ACUTE INFLAMMATION INDUCED BY AGAR: A MODEL FOR IN ...

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Feb 20, 2013 (4 years and 8 months ago)

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ACUTE

INFLAMMATION

INDUCED

BY

AGAR
: A
MODEL

FOR

IN
VIVO

PHARMACOLOGICAL

ANALYSIS


TROVÃO, J. E.
1
, ÁVILA, P. E. S.
2
,

BASTOS, G. N.T
3
,

DO NASCIMENTO,

J. L.M
4
.


1,2,3,4
Laboratório de Neuroquímica Celular e Molecular, Universidade Federal do Pará, Belém
(PA), Brasil

E
-
mail:
josetrovao@gmail.com




Abstract.

Inflammation is a reaction of tissue to injury, expressed by five cardinal points: pain, swelling,
redness, heat and loss of function. There are standard
models for analysis of the pathophysiological process.
One pharmacological model is experimental air
-
pouch used to study the mechanisms of anti
-
inflammatory
drugs. The hydrocolloid agar is a polycationic, consisting agarose and agaropectin used in food,
ph
armaceutical and cosmetics, and together with the carrageenans, It’s a polysaccharide from red algae. In the
present study has adapted the model of the air
-
pouch, replacing the phlogistic agent carrageenan

by the agar.
The objective of this work is to sta
ndardize the air pouch model using the agar in rats Wistar. The pouch were
produced by a subcutaneous injection of sterile air in the intraescapular, forming a cavity. In the first and
second day were injected 10 ml of air in the third day the agar was adm
inistered and after 16 hours the samples
were collected. Five concentrations of agar 1%,

2%, 3%, 4% were tested. Getting the 2% concentration, as
more satisfactory, further tests were performed with groups carrageenan, 2% agar, 2% agar + celebra, 2% agar
+ Aspirina,

for to infer the mechanism of action. All agar concentrations induced inflammation, however, the
group 2% was most evident vasodilation, increased production of nitric oxide, lack of necrosis, greater cell
migration and production of exudate s
imilar to carrageenan. The groups treated with agar and anti
-
inflammatory drugs celebra and aspirina reduced exudation and cell migration. The model produced significant
differences in the induction of inflammation, compared to the carrageenan, as observed

with this signs of
inflammation. Thus, the model of acute inflammation induced by agar demonstrated applicability for use in
experimental studies of drugs with potential anti
-
inflammatory activity that are in any presentation: pure
substances, extracts or

nanoparticle.
.


Keywords:

Inflammation, A
ir
-
pouch,
Á
gar.


INTRODUCTION
.


For new substances with anti
-
inflammatory effects is tested, it is necessary to produce the
inflammation in the tissue in order to prove that their use is effective.

For this, there are several experimental techniques that induce the process of acute
inflammation such as paw edema model [described by Levy (1969)], air
-
pouch, pleurisy
model
[descrito por HENRIQUES et al, (1990)]
, among others, but it requires the use of

a
pro
-
inflammatory agent (phlogistic agent) as carrageenan, LPS, bradykinin, dextran and
zymosan.

However, the phlogistic agent have a high value, which increases the implementation
costs of the study. Thus the process of identifying a pharmacological agent with properties
that result in clinical benefits ends being slow and expensive. The experimental

model of
inflammation air
-
pouch, previously described by Selye (1953) and modified by Ghosh et al
(2000) is used to study the mechanisms of inflammatory responses, besides serving as a
model for various treatments with anti
-
inflammatory drugs (Ellis et al
., 2000). This model
consists of a subcutaneous injection of sterile air in the region intraescapular being
subsequently injected into an inflammatory agent (carrageenan).

The air cavity formed subcutaneously in the non
-
inflamed pocket is lined with a thin

layer of fibroblasts and macrophages, similar to the synovial layer. Injection of carrageenan
into the cavity produces an inflammatory reaction characterized by infiltration of cells, and
increased exudate production of inflammatory mediators such as pros
taglandins, leukotrienes
and cytokines (EDWARDS et al., 1981).

The polycationic hydrocolloid agar is widely used in food, pharmaceutical and cosmetic
industries. The hydrocolloid is carrageenans together with one red seaweed polysaccharide
and consists of
a mixture of agarose and agaropectin (ARMISEN; GALATAS, 2009)..





