European Journal of Pharmacology 700 (2013) 165–172

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Pentoxifylline improves cutaneous wound healing in streptozotocin-induced

diabetic rats
Saeed Babaei, Mohammad Bayat n, Mohsen Nouruzian, Mehrnoush Bayat
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o abstract

Article history: Worldwide, 15% of the 200 million diabetics suffer from diabetic wounds. In 1997, the cost for
Received 12 July 2012 amputation of toes and limbs that resulted from infected diabetic foot ulcers ranged from $25,000–
Received in revised form $40,000 per incident. Increasing numbers of research have shown the positive influence of pentoxifyl-
28 October 2012
line (PTX) on healing skin wounds. In this study, we evaluate the effect of systemic PTX (25 mg/kg bid)
Accepted 14 November 2012
Available online 3 December 2012
on wound healing in 80 diabetic rats (DB) by secondary intention. Wounds (20 mm  5 mm) were
identically inflicted on the skin area of the backs of all rats. On day 15 following surgery, a band of skin
Keywords: (4 mm  60 mm) that contained wound was extracted for biomechanical testing. For histologic
Pentoxifylline analysis, both experimental (DB þ PTX) and control, receiving distilled water (DB þ DW) groups were
Wound healing
further subdivided into day 3 and 7 groups. Rats were sacrificed three and seven days after surgery, and
Streptozotocin
a sample from each wound was taken. All specimens were sectioned stereologically and stained with
Diabetes mellitus
Rat H&E. Cell counts were performed by stereological methods. Semi-quantitative evaluation of matrix
metalloproteinases (MMPs) and inhibitor-1 was performed by Reversed Transcription-PCR and UVI TEC
software. For statistical analysis we used student’s t-test. Collectively, the results of this study
demonstrate that there was significant improvement with PTX in all biomechanical parameters.
Histologically, PTX reduced inflammation by day seven. Quantitatively, by day five, PTX reduced
expression of MMPs and increased TIMP-1 expression. These findings revealed that PTX significantly
improved wound healing indices in streptozotocin-induced DB.
& 2012 Elsevier B.V. All rights reserved.

1. Introduction normoglycemic rats (Andriessen and Oxlund 1987; Bitar, 1998).

The prevalence of diabetes mellitus has increased tremendously
world-wide. Complications arising from diabetes have become
serious public health issues, of which one such complication is
impaired wound healing. Currently, 15% of the 200 million diabetes
suffer from wounds that do not easily heal (De Fronzo et al.,1992;
Groop et al., 1993). Complications arising from diabetes have
become serious public health issues, of which one such complication
is impaired wound healing (Brem and Tomic-Canic, 2007). Healing
impairment in diabetes is characterized by delayed cellular infiltra-
tion and granulation tissue formation, decreased collagen organiza-
tion, diminished blood flow, increased blood viscosity and reduced
angiogenesis (Goodson and Hunt, 1977; Bohlen and Niggl, 1979;
Sebag et al., 1994). Studies have shown that wound healing in
diabetic rats (DB) following full-thickness skin excisions is signifi-
cantly delayed with decreased tensile strength in comparison to
*
Correspondence to: Cellular and Molecular Biology Research Center, Shahid
Beheshti University of Medical Sciences, Tehran, Iran. Tel./fax: þ 98 2122439976.
E-mail addresses: mohbayat@sbmu.ac.ir, bayat_m@yahoo.com (M. Bayat).
Simpson has reported that altered blood viscosity in diabetic
patients produces significant changes in microvascular flow patterns
resulting in ischemia in the absence of a specific anatomical lesion
(Simpson, 1985). These abnormalities are presumed to be the cause
of numerous problems for diabetics such as choroidal blood flow,
neuropathy, and wound healing (Simpson, 1985).
Dose-dependent administration of pentoxifylline (PTX) (Falanga
et al., 1999) interferes with inflammation by increasing blood flow
and modulating or blocking the inflammatory actions of interleukin-1
(IL-1) and TNF-a on neutrophils, monocytes and macrophages
(Poulkaris et al., 1999). PTX inhibits the amount of free intracellular
calcium in polymorphonuclears, improves ocular blood flow in
patients with diabetic retinopathy (Sebag et al., 1994) and possibly
diminishes tissue damage caused by neutrophils under various
conditions (Sullivan et al., 1988).
Matrix metalloproteinases (MMPs) are a group of proteolytic
enzymes that collectively degrade most, if not all, components of
the extracellular matrix (ECM). Recent studies have explained the
potential role for MMPs in inflammation, tissue destruction and
scar formation (Kang et al., 2005). This capability is necessary for
active remodeling of connective tissue in fetal development,
cancer invasion and metastasis, as well as wound healing.

