Reduction of carrageenan-induced acute pulmonary inflammation in mice by novel thiazolidinedione derivative LPSF/RA-4

Article abstract of DOI:10.1016/j.ejphar.2013.08.033

A number of studies have demonstrated the biological activities of peroxisome proliferator-activated receptors. However, few studies have addressed the effects of the agonists of these receptors on lung diseases. The aim of the present study was to evaluate the anti-inflammatory action of a novel synthetic thiazolidine derivative (5Z)-3-benzyl-5-(1H-indol-3-ylmethylene)-thiazolidine- 2,4-dione (LPSF/RA-4) on acute lung inflammation (pleurisy) induced by carrageenan. Forty mice were randomly allocated to the following groups: (I) saline control group (sham); (II) carrageenan (CAR) group; (III) CAR+LPSF/RA-4 group treated with LPSF/RA-4 (60 μmol/kg); and (IV) INDO group treated with indometacin (5 mg/kg). Total cell counts and the measure of nitric oxide (NO) were performed in pleural exudates. Lung fragments were processed for light microscopy, transmission electron microscopy, immunohistochemistry and Western blotting. The influx of leucocytes and NO levels were significantly reduced following treatment with LPSF/RA-4 and INDO. Histopathological and ultrastructural analyses of the CAR group revealed evident tissue alterations, such as oedema, infiltrates of inflammatory cells and emphysema. These alterations were significantly reduced in the groups treated with LPSF/RA-4 or INDO. Immunohistochemistry revealed an increase in inflammatory markers (COX-2, iNOS, TNF-α and IL-1β) in the lung tissue of the CAR group, whereas the groups treated with LPSF/RA-4 and INDO exhibited significant reductions in such immunomarkers. Western blot analysis revealed an increased expression of COX-2 and IL-1 in the CAR group, which was reduced by treatment with LPSF/RA-4. The present findings demonstrate the potent anti-inflammatory action of the novel derivative thiazolidinedione LPSF/RA-4 in acute lung injury induced by carrageenan.

Full text of DOI:10.1016/j.ejphar.2013.08.033

Author's Accepted Manuscript
Reduction of carrageenan-induced acute pul-
monary inflammation in mice by novel
thiazolidinedione derivative LPSF/RA-4
Karla P.S. Barbosa, Laise A.M. Santos, Edlene L.
Ribeiro, Ingrid T. Fragoso, Sura W.S. Rocha, Ana
K.S. Nunes, Maria E.R. França, Bruna S. Silva,
Amanda K.S.e Silva, Mariana A.M. Donato,
Fabiana O.S. Gomes, Teresinha G. Silva, Ivan R.
Pitta, Marina R. Pitta, Maria C.A. Lima, Flávia D.
T. Uchôa, Suely L. Galdino, Christina A. Peixoto
www.elsevier.com/locate/ejphar
PII:
S0014-2999(13)00642-0
http://dx.doi.org/10.1016/j.ejphar.2013.08.033
EJP68793
DOI:
Reference:
To appear in: European Journal of Pharmacology
Received date: 20 November 2012
Revised date: 14 August 2013
Accepted date: 30 August 2013
Cite this article as: Karla P.S. Barbosa, Laise A.M. Santos, Edlene L. Ribeiro,
Ingrid T. Fragoso, Sura W.S. Rocha, Ana K.S. Nunes, Maria E.R. França, Bruna S.
Silva, Amanda K.S.e Silva, Mariana A.M. Donato, Fabiana O.S. Gomes, Teresinha
G. Silva, Ivan R. Pitta, Marina R. Pitta, Maria C.A. Lima, Flávia D.T. Uchôa, Suely
L. Galdino, Christina A. Peixoto, Reduction of carrageenan-induced acute
pulmonary inflammation in mice by novel thiazolidinedione derivative LPSF/
RA-4, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ej-
phar.2013.08.033
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Reduction of carrageenan-induced acute pulmonary inflammation in mice
by novel thiazolidinedione derivative LPSF/RA-4
Karla P. S. Barbosaa, Laise A. M. Santosa, Edlene L. Ribeiroa, Ingrid T. Fragosoa, Sura
W. S. Rochaa, Ana K. S. Nunesa, Maria E. R. Françaa, Bruna S. Silvaa, Amanda K. S. e
Silvaa, Mariana A. M. Donatoa, Fabiana O. S. Gomesa, Teresinha G. Silvac, Ivan R.
Pittac, Marina R. Pittac, Maria C. A. Limac, Flávia D. T. Uchôac, Suely L. Galdinoc†,
Christina A. Peixotoa,b
a Laboratório de Ultraestrutura, Instituto Aggeu Magalhães, FIOCRUZ, Recife, Brazil.
b Laboratório de Microscopia e Microanálise, Centro de Tecnologias Estratégicas do
Nordeste, Recife, Brazil.
c Laboratório de Planejamento e Síntese de Fármacos, UFPE, Recife, Brazil.
*Corresponding author: Karla Patricia de Sousa Barbosa, Departamento de
Entomologia, Centro de Pesquisas Aggeu Magalhães, Av. Moraes Rego s/n, Campus da
UFPE - Cidade Universitária, 50670-420, Recife, PE, Brazil. Fax: 55-81-21012500,
Phone: 55-81-21012551. E-mail address: karlapsb@gmail.com
ABSTRACT
A number of studies have demonstrated the biological activities of peroxisome
proliferator-activated receptors. However, few studies have addressed the effects of the
agonists of these receptors on lung diseases. The aim of the present study was to
evaluate the anti-inflammatory action of a novel synthetic thiazolidine derivative (5Z)-
1