MATERIALS AND METHODS.


Animals
.



For this study, we used 50 male Wistar rats weighing between 150 and 220 grams,
supplied by the vivarium of Universidade Federal do Pará The animals were maintained with
water and balanced ration
ad libitum.




Air
-
Pouch
.



The
air
-
pouchs

were produced

by a subcutaneous injection of 10 ml of sterile air in the
intraescapular forming a cavity (bubble).

On the first day was injected 10 ml of sterile air, the second day was added additional
10 ml of sterile air for maintaining the bag, on the third day the

animals was administered into
the agar and, after 16 hours, there sacrifice.



Experimental Groups
.



The animals were divided into eight groups that were used in different concentrations
of 1% agar, 2%, 3%, 4% and the control group. Subsequently, after
checking the optimal
concentration to be used, an experiment was performed in which animals received 2% agar
and these were treated with anti
-
inflammatory celecoxib, a selective COX
-
2, or acetylsalicylic
acid, COX
-
1 and COX
-
2, to analyze the mechanism of a
ction. Furthermore, analyzes were
performed with animals treated with the substance carrageenan.




Analysis of the
exsudato.


After therapeutic procedures in the treated groups, animals were sacrificed in a
chamber of carbon dioxide (CO
2
). The wells were
washed subcutaneously with 1 ml of saline
(0.9%) and EDTA (1 mM). Then, the exudates were collected and analyzed for volume and
the total number of leukocytes.

The leukocyte count was performed on smears stained with Giemsa staining.


Evaluation of Vasodil
atation of experimental groups.


After removal of the exudate, evaluation was made in vasodilatation this dorsal area,
which was recorded by photography.


Evaluation of nitrergic activity in Inflammatory Exudate
.


The nitric oxide production in the
exsudato
supernatant after treatment with agar was
assessed by measurement of metabolite nitrite, using the method of Griess reagent (1%
sulfanilamide and nafitiletileno 0.1%) (Grenn et al, 1982), after 10 minutes of reaction, the
samples were read in a mi
croplate reader with wavelength of 570 nm.

So that interference does not
happen

accumulation of exudate proteins during the
analysis in the microplate reader, the samples were diluted 1:1 with a solution of 3% zinc
sulfate and centrifuged for 5 minutes at
10,000 rpm, prior to the procedure pattern.


Evaluation of Activity Through the painful test Lick Paw
.


To evaluate the nociceptive activity caused by the agar solution was performed to test
the time of paw licking the adapted model and Hunskaar hole (1987). Was injected in 1%
formalin into the plantar region of the right paw of the animals. The reactions of

licking the
affected paw were recorded in the first phase, which corresponds to the initial 5 minutes and
the second phase that corresponds to the final 15 minutes, corresponding to the nociceptive
and inflammatory pain, respectively.
Each animal was exam
ined for 30 minutes.


Statistical Analysis


The data collected was used variance analysis (ANOVA), followed by the Bonferroni
test. Being used levels of significance of p <0.05 and p <0.001.


RESULTS


Evaluation Process
-
I
nduced Vasodilation in Agar Air
-
Pouch
Model
.


In Figure 1 can be observed in varying degrees of vasodilation different experimental
groups.

In the control group (saline), vasodilation was not observed (Figure 1A). In the group
treated with a solution of 1%

agar was not observed any change in the control group (Figure
1B). While in the group treated with a solution of 2% agar is observed a strong vasodilation
(Figure 1C). In the group administered with a solution of 3% agar, an increase in vasodilation,
with

respect to group 2%, and the onset of ischemic points, which suggests evidence of
necrosis (Figure 1D).

In the group treated with a solution of 4% agar were perceived increase in the number
of points ischemic been observed also foul odor to make the openi
ng of the air pocket (Fig.
1E).

It should be noted an important fact observed in Figures 1D and 1E, which was the
formation of agar gel, caused by the gelation of this polysaccharide.


Figure 1:

(A) only with animals given saline, (B) animals administered with a solution of 1% agar, in (C)
animals administered with a solution of 2% agar, in (D) animals administered with solution

3% agar, in (E)
animals administered with a solution of 4% agar.

The

arrows show the yellow vasodilation, increased according
to the treatment group in question, while arrows indicate green indication of the process of necrosis.

