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Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.
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Clinical Science (2006) 110, 563–573 (Printed in Great Britain) doi:10.1042/CS20050374 563
Use of growth-hormone-releasing peptide-6
(GHRP-6) for the prevention of multiple
organ failure
Danay CIBRI
´
AN
,HussamAJAMIEH, Jorge BERLANGA
,OlgaS.LE
´
ON
,
Jose S. ALBA
, Micheal J.-T. KIM, Tania MARCHBANK§, Joseph J. BOYLE,
Freya FREYRE
, Diana GARCIA DEL BARCO
, Pedro LOPEZ-SAURA
,
Gerardo GUILLEN
, Subrata GHOSH, Robert A. GOODLAD
and Raymond J. PLAYFORD§
Center for Genetic Engineering and Biotechnology, Ave 31 e/158 & 190 Playa 10600, Havana, Cuba, Center for Biological
Studies, Food and Drug Institute, University of Havana, Ave 23 e/44 & 222 La Coronela, La Lisa, Havana, Cuba,
Department of Gastroenterology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road,
London W12 0NN, U.K., §Imperial College School of Medicine, Barts & The London, Queen Mary’s School of Medicine and
Dentistry, Turner Street, London E1 2AD, U.K., and Cancer Research UK, Lincoln’s Inn Fields, London WC2A 3PX, U.K.
ABSTRACT
Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present
study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on
cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer
(HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given
alone and in combination with the potent protective factor EGF (epidermal growth factor)
in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min
of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion).
In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition
caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P < 0.01),
but did not increase proliferation ([
3
H]thymidine incorporation). In vivo studies showed that,
compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage
(histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid
peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 µg/kg
of body weight, intraperitoneally) alone truncated these effects by 50–85 % (all P < 0.05) and an
additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body
weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments.
In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance
its effects, may provide a novel simple approach for the prevention and treatment of MOF
and other injuries of the gastrointestinal tract. In view of these findings, further studies appear
justified.
Key words: epidermal growth factor (EGF), growth-hormone-releasing peptide (GHRP), gut injury, ischaemia/reperfusion, multiple
organ failure, repair, recombinant peptide.
Abbreviations: ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; DMEM, Dulbecco’s modified Eagle’s medium;
EGF, epidermal growth factor; FCS, foetal calf serum; GH, growth hormone; GHRP, GH-releasing peptide; i.p., intraperitoneally;
I/R, ischaemia/reperfusion; 45 min/45 min I/R, 45 min of ischaemia, followed by 45 min of reperfusion; 90 min/120 min I/R, 90 min
of ischaemia, followed by 120 min of reperfusion; MDA, malondialdehyde; MOF, multiple organ failure; MPO, myeloperoxidase;
rhEGF, recombinant human EGF; SOD, superoxide dismutase; TGF, transforming growth factor; THP, total hydroperoxides.
Correspondence: Professor Raymond J. Playford (email r.playford@qmul.ac.uk).
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an and others
INTRODUCTION
MOF (multiple organ failure) is a severe life-threatening
condition that usually occurs as a result of major trauma,
burns or fulminant infections. Whatever the initiating
event, once established, MOF has a high mortality (up to
80%) [1]. The pathophysiological mechanisms underly-
ing this condition are unclear, although important contri-
butory factors probably include hypoxia, increased intes-
tinal permeability, bacterial translocation, endotoxaemia
and uncontrolled systemic inflammatory responses [2].
Several studies suggest that the splanchnic circulation
is particularly vulnerable to hypoperfusion, as occurs
in low-flow states, such as haemorrhagic shock, and
that this hypoperfusion is out of proportion with the
overall reduction in cardiac output [3]. Although it is
obvious that tissue ischaemia initiates a series of events
that can ultimately lead to cellular dysfunction and
necrosis, resumption of blood flow can paradoxically
create more tissue injury, possibly because of production
of oxygen-derived cytotoxic products [4]. The use of
I/R (ischaemia/reperfusion) models of injury, therefore,
not only have relevance to acute vascular disruption
(thrombosis and embolism) and major hepatic surgery,
including transplantation, but also to the pathogenesis of
development of MOF.
Synthetic and recombinant peptides are being used
increasingly for clinical purposes (e.g. human insulin for
diabetes and erythropoietin for anaemia of renal failure),
but assessment of their value for the treatment of luminal
gastroenterological problems is at a much earlier stage [5].
GH (growth hormone) secretagogues compose a
group of heterogeneous synthetic peptides and non-
peptides that, as well as inducing pituitary GH secretion,
also bind to GH secretagogue receptors on peripheral
tissues, such as the myocardium, pancreas and bone
marrow [6,7]. The physiological role of these peripheral
receptors is, however, unclear and the potential value
of GHRP (GH-releasing peptide)-6 administration on
hepatic and gastrointestinal mucosal integrity is untested.
