International Immunopharmacology xxx (xxxx) xxx

Contents lists available at ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Protective effects of piperlongumin in the prevention of inflammatory

damage caused by pulmonary exposure to benzopyrene carcinogen
Tissiane Eid Barbosa Ashino a, b, Monielle Leal Sant ́ Ana b, c, Ariane Harumi Yoshikawa b, d,
Lucas Possebon a, b, Sara de Souza Costa a, b, Melina Mizusaki Iyomasa-Pilon b,
Helena Ribeiro Souza a, b, Giovana Aparecida Gonçalves b, e, Sonia Maria Oliani a, c,
Ana Paula Girol a, b, c, *
a
São Paulo State University, (UNESP), Department of Biology, Laboratory of Immunomorphology, Cristovão Colombo Street, 2265, São José do Rio Preto, SP, Brazil
b
University Center Padre Albino (UNIFIPA), Estudantes Street, 225, Catanduva, SP, Brazil
c
Federal University of São Paulo (UNIFESP), Graduate Program in Structural and Functional Biology, Sena Madureira Street, 1500, São Paulo, SP, Brazil
d
São José do Rio Preto School of Medicine (FAMERP), Brigadeiro Faria Lima Avenue, 5416, São José do Rio Preto, SP, Brazil
e
Federal University of São Paulo, Department of Gynecology, Escola Paulista de Medicina (UNIFESP-EPM), Sena Madureira Street, 1500, São Paulo, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Benzopyrene is one of the main polycyclic aromatic hydrocarbons with carcinogenic capacity. Research has
Lung cancer shown that anti-inflammatory drugs can reduce the incidence of lung cancer. In this scenario, we highlight
Smoking piperlongumin (PL), an alkaloid from Piper longum with anti-inflammatory properties. Therefore, our aim was to
Natural bioactive compound
study the effect of PL administration in a model of pulmonary carcinogenesis induced by benzopyrene in Balb/c
Cytokines
AnxA1
mice. Animals were divided into 3 groups (n = 10/group): sham (10% DMSO), induced by benzopyrene (100
mg/kg, diluted in DMSO) without treatment (BaP) for 12 weeks and induced by benzopyrene and treated with PL
(BaP/PL) (2 mg/kg in 10% DMSO) from the eighth week post-induction. Animals were weighed daily and
pletsmography was performed in the 12th week. Genotoxicity and hemoglobin levels were analyzed in blood and
quantification of leukocytes in bronchoalveolar lavage (BAL). Lungs were collected for histopathological eval­
uation, immunohistochemical studies of annexin A1 (AnxA1), cyclooxygenase 2 (COX-2), anti-apoptotic protein
Bcl-2 and nuclear transcription factor (NF-kB) and also the measurement of interleukin cytokines (IL)-1β, IL-17
and tumor necrosis factor (TNF) -α. Treatment with PL reduced the pulmonary parameters (p < 0,001) of fre­
quency, volume and pulmonary ventilation, decreased lymphocytes, monocytes and neutrophils in BAL (p <
0,05) as well as blood hemoglobin levels (p < 0,01). PL administration also reduced DNA damage and preserved
the pulmonary architecture compared to the BaP group. Moreover, the anti-inflammatory effect of PL was evi­
denced by the maintenance of AnxA1 levels, reduction of COX-2 (p < 0,05), Bcl-2 (p < 0,01) and NF-kB (p <
0,001) expressions and decreased IL-1β, IL-17 (p < 0,01) and TNF-α (p < 0,05) levels. The results show the
therapeutic potential of PL in the treatment of pulmonary anti-inflammatory and anti-tumor diseases with
promising therapeutic implications.