The area in
question is the back of the animals, which were injected doses of sterile air, and
these solutions during the
experiment.


Source: Data from author


Evaluation of nitrergic activity in inflammatory exudate in
to Agar
-
Induced
Model of Air
-
pouch
.


Gra
ph
ic1:
Production of nitrite in the experimental groups treated with a solution of 1%
agar, 2%, 3%, 4% and
a control group (saline). All groups treated with the substance agar showed significant differences when
compared to the control group (** p <0.001).

Source:

Data from author.



In this analysis, we compared the concentrations of
nitrite, a metabolite of NO,
between the control and treated groups with a solution of agar (1%, 2%, 3% and 4%).

In graphic

1, there was a dose
-
dependent increase in the nitrite concentration of
metabolite, but the group treated with 4% agar, decreased by 14% compared to group 3%. All
groups treated with a solution of agar showed highly significant differences (p <0.001) when
compared to the control group (saline).


Evaluation

of Cell Migration into Inflammatory Exudate Agar
-
Induced Model of
Air
-
Pouch
.



Graphic 2:

Total cell count experimental group treated with solution of 1% agar, 2%, 3%, 4% and a control
group (saline). * p <0.05 and ** p <0.001 compared to control group.

Source:

Data from author.


In this test was to assess the total amount of exudates present
in cells induced agar.

In
grapic 2, it can be seen that the agar has promoted an increase in cell migration with
increasing concentration of agar solution administered up to a concentration of 3%.

As seen in
the graph the concentration of 4% promoted a dec
rease in cell migration compared to the
concentration of 3%, but this result was still superior to the group that deal only with saline.


Significant differences between control group and groups treated with a solution of agar (p
<0.05).

The groups which a
re outstanding 3% and 4% (p <0.001) and group 2% (p = 0.0032),
which showed significant differences compared to the control group.



Evaluation of the Volume
-
Induced Inflammatory Exudate Agar Model for
Air
-
pouch
.


Graphic 3:

Volume of exudate (ml) of the experimental groups treated with a solution of 1% agar, 2%, 3% and
4% and group control * p <0.05.

Source:

Data from author.

To evaluate the relationship between dosage and agar effect on the inflammatory
exudate, there was
an analysis of the volume of exudate formed.

In Graphic

3, there is a dose
-
dependent increase in the production of exudate plasma
when compared to control.

Statistical analysis showed a significant difference only in group 4% compared to the
control group.

The groups treated with a solution of agar, when compared, significant
differences compared to the group only 4%.


Count the cells differentiated Induced Inflammatory Exudate Agar 2% in Air
Pouch Model
.



Leukocytes

Eosinophils

Lymphocytes

Monocytes

Neutr
ophils


Average

±

DP

6,00

±

5,0

6,63

±

10,4

27,33

±

2,5

60,33

±

8,3

Table 1:
D
ifferential leukocyte counting.

Source:
Data from author.


Figure 2:
Neutrophils and monocytes present in the inflammatory exudate group of 2% agar.

Source:
Data from author.


The differential count was performed in order to identify cell types which are induced
by 2% agar solution.

As the control group (saline) does not exude cell was not possible to compare group
with 2% agar.

The analysis of the data in Table 1 and Figure 2
show a large population of neutrophils
followed by monocytes and small amount of lymphocytes and eosinophils.


Evaluation of painful activity, induced a 2% agar in Model Lick Paw.


Graf
i
phic 4:

Average duration of paw licking.

Source:

Data from author.


Graphic 5:

Average time licking the paw. The column red expresses the time of the animals under the effect of
the substance 2% agar (with pH adjusted to 2.5).

Source:

Data from author.


To check the activity of nociceptive solution of 2% agar was performed

measuring the
time of paw lick.

Analyz
ing the data, as shown in graphic

4, it is perceived that there was no significant
difference between the substance and saline solution agar (neutral pH) in the induction of pain
in both phases of testing.

As shown in
grapic

5, the change of pH of 2% agar, licking time in the animals treated
with the solution of 2% agar, pH modified with (2:5) showed no significant difference in
nociceptive phase (0
-
5 '). During the inflammatory phase (15
-
30 '), the substan
ce agar also
showed significant difference compared to the control group, treated with saline, and no
difference with the group treated with formalin, a substance which is standard for this test.