In this series of studies, we therefore initially examined
whether GHRP-6 had potentially useful ‘pro-healing’ ac-
tivity using various in vitro models of gut injury. Having
found positive results, we progressed to test the effect
of systemic administration of GHRP-6 in a rat liver I/R
model of hepatic injury and MOF. In addition, as we have
found previously a beneficial effect of the potent growth
factor EGF (epidermal growth factor) when using a re-
lated mesenteric I/R model [8], we also examined the res-
ults of giving EGF alone and in combination with GHRP-
6 (to begin to examine additive/synergistic effects).
MATERIALS AND METHODS
Synthetic and recombinant peptides
GHRP-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH
2
)was
purchased from BCN Peptides. The product in a lyo-
philized form, certified as pyrogen- and contaminant-
free, was stored at 20
C and diluted in sterile saline
just prior to its administration.
rhEGF
152
(recombinant human EGF
152
), expressed
in Saccharomyces cerevisiae, was obtained from Heber-
Biotec in a lyophilized form. This product consists of
a 60:40 mixture of EGF
152
and EGF
151
and is as bio-
logically active as the full length EGF
153
form [9]. Prior
to administration, EGF was diluted in 0.9 % saline under
sterile conditions.
Ethics
Experiments were conducted according to current Local
and National regulatory and ethical guidelines.
Study series 1:
in vitro
models
Effect of exogenous GHRP-6 on an
in vitro
cell
migration model
One of the earliest biological repair responses following
injury to tissue cells is the migration of surviving cells
over the denuded area caused by the injury to re-establish
epithelial integrity. Since it is extremely difficult to study
this effect upon tissue inside a human or animal, cell
culture models are commonly used as surrogate markers
of this pro-migratory response. This method also allows
direct actions of the test peptide on the cells to be
determined.
Cell migration assays were performed using our
methods published previously [10]. Briefly, human colo-
nic carcinoma (HT29) cells or rat intestinal epithelial
(IEC6) cells were grown to confluence in six-well
plates in DMEM (Dulbecco’s modified Eagle’s medium)
containing 10 % (v/v) FCS (foetal calf serum) at 37
C
in 5% CO
2
. The monolayers were then wounded by
scraping a disposable pipette tip across the dishes, washed
with fresh serum-free medium and cultured in serum-
free medium in the presence of various test factors.
The rate of movement of the anterior edges of the
wounded monolayers was then determined by taking
serial photomicrographs at various times after wounding
[10]. Twenty measurements per field were performed
by placing a transparent grid over the photograph and
measuring the distance moved from the original wound
line. All results are expressed as means
+
S.E.M. of three
separate experiments.
The various test factors used were GHRP-6 (10–
400 µg/ml) and EGF (10 µg/ml; used as a positive
control). This dose of EGF was used as we have shown
previously [10a] that this stimulates maximal restitution
responses in this system. The importance of TGF (trans-
forming growth factor) β in any response seen was
analysed by using additional wells which contained
GHRP-6 (40 µg/ml) and a TGFβ-neutralizing antibody
(100 µg/ml; R&D Systems).
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Use of GHRP-6 for multiple organ failure 565
Effect of exogenous GHRP-6 on an
in vitro
cell
proliferation model
Cell proliferation assays were performed using our
methods published previously [10]. Briefly, HT29 and
IEC6 cells were grown in DMEM containing 4 mmol/l
glutamine, 10 % (v/v) FCS and various test factors. Effects
of addition of various doses of GHRP-6 and EGF
(10 µg/ml; used as a positive control) were subsequently
tested under serum-starved conditions.
To assess the degree of proliferation, [
3
H]thymidine
(2 µCi/well) was included 24 h after the addition of the
test factors, and cells were left for a further 24 h. For
each condition, the stimulatory or inhibitory effect of the
solutions was measured in quadruplicate in six separate
wells. Cell viability, determined by the ability to exclude
0.2% Trypan Blue, was always greater than 90 %.
Study series 2:
in vivo
model of I/R
Introduction to method
Having shown that GHRP-6 possesses potentially useful
biological activity in the in vitro systems, we proceeded
to examine its effects when used in an in vivo hepatic
I/R model. Two different timed protocols were used
to examine if any effects seen were applicable to both
relatively short and more prolonged periods of ischaemia.
GHRP-6 was tested alone and also in combination with
EGF, as we have shown previously a beneficial effect
of EGF in a related mesenteric I/R model [8] and we
wanted to determine if any additive/synergistic responses
were apparent. The dose of EGF used in the present study
(1 mg/kg of body weight) was similar to that used in our
study reported previously [8].
Animals
Adult male Wistar rats (200–250 g) were purchased from
the National Center for Laboratory Animals and were
allowed access to food and water ad libitum.
Induction of I/R injury
Animals were anaesthetized with urethane [10 mg/kg of
body weight, i.p. (intraperitoneally)] and placed in a
supine position on a heating pad in order to maintain
body temperature between 36 and 37
C. To induce hep-
atic ischaemia, a midline laparatomy was used and the
blood supply of the right lobe of the liver was interrupted
by placing a bulldog clamp (Fine Science Tools) at the
level of the hepatic artery and the portal vein branches.