Abbreviations: µL, microliter; ATII, alveolar type II cells; ANOVA, variance analysis; AnxA1, annexin A1; BAL, bronchoalveolar lavage; BaP, benzopyrene; Bax, Bcl-
2 associated with protein X; CBR1, carbonil-redutase; COX-2, cyclooxygenase-2; DAB, diaminobenzidine; DNA, deoxyribonucleic acid; DMSO, dimethylsulfoxide;
EDTA, Ethylenediaminetetraacetic acid; GSTP1, glutathione S-tranferase p1; HE, hematoxylin and eosin; Hep-2, epidermoid carcinoma of the larynx; HIF-α, hypoxia-
inducible factor-1; HUVEC, Human umbilical vein endothelial cells; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-17, interleukin 17; ip, intraperitoneal; MAPK,
mitogen-activated protein kinases; MCP-1, Monocyte chemoattractant protein-1, MIP-2, macrophage inflammatory protein; NaCl, sodium chloride; NaOH, sodium
hydroxide; NBC, Natural Bioactive Compound; NF-kB, nuclear factor kappa B; NSCLC, non-small cell lung cancer; PAH, Polycyclic aromatic hydrocarbon; PBS,
Phosphate buffered solution; PL, piperlongumine; ROS, reactive oxygen species; SCLC, small cell lung câncer; Sham, Experimental group tested only with the vehicle
used for drug dilution; TGF- α, tranforming growth fator; TNF-α, tumor necrosis factor; UNIFIPA, University Center Padre Albino.
* Corresponding author at: Department of Physical and Biological Sciences, University Center Padre Albino (UNIFIPA), dos Estudantes Street, 225, Catanduva, SP
15.809-144, Brazil.
E-mail address: anapaula.girol@unifipa.com.br (A.P. Girol).

https://doi.org/10.1016/j.intimp.2021.108285
Received 29 July 2021; Received in revised form 9 October 2021; Accepted 18 October 2021

Please cite this article as: Tissiane Eid Barbosa Ashino, International Immunopharmacology, https://doi.org/10.1016/j.intimp.2021.108285
T.E.B. Ashino et al.
1. Introduction
Lung cancer is among the leading causes of mortality worldwide [58]
and can be classified into two subtypes: non-small cell lung cancer
(NSCLC) and small cell lung cancer (SCLC) [62]. NSCLC represents 85%
of lung cancer cases and is divided into adenocarcinoma, squamous cell
carcinoma (epidermoid) and large cell carcinoma [46].
Tumor cells in the lung cancer microenvironment, chemokines and
inflammatory mediators as cyclooxygenase (COX-2), hypoxia-inducible
factor (HIF)-α, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-α,
and interleukin (IL)-1β, secreted by interstitial cells, interact and acti­
vate pro-inflammatory signaling and cell growth pathways. This inter­
action promotes the malignant biological behavior of lung cancer and
tumor progression [66,1]. Besides, alveolar type II (ATII) cells, that are
essential for lung homeostasis, also play a central role in acute and
chronic illness, being important for the pathogenesis of lung adenocar­
cinoma as a progressive pre-neoplastic change [53].
Even before the appearance of the tumor, the existence of a chronic
inflammatory process acting as a driving force for carcinogenesis is a
well-established factor [25]. Among the critical causes associated with
inflammatory lung diseases, we can highlight tobacco, occupational
exposure, diet, biological factors and environmental pollution [17,71].
Polycyclic aromatic hydrocarbons (PAHs) are chemical products that
contain at least two fused benzene rings, without heteroatoms and are
released into the environment by incomplete combustion of organic
material, such as: coal, oil and organic polymers, and in urban areas,
they are mainly launched by automobiles [68]. In this context, benzo­
pyrene stands out for being one of the main PAHs present in the envi­
ronment and with a high proven carcinogenic capacity in humans [47],
therefore, used in vivo experimental models [23,26].
