study, body weight and daily food intake was measured
each morning. Animal body weight was monitored daily so that drug
doses could be adjusted accordingly. The percentage (%) change in
body weight was calculated by comparing it with the body weight of
day 0. Rats were presented with the sam e am ount of food and their
intake was measured the follow ing day by subtracting the uneaten
food. This was done for 9 consecutive days and calculated as food intake
in g per rat per day.
2.5. Haemoglobin and haematocrit level estimation
Blood sam ples (0.25 ml) were collected from all the animals
from retro orbital sinus under isoflurane anaesthesia in heparinized
1 ml micro centrifuge tubes before starting the dosing as well as 12 h
after the last dose of the test drugs on 10th day, for the determ ina-
tion of the changes in haem oglobin ( Hb) and haem atocrit (HCT)
levels (Wallace et al., 2011). This study w as perform ed in an auto-
m ated haem atology analyser ( VetScan HM-5; Abaxis Inc., Union
City, CA, USA).
2.6. Serum biochemical assays
Blood samples (0.25 ml) were collected from all the animals from
retro orbital sinus under isoflurane anaesthesia in non-heparinized
1 ml micro centrifuge tubes before starting the dosing as well as 12 h
after the last dose of the test drugs on 10th day. Serum levels of albumin,
total protein, ALTand bilirubin (both direct and total) were determined
using fully automated random access clinical chemistry analyser
(Transasia EM 360).
2.7. Tissue collection
After euthanizing the animals, their abdomens were opened imme-
diately and both stom ach and small intestine were removed. The
contents of the stomach and intestine were collected into sterile tubes.
Stom achs were opened along the larger curvature and rinsed with
cold phosphate-buffered saline (PBS,0.01 M, pH = 7.4). Small intestines
were opened longitudinally along the anti-mesenteric border and
rinsed with cold PBS (Mei, Diao, Xu, X-c, & Jin, 2011) and later were
fixed in 10%neutral buffered formalin for 24 h, washed and transferred
to 70%ethanol for 30 min before observation for haemorrhagic damage
(Atchison, West, et al., 2000; LoGuidice, Wallace, Bendel, Redinbo, &
Boelsterli, 2012). All the tissues were subjected to the further experi-
mental procedures as discussed later, where observer was blinded for
respective evaluations and/or measurements.
2.8. Evaluation of gastrointestinal damage (macroscopic evaluation)
Stomachs were inspected for the presence of haemorrhagic lesions
before fixation, because gastric mucosal injury is easier to detect in the
unfixed state. Small intestines were evaluated for lesions after fixation
because intestinal ulcers are easier to see in fixed tissues (Atchison,
West, et al., 2000; LoGuidice et al., 2012). Small intestines were
m easured in length from the pyloric sphincter to the ileocolic valve
and then divided into five equal parts (Atchison, West, et al., 2000).
The third part was cut out and reserved for biochemical studies in all
the groups. Separate gastric and intestinal damage scores (lesion
index) were calculated by summ ing up the lengths in millim etre
(mm ) of all the lesions for each rat (Satoh et al., 2014). An observer
without knowledge of the anim al treatment measured their lengths
with a digital vernier calliper (Absolute AOS Digimatic; Mitutoyo,
Japan).
2.9. Effect of drugs on the gastrointestinal luminal pH
Rats in all the groups were evaluated for alteration in luminal pH.
The contents of the stomach and intestine were collected into separate
sterile tubes. The pH of the contents was measured with a digital pH
metre (CyberScan pH Tutor, Eutech Instruments).
