Artigo Científico

1. Contents lists available at ScienceDirect
Pulmonary Pharmacology & Therapeutics
journal homepage: www.elsevier.com/locate/ypupt
Eucalyptol reduces airway hyperresponsiveness in rats following cigarette
smoke-exposed
Emanuel Kennedy-Feitosaa,1,∗
, Paolo Oliveira-Melob,1
, Eder Evangelista-Costab
,
Daniel Silveira Serrab
, Francisco Sales Ávila Cavalcanteb
, Edson Lopes da Pontec
, Roseli Barbosad
,
Renata Evangelista Rodrigues da Silvad
, Ana Maria Sampaio Assreuyb
,
José Henrique Leal-Cardosob
, Crystianne Calado Limab
a
Departamento de Ciências da Saúde, Universidade Federal Rural do Semi-Árido, UFERSA, Brazil
b
Instituto Superior de Ciências Biomédicas, Universidade Estadual do Ceará, UECE, Brazil
c
Centro Universitário Christus, Unichristus, Brazil
d
Departamento de Química Biológica, Universidade Regional do Cariri, URCA, Brazil
A R T I C L E I N F O
Keywords:
Eucalyptol
Hyperresponsiveness
Lung function
Inflammation
A B S T R A C T
Background: Cigarette smoke is the major cause of airway inflammatory disease, including airway hyperre-
sponsiveness. Eucalyptol (EUC), also named 1.8-cineole, is a monoterpenoid found in essential oil of medicinal
plants, showing several biological effects.
Hypothesis/purpose: Based in the eucalyptol protective activity in respiratory diseases as asthma, our hypothesis
is that eucalyptol is able to reduce the airway hyperresponsiveness and the respiratory mechanic parameters in
rats exposed to cigarette smoke.
Study design: Wistar rats were divided into control and cigarettes smoke (CS) groups. CS group was daily sub-
jected to cigarette smoke and treated by inhalation for 15 min/day with EUC (1 mg/mL) or vehicle during 30
days. After treatment, bronchoalveolar lavage (BAL) was collected to analyze the inflammatory profile, and
tracheal rings were isolated for evaluation of the airway smooth muscle hyperresponsiveness. Lung function was
analyzed in vivo.
Methods: The inflammatory profile was evaluated by optical microscopy performing total (Neubauer chamber)
and differential leukocyte count (smear slides stained in H&E). The hyperresponsiveness was evaluated in tra-
cheal rings contracted with potassium chloride (KCl) carbamoylcholine (CCh), or Barium chloride (BaCl2) in
presence or absence of nifedipine. The lung function (Newtonian resistance-RN) was evaluated by bronco sti-
mulation with methacholine (MCh).
Results: BAL from CS group increased the influx of leukocyte, mainly neutrophils and macrophages compared to
control group. EUC reduced by 71% this influx. The tracheal contractions induced by KCl, CCh or BaCl2 were
reduced by EUC in 59%, 42% and 26%, respectively. The last one was not different of nifedipine activity.
Newtonian resistance (RN) was also reduced in 37% by EUC compared to CS group.
Conclusion: EUC reduces the hyperresponsiveness and the airway inflammatory profile, recovering the lung
function.
1. Introduction
Cigarette smoke is usually associated to chronic obstructive pul-
monary disease (COPD) [1], being characterized by lung parenchyma
destruction, persistent hyperresponsiveness and abnormal
inflammatory response [2,3]. The airway responsiveness is a central
mechanism to the control of air duct tonus, and is associated to ex-
cessive airway smooth muscle contraction, thickness and integrity of
epithelial layer, and bronchial mechanical alterations.
Several studies have suggested that inflammation and airway
https://doi.org/10.1016/j.pupt.2020.101887
Received 22 July 2019; Received in revised form 2 January 2020; Accepted 4 January 2020
∗
Corresponding author. Science Health Department, Federal Rural University of the Semi-Arid (UFERSA), R. Francisco Mota, 572 - Pres. Costa e Silva, Mossoró,
RN, 59625-900, Brazil.