3-benzyl-5-(1H-indol-3-ylmethylene)-thiazolidine-2,4-dione (LPSF/RA-4) on acute
lung inflammation (pleurisy) induced by carrageenan. Forty mice were randomly
allocated to the following groups: I) saline control group (sham); II) carrageenan (CAR)
group; III) CAR + LPSF/RA-4 group treated with LPSF/RA-4 (60 <mu>mol /kg); and
IV) INDO group treated with indometacin (5 mg/kg). Total cell counts and the measure
of nitric oxide (NO) were performed in pleural exudates. Lung fragments were
processed for light microscopy, transmission electron microscopy,
immunohistochemistry and Western blotting. The influx of leukocytes and NO levels
were significantly reduced following treatment with LPSF/RA-4 and INDO.
Histopathological and ultrastructural analyses of the CAR group revealed evident tissue
alterations, such as oedema, infiltrates of inflammatory cells and emphysema. These
alterations were significantly reduced in the groups treated with LPSF/RA-4 or INDO.
Immunohistochemistry revealed an increase in inflammatory markers (COX-2, iNOS,
TNF-ꢀ and IL-1ꢁ) in the lung tissue of the CAR group, whereas the groups treated with
LPSF/RA-4 and INDO exhibited significant reductions in such immunomarkers.
Western blot analysis revealed an increased expression of COX-2 and IL-1 in the CAR
group, which was reduced by treatment with LPSF/RA-4. The present findings
demonstrate the potent anti-inflammatory action of the novel derivative
thiazolidinedione LPSF/RA-4 in acute lung injury induced by carrageenan.
Keywords: Peroxisome proliferator-activated receptor; pleurisy; acute lung injury;
thiazolidinedione
2

1. INTRODUCTION
Lung diseases, such as asthma and chronic obstructive pulmonary disease,
constitute serious public health problems worldwide. While potent anti-inflammatory
drugs, such as the glucocorticoids, are available for treatment, these drugs have
undesired side effects and exhibit limited efficacy. Thus, there is a need for novel forms
of treatment for these diseases (Belvisi et al., 2006).
Peroxisome proliferator-activated receptors (PPARs) are transcription factors
belonging to the family of nuclear receptors related to retinoid, glucocorticoid and
thyroid hormone receptors (Evans, 1988). PPARs have been identified in different lung
tissue cells, including cells associated with inflammation in the lung. Three isotypes
have been identified [alpha (ꢀ), beta/delta (ꢁ/ꢂ) and gamma (ꢃ)]. These isotypes are
encoded by three separate genes, with different tissue distributions and functions
(Desvergne and Wahli, 1999). The suggestion that activation of PPARs may exhibit
potent anti-inflammatory and immunomodulatory activity (Serhan, 1996; Serhan and
Devchand, 2001) has led to increased interest in studying these receptors in different
models of lung inflammation (Belvisi et al., 2006; Crisafulli et al., 2010; Buss et al.,
2012).
In recent years, the anti-inflammatory effect of PPAR agonists has been
evaluated. Selective binders with high-affinity for each of the PPARs and dual
specificity ligands are also available. There is evidence that pioglitazone acts through
the activation of both PPAR-ꢀ and PPAR-ꢃ, while rosiglitazone acts only through
PPAR-ꢃ (Buss et al., 2012).
Acute lung injury (pleurisy) induced by carrageenan is a useful model for the
assessment of the contribution of mediators involved in cell alterations during the
3

inflammatory process (Cuzzocrea et al., 2008). The initial phase of carrageenan-induced
acute inflammation (0 to 1 h) has been attributed to the release of histamine, 5-
hydroxytryptamine and bradykinin, followed by a late phase (1 to 6 h) sustained mainly
by the release prostaglandins and attributed to the induction of cyclooxygenase-2
(COX-2) in the tissue (Salvemini et al., 1996; Nantel et al., 1999; Crisafulli et al.,
2010). The recruitment of polymorphonuclear (PMN) cells from the circulation to the
inflamed tissue plays a key role in the breakdown and remodeling of injured tissue
(Salvemini et al., 1995). Moreover, macrophages participate in the progression of
experimental pleurisy by producing pro-inflammatory cytokines, such as tumor necrosis
factor-ꢀ (TNFꢀ) and interleukin-1 beta (IL-1ꢁ) (Crisafulli et al., 2010).
Nitric oxide (NO) is a simple free radical that acts as a mediator of immune
system function and cell signal transduction (Jorens et al., 1993). Relatively large
amounts of NO can be synthesised by inducible NO synthase (iNOS). Different cells are
capable of synthesising and releasing NO in the lung, including neutrophils,
macrophages, endothelial cells, epithelial cells and vascular smooth muscle cells (Asano
et al., 1994; Moncada, 1994).
The aim of the present study was to investigate the anti-inflammatory action of a
novel thiazolidinedione denominated LPSF/RA-4 in a model of carrageenan-induced
pleurisy through the analysis PMN cell infiltration, NO synthesis (nitrite concentration),
lung injury (histology and ultrastructure) and the expression of the inflammation
mediators / enzymes TNF-ꢀ, IL-1ꢁ, iNOS and COX-2.
2. MATERIAL AND METHODS
2.1 Chemistry
4