Evaluation of Anti
-
Inflammatory Activity Patterns in Proces
s
-
Induced
Vasodilation Agar 2% in Air Pouch Model
.





Figure 3:

In (A) animals administered with carrageenan, (B) administered with a solution of 2% agar, in (C)
animals administered with a solution of 2% agar / Celecoxib, in (D) animals administered with a solution of 2%
agar / ASS. The area in question is the back o
f the animals, which were injected doses of sterile air and the
solutions mentioned above.

Source:

Data from author.

Figure 3 shows the process of vasodilation induced by carrageenan phlogistic agent
(A), 2% agar (B) treated with 2% agar Celecoxib (C) and
2% agar treated with acetylsalicylic
acid (D).

It is observed in Figure 3B vasodilation caused by 2% agar solution, being greater than
the vasodilation induced by carrageenan substance.

Furthermore, it is evident intense redness caused by the increase in v
ascular tone
location higher in the group treated with solution of 2% agar in comparison with the animal
treated with carrageenan.

Figure 3C, celecoxib blocked the activity of 2% agar which promoted the considerable
decrease in vasodilation compared with g
roups 2% carrageenan and agar. In Figure 3D,
aspirin blocked the activity of 2% agar which promoted the reduction of vasodilation and
flushing process, compared to groups carrageenan and agar 2%, yet this group had a greater
vasodilation compared to the gr
oup treated with celecoxib.


Evaluation of Anti
-
Inflammatory Activity in Activity Patterns Induced Nitérgica
Agar 2% in Air Pouch Model
.


Graphic

6:

Concentration of nitrite experimental group treated with solution of 2% agar, carrageenan, agar 2% /
2% agar celecoxib

and / acetylsalicylic acid (AAS
).

Source:

Data from author.


The evaluation of the nitrite concentration was performed to characterize
the effects of
anti
-
inflammatory celecoxib and acetylsalicylic acid in the animals administered with
solutions of 2% agar, and identify the mechanism of action of the substance agar.

The analysis of Graphic

6 shows a reduction in the concentration of metab
olite in the
groups treated with nitrite celecoxib (85%) and ASA (81%) as compared to group that
received treatment with a solution of 2% agar. This fact confirms the reduction in the
vasodilation of those groups, as shown in Figure 10C and 12D.

The group
treated with 2% agar showed higher concentration of nitrite, compared to
the carrageenan group (p <0.05).










Evaluation of Anti
-
Inflammatory Activity Patterns in Cell Migration Induced
Agar 2% in Air Pouch Model




Graphic

7:

Total cell count experimental group treated with solution of 2% agar, carrageenan, agar 2% / 2%
agar celecoxib and / acetylsalicylic acid (AA
S
).

Source:

Data from author.


The analysis of total cell count was performed in order to evaluate the effects of
anti
-
inflammatory celecoxib and acetylsalicylic acid on cell migration and to compare the effect of
agar carrageenan.

Cell migration in the groups treated with celecoxib and
acetylsalicylic acid

showed a
significant reduction when compared to group treate
d with 2% agar (p <0.05).

The groups treated with anti
-
inflammatory celecoxib and
acetylsalicylic acid

reduced
55% and 40% the number of cells, respectively, compared to group 2% agar. The
acetylsalicylic acid

and celecoxib groups showed no significant d
ifferences when compared.
Groups 2% agar and carrageenan did not show significant differences when compared.


Evaluation of Anti
-
Inflammatory Activity Patterns in Volume
-
Induced Exudate
Agar 2% in Air Pouch Model


Graphic

8:
Volume of exudate experimental

group treated with solution of 2% agar, carrageenan, agar 2% / 2%
agar Celecoxib and / Acetylsalicylic Acid (AA).

Source:

Data from author.


The anti
-
inflammatory effects of celecoxib and acetylsalicylic acid were analyzed by
the production of exudate for
med, and comparisons made
with the group 2% agar and
carrageenan.

The production of exudate from the group agar was 86% higher when compared to the
carrageenan. Celecoxib groups and ASA were reduced by 72% and 63% respectively,
compared to group treated
only with 2% agar.

The volume of exudate collected from the agar 2% group showed a significant
difference compared to the carrageenan group (p <0.05). The groups treated with celecoxib
and aspirin had a significant decrease in the volume of the exudate, as

compared to those
administered only with a solution of 2% agar.