E-mail addresses: emanuel.lima@ufersa.edu.br, emanuel.lima@ufersa.edu.br (E. Kennedy-Feitosa).
1
Emanuel Kennedy-Feitosa and Paolo Oliveira Melo contributed equally to this study.
Pulmonary Pharmacology & Therapeutics 61 (2020) 101887
Available online 08 January 2020
1094-5539/ © 2020 Published by Elsevier Ltd.
T

2. remodeling contribute significantly to the development of bronchial
hyper responsiveness. Moreover, among airway structural cells, the
smooth muscle cells are described as promising targets in COPD, due to
its multifunctional role, that include bronchospasm, scarring and local
inflammation [4].
There are several pharmacological approaches to reduce in-
flammation and ameliorate the airway flow, such as anticholinergics,
β2-agonists, phosphodiesterase 4 inhibitors, glucocorticoids or the
combination of various drugs [5,6], but, the choice for the hyperre-
sponsiveness treatment is scarce [7]. In this perspective, several ex-
perimental and clinical studies have been arising with the aim to pro-
pose new therapeutic alternatives to the treatment of COPD [8].
Eucalyptol (EUC) is a monoterpene and the major compound of
Eucalyptus globulus essential oil, traditionally used to treat respiratory
disorders due to its secretolytic property [9].
Experimentally, the anti-inflammatory activity of EUC was demon-
strated in an experimental model of asthma [10]; in mice lungs infected
with influenza, via reduction of IL-6, TNF-α, IL-1β and recover of IL-10
levels [11]; and in acute lung injury induced by lipopolysaccharide
[12]. In patients with stable COPD, EUC reduced exacerbations, ame-
liorated dyspnea and improved lung function [13]. When inhaled EUC
reaches the peripheral airways, extending its beneficial effects to the
entire airway, including the sinuses which are known to be important
source to promote exacerbation-induced infections [14]. Later, EUC
showed anti-inflammatory and antioxidant effects in acute lung in-
flammation induced by cigarette smoke in mice, via reduction of NF-kB
and antioxidant enzymes [15]. The aim of this study was to evaluate the
eucalyptol effect on the airway hyperresponsiveness of rats exposed to
cigarette smoke.
2. Material and methods
2.1. Animals
Male Wistar rats (200–250 g) were fed with chow and had unrest-
ricted access to water in a controlled environment (18–22 °C, 50–70%
relative humidity, 12/12 h light/dark cycle). Rats were allowed to ac-
climatize for two weeks prior to experimental procedures. Animal
handling and the procedures used in this study was previously approved
by the Ethics Committee for Experimental Animals Use and Care of the
Ceara State University (CEUA n° 10462460-4/66).
2.2. Cigarette smoke exposure and procedures
Rats were exposed 3 times per day during 30 days to 12 commercial
cigarettes (10 mg tar, 0.9 mg nicotine and 10 mg monoxide) in in-
halation chamber (40 cm long, 30 cm wide, and 25 cm high), as de-
scribed previously [16] with modifications. A cigarette was coupled to a
60 mL plastic syringe, being 20 puffs (50–60 mL each) of smoke drawn
into the syringe and expelled into the inhalation chamber. Animals
were maintained in this smoke-filled air ( ± 3%) for 6 min. After, the
cover of the inhalation chamber was removed, and the exhaust fan of
the hood was turned on to evacuate the smoke within 1 min. So rats
were exposed to 12 cigarettes 3 times a day for 6 min/each 72 min per
day. Each cigarette produces 300 mg/m3
of total particulate material,
which was collected on Pallflex filters (Pall Corporation, Port Wa-
shington, USA) and weighted. Carboxyhaemoglobin (COHb) levels were
quantified in order to check the toxicity of the procedure (CS-exposed
rats had a range between 7.3% and 12.1%, while control group varied
from 1.1% to 1.9%).