The compound (5Z)-3-benzyl-5-(1H-indol-3-ylmethylene)-thiazolidine-2,4-
dione LPSF/RA-4 was obtained from the Laboratório de Planejamento de Síntese de
Fármacos (LPSF) of the Universidade Federal de Pernambuco (Brazil) and was duly
identified by nuclear magnetic resonance of hydrogen as well as infrared (IR) and mass
spectroscopy (MS). Figure 1 displays the route of LPSF/RA-4 synthesis (Fig. 1). The
starting reagent was thiazolidine-2,4-dione, which was reacted with benzyl chloride
under basic conditions to obtain the 3-benzyl-thiazolidine-2,4-dione LPSF/RA-1
intermediate (Mourão et al., 2005). Laterally, 2-cyano-3-(1H-indol-3-yl)-acrylic acid
ethyl ester LPSF/IP-19 (Brandão et al., 2004) was synthesised through Knoevenagel
condensation between 1H-indol-3-carbaldehyde and ethyl cyanoacetate. A Michael-type
addition was then performed by reacting the ester LPSF/IP-19 with intermediate 3-
benzyl-thiazolidine-2,4-dione to form the final indol-thiazolidine-2,4-dione LPSF/RA-4
compound.
Reactions were monitored with analytical thin layer chromatography in silica gel
60 F254 plates and visualised under UV light (254 nm). Melting points were determined
on a Quimis 340 capillary melting point apparatus and were not corrected. Infrared
spectra were recorded as KBr discs using a BRUKER (IFS 66) infrared
spectrophotometer. Nuclear magnetic resonance 1H NMR and 13C NMR spectra were
recorded in a VMMRS 400 MHz VARIAN spectrometer using tetramethylsilane (TMS)
as the internal standard and DMSO-d6 as the solvent. Chemical shifts (ꢂ, ppm) were
assigned according to the internal standard signal of TMS in DMSOd6 (ꢂ = ppm).
Coupling constants (J) are reported in Hz. 1H NMR spectra are reported in the following
order: chemical shift, multiplicity, number and type of proton and coupling constant(s).
Mass spectra with ESI mass were obtained in a Bruker HCT ultra mass spectrometer.
5

Molecular mass was estimated from the m/z ratio of the deprotonated molecular ions of
the formula (M-H)- in negative ionisation mode.
The LPSF/RA-4 derivative was isolated as a single isomer. X-ray
crystallographic studies and 13C-NMR have demonstrated a preferred Z configuration
for 5-benzylidene-thiazolidinones (Tan et al., 1986; De Simone et al., 1995). The
presence of the arylidene proton peak in the synthesised (5Z)-3-benzyl-5-(1H-indol-3-
ylmethylene)-thiazolidine-2,4-dione LPSF/RA-4 derivatives in 1H NMR confirmed the
completion of the nucleophilic addition reaction. The compound was also confirmed by
MS data in negative mode. The IR spectrum of the compound showed peaks
characteristic of the carbonile group and arilidene (HC=). Preparation of (5Z)-3-benzyl-
5-(1H-indol-3-ylmethylene)-thiazolidine-2,4-dione LPSF/RA-4: Equimolar amounts of
3-benzyl-thiazolidine-2,4-dione (200 mg) and 2-cyano-3-phenyl-acrylic acid ethyl ester
(230 mg) were reacted using absolute ethanol (8 mL) as the solvent and morpholine (1
mL) as the catalyst. The reaction mixture was heated to 50 °C for 8 h and then cooled to
room temperature. The solid that precipitated out was filtered under vacuum and
washed with water and absolute ethanol. MF: C19H14N2O2S; MW: 334.39; MP: 237oC;
Yield: 46%; Rf 0.56 n-hexane/ethyl acetate 7:3. IR (ꢄ, cm-1; KBr): 3418, 1723, 1661,
1594; 1H NMR (400 MHZ, DMSO d6): s (1H, NH indol) 12.19, m (2H, indol) 7.29-
7.18, d (1H indol) 7.93-7.90 J= 7.2, d (1H indol) 7.52-7.50 J= 7.2, s (1H indol) 7.83, s
(1H =CH) 8.22, s (2H NCH2) 4.84, m (5H benzylic) 7.39-7.28. 13C NMR (ꢀ ppm,
DMSO-d6, DEPT): 44.5 (CH2), 110.5 (C), 112.6 (CH), 113.5 (C), 118.5 (CH), 121.3
(CH), 123.3 (CH), 126.3 (CH), 126.8 (C), 127.6 (2CH), 127.8 (CH), 128.7 (2CH), 129.3
(C), 135.9 (CH chain), 136.3 (C), 165.5 (C=O), 167.2 (C=O). MS (m/z) relative
intensity: [M-H]- 332.9 (100), MS2[333.0]-, 241.8 (4), 199.8 (7), 171.8(100) (Fig. 1).
6