Upon completion of the ischaemia time, reperfusion was
initiated by removing the clamp. Reflow was confirmed
by the macroscopic inspection of the target lobe. No
animals were discarded due to non-reflow states. Animals
remained anaesthetized throughout the experiment.
Two different I/R time protocols were used: (i) 45 min/
45 min I/R (45 min of ischaemia, followed by 45 min of
reperfusion; n = 6 per group), and (ii) 90 min/120 min I/R
(90 min of ischaemia, followed by 120 min of reperfusion;
n = 10–12 per group).
Experimental design
For both I/R protocols, rats were randomly assigned
to five experimental groups as follows: group 1 (sham
ischaemia), animals received saline (placebo; 1 ml, i.p.)
and 40 min later underwent all procedures, including
laparotomy, liver exposure and manipulation, but the
hepatic artery and the portal vein branches were not
clamped; group 2 (I/R group), animals received saline
(placebo, 1 ml, i.p.) and 40 min later underwent I/R;
group 3 (I/R with GHRP-6), animals received GHRP-
6 (120 µg/kg of body weight, i.p.) and 40 min later
underwent I/R; group 4 (I/R with EGF), animals received
rhEGF (1 mg/kg of body weight, i.p.) and 40 min later
underwent I/R; group 5 (I/R with GHRP-6 + EGF),
animals received GHRP-6 (120 µg/kg of body weight,
i.p.) and rhEGF (1 mg/kg of body weight, i.p.) and 40 min
later underwent I/R.
Autopsy and sample processing
At the end of the study periods, blood samples were
obtained from the abdominal aorta for biochemical deter-
minations. Serum was obtained, aliquoted and stored at
20
C until processing. Rats were subjected to autopsy,
and samples of different regions from the right ischaemic
lobe were collected for subsequent histopathological
examination and tissue homogenization. In addition, re-
presentative samples were collected from lungs, kidneys,
jejunum and ileum. Samples to be processed for histo-
logical study were immediately placed in 10 % buffered
formalin and subsequently paraffin-embedded and
stained with haematoxylin/eosin.
Blood analyses
Serum levels of ALAT (alanine aminotransferase) and
ASAT (aspartate aminotransferase), used as markers of
hepatocyte injury, were determined using a commercial
kit according to the manufacturer’s instruction (Sigma).
Serum creatinine levels, used as a marker of renal function,
were determined using standard colorimetric methods.
Tissue biochemical analyses
The oxidative state of the liver was analysed by meas-
urement of both enzyme activities [SOD (superoxide
dismutase) and catalase] and chemical components [THP
(total hydroperoxides) and MDA (malondialdehyde)
levels]. MDA levels are a commonly used marker of
lipid peroxidation. In addition, liver and intestinal MPO
(myeloperoxidase) activities were measured as a marker
of neutrophilic infiltration.
For liver tissue biochemical studies of MDA, THP and
SOD, tissue was homogenized [1:10 (w/v)] in 50 mmol/l
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566 D. Cibri
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an and others
KCl/5 mmol/l histidine buffer (pH 7.4), followed by
centrifugation at 5000 g for 20 min at 4
C. The super-
natants were collected, aliquoted and stored at 20
C
until assay. All the biochemical parameters were deter-
mined by spectrophotometric methods. MDA content
was assessed using the Bioxytech LPO-586 kit (Bio-
Rad Laboratories), and THP were determined using
the Bioxytech H
2
O
2
-560 kit (Bio-Rad Laboratories).
SOD activity was determined by following changes in
autoxidation of pyrogallol in response to adding the
homogenate [11]. MPO activity was determined using
a modification of the method described by Krawisz
et al. [12], and 1 unit of MPO activity was defined as the
quantity of enzyme that degrades 1 µmol of H
2
O
2
/min
at 25
C. Biochemical data were adjusted to reflect total
protein concentration using a commercial spectrophoto-
metric protein dye kit (Bio-Rad Laboratories).
Histological assessment
All tissues were assessed in a blinded manner.
Small intestine The total lengths of the small intestine
were measured and then split longitudinally to allow a
macroscopic assessment of the percentage injured area.
The percentage of damage was calculated by measuring
(cm) all the regions showing gross macroscopic changes,
such as petechiae and haemorrhagic areas, and con-
sidering the whole length of the small intestine (in cm) as
100%. Eight equal-spaced 2 cm segments from the length
of the small bowel were then collected for histological
assessment.
For the microscopic assessment, mucosal damage of
the small intestine was quantitatively assessed according
to the grading system of Chiu et al. [13]. This system uses
a scale of 0–5, where 0 is normal mucosa; 1 is development
of subepithelial (Gruenhagen’s) spaces; 2 is extension of
the subepithelial space with moderate epithelial lifting
from the lamina propria; 3 is extensive epithelial lift-
ing with occasional denuded villi tips; 4 is denuded villi
with exposed lamina propria and dilated capillaries, and 5
is disintegration of the lamina propria, haemorrhage and
ulceration. The mean scores of 30–40 villi from each of the
eight segments for each animal were pooled to provide an
average score for the intestine of that animal.