The groups celecoxib, aspirin and carrageenan group showed no significant
differences when compared.


DISCUSSION


The purpose of this study was to assess the activity of the phlogistic agar h
ydrocolloid
in an experimental model of air pouch in Wistar rats.

As well as its ability to observe its
mechanism nociceptive and flogistic The results demonstrate that the substance agar, 2%
concentration, phlogistic activity produced by activating the pr
ostaglandin pathway, it has not
been able to produce a nociceptive response in a short time.


In the present study we used the experimental model of air bag to prove the efficacy of
hydrocolloid agar as phlogistic agent, using the pharmacological model of
air pouch, which
can demonstrate the four cardinal signs of inflammation: swelling, redness, heat and
loss

function.


Inflammation may be considered as classical biological response of an organism, in
injury.

The expression of the inflammatory process is c
haracterized by five cardinal signs:
pain, heat, swelling, erythema and loss of function (RUSSEL, 2005).


To assess the amount of agar suitable for use solutions were tested with different
concentrations of agar and were assessed for cell migration, nitrit
e concentration and volume
of exudate to the cardinal signs of inflammation were shown.

The ON increases vascular permeability and an intermediate in the production of
prostaglandins, for lipid oxidation, and is a potent vasodilator (DAVIS et al., 2001). T
he
results show that injection of agar in the air pouch cavity, causes an increase in the
concentration of nitrite, as the concentration of t
he solution administered (Graphic

1) induced
inflammation in agar. These results are confirmed by Szabo, 1996, 1998
, which showed that
during the first hours after an insult capable of initiating an inflammatory process, production
of NO by iNOS
-
mediated begins to be upregulated, resulting in a burst release of NO, which
exceeds the basal levels of free radicals. This
production, increased NO, leads a cellular
damage. First, NO may directly promote an exacerbation of peripheral vasodilation, resulting
in a vascular decompensation, NO may also positively regulate NF
-
κ
B by initiating an
inflammatory signaling pathway that

culminates in the production of proinflammatory
cytokines (SZABO, 1996, 1998 , cited, Horton, 2003).

This

production

of

nitric

oxide

induced

by

agar

may

be

influencing

the

process

of

vasodil
ation

and

cell

migration

(Graphic

2

and

3).

According

to

Szabo

and

Bechara,

2006,

during

the

acute

inflammatory

process

is

no

change

in

vessel

diameter,

blood

flow

and

vascular

permeability

which

triggers

cell

migration.

Salvemini

and

colleagues

(1995)

when

used

for

iNOS

inhibitors

in

inf
lammatory

model

of

air

bag

and

found

that

there

was

an

anti
-
inflammatory

action,

not

only

by

blocking

iNOS,

but

also

by

decreased

cellular

infiltration

and

prostaglandins

in

the

area

where

he

was

going

the

inflammatory

process.

One

of

the

first

signs

of

in
flammation

in

the

microcirculation

is

the

elevation

of

endothelial

permeability.

The

endothelium

becomes

fragile

for

practically

all

the

components

contained

in

the

plasma,

water

and

ions,

and

a

large

number

of

protein

species.

The

mechanisms

by

which

perm
eability

occurs

involving

transmembrane

signaling

and

responses

in

endothelial

cells,

with

separation

of

proteins

in

the

VE
-
cadherin

junctions

(Schonbein,

2006).

To

prove

this

cardinal

sign

was

measured

the

volume

of

exudate

formed

in

the

bag

(Figure

3)

wh
ich

showed

that

the

agar

promoted

an

increase

in

vascular

permeability,

as

the

increase

in

concentration

of

the

solution

administered.

During

exudation,

activated

enzymes

in

plasma

or

interstitial

cells

exsudated

enzymes

act

on

the

ground

substance

of

prot
eoglycans

and

break

molecules,

increasing

the

hydrophilicity

site.

Due

to

the

abundance

of

hydroxyl

groups,

carboxyl

and

sulphate

chain

carbohydrate

in

most

glisosaminoglicanas,

proteoglycans

are

strongly

hydrophilic

and

act

as

polyanions

(BOGLIOLO,

2006;

JUNQUEIRA,

CARNEIRO,

2004).