2.3. Eucalyptol treatment
Rats were randomly divided into control (sham; n = 4) and cigar-
ette smoke (CS) groups. CS groups were exposed to cigarette smoke and
treated with EUC (1 mg/mL) (n = 6) or vehicle (saline) (n = 4) for
15 min per day, by nebulizer (Max Breathe ® inhaler ultrasonic NS, the
Medical Device Industry, Ltd., SP) and another to serve as an outlet
[10]. Control groups received EUC (n = 6) or vehicle (n = 4), but were
not exposed to cigarette smoke.
2.4. Experimental protocols
2.4.1. Bronchoalveolar lavage
The lung air spaces were harvested three times with 3.5 mL buffered
saline for total and differential leukocytes count [17].
2.4.2. Tracheal tissue preparation and evaluation of mechanical activity
Trachea was dissected, cut in 4–5 cylindrical rings, which were
mounted in organ baths containing 5 mL Krebs-Hanseleit (95% O2/5%
CO2 at 37 °C, pH 7.4) under a passive tension of 1 g and left during 1 h
for equilibrium. The contractile response was measured by the use of an
isometric force transducer (Grass model FTO3, Quincy, MA, USA)
coupled to a data acquisition system (Dataq instruments, PM-1000,
CWE Inc., Akron, OH, USA). The control contractions were induced by
potassium chloride (KCl; 60 mM). The contractile amplitude was
measured at the peak upward deflection.
2.4.3. Airways hyperresponsiveness
Series 1: performed to evaluate the EUC effect on spontaneous tonus
of tracheal rings by cumulative EUC concentrations (6.5 × 10−6
to
2 × 10−2
M) or vehicle.
Series 2: performed to evaluate the EUC inhibitory effect on elec-
tromechanical coupling induced by cumulative concentration of po-
tassium chloride (KCl) (10–80 mM).
Series 3: performed to evaluate the EUC inhibitory effect on phar-
macological coupling induced by cumulative concentrations of carba-
mylcholine (CCh; 0.001–100 μM).
Series 4: performed to evaluate the EUC effect on voltage-operated
calcium channels (VOCCs). Tracheal rings were maintained in Ca2+
-
free Krebs-Henseleit solution in the presence of high concentration of
K+
(60 mM) and ethylene glycol-bis (2-aminoethylether)-N,N,N′,N'-
tetraacetic acid (EGTA; 0.2 mM) to remove extracellular Ca2+
.
Contractions were induced by Barium chloride (BaCl2; 0.03–10 mM) in
List of abbreviations
BaCl2 Barium chloride
BAL Bronchoalveolar lavage
Ca2+
Calcium
CCh Carbamylcholine
COHb Carboxyhaemoglobin
COPD Chronic Obstructive Pulmonary Disease
CS Cigarette smoke
EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N'-tetraacetic
acid
EUC Eucalyptol
IL-10 Interleukin 10
IL-1β Interleukin 1β
IL-6 Interleukin 6
KCl Potassium chloride
NF-κB Nuclear factor kappa B
PEEP Positive end-expiratory pressure
TNF-α Tumor necrosis factor alpha
VOCCs Voltage-operated calcium channels
K+
Potassium
E. Kennedy-Feitosa, et al. Pulmonary Pharmacology & Therapeutics 61 (2020) 101887
2

3. tracheal segments depolarized with K+
under Ca2+
–free conditions.
Series 5: to compare EUC effect on VOCCs, using nifedipine as
standard. Tracheal rings from rats exposed to cigarette smoke, but
without treatment by nebulization with EUC. After verifications of
tissue responsiveness, the preparation was maintained in the same
conditions of series 4. KCl (60 mM) was added 5 min before addition of
EUC (6.5 × 10−3
M) or nifedipine (10 μM). After stabilization a con-
centration-effect curve of BaCl2 (0.03–10 mM) was performed.