2.10. Estimation of lipid peroxidation
Stomach and intestinal tissues were taken separately in 5 ml
of Hank's balanced salt solution (HBSS, pH 7.4) and homogenized at
5000 rpm, using a Polytron homogenizer (3 cycles of 30 s each;
Kinematica, Switzerland). The hom ogenate was then centrifuged
at 3500 rpm for 10 m in using Sorvall, legend X1R centrifuge. The
pellet was re-suspended in 0.1 ml of HBSS and used for estimation of
lipid peroxidation. Lipid peroxidation was m easured in term s of
malondialdehyde (MDA):thiobarbituric acid (TBA) reaction as reported
by Ohkawa and co-workers (Ohkawa, Ohishi, & Yagi, 1979). The reac-
tion m ixture contained 0.1 m l of tissue hom ogenate (as described
above), 0.2 ml of 8.1%sodium dodecyl sulphate,1.5 ml of 20%acetic
acid (pH adjusted to 3.5 with 1 M NaOH), and 1.5 ml of 0.8%aqueous
solution of TBA. The reaction mixture was made to a volume of 4 ml
with the addition of 0.7 m l of double distilled water and heated at
95 °C for 1 h in a water bath. After heating, 1 ml of double distilled
water and 5 ml of a mixture of nbutanol and pyridine (15:1 v/v) were
added and the mixture was shaken vigorously on a vortex mixer for
5 min. This mixture was then centrifuged at 3000 rpm for 7 min. After
centrifugation the upper organic layer was separated and the amount
of MDA form ed in this layer was measured at 532 nm using an ultra
violet/visible spectrophotom eter (Shim adzu UV-1800). Appropriate
controls were used at different steps during this estimation (extinction
coefficient of MDA is 1.45 × 10− 5
/min/cm).
2.11. Determination of intestinal permeability by Evans blue
The intestinal epithelial permeability was examined by Evans blue
(EB) assay as previously described (Lange, Delbro, & Jennische, 1994;
Mei et al., 2011). Non-absorbed macromolecules, such as EB, are often
used as probes in intestinal perm eability (IP) tests. Briefly, a small
bowel sac of 5 cm length beginning at 3 cm proximal to the ileocecal
valve was prepared. The proximal and distal portion was ligated and
0.2 ml of 1.5%(w/v) EBin PBS was infused into the lumen. The intestinal
sacs were incubated in 20 m l of Krebs buffer at 95% O2 and 37 °C.
They were removed 30 min later, washed three times in 6 m mol/L
acetylcysteine, dried on filter paper at 37 °Cfor 24 h, and later incubated
with 1 ml of formamide for 24 h. The amount of dye eluted was estimat-
ed using a wavelength of 620 nm using an ultra violet/visible spectro-
photometer (Shimadzu UV-1800). The amount of EB permeating into
the intestinal wall was calculated based on a standard curve of EB in
formamide.
2.12. Histopathological studies
To confirm the formation of gastric and intestinal ulcers, at the end
of the study, samples of stomach and intestine bearing the representa-
tive lesions were excised and maintained in 10%neutral buffered forma-
lin for 24 h. Serial paraffin sections were then prepared from the fixed
tissues and stained with haem atoxylin (H) and eosin (E). The slides
were observed and photo-documented by optical microscopy (IX 51;
Olympus, Tokyo, Japan) equipped with a digital camera (TL4) in order
to confirm the presence of GI damage.
2.13. Statistical analysis
Data were expressed as the mean ± standard deviation (SD) (N =
6). Statistical analysis was performed using 1-way analysis of variance
68 D.P. Singh et al. / Journal of Pharmacological and Toxicological Methods 78 (2016) 66–75

(ANOVA) followed by the Holm-Sidak's Multiple Com parison test to
determine the significance of differences among groups. Post hoc test-
ing was done after ANOVA only if F was significant. Mean values were
considered statistically significant at P b 0.05. Graph Pad Prizm 6.0
was used for statistical calculations.
3. Results
3.1. Changes in food intake and body weight
The average daily food intake of rats in PTZ, DCF and DCF + PTZ
groups was significantly lower com pared to NC group (P b 0.05)
(Fig. 1). A steep decrease in daily food intake was observed in DCF and
DCF + PTZ groups, when DCF treatment was initiated on 5th day. The
average food intake in PTZ group kept on declining over the period of
9 days. Largest decline in food intake was observed in DCF + PTZ
group and was significantly different compared to PTZ and DCF groups
(P b 0.05).
Similar changes were observed in body weight of rats in all the
groups. The percentage (%) change in body weight of rats in PTZ, DCF
and DCF + PTZ groups differed significantly com pared to NC group
(P b 0.05) (Fig. 2). An increasing trend in body weight was observed
in rats of NC group, while the body weight of rats in all other groups
decreased over the period of 10 days. Largest decline in body weight
was observed in DCF + PTZ group, which was significantly different
compared to PTZ and DCF groups (P b 0.05) respectively (Fig. 2).