The

agar

hydrocolloid

possesses

the

property

of

retaining

water,

and

colloidal

substances

forming

water

activity

controlling

a

system,

being

formed

by

agarose

with

a

small

amount

of

sulfate

and

agaropectin,

which

is

sulfated

and

has

acid

residues.

(Hanus

et

al.,

1967;

Raven,

2005).

With

this

we

can

infer

that

the

increase

in

the

volume

of

exudate

formed,

induced

by

agar,

due

to

the

action

of

NO

in

relaxation

of

blood

vessels

associated

with

the

hydrophilic

property

of

the

agar

in

the

interstitial

space.

For

confirmation

of

this

event

cell

counts

were

performed

in

cells

present

in

the

exudate

collected.

All

experimenta
l

groups

(Graphic

2)

treated

with

a

solution

of

agar

significant

differences

in

cell

migration,

confirming

that

the

agar

substance

induces

the

production

of

chemokines,

with

consequent

cell

migration

to

the

site

of

inflammation.

Moreover, the leukocyte differential count agar 2% group showed a prevalence of
neutrophils followed by monocytes and small amounts of
eosinophils and lymphocytes (Table
2). Studies by Ramalho
-
Garcia et al., 2002 showed the role of inflammation resident cells
which when stimulated release TNF
-
α
, responsible for the synthesis of chemokines and,
consequently, the migration of leukocytes. Ot
her cytokines such as IL
-
1 and IL
-
6 also
participate in this process. During the development of inflammation, cell migration of
leukocytes is initiated by a significant number of polymorphonuclear neutrophils, followed by
migration of monocytes. One of the

main points on which we can evaluate the activity of the
agar is phlogistic cell migration of polymorphonuclear leukocytes (PMN) and monocytic cells
to air pocket region (Table 1
). This increase in leukocyte migration could be due to induction
of expressi
on of adhesion molecules that are responsible for the "rolling" of leukocytes over
endothelial. This process can be explained by the activation of nuclear transcription factors
such as, NF
-
κ
B part which is responsible for transcription of genes iNOS, COX
-
2

and
vascular cell adhesion molecule 1 (VCAM
-
1) (
MARTIN

et al., 2000; KIM et al., 2010). The
binding of inflammatory mediators to receptors present on macrophages results in the
phosphorylation and degradation of I
κ
B and translocation of NF
-
κ
B to the
nucleus and binds
the promoter regions of DNA which will initiate the transcription of other inflammatory
mediators (
LASKIN

et al. 2001).

As pain is involved in the inflammatory process in the experimental pharmacology
paw licking test was introduced in 19
67, which is performed in the subcutaneous injection of
formalin produced a biphasic response (
SHIBATA
, 1989). The formalin test has two distinct
phases of nociception, the first phase takes place immediately after intraplantar application of
formaldehyde
solution (the first five minutes) and the second stage corresponds to twenty
minutes after injection. Thus, to evaluate the action of painful solution of agar used model
paw lick. After tissue injury, inflammatory mediators are released promoting a synergi
stic
way, a change in the transduction mechanism of the peripheral nociceptive stimulation, which
increases the sensitivity of the transduction of high threshold nociceptors, exaggerated
response to suprathreshold noxious stimuli and spontaneous pain (
CARV
ALHO
;
LEMÔNICA, 1998). Although the solution phlogistic activity agar 2%, not shown
nociceptive activity, probably due to the time frame. Thus, the test was repeated with pH
adjustment the solution of 2% agar, to 2.5. According to Burke, 1933 formalin sol
ution has a
pH due to the biphasic response of the formalin solution which promotes an injury site
injected.
This observation confirms our results demonstrate that activation of nociceptors after
the change of pH of the solution of agar used.

Given the res
ults above, was chosen solution of 2% agar as the optimal concentration
for our model of inflammation. However, OKOLI et al., 2005, agar used in the model of
induced paw edema. However, the described solution used was the concentration of 3% (v /
v). In th
e experiments of this work were used solutions 1
-
4%, and it was observed that the
highest concentration hydrocolloid has the ability to form a gel solution which needs to be
constantly at a temperature of 37 ° C as the solution viscosity hampers administra
tion of the
substance, leading to easy clogging of the syringe.