2.2 Animals
Male Swiss mice weighing 18 to 25 g were lodged under standardised conditions
(22 2 °C, alternating 12-h periods of light and dark and 50% to 60% relative
humidity) and allowed free access to standard mouse chow and water prior to the
experiment. This study was approved by Ethics Committee for Animal Experimentation
of the Centro de Pesquisas Aggeu Magalhães/Fundação Oswaldo Cruz (Brazil) under
process number Nꢁ 12/2010.
2.3 Experimental Design
The mice were randomly allocated to the following groups: I) sham group,
which received only saline [0.1 ml, intrapleural (i.pl.) route] (N = 10); II) CAR group,
subjected to carrageenan-induced pleurisy (N = 10); III) CAR + LPSF/RA-4 group,
which received LPSF/RA-4 [60 <mu>mol /kg, intraperitoneal (i.p.) route, solubilised in
3% cremophor and diluted in sterile 0.9% saline] 0.5 h prior to carrageenan-induced
pleurisy (N = 10); and IV) CAR + INDO group, which received indometacin (5 mg/kg,
i.p. route) 0.5 h prior to carrageenan-induced pleurisy (N = 10). Indometacin (INDO)
was used as the reference anti-inflammatory drug.
Dose-response assays were first performed to evaluate the optimum LPSF/RA-4
dosage. Prior to pleurisy induction, the animals were treated with different doses of
LPSF/RA-4 (10, 30 or 60 <mu>mol /kg, i.p. route). Inflammatory parameters were
analysed 4 h after carrageenan injection, based on previous studies (Guiara et al., 1986;
Henriques et al., 1990). Subsequently, all procedures as well as the
immunohistochemical and western blot analyses were performed only with the optimum
dose.
7

2.4 Induction of pleurisy
Mice were anaesthetised with an intramuscular injection of a combination of
ketamine hydrochloride 10% (115 mg/kg) and xylazine 2% (10 mg/kg). Following
confirmation of anaesthesia, the animals received a single intrapleural (i.pl.) injection of
0.1 mL sterile saline (NaCl 0.9%) or saline sterile containing ꢅ-carrageenan (1%) on the
right side of the chest. Four hours after the injection of carrageenan, the animals were
killed by CO2 inhalation. The chest was carefully opened and the pleural cavity was
washed with 1.0 mL of sterile phosphate buffered saline (PBS), pH 7.6 (Fröde et al.,
2009). The exudate and washing solution were removed by aspiration. Any exudate
contaminated with blood was discarded. Fluid samples were collected from the pleural
cavity to determine the total leukocyte content in a Neubauer chamber with the dilution
of the exudate in Trypan blue stain (1:20). Fluid leakage samples were collected for
further determination of total leukocytes and NO levels.
2.5 Measurement of NO
The Griess colorimetric reaction was employed for the measurement of NO.
This method allows the detection of the oxidation of NO into nitrite (NO2-) in the
pleural fluid. Briefly, 50 ꢆl of pleural fluid were added to a 96-well ELISA plate,
followed by the same volume of Griess reagent, which is composed of 1%
sulphanilamide diluted in 2.5% H3PO4 (solution A) and N-1-naphtyl-ethylenediamine
also diluted in 2.5% H3PO4 (solution B). Assays were performed in duplicate. To
prepare a standard curve, a solution of sodium nitrite at an initial concentration of 100
ꢆM was serially diluted in PBS. After incubation for 10 minutes in the dark, reading in
the spectrophotometer was performed at 490 nm. The absorbance of different samples
8

was compared with the standard curve and the results were expressed as mean
standard error of the duplicate, using GraphPad Prism software programme (v. 5.0).
2.6 Histological examination
Lung fragments were washed twice in PBS, pH 7.6, fixed in Bouin’s solution
(75% picric acid, 20% formaldehyde and 5% glacial acetic acid) for 8 hours, dehydrated
in an increasing ethanol series, cleared in xylene and embedded in purified paraffin
(VETEC, São Paulo, SP, Brazil). Sections measuring 5 μm were cut using a microtome
(Leica RM 2125RT), deparaffinised with xylene, stained with haematoxylin-eosin and
evaluated under magnification (400 x) using an inverted microscope (Observer Z1,
Zeiss MicroImaging GmbH) equipped with a camera and the 4.7.4 Image Analysis
programme (AxionCam MRm Zeiss).
2.7 Electron transmission microscopy
Lung fragments were fixed overnight in a solution containing 2.5%
glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer. The specimens
were then washed twice in the same buffer and post-fixed in a solution containing 1%
osmium tetroxide, 2 mM calcium chloride and 0.8% potassium ferricyanide in 0.1 M
cacodylate buffer, pH 7.2, dehydrated in acetone and embedded in Embed 812.
Polymerisation was performed at 60 °C for three days (Florêncio et al., 2005). Ultrathin
sections were collected on 300-mesh nickel grids, counterstained with 5% uranyl acetate
and lead citrate and examined using a Morgani FEI 268D transmission electron
microscope.
2.8 Immunohistochemical localisation of COX-2, iNOS, TNF-ꢀ and IL-1ꢁ
Five sections (thickness: 5 μm) from each group were cut and adhered to slides
9