3.2. Evaluation of gastrointestinal damage (macroscopic evaluation)
DCF administration over 5 days (twice daily) resulted in high levels
of haemorrhagic damage in the stomach and small intestine. When DCF
was administered to rats receiving PTZ i.e. in DCF + PTZ group, gastric
damage was significantly attenuated, but intestinal damage was signif-
icantly worsened (Figs. 3-6). Indeed, Lesion indices (indexes) of
stomach in DCF and DCF + PTZ groups were 14.60 ± 3.95 m m and
3.44 ± 1.57 mm respectively and intestinal lesion indices of DCF and
DCF+ PTZgroups were 31.36 ± 4.25 mm and 43.68 ± 7.32 mm respec-
tively (Fig. 6). While, there was no gastric injury in rats of NC group,
little inflam m ation w as observed in th e stom ach of rats in PTZ
group, but ulceration w as absent ( Fig. 3). Gastric lesion index of
DCF group w as significantly m ore com pared to NC, PTZ and
DCF + PTZ groups (P b 0.05) respectively. While, there was no signif-
icant difference betw een the gastric lesion indices of NC, PTZ and
DCF + PTZ groups indicating that PTZ significantly attenuated the
DCF induced gastric injury (Fig. 6).
Intestinal lesion indices of rats in DCF and DCF + PTZ groups were
significantly different compared to NC and PTZ groups (P b 0.05).
However, intestinal lesion index of DCF + PTZ group was significantly
more compared to DCFgroup (P b 0.05) indicating that PTZsignificantly
exacerbated the DCFinduced enteropathic damage. While, there was no
significant difference between the lesion indices of NC and PTZ groups
(Fig. 6).
3.3. Effect of drugs on the gastrointestinal luminal pH
As shown in Fig. 7, administration of DCF alone as expected resulted
in significant reduction in gastric pH but, interestingly we observed that
intestinal luminal pH was also decreased. In contrast, PTZ administra-
tion significantly increased the gastric pH (P b 0.05) but intestinal lumi-
nal pH was not altered significantly compared to NCgroup. Indeed, both
gastric and intestinal luminal pH in DCF group was significantly lower
compared to NC and PTZ groups (P b 0.05). Gastric luminal pH in DCF
group was significantly lesser compared to PTZ group (P b 0.05). How-
ever, co-administration of DCFand PTZresulted in significant increment
in gastric and surprisingly in the intestinal luminal pH also. While, com-
pared to NC and DCF groups, both gastric and intestinal luminal pH of
DCF + PTZ group was significantly higher (P b 0.05). Intestinal luminal
pH in DCF + PTZgroup was significantly higher compared to PTZ group
also (P b 0.05), while, there was no significant difference between the
intestinal luminal pH of PTZ and NC groups.
3.4. Haemoglobin and haematocrit level estimation
After sacrifice, during necropsy, blood was evident in the lumen of
all the rats of DCF and DCF + PTZ groups and ulcers were clearly
evident, except in the stomachs of rats in DCF + PTZ group (Figs. 3-5).
Consistent with the presence of luminal blood, administration of DCF
and its co-adm inistration with PTZ in DCF and DCF + PTZ groups
respectively; resulted in a significant decrease in Hb and HCT levels
compared to NC and PTZ groups (P b 0.05) (Fig. 8). While, compared
with the DCF group, there was a significant decrease in the Hb and
HCT levels in the DCF + PTZ group (23.2 ± 4.3%vs 16.8 ± 1.8%, and
16.2 + 1.9%vs 10.5 ± 2.2%, respectively, P b 0.05). These observations
indicate that co-administration of PTZ with DCF exacerbated the DCF-
induced small intestinal damage and subsequently enhanced the loss
of blood; even though PTZ significantly attenuated DCF-induced gastric
injury. However, PTZ alone had no significant effect on either Hb or HCT
levels.
Fig. 1. Effect of different treatments on average daily food intake of rats in various
groups. The average daily food intake of rats in PTZ, DCF and DCF + PTZ groups was sig-
nificantly lower compared to NC group and largest decline was observed in DCF + PTZ
group, which was significantly different compared to PTZ and DCF groups (*P b 0.05).
Fig. 2. Effect of different treatments on average body weight of rats in various groups
(N = 6). The percentage change in body weight in PTZ,DCFand DCF+ PTZgroups differed
significantly compared to NCgroup, while, largest decline in body weight was observed in
DCF+ PTZgroup,which was significantly different compared to PTZand DCFgroups (⁎P b
0.05). Data expressed as mean ± SD.