Liver For each animal, the degree of liver damage was
determined in at least five different lobar regions and
graded using the modified Suzuki scoring system [14].
Briefly, the various changes noted are sinusoidal conges-
tion, hepatocyte necrosis and ballooning degeneration.
The specimen was then graded from 0–4, where no nec-
rosis or congestion/centrilobular ballooning was given a
score of 0, and severe congestion/ballooning degeneration
as well as > 60% lobular necrosis was given a value of 4.
Kidney Each sample was classified in a blinded fashion
into one of three groups: 0, essentially normal histology;
1, moderate, probably reversible, changes (hydropic cyto-
plasmic changes); and 2, severe changes (nuclear break-
down or cellular detachment from the tubule basement
membrane).
Lungs Lung interstitial damage ranged from normal
to showing varying degrees of septal thickening, hyper-
cellularity, neutrophilic recruitment, interstitial adhesion
and alveolar luminal reduction. Each sample was classi-
fied in a blinded fashion into one of three groups: 0, essen-
tially normal histology; 1, abnormal showing some of
the changes described above, and 2, grossly abnormal
showing all of the changes described above.
Data analysis
Data were analysed using ANOVA with treatment
as factor. Where significant effects were seen on the
ANOVA (P < 0.05), individual comparisons based on
the group mean square error and residual were per-
formed, a method equivalent to multiple comparisons
analyses.
RESULTS
Study series 1:
in vitro
studies
Restitution assays
GHRP-6 caused pro-migratory activity of wounded
monolayers in both HT29 and IEC6 cells in a dose-
dependent manner. Maximal effects were observed at
40 µg/ml for HT29 cells (Figure 1A) and 160 µg/ml for
IEC6 cells (Figure 1B).
The addition of a neutralizing anti-TGFβ antibody did
not affect the cell migration response caused by GHRP-6
(Figure 1C), suggesting that cell migration in response to
GHRP-6 is independent of TGF-β production.
Proliferation assay
GHRP-6 did not induce increased thymidine uptake in
HT29 or IEC6 cells at any of the doses tested (Figure 1D).
Study series 2:
in vivo
model of I/R
For both of the short (45 min/45 min I/R)- and longer
(90 min/120 min I/R)-timed protocols, the results were
essentially the same. The results from the 90 min/120 min
I/R protocol are therefore discussed in detail and shown
in the Figures and Table 1. The main results from the
45 min/45 min I/R protocol are shown in Table 2 and,
in the few instances where the results differ from the
90 min/120 min protocol, these are mentioned in the text.
Liver
Biochemical analyses I/R caused an approx. 10-fold
increase in serum ASAT and ALAT. Pre-administration
of either GHRP-6 or EGF alone reduced this rise by
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Use of GHRP-6 for multiple organ failure 567
Figure 1
Effect of GHRP-6 on the rate of migration (restitution) or proliferation of various gastrointestinal cell lines
The addition of GHRP-6 to wounded monolayers of (A) HT29 cells or (B) IEC6 cells caused a dose-dependent increase in the rate of migration compared with the
negative control (, no GHRP-6 added). The various doses tested were 1 µg/ml (+), 20 µg/ml (
), 40 µg/ml (
) and 160 µg/ml (). Maximum effects were
seen at 40 µg/ml in HT29 cells and 160 µg/ml in IEC6 cells. Cells were treated with 10 % (v/v) FCS as positive control ().
P
< 0.01 compared with the
negative control at all doses above 1 µg/ml at each time point after 4 h. (C) The pro-migratory effect of GHRP-6 on HT29 cells was not affected by co-incubating
with a neutralizing anti-TGFβ antibody. , Negative control (no GHRP-6);
, cells incubated with 40 µg/ml GHRP-6; and X, cells incubated with 40 µg/ml
GHRP-6 and a neutralizing anti-TGFβ antibody. Similar results were seen using IEC6 cells (results not shown). (D) HT29 cells incubated in DMEM alone (negative
control; ve) had a [
3
H]thymidine uptake of approx. 400 000 c.p.m. Addition of EGF (10 µg/ml, positive control; + ve) caused an approximate doubling of
[
3
H]thymidine uptake, whereas GHRP-6 (50–400 µg/ml) did not increase [
3
H]thymidine uptake above baseline. Similar results were seen with IEC6 cells (results
not shown).
approx. 50 % and combination treatment resulted in a
further reduction in enzyme levels (Figure 2 and Table 1).
I/R caused the MDA levels (marker of lipid per-
oxidation) to increase by approx. 4-5 fold (Figure 2).
In the 90 min/120 min protocol, this rise was truncated
by approx. 50 % in animals that had received GHRP-
6 or EGF alone and virtually completely prevented by
pre-treatment with GHRP-6 + EGF together (Figure 2).