2.4.4. Lung function analysis
After 30 days of CS exposition and treatment with EUC, animals
were sedated with diazepam (1 mg/kg, i.p.), anesthetized with sodium
pentobarbital (50 mg/kg, i.p., Hypnol® 3%, Syntect, Brazil), tracheos-
tomized and intubated with a 18-gauge cannula (Eastern Medikit,
Delhi, India), that was connected to a computer controlled ventilator
for small animals (Scirec© -flexVent®, Montreal, QC, Canada). Animals
were ventilated at baseline settings: respiratory frequency (150
breaths/min); tidal volume (10 mL/kg); limiting pressure (30 cmH2O);
positive end-expiratory pressure (PEEP; 3 cmH2O) before receiving
pancuronium bromide (0.5 mL/kg, i.p., Cristália, Lindoia, MG, Brazil).
The mechanical conditions of respiratory system were established
by application of two deep breaths under pressure limit (30 cmH2O;
3 s). To reduce the bronchial effect, animals were ventilated during
20 min in baseline standards. The experiment was fitted to the constant
phase model [18].
The impedance of respiratory system was assessed by forced oscil-
lation technique, at intervals of 30 s for 6 min and collected the fol-
lowing data: Newtonian Resistance (ΔRN), Tissue Resistance (ΔG) and
Tissue Elastance (ΔHe). After collecting the initial data, two additional
deep breaths were applied followed by 20 min of ventilation. The hy-
perresponsiveness of the airway smooth muscle was assessed by in-
halation of methacholine (MCh; 30 mg/mL; 1,2 mg/kg/animal) during
30 s using ultrasonic nebulizer (Inalasonic®, NS) coupled to inspiratory
branch of the mechanical ventilator [18]. Immediately after nebuliza-
tion, the forced oscillation technique was used in sequential way, at
intervals of 30 s for 6 min. At the end of the data collection (ΔRN, ΔG,
ΔHe). two additional deep breaths were applied 30 s after, the forced
oscillation technique was repeated in order to check the return to initial
parameters (pre-nebulization with MCh).
2.4.5. Statistical analysis
Data are expressed as mean ± S.E.M. GraphPad Prism 5 software
was used for the statistical analysis (San Diego, CA, USA). Results
considered statistically significant had a null hypothesis probability of
less than 5% (p < 0.05). Student t tests and analysis of variance (one-
way ANOVA) were used, followed by Holm-Sidak or
Student–Newman–Keuls multiple comparisons method, when appro-
priate.
3. Results
EUC reduces leukocyte infiltrate in the bronchial alveolar lavage CS
stimulated in 9 fold the migration of total leukocytes, mainly neu-
trophils (56 fold), into BAL compared to controls (total leukocytes:
6425 ± 1546; neutrophils: 1058 ± 249.4 cells/mL). CS also stimu-
lated macrophages migration in 8 fold compared to controls
(3553 ± 742.4 cells/mL) (Fig. 1A/B/C). EUC reduced the migration of
total leukocytes (1425 ± 322.7 vs. CS: 6425 ± 1546 cells/mL)
(Fig. 1A), neutrophils (188.6 ± 36.76 vs. CS: 1058 ± 249.4 cells/mL)
(Fig. 1B) and macrophages (1128 ± 194.8 vs. CS:
3553 ± 742.4 cells/mL) (Fig. 1C) by 80%, 71% and 82%, respectively.