3.5. Serum biochemical assays
Consistent with the declines in Hb and HCT levels, treatm ent-
associated significant declines in serum levels of albumin and total
protein were observed in DCF and DCF + PTZ groups compared to NC
and PTZ groups (P b 0.05) (Fig. 9). Whereas, compared with the DCF
group, there was a significant decrease in the serum albumin and total
protein levels in the DCF + PTZ group (26.2 ± 4.1%vs 15.9 ± 6.1%
and 54.8 ± 1.6%vs 42.8 ± 2.4%respectively, both P b 0.05). However,
PTZ alone had no significant effect on either albumin or total protein
levels. Neither serum ALT or bilirubin levels increased after treatment
(data not shown). These negative observations indicate that our animal
model of NSAID-induced gastroenteropathy is not confounded by he-
patic leakage or dysfunction.
Fig. 3. Macroscopic observation of gastropathic damage in various treatment groups. (A) NC (B) PTZ (C) DCF (D) DCF + PTZ. DCF administration over 5 days (9 mg/kg twice daily)
resulted in high levels of haemorrhagic damage in the stomach. When DCF was administered to rats receiving PTZ i.e. in DCF + PTZ group, gastric damage was significantly attenuated.
PTZ alone did cause little inflammation but no ulcers were seen (B). Arrows indicate macroscopic damage.
Fig. 4. Macroscopic observation of enteropathic damage in various treatment groups (before fixation). (A) NC (B) PTZ (C) DCF (D) DCF + PTZ. DCF administration over 5 days (9
mg/kg twice daily) resulted in high levels of haemorrhagic damage in the intestine. When DCF was administered to rats receiving PTZ i.e. in DCF + PTZ group, enteropathic damage
was significantly exacerbated. PTZ alone did cause little inflammation but no ulcers were seen (B). Arrows indicate macroscopic damage.

3.6. Estimation of lipid peroxidation
As shown in Fig. 10, the gastric MDA level of DCF group was signifi-
cantly higher compared to NC, PTZ and DCF + PTZ groups (P b 0.05).
Additionally, it is evident from results that co-adm inistration of DCF
and PTZ resulted in significant reduction in the gastric MDA levels of
DCF + PTZ group. How ever, no significant difference was observed
between the gastric MDA levels of NC, PTZ and DCF + PTZ groups. In
contrast, the intestinal MDA level of the DCF + PTZ group was signifi-
cantly higher compared with the DCF group (P b 0.05), indicating that
DCF caused oxidative dam age and inflamm ation in sm all intestinal
mucosa and PTZ exacerbated this damage when combined with DCF.
Indeed, the small intestinal MDA levels of DCF and DCF + PTZ groups
were significantly higher compared to NC and PTZ groups (P b 0.05).
PTZ alone did not elicit any significant change in MDA levels in both
stomach and intestinal tissues.
3.7. Effect of drugs on intestinal permeability
As discussed earlier, adm inistration of either DCF alone or with
PTZ resulted in significant injury in intestine, accordingly, the amount
of EB which had perm eated into the intestinal wall in the DCF and
Fig. 5. Macroscopic observation of enteropathic damage in varioustreatment groups (after fixation in 10%neutral formalin for 24 h followed by treatment with 70%ethanol for
30 min). (A) NC (B) PTZ (C) DCF (D) DCF + PTZ. DCF administration over 5 days (9 mg/kg twice daily) resulted in high levels of haemorrhagic damage in the intestine. When DCF was
administered to rats receiving PTZi.e.in DCF+ PTZgroup,enteropathic damage was significantly exacerbated. PTZalone did cause little inflammation but no ulcers were seen (B).Arrows
indicate macroscopic damage.
Fig. 6. Effect of various treatments on the gastrointestinal lesion index of rats in var-
ious groups (N = 6). DCF administration over 5 days (9 mg/kg twice daily) resulted in
high levels of gastroenteropathic damage. Gastric lesion index of DCF group was signifi-
cantly more compared to NC, PTZ and DCF + PTZ groups (*P b 0.05) respectively. Co-ad-
ministration of PTZ with DCF significantly attenuated the DCF induced gastric injury
while intestinal damage was significantly worsened. Intestinal lesion indices of rats in
DCF and DCF + PTZ groups were significantly higher compared to NC and PTZ groups
(*P b 0.05). Additionally, intestinal lesion index of DCF + PTZ group was significantly
higher compared to DCF group (*P b 0.05). However, there was no significant difference
between the gastric lesion indices of NC, PTZ and DCF + PTZ groups and intestinal lesion
indices of NC and PTZ groups. Data expressed as mean ± SD.