treated with 3-aminopropyltriethoxysilane (Sigma, USA). Briefly, sections were de-
paraffinised with xylene and rehydrated in graded ethanol (100 to 70%). To minimise
endogenous peroxidase activity, the slides were treated with 10% (v/v) H2O2 in water
for fifteen minutes. The sections were washed with 0.01 M Phosphate Buffered Saline
(PBS), pH 7.2, and blocked with 1% Bovine Serum Albumin (BSA), 0.2% Tween 20 in
PBS for 1 h at room temperature. The sections were incubated overnight at 4 ºC with
anti-COX-2 antibody (ABCAM, CA, USA, 1:400), anti-iNOS, anti-TNF-ꢀ antibody
(ABCAM, CA, USA, 1:250) and anti-IL-1ꢁ antibody (GenWay, San Diego, CA, 1:250).
The antigen-antibody reaction was visualised with avidin-biotin peroxidase (Dako
Universal LSAB ® + Kit, Peroxidase) using 3.3-diaminobenzidine as the chromogen.
The slices were then weakly counterstained with Harris’ haematoxylin and mounted in
entellan (Merck, catalogue number: 1079610100). Positive staining resulted in a brown
reaction product. Five pictures at the same magnification were quantitatively analysed
using the Gimp 2.6 software (GNU Image Manipulation Program, UNIX platforms).
2.9 Western blotting
Lungs were quickly dissected and homogenised in an overhead stirrer (Wheaton,
nº 903475) in an extraction cocktail (10 mM ethylenediaminetetraacetic acid, 2 mM
phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, 10 mM sodium
pyrophosphate, 10 mM sodium orthovanadate, 10 mg of aprotinin and 100 mM
Tris(hydroxymethyl)aminomethane, pH 7.4). Homogenates were centrifuged at 3000 xg
for 10 min. The supernatant was collected and stored at -70 ºC until use for
immunoblotting. Protein levels were determined using the Bradford method with BSA
as the standard (Bradford, 1970). Proteins (40 μg/μl) were separated on 10% (COX-2)
or 14% (IL-1ꢁ) sodium dodecyl sulphate polyacrylamide by gel electrophoresis under
10

reduced conditions and were electrophoretically transferred onto nitrocellulose
membranes (Bio Rad, CA, USA, Ref. 162-0115). After blocking overnight at 4 ºC with
5% non-fat milk in TBS-T (Tris-buffered saline 0.1% plus 0.05% Tween 20, pH 7.4),
the membranes were incubated at room temperature for three h with rabbit polyclonal
antibody against COX-2 (1:1,000 dilution; ABCAM, CA, USA) and IL-1 ꢁ (1:2.000
dilution, Genway, San Diego, CA) diluted in TBS-T buffer solution containing 3% non-
fat milk. After washing (six times, 10 min each) in TBS-T, the membranes were further
reacted with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary
antibody (1:80,000 (Ref. A6154) and 1:80,000 (Ref. A5420), respectively, Sigma,
USA) diluted in TBS-T with 1% non-fat milk for 1 h 30 min at room temperature. An
enhanced chemiluminescence reagent (Super Signal, Pierce, Ref. 34080) was used for
the visualisation of the labelled protein bands and the blots were developed on X-ray
film (Fuji Medical, Kodak, Ref. Z358487-50EA). For quantification, the pixel density
of each band was determined using the ImageJ 1.38 programme (available at
http://rsbweb.nih.gov/ij/download.html; developed by Wayne Rasband, NIH, Bethesda,
MD, USA). The results were confirmed in three sets of experiments for each protein
investigated. Immunoblotting for ꢁ-actin was performed as a control for the above
protein blots. After visualisation, the protein antibodies were stripped from the
membranes, which were re-probed with monoclonal anti-ꢁ-actin antibody (1:2.000
dilution, Sigma, USA). Protein densitometry was then performed.
2.10 Statistical analysis
The GraphPad Prism software programme (version 5.0) was used for the
statistical analysis. Data were expressed as mean standard error. For the in vivo
studies, n represents the number of animals studied. Differences between the control and
11

treated groups were analysed using analysis of variance (ANOVA), followed by either
Dunnett’s or Tukey’s post hoc test. All p-values less than 0.05 were considered
significant.
3. RESULTS
3.1 Effect of LPSF/RA-4 on leukocyte counts
In the CAR group, the injection of carrageenan induced an acute inflammatory
response characterised by the accumulation of fluid containing a large amount of total
leukocytes (338.8 24.54 x 106 cells/cavity). However, a significantly lower number of
total leukocytes were found in the LPSF/RA-4 and INDO groups (70.16 3.23 x 106
and 58.78 6.50 x 106 cells/cavity, respectively) (Fig. 2A). The present results also
revealed a significant reduction in cellular infiltrate in the lung tissues at both
LPSF/RA-4 (10 and 30 <mu>mol /kg). However, the animals these groups exhibited
congestion, haemorrhage, oedema and emphysema. The choice of the LPSF/RA-4 dose
used in this study (60 <mu>mol /kg) was based on histopathological studies carried out
in our laboratory.
3.2 Effect of LPSF/RA-4 on NO levels in pleural exudate
NO levels in the exudate of the CAR group were significantly higher than those
in the sham group, whereas no significant differences were found between sham group
and either the LPSF/RA-4 or INDO group. Treatment with LPSF/RA-4 caused a
reduction of 72.46 7.51 in NO in comparison to the CAR group (p < 0.05). Treatment
with either LPSF/RA-4 or indomethacin prevented the increases in NO levels in CAR
group.
12