3.1. EUC does not alter the tracheal basal tonus of rats exposed to cigarette
smoke
EUC did not affect the basal tonus of tracheal rings of rats exposed
to cigarette smoke up to the dose of compared to vehicle (saline)
(Fig. 2).
a
b
c
C
ontrol
C
S
EU
C
C
S+EU
C
0
2000
4000
6000
8000
10000
Leucocytes/mL
****
####
C
ontrol
C
S
EU
C
C
S+EU
C
0
500
1000
1500
2000
Neutrophils/mL
****
####
C
ontrol
C
S
EU
C
C
S+EU
C
0
2000
4000
6000
Macrophages/mL
****
####
Fig. 1. EUC reduces leukocyte infiltrate in the bronchial alveolar lavage of rats
exposed to cigarette smoke. a) Total leukocytes; b) Neutrophils; c)
Macrophages. ####
means significant differences between Control group and
CS group (p < 0,001). **** means significant difference between CS + EUC
and CS group (p < 0,001). Values are expressed as mean ± S.E.M.; (Control:
n = 4; CS: n = 5, CS + EUC: n = 6; EUC: n = 4); (p < 0.05, one-way ANOVA
followed by Holm-Sidak).
E. Kennedy-Feitosa, et al. Pulmonary Pharmacology & Therapeutics 61 (2020) 101887
3

4. 3.2. Effects of EUC on KCl-induced contractions in tracheal segments
The mean of the maximal responses induced by KCl in the CS group
was 3.77 ± 0.77 g, being higher than the control group
(1.69 ± 0.38 g) statistically significant at concentrations of 30 mM
(p = 0.002; one-way ANOVA). The CS + EUC group showed maximal
response 1.52 ± 0.31 g (p = 0.015; one-way ANOVA) inferior to the
maximum response of CS group (Fig. 3).
3.3. Effects of EUC on CCh-induced contractions in tracheal segments
The mean of the maximal responses induced by CCh in the control
group was 3.07 ± 0.12 g, however, the CS group showed an increase
in the contractile response (5.06 ± 0.29 g) differing significantly from
0,3 μM (p = 0.0004; one-way ANOVA). The maximal response was
reduced to 2.92 ± 0.43 g (p = 0.0001; one-way ANOVA) to the
CS + EUC group (Fig. 4).
3.4. Effects of EUC on Ba2+
-induced contractions in tracheal segments
depolarized with K+
under Ca2+
–free conditions
The mean of the maximal responses induced by BaCl2 (10 mM) was
1.09 ± 0.07 g in the control group and 2.50 ± 0.16 g in the CS group
(p = 0.0001; one-way ANOVA). The CS + EUC group showed a re-
duction of this mean to 1.84 ± 0.18 g (p = 0.017; one-way ANOVA)
(Fig. 5). There is no difference between the EUC and nifedipine effect in
the calcium channels block (Fig. 6).
3.5. Lung function
The values of lung function are in Table 2. The RN was increased on
CS group when compared to control group (p = 0.015), but the
treatment with EUC reduced this alteration (p = 0.03). We didn't ob-
serve significant differences in G in any of the groups. The H and hys-
teresivity were reduced on CS group when compared to control group,
but EUC did not recover these alterations. The Inspiratory Capacity (IC)
increased on CS group, but this parameter was reduced when the ani-
mals were treated with EUC (p = 0,03). We didn't observe significant
alterations on Static Compliance and Pressure-Volume (PV) Loop Area.
4. Discussion
This study showed original results in respect to EUC effect on an
animal model of airway hyperresponsiveness induced by cigarette
smoke inhalation.
Pharmacological treatment in many airway inflammation diseases
includes the use of bronchodilators and corticosteroids. However, be-
sides high costs and several associated side effects by these medicines,
studies suggest the need to seek alternative therapies that minimize
these difficulties [19]. Here we showed that EUC is able to reduce in-
flammatory cells migration to the lung, hyperresponsiveness and im-
prove the lung function.