Fig. 7. Effect of various treatments on gastrointestinal luminal pH of rats in various
groups (N = 6). Gastric and intestinal luminal pH in DCF group was significantly lower
compared to NC group, in contrast, PTZ administration significantly increased the gastric
pH compared to NC group (*P b 0.05) but, intestinal luminal pH was not altered signifi-
cantly. However, compared to NC and DCF groups, both gastric and intestinal luminal
pH of DCF + PTZ group was significantly higher (*P b 0.05). However, there was no signif-
icant difference between the intestinal luminal pH of PTZ and NCgroup. Data expressed as
mean ± SD.

DCF + PTZ group was also significantly higher compared to the NC and
PTZ groups with largest increase in IP observed in DCF + PTZ group,
which was significantly higher compared to DCF group (P b 0.05)
whereas, PTZ alone had no significant effect (Fig. 11). These results
further substantiated that, DCF damaged the small intestinal mucosal
barrier and addition of PTZ further exacerbated it.
3.8. Histopathological study
Consistent with the macroscopic observations (Figs. 3-6), observa-
tion of histological photom icrographs revealed focal erosions of the
superficial epithelium, epithelial stratification and perforations, basal
lamina degeneration, and infiltration with neutrophils in stomach
sections of rats in DCF and intestinal sections of DCF and DCF + PTZ
groups. From histopathological exam ination it was evident that PTZ
attenuated DCF induced gastric injury but, exacerbated the intestinal
injury caused by DCF. Microscopic observation revealed no ulceration
in PTZ and NC groups (Fig. 12).
4. Discussion and conclusion
GI toxicity of NSAIDs often results in poor patient compliance and
limits the clinical utility of these drugs (Scarpignato & Hunt, 2010).
Recent reports suggest that NSAIDs pose equal risk of developing
gastropathic as well as enteropathic damage, that is, gastroenteropathy
in humans (Lanas et al., 2009; Lim & Yang, 2012; Sostres, Gargallo, &
Lanas, 2013). Currently, there are no approved therapeutic strategies
to prevent NSAID induced enteropathic damage (Blackler et al., 2015)
and although antisecretory drugs such as PPIs are highly effective in
preventing NSAID induced gastropathic damage but emerging evidence
indicates that these drugs rather worsen the small intestinal damage
caused by NSAIDs (Satoh et al., 2014; Wallace et al., 2011; Watanabe
et al., 2013).
Fig. 8. Effect of various treatments on blood haemoglobin and haematocrit of rats in
various groups (N = 6). Adm inistration of DCF alone and its co-administration with
PTZ in DCF and DCF + PTZ groups respectively; resulted in a significant decrease in Hb
and HCT levels compared to NC and PTZ groups (*P b 0.05). While, compared with the
DCF group, there was a significant decrease in the Hb and HCT levels in the DCF + PTZ
group (*P b 0.05). However, PTZ alone had no significant effect on either Hb or HCT levels.
Data expressed as mean ± SD.
Fig. 9. Effect of various treatments on various biochemical parameters of rats in vari-
ous groups (N = 6). Treatment-associated significant declines in serum levels of albumin
and total protein were observed in DCF and DCF + PTZ groups compared to NC and PTZ
groups (*P b 0.05). Whereas, compared with the DCF group, there was a significant de-
crease in the serum albumin and total protein levels in the DCF + PTZ group (*P b 0.05).
However, PTZ alone had no significant effect on either albumin or total protein levels.
Data expressed as mean ± SD.
Fig. 10. Effect of various treatments on lipid peroxidation (LPO; MDA levels) in the
gastrointestinal tissues of rats in various groups (N = 6). Gastric MDA level of DCF
group was significantly higher compared to NC, PTZ and DCF + PTZ groups (*P b 0.05).
While, MDA levels of the small intestinal homogenates of DCF and DCF + PTZ groups
were significantly higher compared to NC and PTZ groups and the intestinal MDA level
of the DCF + PTZ group was significantly higher compared with the DCF group (*P b
0.05). PTZ alone did not elicit any significant change in MDA levels in both stomach and
intestinal tissues. Data expressed as mean ± SD.