3.3 Histopathology of lung fragments
Histological analysis of the lung fragments from the sham group revealed well-
preserved tissue (Fig. 2C). In contrast, the animals with carrageenan-induced pleurisy
exhibited patchy areas of inflammation composed of lymphocytes, neutrophils and
macrophages. Mild haemorrhage, congestion, discrete alveolar thickening due to
increased cellularity, oedema and emphysema were also observed (Fig. 2D). The lungs
from mice pre-treated with LPSF/RA-4 revealed a significantly lower number of the
inflammatory cells, lesser alveolar thickening, fewer areas of haemorrhage and a lesser
degree of emphysema (Fig. 2E). Treatment with INDO also prevented lung injury (Fig.
2F).
3.4 Effect of inflammation on ultrastructure of lung tissue
The ultrastructural analysis of the lung fragments from the sham group revealed
a preserved morphological pattern, with the alveolar epithelium composed of
pneumocytes (Fig 3A). Carrageenan-induced pleurisy led to ultrastructural alterations,
such as type II pneumocytes with evident lamellar bodies, electrodense granules,
vacuoles and myelin bodies (characterising cell damage), vasodilatation in capillary
vessels, numerous collagen fibres in the interstitial space (increasing its thickness) and
discrete interstitial haemorrhage. Inflammatory cells and activated alveolar epithelial
cells were also observed (Fig. 3B). The animals treated with LPSF/RA-4 and INDO
(Fig. 3C and 3D, respectively) exhibited a preserved alveolar epithelium similar to that
of the sham group, thereby demonstrating attenuated tissue damage.
3.5 Effect of LPSF/RA-4 on IL-1ꢁ, TNF-ꢀ, COX-2 and iNOS levels
13

In the present study, no significant staining for IL-1ꢁ was found in the lung
tissues from the sham group (Fig. 4A). In contrast, lung tissue from the CAR group
exhibited a positive reaction for IL-1ꢁ in leucocytes, alveolar macrophages, endothelial
cells and epithelial cells (Fig. 4B). Treatment with both LPSF/RA-4 and INDO (Fig. 4C
and D, respectively) led to a significant reduction in IL-1ꢁ staining in comparison to the
CAR group (see densitometry analysis, Fig. 4E).
Immunohistochemical analysis for TNF-ꢀ revealed no significant labelling in the
sham group (Fig 4F). In contrast, lung tissue from the CAR group exhibited a positive
staining for TNF-ꢀ, especially in pneumocytes, macrophages, infiltrate cells and the
vascular wall (Fig. 4G). Treatment with both LPSF/RA-4 and INDO (Fig. 4H and I,
respectively) led to a reduction in IL-1ꢁ staining in comparison to the CAR group (see
densitometry analysis, Fig. 4J).
Lung tissue from the sham group expressed baseline levels of COX-2 (Fig. 5A),
whereas lung tissue from the mice treated with carrageenan demonstrated greater COX-
2 expression (Fig. 5B). This expression was significantly reduced in the LPSF/RA-4
group (Fig. 5C). The treatment with INDO also inhibited COX-2 expression, but to a
lesser extent than that found with LPSF/RA-4 (Fig. 5D, densitometry analysis 5E).
The sham group demonstrated baseline levels of iNOS (Fig. 5F), whereas the
CAR group exhibited significant iNOS labelling in alveolar epithelial cells (Fig 5G).
Treatment with LPSF/RA-4 and INDO led to a significant reduction in staining for
COX-2 (Fig. 5H and I, respectively) in comparison to the CAR group (see densitometry
analysis, Fig. 5J).
3.6 Western blotting analysis for COX-2 and IL-1 ꢁ
14

Baseline levels of COX-2 were detected in the sham group, whereas a significant
increased in COX-2 was found in the CAR group. However, treatment with LPSF/RA-4
significantly reduced the expression of COX-2 (Fig. 6A and B), confirming the results
obtained in the immunohistochemical analysis performed with lung fragments. In the
present study, the entire lung was used in this analysis, which may explain the small
increase in the expression of COX-2 found with this technique.
Baseline levels of IL-1ꢁ were detected in the sham group, whereas the CAR
group exhibited increased levels of IL-1ꢁ. As expected, LPSF/RA-4 led to a significant
reduction in IL-1ꢁ levels in comparison to the CAR group (Fig. 6C and D).
4. DISCUSSION
The present study evaluated the effects of a novel derivative of
thiazolidinedione, denominated LPSF/RA-4, on lung acute inflammation induced by
carrageenan in mice. The inflammatory process is characterised by the production of
histamine, leukotrienes, prostaglandins, platelet-activating factor and bradykinin as well
as the release of chemicals from cells (Tomlinson et al., 1994).
Carrageenan-induced pleurisy is a well-characterised experimental model of
inflammation commonly used to evaluate cell migration and other inflammatory
parameters. Non-steroidal anti-inflammatory drugs are effective in inhibiting both cell
migration and exudation (Saleh et al., 1999). In the present study, both LPSF/RA-4 and
indometacin were effective in significantly reducing the migration of neutrophils and
the influx of mononuclear cells. These findings are consistent with data described in
previous studies that also used the pleurisy model for the evaluation of the PPARg
agonist rosiglitazone (Buss et al., 2012) and the PPARa agonist pioglitazone (Fröde et
al., 2009), demonstrating that glitazones are effective in inhibiting the migration of
15