Similar results were seen in animals undergoing the
45 min/45 min I/R protocol, although the rise in MDA
was slightly less marked and either peptide given
alone was sufficient to prevent an increase in MDA
levels (Table 2). Similarly, animals that received placebo
and underwent the 90 min/120 min I/R protocol had a
3–4-fold increase in THP (Table 1). GHRP-6 or EGF
alone truncated this response by approx. 75 % with
combination treatment preventing the rise completely
(Table 1). Similar results were seen in animals that under-
went the 45 min/45 min I/R protocol, although the
amount of THP produced was less (I/R + saline-treated
animals having an approx. 2-fold increase above sham-
operated animals; Table 2).
I/R caused an approx. 60 % fall in hepatic SOD levels,
and this change was partially reversed by pre-treatment
with either GHRP-6 or EGF alone. A further improve-
ment was seen in animals that had received the com-
bination treatment (Figure 2 and Table 2).
Catalase activity was increased by approx. 30-fold in
response to I/R. This increase was markedly truncated
in animals that had received either GHRP-6 or EGF
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Figure 2
Influence of pre-administration of GHRP-6 and EGF alone or in combination on injury sustained in various organs
Rats (10–12 per group) were pre-treated with GHRP-6 (120 µg/kg of body weight, i.p.) and EGF (1 mg/ml, i.p.) and then underwent organ injury induced by 90 min
of hepatic vessel clamping, followed by 120 min of reperfusion. Animals were then killed and blood and tissue collected for various assays of tissue injury. ALAT is a
marker of hepatic injury, MDA is a marker of lipid peroxidation and, along with SOD, allows assessment of the oxidative state of the liver. Liver and intestinal MPO
activity was measured as a marker of neutrophilic infiltration. Serum creatinine was used as a marker of renal function. Values are means
+
S.E.M.
P
< 0.05 and
∗∗
P
< 0.01 compared with the equivalent value in sham-operated animals.
+
P
< 0.05 and
++
P
< 0.01 compared with the equivalent value in animals treated
with I/R + saline.
$
P
< 0.05 and
$$
P
< 0.01 when the values in animals given combination therapy (GHRP-6 + EGF) are compared with the values in animals
given the same dose of either GHRP-6 or EGF alone.
Table 1
Effect of GHRP-6 and EGF on injury induced by 90 min/120 min of hepatic I/R
Values are means
+
S.E.M.,
n
= 10–12 per group. Also see Figure 1.
P
< 0.05 and
∗∗
P
< 0.01 compared with the equivalent value in sham-operated animals.
++
P
< 0.01 compared with the equivalent value in I/R animals.
$
P
< 0.05 and
$$
P
< 0.01 when the values in animals given combination therapy (GHRP-6 + EGF)
are compared with those in animals given the same dose of either GHRP-6 or EGF alone. IU, international units.
Sham operation (laparotomy) I/R I/R + GHRP-6 I/R + EGF I/R + GHRP-6 + EGF
ASAT (IU/l) 34
+
4 1452
+
308
∗∗
543
+
123
∗++
404
+
82
++
115
+
33
++
Catalase (units · min
1
· mg
1
of protein) 16
+
4 581
+
57
∗∗
31
+
4
++
58
+
13
++
20
+
4
++
THP (µmol/mg of protein) 27
+
3 109
+
16
∗∗
51
+
2
∗++$$
43
+
2
++$
21
+
2
++
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Use of GHRP-6 for multiple organ failure 569
Table 2
Effect of GHRP-6 and EGF on injury induced by 45 min/45 min of hepatic I/R
Values are means
+
S.E.M.,
n
= 6 for each group.
P
< 0.05 and
∗∗
P
< 0.01 compared with the equivalent value in sham-operated animals.
++
P
< 0.01
compared with the equivalent value in I/R animals.
$
P
< 0.05 and
$$
P
< 0.01 when the values seen in animals given combination therapy (GHRP-6 + EGF)
are compared with those in animals given the same dose of either GHRP-6 or EGF alone. IU, international units.
Sham operation
(laparotomy) I/R I/R + GHRP-6 I/R + EGF I/R + GHRP-6 + EGF
MPO intestine (units · min
1
· mg
1
of protein) 26
+
3 148
+
13
∗∗
60
+
8
∗∗++ $$
40
+
5
++$
16
+
2
++
MDA liver (nmol/mg of protein) 0.31
+
0.01 1.17
+
0.11
∗∗
0.37
+
0.01
++$
0.29
+
0.01
++
0.19
+
0.01
++
MPO liver (units · min
1
· mg
1
of protein) 19
+
3 105
+
9
∗∗
38
+
6
++$
34
+
10
++$
8
+
1
++
ASAT (IU/l) 13
+
2 116
+
11
∗∗
62
+
7
∗∗++ $
57
+
2
∗∗++ $
33
+
4
∗++
ALAT (IU/l) 21
+
3 157
+
19
∗∗
69
+
16
∗∗++
82
+
13
∗∗++
61
+
8
∗++
Catalase (units · min
1
· mg
1
of protein) 9
+
2 288
+
28
∗∗
106
+
7
∗∗++ $$
63
+
2
∗∗++
47
+
5
∗++
THP (µmol/mg of protein) 182
+
16 295
+
13
∗∗
168
+
5
++$
123
+
20
∗++
108
+
8
∗∗++
10
3
× SOD (units · min
1
· mg
1
of protein) 32.5
+
1.3 14.2
+
1.8
∗∗
22.6
+
1.0
∗∗++ $
20.2
+
0.5
∗∗++ $$
26.5
+
0.9
∗∗++
Creatinine (µmol/l) 43
+
584
+
8
∗∗
67
+
16 70
+
5* 79
+
11
∗∗
alone (causing a 60–70 % reduction), with GHRP-
6 + EGF combination treatment truncating this response
by approx. 90% (Tables 1 and 2).