Fig. 11.Effect of varioustreatmentson alteration in intestinal permeability (IP) of rats
in variousgroups(N = 6). IP of rats in DCFand DCF+ PTZgroup was significantly higher
compared to the NC groups (*P b 0.05), whereas, PTZ alone had no significant effect. IP in
DCF + PTZ group was significantly higher compared to DCF group (*P b 0.05). Data
expressed as mean ± SD.

Thus, it is imperative to find ideal therapeutic interventions
which are not only effective in preventing NSAID-induced gastric as
well as enteropathic damage but are also GI safe. Thus, undoubtedly,
these therapeutic interventions should also be tested for their complete
GI safety and efficacy alone as well as in com bination with PPIs
and other such drugs at preclinical level. In this purview, a more simu-
lating rat model to the clinical situation of NSAID use could be highly
useful.
While, in earlier studies, researchers have mostly used different dose
and duration of NSAID treatm ent to induce gastropathic and/or
enteropathic damage in different groups of rats (Atchison, West, et al.,
2000; Cheung et al., 2014; Fornai et al., 2014; Kim et al., 2005; Saud
et al., 2005), we thought of a single rat model for NSAID-induced
gastroenteropathic injury simulating the clinical scenario of NSAID use
in humans. In addition, we modelled the common clinical scenario of
co-administration of NSAID and PPI to explore if PPI contribute to exac-
erbation of NSAID-enteropathy.
In the present work, to induce gastroenteropathic damage in rats,
we administered DCF at a dose of 9 mg/kg twice daily for 5 days and
indeed,all the rats developed significant gastric and enteropathic injury.
We also observed that pre-treatment with PTZ for 5 days followed by
its co-adm inistration with DCF for the next five days resulted in
significant exacerbation of enteropathic damage; while DCF induced
gastropathy was significantly attenuated. Our results were similar to
the NSAID-induced enteropathic damage and its exacerbation by PPIs
as reported by Wallace et al. (Satoh et al., 2014; Wallace et al., 2011)
and gastropathic damage and its attenuation by PPIs as reported by
Blandizzi and co-workers (Blandizzi et al., 2005). In clinical settings,
both Goldstein and co-workers (Goldstein et al., 2005) and Hawkey
et al. (Hawkey et al., 2008) observed an increase in small intestinal
lesions in healthy volunteers taking a combination of PPI and NSAID.
However, our results are contrary to those reported by others, where
lansoprazole was reported to prevent the NSAID induced enteropathic
damage (Higuchi et al., 2009; Kuroda et al., 2006). This anom aly in
results needs to be further investigated.
However, this PPI induced exacerbation of NSAID-induced intestinal
injury has often been linked to dysbiosis (Wallace et al., 2011); the
phenomenon often described in humans with the use of antisecretory
drugs such as PPIs (Compare et al., 2011; Lombardo, Foti, Ruggia, &
Chiecchio, 2010; Spiegel, Chey, &Chang, 2008). It has also been reported
that administration of NSAIDs too results in significant alterations in the
composition of small bowel microbiota, often with increases in the
numbers of Gram-negative bacteria (Hagiwara, Kataoka, Arim ochi,
Kuwahara, & Ohnishi, 2004; Wallace et al., 2011). However, since pH
variability along the distal intestine could also affect microbiota compo-
sition (DiBaise et al., 2008; Zhang, Sparks, Karyala, Settlage, & Luo,
2015), thus, looking into this aspect, we were curious whether drug
treatments in various groups altered GI pH also. Indeed, we found
while, PTZ alone increased the gastric pH but it did not alter the intesti-
nal pH significantly. How ever, DCF alone significantly decreased the
gastric and intestinal luminal pH, and its co-administration with PTZ
resulted in significant elevation of intestinal luminal pH. Indeed, such
an effect may play important role in the alteration of gut microbiota
(Zhang et al., 2015), but it is quite a possibility that the altered pH
could be a consequence of the changes in the microbiota with the use
of PPIs and NSAIDs. How ever, this aspect needs to be elucidated in
future studies.
During the duration of study, we also observed significant reduction
in food intake and body weight of rats according to the model
progression in all the groups except those in the NC group. Though,
the decline in food intake and bodyweight of rats in DCF and
DCF + PTZ group was understandable but in PTZ group it might be
due to the ability of PPIs to cause gastrointestinal inflammation limiting
intake of food (Graham &Genta, 2008; Kuipers, 2006). Recently, PPI use
in humans have been reported to increase the faecal calprotectin levels,
which is an important inflammatory biomarker (Andréasson, Scheja,
Saxne, Ohlsson, & Hesselstrand, 2011; Poullis, Foster, & Mendall,
2003). Indeed, we observed little inflammation in gastric as well as
intestinal region of rats which were fed PTZ alone, but, there was no
ulceration (Fig. 3).