leukocytes following the administration of carrageenan in the pleural cavity of a murine
model.
Light microscopy revealed that the carrageenan-induced pleurisy was
characterised by cellular infiltrate surrounding blood vessels, extravasated erythrocytes
in the perivascular interstitium of arterioles and alveolar haemorrhage. The histological
analyses demonstrated that, when administered 0.5 hour prior to CAR, LPSF/RA-4
significantly suppressed morphological changes in the lung, such as oedema, the
accumulation of inflammatory cells and alveolar thickness, thereby reducing lung
injury. Similarly, the ultrastructural analyses revealed that carrageenan-induced pleurisy
led to damage to type II pneumocytes and vasodilatation of capillary vessels. However,
treatment with either LPSF/RA-4 or INDO inhibited lung injury, as the treated groups
exhibited preserved alveolar epithelium similar to that of the sham group.
In the present study, TNF-ꢀ and IL-1ꢁ levels were significantly reduced
following treatment with LPSF/RA-4 and similar effects were obtained with
indometacin, which is the drug of reference for the attenuation of acute pulmonary
inflammation. Therefore, this study provides evidence of the beneficial effect of the
novel thiazolidinedione-derived LPSF/RA-4 and demonstrates that this effect is
associated with an inhibitory effect on the pro-inflammatory cytokines IL-1ꢂ and TNF-
ꢃ, which are potent mediators of the chemotaxis of leukocytes in inflammatory
processes (Lampinen et al., 2004). These findings confirm and extend those obtained by
Buss et al. (2012), who found that roziglitazone treatment decreased the levels of these
cytokines in a murine pleurisy model.
Previous studies have demonstrated that nitric oxide plays a critical role in acute
and chronic inflammatory responses (Medeiros et al., 1995). However, treatment with
LPSF/RA-4 significantly reduced NO products in the exudates of the lungs affected
16

with pleurisy. Similarly, immunohistochemical analyses of the lung tissues also
indicated that LPSF/RA-4 reduced levels of iNOS. These effects were similar to those
obtained with indometacin. Studies have indicated that inhibitors of NOS activity
reduce the development of carrageenan-induced inflammation, which suggests a role for
NO in the pathophysiology associated with this model of inflammation (Cuzzocrea et
al., 1998; Rubbo et al., 1994).
The NO pathway has similarities with the COX pathway, since both are key
regulators of inflammatory responses and have both constitutive and inducible isoforms
of their enzymes (Sano et al. 1992; Clancy et al., 2000).
PPARꢃ agonists reduce iNOS expression and NO production in a dose-
dependent manner in macrophages and other inflammatory cells (Crosby et al., 2006)
and PPARꢃ inhibits the induction and production of cytokines, such as IL-1ꢁ and TNF-
ꢀ, by macrophages (Buss et al., 2012).
Cuzzocrea et al. (2004) showed that the PPAR-ꢄ agonist rosiglitazone reduced
the expression of COX-2 and iNOS in mouse lungs submitted to carrageenan. The
results of present study demonstrate that treatment with LPSF/RA-4 was more effective
in inhibiting the expression of COX-2 than treatment with indometacin. However, the
difference did not achieve statistical significance, which is likely related to the non-
selectivity of indometacin for COX isoforms, as reported in previous studies (Nakano et
al., 2007).
The present study shows evidence that the novel thiazolidinedione denominated
LPSF/RA-4 has an anti-inflammatory profile, as it inhibits the influx of inflammatory
cells and leads to a decrease in lung injury in a pleurisy model induced by carrageenan.
This effect is linked to the inhibition of the expression of TNF-ꢀ, IL-1ꢁ, iNOS and
COX-2. The findings suggest that the PPAR ligands may represent novel therapeutic
17

agents for lung inflammation. New experiments are currently being designed to evaluate
whether this effect is related to the transcriptional regulation of PPAR-ꢃ, PPAR-ꢀ or the
activation of both receptors.
5. ACKNOWLEDGMENTS
This study was supported by the Brazilian fostering agencies Pernambuco Foundation
for Assistance to Science and Technology (FACEPE), Aggeu Magalhães Research
Centre of the Oswaldo Cruz Foundation in Recife (CPqAM/FIOCRUZ), Centre for
Strategic Technology in the Northeast (CETENE) and the National Institute of
Structural Biology and Bioimaging (INBEB).
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FIGURE LEGENDS:
Figure 1: Synthesis route of (5Z)-3-benzyl-5-(1H-indol-3-ylmethylene)-thiazolidine-2,4-dione
compound LPSF/RA-4
Figure 2: (A) Effect of thiazolidinedione-derived LPSF/RA-4 (10, 30 or 60 ꢅ<mu>mol /kg) on
total cell migration in initial phase (4 h) of carrageenan-induced pleurisy in mice. Data are
expressed as mean S.E.M. of 10 mice in each group. *** P < 0.0001 compared with CAR
group. (B) Effect of LPSF/RA-4 (60 ꢅ<mu>mol /kg) on NO quantification following
carrageenan-induced pleurisy in mice. * P < 0.013, LPSF/RA-4 and INDO groups in
22