Histology Sham-operated animals had an essentially
normal liver histology (Figure 3). Animals that had
undergone I/R with placebo (saline) injection had severe
changes, consisting of areas of necrosis, haemorrhage,
cytoplasmic ballooning and sinusoidal distension. Ani-
mals that had been pre-treated with GHRP-6 alone, EGF
alone or the GHRP-6 + EGF combination therapy all
showed improvements compared with the I/R group,
with the combination therapy appearing to have the most
protective effect (Figure 3). Assessment using the micro-
scopic scoring system confirmed these results; all animals
that underwent I/R and received placebo had scores of
3 or 4, whereas six out of ten animals that had received
combination therapy had scores of 0 (Figure 4).
Intestine
I/R alone resulted in macroscopically obvious injury
affecting 73
+
4% of the intestinal length. Pre-treatment
with either peptide alone significantly decreased (P <
0.01) the degree of macroscopic injury (27
+
3and
30
+
2% for GHRP-6- and EGF-treated animals res-
pectively), with the most beneficial effect being seen
in animals that had received both GHRP-6 and EGF
(19
+
2%; P < 0.01 compared with I/R alone or I/R plus
either peptide given alone).
Histological assessment showed I/R caused severe
mucosal damage, with most animals showing complete
loss of villous architecture and extensive areas of
mucosal infarction (Figure 3). These changes were much
less prominent in animals that had received GHRP-6
alone, EGF alone or the GHRP-6 + EGF combination
treatment (Figure 3). Quantitative assessment showed
similar effects; all animals that underwent I/R and
received placebo had scores of 4 or 5, whereas six out
of ten animals that had received combination therapy had
scores of 0 (Figure 4).
Kidney
Biochemical analysis In the animals undergoing the
90 min/120 min I/R protocol, serum creatinine levels rose
from 45 to 70 µmol/l in response to I/R. Pre-treatment
with GHRP-6 was associated with a 30% (non-sig-
nificant) fall in creatinine levels, whereas pre-treatment
with EGF either alone or in combination with GHRP-6
resulted in the creatinine levels remaining in the normal
(sham-operated) range (Figure 2). A similar trend was
seen in animals that underwent the 45 min/45 min I/R
protocol, although the beneficial effects were less marked
and non-significant (Table 2).
Histology Animals that had undergone I/R but not re-
ceived GHRP-6 or EGF all showed moderate or severe
renal injury comprising nuclear breakdown or cellular
detachment from the tubule basement membrane. Ad-
ministration of GHRP-6 or EGF given alone, or in
combination, tended to reduce the degree of injury;
GHRP-6 + EGF combination treatment had the most
beneficial effect with nine out of ten animals having
essentially normal renal histology by semi-quantitative
scoring (Figure 4).
Lung
Histology Animals that had received I/R without
GHRP-6 or EGF had severe changes comprising septal
thickening, hypercellularity, neutrophilic recruitment,
interstitial adhesion and alveolar luminal reduction (Fig-
ure 3). None of the animals that had received I/R without
GHRP-6 or EGF had normal lung histology, whereas
nine out of ten animals that had received both peptides
had normal histology. Animals that had received either
peptide alone occupied intermediate positions (Figures 3
and 4).
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2006 The Biochemical Society
570 D. Cibri
´
an and others
Figure 3
Histopathology of rats given placebo (saline), or GHRP-6 and EGF alone or in combination prior to 90 min hepatic
vessel clamping followed by 120 min reperfusion
Compared with sham-operated animals, rats that underwent I/R, but did not receive GHRP-6 or EGF, had severe changes. Administration of either peptide alone
improved histological appearances with the most improvement being seen in animals that received the combination of GHRP-6 and EGF. Original magnification of
intestine, lungs and liver was ×10, ×10 and ×40 respectively.
DISCUSSION
Using in vitro models of injury and repair, we have
shown that GHRP-6 stimulates gut epithelial restitution,
but not proliferation. In vivo studies have shown that
pre-administration of GHRP-6 reduced the amount of
intestinal and extra-intestinal injury caused by hepatic
vessel I/R and that added benefit was observed if EGF
was co-administered with GHRP-6.