The colour of the faeces also changed from brown (5th day) to black
(10th day) in DCF and DCF + PTZ groups, which might be due to signif-
icant GI bleeding (Lanas et al., 2012; Rainsford, Stetsko, Sirko, &Debski,
2003). Indeed, in our study, we observed a significant decline in Hb,
HCT, serum albumin and serum total protein levels in DCF and
DCF + PTZ groups, indicating GI bleeding, with maximum decline in
DCF + PTZ group. However, neither serum ALT or bilirubin levels
increased after treatment, indicating that our animal model of NSAID-
induced gastroenteropathy is not confounded by hepatic leakage or
dysfunction. These observations are in line with the previous reports
(Kim et al., 2005; Ramırez-Alcántara, Castaneda-Hernández, Rampy,
Aronson, & Treinen-Moslen, 2005).
Fig. 12. Typical microscopic observations of histological sections of the stomach (A-D) and small intestine (E-H) of rats in different treatment groups. H&E-stained sections of
stomach and small intestine under 10X magnification. Histology revealed the focal erosions of the superficial epithelium, epithelial stratification and perforations, basal lamina degener-
ation, and infiltration with neutrophils in stomach sections of DCF and intestinal sections of rats in DCF and DCF + PTZ groups. PTZ attenuated DCF induced gastric injury while DCF-in-
duced intestinal injury was exacerbated. Arrows indicate mucosal erosions and ulceration.

Our results also demonstrated that administration of DCFresulted in
significant elevation of MDA; a marker of LPO or oxidative stress (Mei
et al., 2011). While co-administration of PTZ with DCF reduced gastric
MDA level, but, intestinal MDA level was comparatively elevated.
Higher MDA levels indicate higher reactive oxygen species (ROS) in GI
tissues (Kamanlı, Nazıroğlu, Aydılek, & Hacıevlıyagil, 2004; Mei et al.,
2011), which in turn may result in mitochondrial dysfunction, leading
to epithelial tight junction dysfunction resulting in an enhanced IP
(Mei et al., 2011). Indeed, in our study, we found while DCF alone
caused significant increment in IP, its co-administration with PTZ
further enhanced it. These observations were quite evident as there
was highest intestinal m ucosal ulceration in DCF + PTZ group. Over
the years, altered IP has been identified as an important contributor in
the pathogenesis of NSAID induced GI damage (Bjarnason & Takeuchi,
2009), since resultant increased IP that ensues exposes the stressed
and ulcerated mucosa to noxious agents like bile, bacteria and NSAIDs
resulting in severe GI damage (Bjarnason & Takeuchi, 2009).
In conclusion, this model was set up to simulate the human clinical
situation of NSAID therapy and indeed the present DCF regimen caused
both gastric and sm all bowel alterations, such as multiple erosive
lesions, together with a decrease in haemoglobin, haematocrit, serum
albumin, serum total protein levels and IP alteration, known to occur
in patients receiving NSAIDs (Lanas et al., 2012; Maiden et al., 2005;
Seo et al., 2012; Wallace, 1997; Watanabe et al., 2013). Additionally,
we have provided yet another evidence for PPI induced exacerbation
of NSAID enteropathy. Indeed, the proposed model could be highly
useful in establishing the efficacy of novel therapeutic interventions
for the management of NSAID induced gastroenteropathy and will aid
in simultaneously assuring their gastric as well as intestinal safety either
alone or in combination with PPIs and/or other drugs as well. Transla-
tion of this knowledge into clinically relevant therapeutic interven-
tions/strategies is eagerly awaited.
Conflict of interest
The authors have no potential conflict of interest to declare.
Acknowledgements
The authors are thankful to Dr. John L Wallace, Professor, Depart-
ment of Physiology and Pharmacology, University of Calgary, Calgary,
Alberta, Canada for his advice during the design of experiments.Authors
are highly thankful to B.V. Patel Pharmaceutical Education and Research
Development (PERD) Centre, Ahmedabad, for providing all the facilities
for the successful completion of the work and NIRMA University.
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