Inflammatory changes triggered by intrapulmonary instillation of
benzopyrene such as hyperplasia and formation of bronchus-associated
lymphoid tissue (BALT) are characteristic in the formation of neoplasms
[54,51]. Research has shown that substances with anti-inflammatory
activity can reduce the incidence of lung cancer [4,66]. In this sce­
nario, natural bioactive compounds (NBCs) such as terpenes and flavo­
noids that have many properties such as reducing inflammation and
stimulating the immune system [6], may represent a possibility for
preventive treatment for lung cancer.
Among NBCs we highlight piperlongumin (PL) (piperlongumin, 5,6-
di-hidro-1-[(2E)-1-oxo-3 (3,4,5-trimetoxifenil)-2-propenil]-2(1H) pir­
idinona), the biologically active component of the Piper species
(Piperaceae) with great economic and medicinal importance [5] and
different pharmacological actions [6,61]. Several studies have shown
that PL has antitumor, antifungal, anxiolytic, antidepressant, cytotoxic,
antibacterial, antiplatelet and anti-inflammatory properties, among
others [28,5,31,52,16,60,24].
Raj and collaborators [52] reported that PL induces apoptosis by
interfering with homeostatic regulators, redox and reactive oxygen
species (ROS), such as glutathione S-transferase pi 1 (GSTP1) and
carbonyl reductase (CBR1). The antitumor activity of PL involves cell
cycle arrest, activation of caspases and mitogen-activated protein ki­
nases (MAPKs), deregulation of anti-apoptotic proteins such as Bcl-2 and
nuclear transcription factor kB (NF-kB) [34,15,36]. In breast cancer
cells, PL reduced the expression of Bcl-2 and stopped the cell cycle by
destabilizing the tubulin microtubules [42]. PL has also been shown to
be a potent inhibitor of NF-kB activation induced by carcinogens and
inflammatory stimuli acting selectively in tumor cells of different strains
and absence of toxic effect in normal strains [22]. In addition, in­
vestigations have used anticancer drugs in combination with PL in order
to reduce the mechanisms of resistance to chemotherapy. Jyothi and
collaborators [31] evaluated the concomitant treatment of PL with
diferuloylmethane, an anti-inflammatory and anticancer agent. This
treatment increased the cytotoxicity induced by PL and the antitumor
activity of the chemotherapeutic agent. The potential of PL as a thera­
peutic immunomodulator for cancer prevention and progression was
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reinforced by analyzing PL administration in epidermoid carcinoma of
the larynx (Hep-2) cells and human umbilical vein endothelial cells
HUVEC) in which PL modulated the expression of genes involved in
inflammatory processes [24]. In another investigation by our research
group on an experimental model of respiratory inflammation, PL treat­
ment recovered lung tissues, increased the expression of the anti-
inflammatory protein annexin-A1 (AnxA1) while reduced cyclo­
oxygenase (COX-2), nuclear factor kappa B (NF-kB) and neutrophil
elastase expressions, controlling the inflammation induced by cigarette
smoke [55].
However, there are still few reports of the actions of PL in inflam­
matory and neoplastic lung conditions. For this reason, in this study, we
sought to investigate the effect of PL administration on the treatment of
precancerous inflammatory processes in the lung, induced by the
administration of benzopyrene in an experimental model, as a possible
preventive therapeutic alternative in the treatment of lung cancers. To
verify the effectiveness of the treatment, physiological and histopatho­
logical parameters, lymphocyte genotoxicity; inflammatory influx and
levels of inflammatory mediators were studied.
2. Materials and methods
2.1. Animals
Male Balb/c mice (n = 30) (20–25 g body weight) were obtained
from the Didatic and Experimental Research Unit of the University
Center Padre Albino (UNIFIPA) and kept in individual cages in a
controlled environment (24–25 ◦ C, 12 h light/dark cycle) with water
and food ad libitum.