0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2012.11.024
166 S. Babaei et al. / European Journal of Pharmacology 700 (2013) 165–172

PTX may affect some cytokines such as,Tumor necrosis factor-

a (TNF-a), IL-1, interleukin-6 (IL-6), interleukin-8 (IL-8), VEGF and
TGF-b1 (Poulkaris et al., 1999; Zhou et al., 2009; de Campos et al.,
2008; Sullivan et al., 2001; Vuković and Lapcevic, 2006). These
cytokines impact the quantitative expression of MMPs and their
inhibitors (Subramaniam et al., 2008; Yager et al., 1996; Blakytny
and Jude, 2006; Ferrari et al., 2010). In this study we investigate
the effects of PTX on wound healing in streptozotocin-induced
diabetic rats.

2. Materials and methods

This study was approved by the Medical Ethical Committee at

Shahid Beheshti University of Medical Sciences, Tehran, Iran
Fig. 2. Pattern of open wounds on the backs of the rats.
(protocol no. 89-01-91-7397). Eighty male Wistar rats that
weighed 250–350 g were obtained from Pasteur Institute of Iran.
Rats were maintained one per cage with free access to food and
water in a room with controlled humidity and temperature (22–
24 1C) on a 12-h light/dark cycle.

2.1. Induction of type 1 diabetes mellitus

Eighty rats, after a 12-h fast, each were given single intraper-
itoneal injections of streptozotocin (Zanosar Pharmacia & Upjohn
Co., Kalamazoo, Ml 49001, USA) at a dose of 55 mg/kg body
weight in distilled water (DW). Seven days after the streptozoto-
cin injection, blood glucose measurements (Biomine, Rightesttm
GM300, Biomine Corporation, Switzerland) were taken from tail
blood. Type one diabetes was defined for each animal if blood
glucose levels were consistently above 300 mg/100 ml one week
after the injection (Karasoy et al., 2002; Vuković and Lapcevic, Fig. 3. Excised skin strip (4 mm width and 60 mm length) for biomechanical
2006). During this period diabetic rats (DB) showed clinical signs testing.
of diabetes mellitus such as polyuria, polyphagia and weight loss.
After 30 days of consistent hyperglycemia, animals were consid- 2.3. Pentoxifylline (PTX) administration
ered eligible for the remainder of the study. During the study
blood glucose levels for all rats were measured every three days. PTX (Sigma–Aldrich, St. Louis, MO, USA) was administered at a
dose of 25 mg/kg (bid) until the end of each analysis (biomecha-
2.2. Wounding model nical test, histologic examination,and semi-quantitative evalua-
tion of MMPs-1, -3 and TIMP-1 expressions) in this study. PTX
To provide the incisional wound (day 0) each animal was was resumed seven days before the onset of the above mentioned
anesthetized with ketamine (50 mg/kg) and daizepam (5 mg/kg). analysis of the study.
The backs of the rats were shaved and a full-thickness incisional
wound (20 mm  5 mm; Fig. 1) was made to the level of the
panniculus carnosus muscle (Figs. 1 and 2). Wounds were not 2.4. Macroscopic assessment
sutured or covered, but were allowed to heal by secondary
intention. The wounds were inflicted in the same manner for Body weight and blood glucose levels were measured on the
all rats. day of surgery (day 0) and every three days after surgery until the
end of each analysis (biomechanical, histologic, semiquantifica-
tion of MMPs and TIMP-1 gene expression).