comparison to CAR group. (C-F) Histopathology of lungs from mice following carrageenan-
induced injury. (C) Lung sections from sham group. (D) Tissue damage caused by carrageenan-
induced pleurisy evidenced in increased cellularity and polymorphonuclear infiltration (white
arrow), haemorrhage (asterisk) and thickening of alveolar wall (black arrow). (E) Treatment
with LPSF/RA-4 and (F) INDO administered 0.5 hours prior to pleurisy led to reduced lung
injury and neutrophil infiltration. Figure is representative of at least 3 experiments performed on
different experimental days. Data are expressed as mean S.E.M. of 10 mice in each group.
Scale bar = 20 μm.
Figure 3: Ultrastructural analysis of lung after carrageenan-induced injury and LPSF/RA-4
treatment. (A) Lung sections from mice in sham group (note preserved lung architecture). (B)
Lung with carrageenan-induced pleurisy showing inflammatory cells (asterisk) and activated
alveolar epithelial cells (arrows) (note cellular debris). (C) Lung treated with LPSF/RA-4
exhibiting well-preserved pneumocytes and alveoli. (D) Ultrathin sections of lungs from INDO
group demonstrating reduction in lung injury. Bar = 5 μm.
Figure 4: Effects of LPSF/RA-4 on immunohistochemical localisation of IL-1ꢁ and TNF-ꢃ. (A)
Baseline levels of IL-1b detected in sham group. (B) At 4 h after carrageenan injection, staining
intensity for IL-1ꢁ substantially increased in alveolar macrophages and epithelial cells. (C)
Reduction of staining for IL-1 following administration of LPSF/RA-4 or (D) INDO 0.5 hour
prior to carrageenan injection. (E) Densitometry analysis of photographs of
immunohistochemistry for IL-1-ꢁ from lung tissues. *** P < 0.002 in comparison to CAR
group. (F) Baseline levels of TNFꢃ in sham group. (G) Positive staining for TNF-ꢀ in tissue
sections from CAR group mainly located in pneumocytes and inflammatory cells. (H) After
LPSF/RA-4 or (I) INDO treatment, reduced degree of positive staining for TNF-ꢀ in lung
tissues. (J) Densitometry analysis of photographs for TNF-ꢀ from lung tissues. *** P < 0.001 in
comparison to CAR group. Figures are representative of at least 3 experiments performed on
23

different experimental days. Data are expressed as mean S.E.M. of n = 6 mice in each group.
Scale bar = 20 μm.
Figure 5: Effect of LPSF/RA-4 on immunohistochemical localisation of COX-2 and iNOS in
lung tissue after carrageenan-induced pleurisy. (A) Baseline levels of COX-2 in sham group.
(B) Positive labelling detected in type II pneumocytes in CAR group. (C) Treatment with
LPSF/RA-4 significantly reduced COX-2 staining in comparison to CAR group, achieving
similar levels to sham group. (D) Indometacin treatment also reduced COX-2 staining. (E)
Densitometry analysis of photographs of immunocytochemistry for COX-2 from lung tissues.
*** P < 0.006 in comparison to CAR group. (F) Low levels of iNOS in sham group. (G)
Positive labelling detected in alveolar epithelial cells in CAR group. (H) Treatment with
LPSF/RA-4 significantly reduced iNOS staining in comparison to CAR group. (I) Indometacin
treatment also reduced iNOS staining. (J) Densitometry analysis of photographs of
immunohystochemistry for iNOS in lung tissues. *** P < 0.0001 in comparison to CAR group.
Figures are representative of at least 3 experiments performed on different experimental days.
Data are expressed as mean S.E.M. of n = 6 mice in each group. Scale bar = 20 μm.
Figure 6: Western blot analysis of COX-2 and IL-1ꢂ expression in lung tissues. (A and B)
Baseline expression of COX-2 detected in sham group, with significant difference in
comparison to CAR group. LPSF/RA-4 treatment reduced expression of COX-2. (A)
Representative blot of lysates obtained from pool of 5 animals per group. (B) Data are
expressed as mean S.E.M. of 3 replicates for each group. (C and D). Baseline expression of
IL-1ꢂ detected in lung samples of sham group, with significant difference in comparison to
CAR group. LPSF/RA-4 treatment reduced expression of IL-1ꢂ. (C) Representative blot of
lysates obtained from pool of 5 animals per group. (D) Data are expressed as mean S.E.M. of
3 replicates for each group. Data analysed by one-way ANOVA, followed by Dunnet’s test and
Tukey’s test. ***P < 0.0001 in comparison to CAR group.
24

Figure 1
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