An important feature present in some lung inflammatory diseases is
the migration of many cells and cellular elements such as mastocytes,
eosinophils, lymphocytes or neutrophils play key roles [20]. The lit-
erature has been reported that chronic exposure to cigarette smoke
induces significant lung inflammation [16] in line with our present data
in which it was found lung leukocyte influx in CS group. The neutrophil
migration to the site of the inflammation is a critical factor to the re-
lease of chemotactic factors responsible by mononuclear influx with
differentiation on macrophage [21]. Accordingly, we demonstrated
both macrophage and neutrophil influx to the lung in the CS group with
that of control group. But this influx was reduced by the treatment with
EUC. Other studies showed the EUC as an anti-inflammatory compound
to reduce the inflammatory cells on lung inflammation [10,12,15]. This
effect is probably because the EUC is able to inhibit the NF-kB nucleus
translocation and transcription to cytokines and chemokine such as KC
[15].
The airway hyperresponsiveness is a clinical feature present in
several airway inflammatory diseases [20]. In this study we figure out
evidence of a smoke induced increase in maximal contraction ampli-
tude when the segments were stimulated with CCh. Similarly, con-
tractions induced by CCh of tracheal smooth muscle on guinea-pig
challenged with ovalbumin a classical method to induce airway hy-
perresponsiveness [10]. In this study we figure out evidence that EUC
reduced this contraction amplitudes. It also was found in the studies
that the guinea-pig challenged with ovalbumin and treated with EUC
1 mg/mL by inhalation [10,22] as in our study. This is correlated with
the in vivo results figure out on RN of animals challenged with MCh. The
airway resistance (RN) of CS group treated with EUC was reduced when
compared to CS group. We believe that the RN reduction in our model is
because the EUC reduce the airway hyperresponsiveness and conse-
quently ameliorates the inspiratory capacity, as previously suggested by
Ref. [19].
The cigarette smoke exposure has a key role on exacerbation of
airway smooth muscle (ASM) contraction due to some chemical pro-
ducts and pollutants generated by smoke that directly affect the
Fig. 2. Concentration–response curves to EUC on tracheal segments of rats
exposed to cigarette smoke. Trachea (4–5 rings) was dissected and maintained
in physiological solution (95% O2; 5% CO2, 37 °C, pH 7.4) under passive ten-
sion of 1 g. EUC was added to tissue in cumulative concentrations (6.5 × 10−6
to 2 × 10−2
M). Values are expressed as mean ± S.E.M.; (Control: n = 4; CS:
n = 5, CS + EUC: n = 6; EUC: n = 4); (p < 0.05, one-way ANOVA followed
by Holm-Sidak).
Fig. 3. Concentration–response curves to KCl (10–80 mM) in tracheal segments
from rats exposed to cigarette smoke and EUC effect on the hyperresponsive-
ness of tracheal segments in rats exposed to cigarette smoke. #
means sig-
nificant differences between Control group and CS group. * means significant
difference between CS + EUC and CS group. Values are expressed as
mean ± S.E.M.; (Control: n = 4; CS: n = 5, CS + EUC: n = 6; EUC: n = 4);
(p < 0.05, one-way ANOVA followed by Holm-Sidak).
E. Kennedy-Feitosa, et al. Pulmonary Pharmacology & Therapeutics 61 (2020) 101887
4

5. contractility [23]. This is a classic airway hyperresponsiveness me-
chanism, either by muscarinic agonists or by membrane plasmatic de-
polarization [24]. However, in the changes in intracellular Ca2+
con-
centration ([Ca2+
]i) that regulate contraction in ASM may contribute
to the key features of asthma or COPD [25]. Animal data indicates that
cigarette smoke exposure increases ASM responsiveness to acetylcho-
line (ACh) and high K+
depolarization in rats [10]. In our model, we
found evidence of the cigarette smoke exposition increase in maximal
contraction amplitude when the segments were stimulated with KCl.