2.5. Biomechanical test

On day 15, fourteen diabetic (experimental: n ¼7; control:

Fig. 1. White line shows the place of open wound which was consistent for
all rats.
n¼7) rats were sacrificed. Strips of skin that included the healed
wounds (60 mm  4 mm) oriented perpendicular to the long axis
of the body were uniformly excised (Fig. 3) All skin strips were
immediately placed on a material testing machine (Zwick-Roell, Z
25-ph1F, Germany) for evaluation of the biomechanical para-
meters. Specimens were held in jaws and stretched at a constant
speed of 6 cm/min until the skin strips were ruptured.
Load–deformation curves and related parameters [work-up to
maximum force (W up to F max-Nmm) and maximum stress
(Rm-N/mm2)] were analyzed by computer to compare biomecha-
nical test parameters in the experimental (DMþ PTX) and control
(DMþDW) groups.
 S. Babaei et al. / European Journal of Pharmacology 700 (2013) 165–172
2.6. Tissue extraction and histologic examination
On days three and seven rats were sacrificed by intraperitoneal
overdoses of pentobarbital. The complete area of the wound that
consisted of the incision area and adjacent normal skin was
excised. Excised tissues were fixed in formalin (pH 6.8) and
embedded in paraffin. Sectioning was performed according to
stereological methods (Amadeu et al., 2003; Kristiansen and
Nyengaard, 2012; Nepomnyashchikh et al., 2011). Stereological
methods are precise techniques used to obtain information about
three-dimensional structures based on observation of two-
dimensional sections. In this study we obtained 20  2 (doubled)
(5 mm thickness) sections from the healing wounds of each rat.
The 5 mm sections were stained with H&E and observed by
stereological methods. We chose 150 random fields for each
group (n ¼7) (totally 3.15 mm2) and used a 10  100 objective
lens (NIKON Eclipse E2000-Video camera DS-Fi1, Japan) to cap-
ture about 5000 photos that were used to count cells by stereo-
logical methods (Fig. 4). Quantitative evaluation of tissue
elements (neovasculars and blood vessels) were based on counts
obtained in vertical sections (Amadeu et al., 2003). Blood vessels
and neovasculars were identified by the presence of blood cells in
their lumens. We used the camera’s scale to measure the thick-
ness of the regenerated epithelium.
2.7. Semi-quantitative evaluation of MMP-1, -3 and TIMP-1
expressions
Full-thickness adjacent wound tissue from all experiment and
control groups were extracted (1 mm3) on days-one, -three and -
five following surgery and maintained in tagged 1.5 ml eppendorf
tubes at 20 1C. Extracted tissues were homogenized in 4 M
guanidinium thiocyanate (Merck, Germany) for protein denatura-
tion; the RNA was kept soluble.
Total RNA was purified by centrifugation at 35,000  g (Beck-
man Instruments, CA) through 3 ml of 5.6 M chloroform and
extracted by the single-step method (Chomczynski and Sachi,
1987). RNA was precipitated by the addition of alcohol, after
which the supernatant was extracted. We used the extracted RNA
Fig. 4. Stereological method used to obtain information about three-dimensional
structures based on observation of the two-dimensional sections. Photography of
a random histologic section is conformed by a special grid (containing plenty of