The control of release of endogenous GH from the
pituitary gland is thought to be partially mediated by
the presence of GHRP receptors acting via a specific
G-protein-coupled receptor pathway, the natural ligand
of which is probably the 28-amino-acid peptide ghrelin
[15,16]. During the course of research into the control of
GH release, several peptides that induce GH secretion
were developed and one of the most potent was the
hexapeptide GHRP-6 [15,16] used in the present study.
Using a variety of GH secretagogue molecules, it is
now known that, in addition to being present within
the pituitary gland, GHRP receptors are also present in
several peripheral tissues, including bone marrow, spleen,
pancreas, thyroid and myocardium [6,7], suggesting
additional roles for GHRP ligands that extend beyond
GH release.
GHRP-6 stimulated cell migration of the human
colonic cell line HT29 and the rat intestinal cell line IEC6,
showing that these effects were not species specific and
that GHRP-6 was able to influence gut epithelial function
by acting directly on the cells. The pro-migratory effects
of some of the well established pro-migratory ‘growth
factors’, such as IFNγ (interferon γ ), TGFα and EGF,
are dependent upon their ability to induce TGFβ release
into the medium [17]. It is, therefore, of interest that
we found that the pro-migratory activity of GHRP-6
was not blocked by adding a neutralizing anti-TGFβ
antibody. Caution always has to be shown, however, in
extrapolating from the in vitro situation (utilizing cancer
cell lines) to the in vivo situation.
GHRP-6 has been reported previously to stimulate
proliferation of the hepatoma cell line HepG2, human
pancreatic and prostate cancer cell lines and rat pitui-
tary somatotrophs, possibly acting through the MAPK
(mitogen-activated protein kinase) and ERK (extra-
cellular-signal-regulating kinase) pathways [18,19]. In
contrast, GHRP-6 possessed anti-proliferative activity
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2006 The Biochemical Society
Use of GHRP-6 for multiple organ failure 571
Figure 4
Histomorphometric assessment of histological injury in various organs
Quantitative assessment, using well-validated histological scoring systems, was performed on the livers (modified Suzuki scoring scheme [14]) and intestines (Chiu
scoring scheme [13]). In addition, semi-quantitative assessments of lungs and kidneys (scale: 0, normal; and 2, grossly abnormal) were also performed. See text for
details of the parameters of assessment.
when added to the human lung cancer cell line CALU-1
[20]. To the best of our knowledge, studies on the effect
of GHRP-6 on luminal gut epithelial cells have not been
assessed previously. We found that GHRP-6 had no effect
on proliferation using either HT29 or IEC6 cells, even
though GHRP-6 receptors are presumably present (based
on the pro-restitutive activity in the same cells).
The use of arterial occlusion followed by reperfusion
is a well-established model of injury resulting from acute
vascular occlusion as occurs following embolism or
thrombosis. In addition, it is used as a model for loss of the
intestinal barrier function associated with haemorrhagic
shock, major burns and multiple traumas, which can
result in MOF [21]. Several models have been used to
mimic the early stages of MOF. I/R has the advantage of
being more physiologically relevant than administration
of toxic agents, such as thioacetamide [22], as the major
factors causing injury are probably internally generated
pro-inflammatory cytokines and free radical production
[4,23,24], rather than resulting from metabolism of an
external damaging agent. Mesenteric artery occlusion
is one of the most popular models used (for example,
[8]), but suffers from the drawback that much of the
intestinal injury is induced directly. The mesenteric I/R
model, therefore, although of direct relevance if studies
are being performed in relation to therapies of mesenteric
thrombosis, has limitations if therapeutic interventions
are being studied in relation to gut changes in MOF, where
complete occlusion of the mesenteric vessels usually does
not occur. It was because of these issues that we decided
to use the liver vessel clamping technique.
I/R caused marked hepatic necrosis as demonstrated by
histology and elevated ALAT and ASAT plasma levels.
Addition of GHRP-6 markedly truncated the degree
of damage determined using all of these parameters.
The mechanisms underlying I/R-induced injury and the
protective effects of GHRP-6 are likely to be complex
and multi-factorial. During hypoxic conditions, there is
up-regulation of cell adhesion molecules [25], facilitating
recruitment of inflammatory cells to ischaemic areas.
Our studies confirmed a marked influx of inflammatory
infiltrate within the liver, along with a rise in its associated
marker, MPO. Although GHRP-6 has not been directly
assessed, administration of ghrelin, the natural receptor
ligand homologue of GHRP-6, has been shown to reduce
the adhesion of mononuclear cells to endothelial cells
activated with TNFα (tumour necrosis factor α) [26]. It
is important to note, however, that the influx of inflam-
matory cells was not restricted to the intestine, but also
affected distant organs such as the lungs. This must either
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2006 The Biochemical Society
572 D. Cibri
´
an and others
be due to an alteration in circulating factor(s), such as pro-
inflammatory cytokines, or to the priming and activation
of inflammatory cells (mainly neutrophils) at the hepatic
site that subsequently migrate to distant organs.