In the second stage of labor, with the agar solution chosen, we tested the activity of
anti
-
inflammatory patterns, celecoxib, and acetylsalicylic acid on the type of air bag. The
results demon
strated that aspirin an anti
-
inflammatory estereoidal and celecoxib, a selective
inhibitor of COX
-
2 inhibited the activity

of the phlogistic agar (Graphics

6, 7 and 8).

The anti
-
inflammatory action of acetylsalicylic acid is due to the inhibition of
prosta
glandins and thromboxane by acetylation and blocking the catalytic activity of COX
-
1.
Aspirin and other derivatives of the anti
-
inflammatory drugs (NSAIDs) lipooxigenaxe not
inhibit, and thereby do not suppress the formation of leukotrienes (CARVALHO,
LEMÔ
NICA, 1998; SERHAN et al., 2008).

Celecoxib exhibits anti
-
inflammatory, antipyretic and analgesic properties attributed to
selective inhibition of COX
-
2. At therapeutic concentrations, celecoxib does not inhibit COX
-
1 does not alter platelet function (GILM
AN, 2004).

The vasodilation is effected by mediators, including histamine, ON, prostaglandin
(PGE2) and prostacyclin (PGI2). Furthermore, PGE2 PGI2 and cause redness and heat the
tissue due stimulating increase local blood flow (BOGLIOLO, 2006; COTRAN 2001
; RANG
et al., 2006).

In the qualitative test of vasodilation was greater vasodilation and flushing the group
agar 2% compared to the carrageenan group, suggesting an increased release of inflammatory
mediators by substance agar. In groups treated with cel
ecoxib and acetylsalicylic acid
vasodilation and redness were removed by the
action of these drugs (Figure 3
). The activity of
celecoxib suggests that the agar can be induced COX
-
2 in inflammation (HILARIO et al.,
2006).

The results demonstrate that the ac
tivity induced by nitrergic agar is blocked by anti
-
inflammatory patterns used (
Graphic

6). Jung et al (2010) analyzed the anti
-
inflammatory
action of n
-
Propyl Gallato through the negative regulation of NF
-
κ
B in cultures of
macrophage line RAW 264.7,
coming to the conclusion that there is a decrease in
concentration of nitrite in the treated cells with substances that have anti
-
inflammatory
activity, this decrease is probably a result of inhibition of nuclear transcription factors (NF
-
κ
B). In experimen
tal models of inflammation induced by carrageenan in air bag, there is an
increased activation of NF
-
κ
B (Crippen, 2006), and when animals are treated with
dexamethasone (anti
-
inflammatory steroid) for a reduction in the markup for NF
-
κ
B has been
made when

an immunohistochemistry assay for tissue removed from the stock area (Ellis et
al., 2000). Analyzing the results of concentration of nitrite, it was observed that the agar could
be inducing the activation of NF
-
κ
B.

Several studies have suggested that NSAI
Ds may act directly on the surface of
mononuclear cells by preventing the migration of these cells to sites of inflammation.
Interestingly, immunosuppressive agents may exert an anti
-
inflammatory activity by reducing
the infiltration of PMNs (DUKE et al.,
1973, apud, MEACOCK & KITCHEN, 1976).
MEACOCK & KITCHEN (1976) testing the performance of various NSAIDs such as
indomethacin, phenylbutazone, ketoprofen, ibuprofen, acetylsalicylic acid a, fenoprofen and
naproxen in the process of cell migration, they con
cluded that none of the tested drugs
prevented the migration of PMNs in a carrageenan
-
induced inflammation, however, certain
NSAIDs tested suppressed the migration of mononuclear cells (monocytes).

The decrease in leukocyte migration

observed in the result
s (Graphic

7) may be due to
blocking the expression of adhesion molecules that are responsible for "rolling" of leukocytes
along the endothelium. This blocking the expression of these molecules can be demonstrated
by inhibition of nuclear transcription fac
tors such as, NF
-
κ
B part which is responsible for
transcription of genes for iNOS, COX
-
2 and vascular cell adhesion molecule 1 (VCAM
-
1)
(Martin et al ., 2000, Kim et al., 2010).


CONCLUSION


The present study demonstrated that the hydrocolloid agar can be
used as template in
phlogistic agent air bag (air
-
pouch), since this promotes the plasma and leukocyte exudation
into the cavity of the bag, induction of vasodilation and redness.


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