rectangular frames) for ease in counting and distinguishing the tissue elements. In
this method only all elements that are within each frame or conformed with
continued lines of frame would be counted.
167
TM
to construct a cDNA library with the Uni-ZAP XR Vector Kit
(Stratagene, Los Angeles, CA). This library was screened with size-
selected (  195–220 bp) RT-PCR products.
For RT-PCR, poly (A)þRNA from the wound area was reverse-
transcribed using an oligo (dt) primer. The provided cDNA was
amplified using a set of degenerated primers that corresponded to
the cysteine-switch and zinc-binding domains of rat-related MMPs
and TIMP-1. In brief, the reaction solution was composed of
premixed PCR buffer and a final concentration of 300 nM each of
the following forward and reverse primers: TIMP-1 forward
(50 -TTTGCATCTCTGGCCTCTG-30 ) and reverse (50 -CATCTTGATCTCA-
TAACGC-30 ); MMP-1 forward (50 -TTTGATGGACCTCAATAT-30 ) and
reverse (50 -CATTAGTGCTCCTACA-30 ); MMP-3 forward (50 -GAT-
TAATGGAGATG-30 ) and reverse (50 -CAGCATTGGCTGAGTG-30 ); and
GAPDH forward (50 -AAACCTGCCAAGTATGATG-30 ) and reverse
(50 -GCATCAAAGGTGGAAGAATG-30 ) plus the cDNA mixture (25 ng),
which was mixed to a total volume of 25 ml. The conditions for RT-
PCR were as follows: 94 1C for 5 min; 94 1C for 30 min; followed by
30 cycles of shuttle heating at different temperatures for the
different genes; 72 1C for 1 min; and 72 1C for 5 min. All PCRs were
run in duplicate and the relative quantify of each mRNA was
normalized to the relative quantify of GAPDH determined by the
specific endogenous control primers.
For electrophoresis, we used agarose gel and cybergreen staining.
After electrophoresis, gels were photographed with an SYN GENE
instrument. UVITEC software (www.uvitec.co.uk/products/geldoc.
html) was used for calibration and semi-quantification of gene
expression.
2.8. Statistical analysis
For all experiments we used SPSS version 16 (Kolmogorov
Smirnov, Leven’s test and student’s t-test for independent samples)
for statistical analysis of the macroscopic, biomechanical and histo-
logic tests, and semi-quantitative evaluation of MMP-1,3 and TIMP-1
expressions. P-values less than 0.05 were considered statistically
significant. Data were expressed as mean7standard error of mean
(S.E.M.).
3. Results
3.1. Macroscopic analysis
Rats’ weights and blood glucose levels were measured every three
days. In both the experimental and control groups their weights and
blood glucose levels changed permanently, but were not significant
(P40.05). A total of 5 DB died during the first week following
streptozotocin injections and were replaced by new animals.
3.2. Biomechanical test
PTX administration improved the skin biomechanical indices
(W up to Fmax-Nm and Rm-N/mm2) 15 days after surgery in the
experimental groups compared to the control groups. Table
1 shows the results of the biomechanical test. Biomechanical
indices between experimental and control groups were signifi-
cantly different (Po0.05).
3.3. Microscopic analysis
Microscopic pictures of the wound areas and adjacent normal
skin are shown in Figs. 5–8.
168 S. Babaei et al. / European Journal of Pharmacology 700 (2013) 165–172