Production of highly reactive oxygen species and
other free-radical-damaging metabolites is known to
occur during I/R [23,24,27]. Uncontrolled production of
such factors results in cellular damage, including lipid
peroxidation, as well as induction of both apoptosis
and necrosis [28,29]. We found excessive free radical
production in I/R-treated animals, measured indirectly as
markedly raised hepatic MDA levels (indicating increased
lipid peroxidation) and a general shift in the redox state, as
demonstrated by changes in both the enzyme constituents
(SOD and catalase) and chemical components (TPH
and MDA). The molecular mechanisms underlying the
reduction in MDA levels may be due to several factors,
including immune modulation. In support of this idea
is the finding that ghrelin can directly reduce the pro-
inflammatory response of stressed endothelial cells [26],
which normally results in a pro-inflammatory cascade
and increased free radical production. In addition,
GHRP-6 may also have up-regulated the production of
cellular antioxidant enzymes. Further studies in this area
could potentially measure changes in antioxidant enzyme
levels in various hepatic and gastrointestinal cell lines.
GHRP-6 has been shown to reduce the amount of
apoptosis in the cerebellar cells of aged rats [30]. The
changes seen in our present studies may have been par-
tially mediated by alteration in apoptosis within the liver
and other tissues, although the predominant histological
feature seen in the liver and intestine was of necrosis.
Further investigation into these mechanisms is complex,
however, as single cell-culture model systems do not
contain inflammatory cells and these are likely to be of
major importance in the damaging process in vivo (as
demonstrated in the present study by raised MPO levels
and histology). Similarly, there are major difficulties in
attempting to measure the degree of apoptosis within
tissues containing large amounts of necrotic tissue. Less
damaging models will probably have to be developed to
address this question.
Over the last few years, recombinant peptides have
been introduced increasingly into the clinical arena
(e.g. colony-derived growth factor for bone marrow
support and interferon therapy for viral hepatitis). We
have examined the effects of EGF in rats undergoing
mesenteric I/R previously [8] and also in a clinical trial
when administered via enema to patients with colitis [31].
In view of the positive nature of these studies, we also
examined and compared the effect of EGF given alone and
in combination with GHRP-6 in the present model. We
found that EGF given alone was approximately similar
in its beneficial effects to those seen with GHRP-6 given
alone (although the dose used was 8 times that of GHRP-
6). Administration of both peptides together gave additive
or synergistic responses, suggesting that, in the clinical
arena, use of multiple therapies may have advantages and
deserve further research.
In conclusion, our present studies provide preliminary
evidence that the synthetic hexamer GHRP-6 which,
because of its small size, is relatively simple and cheap to
make may be of benefit for injury associated with visceral
vascular hypoperfusion. If patients at high risk of MOF
can be identified at an early stage of their admission to
hospital, rapid intervention with GHRP-6 may maintain
organ viability. Further studies of GHRP-6 given alone,
or possibly in combination with EGF to enhance effects,
in additional models that allow administration of the
peptides after MOF has been induced therefore appear
justified.
ACKNOWLEDGMENTS
This work was partially funded by the Wellcome
Trust (grant number 054787/B/98/Z), Wexham Park
Gastrointestinal Trust (grant number 2004/6772), and
a DDF/Belmont Trust Award. The EGF used in this
study was produced by Heber-Biotec (Havana, Cuba),
which is the commercial arm of the Center for Genetic
Engineering and Biotechnology.
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Received 20 December 2005; accepted 17 January 2006
Published as Immediate Publication 17 January 2006, doi:10.1042/CS20050374
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2006 The Biochemical Society
... En la actualidad, este péptido, así como otros análogos sintéticos, se emplean como agentes secretagogos para el diagnóstico clínico diferencial de las diferentes formas de enanismo (4). El GHRP-6 posee demostradas propiedades anti-inflamatorias, anti-oxidantes y citoprotectoras y un buen perfil de seguridad (5). Un hallazgo fortuito de nuestro grupo de trabajo, estableció las primeras observaciones de que el GHRP-6 instaura un programa celular de degradación o remoción del exceso de MEC en órganos parenquimatosos. ...
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... Moreover, the molecular mechanism mediating the action of GHRP-6 peptide was shown to involve the phosphatidylinositol 3-kinase/RAC-alpha serine/threonineprotein kinase (PI-3K/AKT1) pathway, as the induction of the hypoxia-inducible factor-1 alpha (HIF-1α) all committed in cellular survival. 51 Subsequently, Granado et al 52 examined the potential anti-inflammatory impact of GHRP-2 in lipopolysaccharide (LPS)-challenged rats. GHRP-2 administration attenuated the effects of LPS on the elevation of circulating levels of transaminases, nitrites/nitrates, and tumor necrosis factoralpha (TNF-α), via direct interaction with liver nonparenchymal cells. ...
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