However, the treatment with EUC was able to reduced these contrac-
tion amplitudes as showed similarity on ASM from guinea-pig chal-
lenged with ovalbumin [10]. We suggest that EUC has effect in re-
ceptors or channel membrane, since this compound probably did not
interfere with the sarcoplasmic reticulum function on rat cardiac tis-
sues, but, blocked the inwards calcium flux across the cell membrane,
as previously suggested by Ref. [26]. Additionally, recent studies also
demonstrated that the EUC promotes ASM relaxation through of direct
action on the pore of the L-type VOCCs, which channels that are not
Fig. 4. Concentration–response curves to CCh (0.001–100 mM) on tracheal
segments of rats exposed to cigarette smoke and EUC effect on hyperrespon-
siveness of tracheal segments in rats exposed to cigarette smoke. #
means sig-
nificant differences between Control group and CS group. * means significant
difference between CS + EUC and CS group. Values are expressed as
mean ± S.E.M.; (Control: n = 4; CS: n = 5, CS + EUC: n = 6; EUC: n = 4);
(p < 0.05, one-way ANOVA followed by Holm-Sidak).
Fig. 5. Concentration–response curves to BaCl2 (0.03–10 mM) of tracheal
segments from rats exposed to cigarette smoke and EUC effect on the hy-
perresponsiveness of tracheal segments from rats exposed to cigarette smoke. #
means significant differences between Control group and CS group. * means
significant difference between CS + EUC and CS group. Values are expressed as
mean ± S.E.M.; (Control: n = 4; CS: n = 5, CS + EUC: n = 6; EUC: n = 4);
(p < 0.05, one-way ANOVA followed by Holm-Sidak).
Fig. 6. Concentration–response curves to BaCl2 (0.03–10 mM) in the tracheal
segments of rats exposed to cigarette smoke. EUC (1 mg/mL) effect on channels-
voltage operated (VOCCs). Values are expressed as mean ± S.E.M.; (Control:
n = 4; CS: n = 5); (p < 0.05, one-way ANOVA followed by Holm-Sidak).
Table 1
Values of EC50 concentration–response curves to KCl, to CCh, or BaCl2 in iso-
lated tracheal segments from rats exposed to cigarette smoke.#
means sig-
nificant differences between Control group and CS group. * means significant
difference between CS + EUC and CS group. Values are expressed as
mean ± S.E.M.; (Control: n = 4; CS: n = 5, CS + EUC: n = 6; EUC: n = 4);
(p < 0.05, one-way ANOVA followed by Holm-Sidak).
Control CS EUC CS + EUC
KCl (mM) 37,0 ± 0,49 24,9 ± 0,68#
28,5 ± 0,50 20,9 ± 0,30*
CCh (mM) 0,28 ± 0,04 0,16 ± 0,01#
0,22 ± 0,01 0,05 ± 0,01*
BaCl2 (mM) 4,45 ± 0,37 0,75 ± 0,13#
1,97 ± 1,00 0,52 ± 0,06*
Table 2
Values of lung function. Newtonian Resistance (RN); Tissue Resistance (G);
Tissue Elastance (H); Inspiratory capacity (IC); static compliance (CST);
Hysteresivity (η). PV Loop Area (A). Values expressed in mL.cmH2O or mL.#
means significant differences between Control group and CS group. * means
significant difference between CS + EUC and CS group. Values are expressed as
mean ± S.E.M.; (Control: n = 4; CS: n = 5, CS + EUC: n = 6; EUC: n = 4);
(p < 0.05, one-way ANOVA followed by Student–Newman–Keuls test).