Table 1
Comparing of biomechanical indexes between experimental and control groups
based on Mean 7SEM, control group (DBþ DW) experimental group (DB þPTX).
Biomechanical indices between experimental and control were significantly
different (independent sample t-test, P o 0.05).

GROUPS Work up to Fmax- Student Rm-N/mm2 Student

Sample Nmm Sample

(mean7 SEM) t-Test (mean 7 SEM) t-Test

Group I 101.99 746.7 P ¼ 0.04 1.84 7 0.44 P ¼ 0.000

(DB þPTX)
GroupII 17.77 79 0.57 7 0.14
(DB þDW)

Fig. 7. Day seven after infliction of the would shows evidence of wound
contraction with healing tissue that includes fibroblasts and blood vessel pro-
liferation, which are predominant in H&E-stained section. Arrow notes the
wound’s surface.

Fig. 5. Day three after infliction of wound shows complete formation of healing
tissue. (H&E; arrow refers to the wound’s surface).

Fig. 8. Day seven after infliction of the would shows lack of wound contraction in
comparison to the DB þ PTX group, with less healing tissue (including fibroblast
and blood vessels). (H&E; arrow refers to the surface of the wound).

Fig. 6. Day three after infliction of the wound shows the presence of a fibrin mesh
and incomplete formation of healing tissue. (H&E; arrow refers to the wound’s
surface).
3.3.1. Quantification of fibroblasts
On the third day following surgery, stereological quantification
of fibroblasts showed increased numbers of these cells in the
control group (DBþDW), which was significant (P ¼0.000; Fig. 9).
By the seventh day post-surgery, stereological quantification of
fibroblasts showed increased numbers of these cells in the experi-
mental group (DBþPTX), which was significant (P¼0.000; Fig. 10).
Fig. 9. Statistical analysis of histologic quantification of different elements of
wounded area and adjacent normal skin on 3rd day after surgery in experiment
(PTX) and control (DW) groups, provided by stereological methods and based on
Mean7 S.E.M.
3.3.2. Quantification of neutrophils
By the third day after surgery, stereological quantification of
neutrophils showed increased numbers of these cells in the
control group (DBþDW), which was significant (student sample
t-test, P¼0.000; Fig. 9).
 S. Babaei et al. / European Journal of Pharmacology 700 (2013) 165–172 169

Fig. 11. TIMP-1 on day-one in the control and experiment groups. Gene bands
expressed in the 198 bp region, comparing rainbow marker.

Fig. 10. Statistical analysis of histologic quantification of different elements of the

wound area and adjacent normal skin seven days after surgery. (stereological
methods, based on mean 7 S.E.M.).

Fig. 12. MMP-1 on day-one in the control and experiment groups. Gene bands
expressed in the 200 bp region, comparing rainbow marker.
By the seventh day following surgery, stereological quantification
of neutrophils showed increased numbers of these cells in the
control group (DBþDW), which was significant (P¼ 0.873; Fig. 10).

3.3.3. Quantification of macrophages

Stereological quantification of neutrophils showed increased Fig. 13. MMP-3 on day one in the control and experiment groups, showing gene
numbers of these cells in the control group (DBþDW) on the third bands expressed in the 134 bp region, comparing rainbow marker.
and seventh days following surgery, both of which were significant
(P¼0.000 for day 3; P¼0.04 for day 7; Figs. 9 and 10).

3.3.4. Quantification of neovasculars and blood vessels

Stereological quantification of neovasculars and blood vessels
Fig. 14. GAPDH gene bands on day-one in the control and experiment groups that
from the third and seventh days following surgery showed
expressed in 150 bp regions, comparing rainbow marker.
increased numbers of these cells in the control group (DBþDW),
both of which were significant (P¼0.000; Figs. 9 and 10).

Table 2
Effect of pentoxifylline administration on MMP-1 mRNA expressin in experimen-
3.3.5. Quantification of epithelium thickness tal and control diabetic rats on days 1, 3 and 5. All mRNA expression levels were
On the third day post-surgery, stereological quantification of normalized by GAPDH. All data are shown as mean 7 S.E.M., control group
(DBþ DW) experimental group (DBþ PTX). There was a significant difference in
epithelium thickness showed either decreased or no epithelium in
quantitative expression of MMP-1 mRNA in the DB þ PTX and DBþ DW groups on
the control group (DBþDW), which was not significant (P ¼0.27; day five (independent sample t-test, P ¼0.031).
Fig. 9).
However, on day seven following surgery, stereological quan- Day-mRNA Groups Volume
tification of the epithelium thickness showed a significant
Mean7SEM
increase in thickness in the experimental group (DBþPTX), which
was significant (P ¼0.000; Fig. 10). Day 1, MMP-1 DBPTX 29.85 73.52
DBDW 20.01 72.62
Day 3, MMP-1 DBPTX 13.89 756.99
DBDW 157.11769.38
3.4. Quantification of MMPs-1, -3, and TIMP-1 Day 5, MMP-1 DBPTX 49.53 71.68
DBDW 68.37 74.59
Figs. 11–14 show mRNA from different genes (MMP-1, MMP-3
and TIMP-1) following RT-PCR.
Semi-quantitative expressions of mRNA from different genes
Table 3
as measured by RT-PCR and UVITEC software are presented in Effect of pentoxifylline administration on MMP-3 mRNA expressin in experimen-
Tables 2–4. tal and control rats on days 1, 3 and 5. All mRNA expression levels were
A significant difference existed in quantitative expression of normalized by GAPDH. All data are shown as mean 7S.E.M. Significant difference
MMP-3 mRNA between the experimental (DBþ PTX) and control existed in quantitative expression of MMP-3 mRNA between the experimental
(DBþ PTX) and control (DBþ DW) groups on days three (independent sample t-
(DBþDW) groups on days three (independent sample t-test;
test; P ¼ 0.025) and five (independent sample t-test; P ¼0.034).
P¼0.025) and five (independent sample t-test; P¼ 0.034).
There was a significant difference in quantitative expression of Day-mRNA Groups Volume
MMP-1 mRNA in the DBþ PTX and DBþDW groups on day five
Mean7 SEM
(independent sample t-test, P¼0.031).
There is significant difference in quantitative expression of Day 1, MMP-3 DBPTX 49.05 7 2.93
MMP-3 mRNA in experimental (DBþPTX) and control (DBþDW) DBDW 51.72 7 2.88
groups on day five (P ¼0.03). Day 3, MMP-3 DBPTX 77.51 7 6.41
There was a significant difference in quantitative expression DBDW 11.55 7 1.28
Day 5, MMP-3 DBPTX 44.55 7 1.72
of TIMP-1 mRNA in the experimental (DBþPTX) and control DBDW 65.87 7 5.04
(DBþDW) groups on day five (P¼0.021).
170 S. Babaei et al. / European Journal of Pharmacology 700 (2013) 165–172
Table 4
Effect of pentoxifylline administration on TIMP-1 mRNA expressin in experimental
and control diabetic rats on days 1, 3 and 5. All mRNA expression levels were
normalized by GAPDH. All data are shown as mean 7S.E.M. There was a significant
difference in quantitative expression of TIMP-1 mRNA in experimental (DBþ PTX)
and control (DBþDW) groups on day five (independent sample t-test, P¼0.021).
Day-Gene Groups Volume
Mean7 SEM
Day 1, TIMP-1 DBPTX 20.29 7 2.31
DBDW 20.61 7 2.22
Day 3, TIMP-1 DBPTX 56.98 7 5.14
DBDW 46.59 7 5.37
Day 5, TIMP-1 DBPTX 77.007 5.13
DBDW 56.56 7 5.38
Note: Control group (DB þ DW), experimental group (DB þPTX)
4. Discussion
4.1. Biomechanical test
The results of numerous recent studies have shown the
distinct effects of diabetes mellitus on the wound healing process.
Most importantly, these results have shown reductions in col-
lagen organization; diminished blood supply; defects in leukocyte
function; unbalanced production of growth factors, cytokines and
proteases; increased blood viscosity; delays in inflammatory cell
infiltration; and the conversion from acute to chronic inflamma-
tion (Goodson and Hunt, 1977; Bohlen and Niggl, 1979; Sebag
et al., 1994; Pradhan et al., 2011; Jeffcoate et al., 2004).
Recent studies in DB have shown significant delays in wound
healing and the biomechanical parameters of skin after wounding
in comparison to healthy rats (Andriessen and Oxlund, 1987;
Bitar, 1998; Karasoy et al., 2002; Brem et al., 2009). In some
studies, diabetic patients had decreased blood viscosities that
resulted in serious problems with tissue microcirculation and
caused hidden ischemia in tissues which has been noted to be the
source of diabetic retinopathy, defects and delays in wound
healing, and quantitative reduction in biomechanical indices
(Tamariz et al., 2008; Andriessen and Oxlund, Bitar, 1998).
In the current study, PTX significantly improved the biomechani-