Measure Group Value
Newtonian Resistence (RN ) (cmH2O·s·mL−1
) Control 0.0534 ± 0.0223
CS 0.0881 ± 0.0275#
EUC 0.0469 ± 0.0192
CS + EUC 0.0549 ± 0.0251*
Tissue Resistence (G) (cmH2O·mL−1
) Control 0.656 ± 0.226
CS 0.580 ± 0.156
EUC 0.687 ± 0.363
CS + EUC 0.610 ± 0.291
Tissue Elastance (H) (cmH2O·mL−1
) Control 5.387 ± 2.200
CS 2.764 ± 0.907#
EUC 4.586 ± 2.185
CS + EUC 3.589 ± 1.542
Hysteresivity ( ) Control 0.145 ± 0.075
CS 0.215 ± 0.036#
EUC 0.160 ± 0.062
CS + EUC 0.200 ± 0.114
Inspiratory Capacity (IC) (mL) Control 9.604 ± 0.821
CS 12.409 ± 1.776#
EUC 9.544 ± 1.788
CS + EUC 10.106 ± 1.951*
Static Compliance (CST) (mL·cmH2O−1
) Control 0.849 ± 0.270
CS 1.105 ± 0.289
EUC 0.813 ± 0.185
CS + EUC 0.909 ± 0.104
PV Loop Area (A) (mL·cmH2O) Control 77.90 ± 30.57
CS 98.77 ± 38.82
EUC 75.26 ± 36.02
CS + EUC 80.19 ± 19.55
E. Kennedy-Feitosa, et al. Pulmonary Pharmacology & Therapeutics 61 (2020) 101887
5

6. involved directly in the maintenance of basal tonus, when the rest po-
tential values are still very negative [27]. This explains why we found
no effect of EUC on basal tonus.
When analyzed the EC50 (Table 1) of the pharmacological and
electromechanical coupling these date suggest that EUC is able to re-
duce the hyperresponsiveness in both type of coupling. This is probably
because the EUC relax rat and guinea-pig ASM by a nonspecific me-
chanism [28]. But on other hand, EUC increased the EC50 to KCl on
effect curve-concentration showed a reduction on airway hypersensi-
tivity caused by cigarette smoke expose in the electromechanical cou-
pling. This effect supports the hypothesis that EUC is a compound
which affects both signaling pathways, but in this model, it is more
specific to membrane plasmatic depolarization, probably because the
EUC has inhibition effect of phasic contractions induced by the high-
potassium solution in vitro, suggesting that it have an antagonist action
on the calcium influx across the cell membrane [22].
The increase on calcium channel activity can be one of the most
important factors responsible for hyperresponsiveness on ASM in
asthma [11]. In this study the increase of the contraction induced by
BaCl2, suggests that the cigarette smoke exposure induced the hy-
perresponsiveness in this model of occurs via VOCCs. Barium chloride is
an ion that permeates the membrane mainly via VOCCs, that via ligand-
gated calcium channel [29]. Although the treatment with EUC does not
reverse the hypersensitivity induced by cigarette smoke, this compound
reduced the hyperresponsiveness. EUC showed selective action on
VOCCs [22], and inhibited the contraction ventricular papillary mus-
cles from rats, by the blockage of calcium channels [26]. Moreover, the
increase of contractility in tracheal smooth muscle from rats is related
to be probability for increase the permeability of Ca2+
and Cl−
chan-
nels [30]. When compared with the L-type calcium channel blocked by
nifedipine, EUC did not show any difference in effect. It suggests that
EUC can reduce the hyperresponsiveness, probably by blocking the
signaling pathway of voltage-gated calcium channel.
In conclusion, EUC presents myorelaxant effect in rat airway hy-
perresponsiveness induced by cigarette smoke, probably by the
blockade of voltage-gated calcium channels, along with an anti-in-
flammatory effect reducing inflammatory cells. Probably, these effects
occurs in a synergic fashion, suggesting potential pharmacological ap-
plication to inflammation and airway hyperresponsiveness induced by
cigarette smoke.
Declaration of competing interest
We wish to confirm that there are no known conflicts of interest
associated with this publication and there has been no significant fi-
nancial support for this work that could have influenced its outcome.
Acknowledgment
Funcap - Fundação Cearense de Apoio ao Desenvolvimento
Científico e Tecnológico
CNPq - Conselho Nacional de Desenvolvimento Científico e
Tecnológico
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