cal indicies of the experimental group. In a study by Karasoy et al.
(2002), the administration of 10 mg/kg PTX did not improve biome-
chanical indices in the diabetic group. According to research the effect
of PTX on inflammation has been determined to be dose-dependent
(Sebag et al., 1994; Sullivan et al., 1988; Lin and Yeh, 2010).
A study by Tireli et al. (2003) used the same dose as PTX in this
study. Those researchers noted that administration of PTX improved
healing indices of ischemic tissue of small intestine anastomosis. The
reason for this improvement was a reduction in neutrophils, followed
by a reduction in free radicals and proteolytic enzymes that degrade
the ECM. A study by Bayat et al. evaluated the effect of PTX on skin
flaps by comparing electron microscope sections between the experi-
mental and control groups. In the control group inflamed mitochon-
dria, numerous cytoplasmic pinocytotic vesicles, and thickened
cytoplasmic membrane were visualized, however these changes were
not seen in the PTX-treated groups (Bayat et al., 2006). The results of
different studies have shown that PTX induces peripheral blood
monocytes and alveolar macrophages that inhibit production of
TNF-a and increase production of TIMPs (Tireli et al., 2003). In a
recent study PTX reduced production of TGF-b1. This reduction has
been shown to prevent the conversion of an acute inflammation into
a chronic inflammation (Zhou et al., 2009). This study has reported
that PTX reduced neutrophil chemoattractants by 50%.
As with a recent study we found that PTX diminished mast cell
degranulation (unpublished data), thus inhibiting the secretion of
growth factors and fibroblasts extra proliferation it may has an
important role in reorganization of fibroblasts and collagen fibers,
preventing scar formation. These effects may be the reason for
improvement in biomechanical parameters seen in the current
study (Karasoy et al., 2002).
4.2. Histologic analysis
Based on stereological methods, PTX caused significant reduc-
tions in fibroblasts on both the third and seventh days following
surgery. Therefore it is possible that PTX may inhibit fibroblast
proliferation and migration. As mentioned above PTX induces
reduction of TGF-b1 production, this reduction inhibits progression
of fibrosis and MMP activity (Zhou et al., 2009). It has been reported
that neutrophils and activated monocytes release TNF-a with strong
mitogenic effects on fibroblasts (Mirshahi et al., 1995) that may
either directly or indirectly inhibit production and secretion of IL-1
and TNF-a, inhibit calcium release in neutrophils and inhibit
neutrophil degranulation, which is followed by a reduction in
secretion of both TNF-a and TGF-b1. Both are important motivators
for fibroblast proliferation (Sebag et al., 1994; Sullivan et al., 1988;
Lin and Yeh, 2010). These cytokines are the main mediators in the
production of granulomatosis (with signs of fibrosis) in sarcoidosis
(Vuković and Lapcevic, 2006; Lin and Yeh, 2010).
In this study PTX significantly reduced the number of macro-
phages in the experimental group. Activated tissue macrophages are
produced in response to chemokines, cytokines, growth factors, and
soluble fragments of the ECM components that have been produced
by proteolytic degradation of collagen and fibronectin (Diegelmann
and Cohen, 1997; Diegelmann and Evans, 2004). The long-term
presence of macrophages in the wound area causes unwanted
degradation of the ECM and a delay in wound healing. Cytokines,
in addition to interleukins and growth factors, are important in the
progression of inflammation, release of mediators and their activa-
tion, and chemotaxi (Liechty et al., 1998). Among these, IL-6 is
responsible for monocyte and macrophage chemotaxi and activation
(Liechty et al., 2000). TGF-b1 is known as a macrophage chemotaxi
modulator (Nall et al., 1996) and is released by platelets. Based on
research, PTX is a TGF-b1 inhibitor that reduces IL-6 secretion (Zhou
et al., 2009). These results enable an understanding of why PTX
reduced macrophage condensation in the experimental group
(Vuković and Lapcevic, 2006; Burgeson and Christiano, 1997).
Despite factors that motivate vascularization (oxygen pres-
sure, reduced pH, and increased lactate) (Brem et al., 2009), VEGF
that is released by platelets and other inflammatory cells plays an
important role in neovascular formation. In this study we have
noted a reduction in blood vessels and neovasculars, which was
significant on day seven.
Recent studies have shown that on days 7 and 14 after PTX
administration, there was an increased half-life of the variety of
VEGF mRNA isomers (120, 164 and 188) and their proteins (Zhou
et al., 2009). In another study PTX increased the peritubular
capillary network (Woessner and Nagase, 2000). In the current
study, PTX decreased angiogenesis in DB.
It is possible that wound healing in diabetes is characterized
by delays in cellular infiltration and granulation tissue formation,
and reduced angiogenesis (Goodson and Hunt, 1977; Bohlen and
Niggl, 1979; Sebag et al., 1994). This necessitates a need for
additional research on the effect of PTX administration on
diabetic wounds. PTX should be administered for a longer period
of time or a higher dose with the intent to determine the distinct
effect of PTX on angiogenesis.
In the present study PTX reduced the thickness of the epithelium
on the third day after surgery. This was expected because PTX,
according to recent studies, reduced expressions of TNF-a, IL-1, and
IL-6 (Burgeson and Christiano, 1997; Vuković and Lapcevic, 2006).
 S. Babaei et al. / European Journal of Pharmacology 700 (2013) 165–172
The latter has been shown to reduce KGF expression (Vuković and
Lapcevic, 2006). On day seven PTX increased the thickness of the
epithelium to the same amount as normal re-epithelialization in
healthy rats. Understanding the mechanism of this function needs
additional research.
4.3. Semiquantification of MMP-1, -3, TIMP-1
MMPs are produced in zymogen form; their activation is an
important step for the beginning of their function as proteinases
(Shiomi et al., 2010). MMP expression is controlled at different
levels of transcription, secretion, activation and even inhibition of
the activated enzyme. Preactivated and activated forms of MMPs
are controlled by special stoichiometric connections and/or by
their inhibitors, TIMPs (Brunner and Blakytiny, 2004). MMP-1 is
constantly expressed by migrating keratinocytes that have been
extracted from basal layers and by macrophages (Parks, 1999).
MMP-3 is expressed in basal keratinocytes in acute wounds and is
necessary for re-epithelialization and basal layer remodeling.
MMP-3 is expressed in the cell population adjacent to hyperpro-
liferating keratinocytes (Fulgueras et al., 2004). Null mices for
MMP-3 secretion show significant delays in wound healing
(Gawronska, 2011). MMP expression is related to cell–cell and
cell–matrix interactions, and the presence of growth factors and
cytokines (Fulgueras et al., 2004). Numerous researches have
shown that skin fibroblasts are able to secret IL-1 and TNF-a
which inspire the MMP-1 protein (Subramaniam et al., 2008).
TIMP-1 may be found in keratinocytes of borderline normal acute
wounds, spindle-like fibroblasts and macrophage-like stromal cells
(Kahari and Saarialhokere, 1997; Vaalamo et al., 1999). As previously
mentioned, PTX in a dose-dependent manner influences different
aspects of inflammation (Falanga et al., 1999) such as inhibition of the
production and secretion of IL-1, TNF-a, TGF-b1 and IL-6 in fibro-
blasts, neutrophils, monocytes, macrophages, peripheral blood mono-
cytes and alveolar macrophages. Reduced production of TNF-a causes
increased secretion of TIMPs, which reduces ECM degradation (Sebag
et al., 1994; Sullivan et al., 1988; John et al., 1999; Zhou et al., 2008;
Ferrari et al., 2010; Burgeson and Christiano, 1997; Vuković and
Lapcevic, 2006; Lin and Yeh, 2010). There are few studies that have
investigated the effect of PTX on quantitative expression of MMPs and
TIMPs. However numerous studies have shown the effects of PTX on
transcription, secretion, and activation of cytokines, growth factors
and chemokines (de Campos et al., 2008; Sullivan et al., 1988; Zhou
et al., 2009; Ferrari et al., 2010). This indirect relationship between
PTX administration and release of cytokines and growth factors may
be able to express the relation between PTX and MMPs and TIMPs
expression. In the present study there were significant differences in
PTX effectiveness on MMPs-1, 3 and TIMP-1 expressions on day five.
5. Conclusion
PTX significantly improved all biomechanical parameters in this
study. A reduction in inflammation in the experimental group was
mostly noted on day seven. On day five, PTX reduced MMPs
expression, but increased expression of TIMP-1. PTX was shown
to significantly affect wound healing process in streptozotocin-
induced diabetes in rats. However additional research is warranted
in order to clarify the direct effect of various doses of PTX on
wound healing and those factors that affect wound healing.
Acknowledgment
We wish to thank the Vice Chancellor of Research at Shahid
Beheshti University of Medical Sciences, Tehran, Iran for financial
support.
171
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