All experimental procedures were conducted according to the
guidelines for biomedical research stated by the Brazilian Societies of
Experimental Biology and approved by the Ethics Committees on Ani­
mal Use at UNIFIPA (Certificate n◦ 10/18), Brazil. The animals were
divided into 3 groups: sham (vehicle – 10% DMSO), induced by ben­
zopyrene without treatment (BaP) and benzopyrene-induced and
treated with piperlongumin (BaP/PL) (n = 10/group).
The experiments were designed to minimize the number of animals
used and their suffering during the execution of the protocols. For this
reason, PL control group was not included as the safety of PL has already
been demonstrated even at higher concentrations (10 mg/kg) [39]. All
animals were daily evaluated by the institution’s veterinarian.
2.2. Benzopyrene exposure and PL treatment protocol
For the benzopyrene exposure, two groups of animals were anes­
thetized intraperitoneally (ip) with 0.2 ml/100 g of ketamine and 0.05
ml/100 g of xilasine and administered ip with 0.2 ml of the benzopyrene
solution (Sigma – B1760) (100 mg/kg) diluted in dimethylsulfoxide
(DMSO) 10% [69].
The therapeutic efficacy of PL (Sigma - SML 0221), against inflam­
matory and pre-carcinogenic processes caused by exposure to benzo­
pyrene, was evaluated in one of the induced groups, from the eighth
week post-induction, through the ip administration of PL (2.0 mg/Kg,
diluted in DMSO 10%), 3× a week, on alternate days, [52,55], for 5
weeks.
The animals of all groups were euthanized after 12 weeks post-
induction by excessive dose of anesthetic.
2.3. Physiological evaluations of plethysmography and weight
All animals were weekly weighed. At the end of the experiment, the
animals were evaluated for plethysmography by measuring breathing
capacity, lung ventilation, frequency and inspired air volume by a spe­
cific device and adapted for use (PowerLab, AD Instruments-Gas
Analyzer, Sydney, Australia).
T.E.B. Ashino et al.
2.4. Biochemical blood assays
Blood was collected by cardiac puncture in heparinized syringes and
separated in aliquots for the analysis of hemoglobin by a commercial Kit
(LAB test, Minas Gerais, Brazil) and reading on the spectrophotometer.
2.5. Comet assay
For the Comet Assay, two slides per animal were prepared using a
mixture of agarose (1.5% in PBS) boiled for one minute, cooled and
solidified three times. Then, the slides were bathed and placed under
refrigeration for later use. One aliquot of 10 µl of blood was added to
1000 µl of saline in a microtube and reserved. A second Low Melting
agarose (0.5%) was prepared and packed 120 µl per sample in a 37 ◦ C
water bath together with 10 µl of saline/blood solution and this mixture
was placed on the slides, covered with coverslips and taken to the
refrigerator by at least thirty minutes to solidify the Low Melting
agarose. Then, the coverslips were removed and the slides were placed
in a glass vat to be immersed in pH 10 lysis solution (2.5 M NaCl, 100 nM
EDTA, 10 mM Tris, 35 mM Lauryl) at 4◦C for one hour in the refrigerator
and at room temperature shelter of light. Afterwards, the slides were
placed in the pH13 electrophoresis buffer solution (200 mM EDTA, 10 N
NaOH) for about 30 min. Then, the slides were neutralized and washed
for 3x of five minutes with 5 ml of neutralization solution pH 7.5 (0.4 M
Tris). Subsequently, the slides were immersed in 100% ethanol for 10
min for fixation. After drying, 100 µl of solution for staining with
ethidium bromide was added and the slides were covered with a
coverslip for analysis under a fluorescence microscope (Fluo3, Bel).
The cells were classified according to tail length: class 0, undamaged,
without tail; class 1: with short tail, smaller than the diameter of the
nucleus; class 2, with a tail corresponding to one or two times the
diameter of the nucleus; class 3, tail length greater than twice the
diameter of the nucleus [5].
Fifty lymphocytes per group were counted and DNA damage (tail
length, percentage of DNA damage and tail moment) was determined
using the CometScore software version 1.5 [33].
2.6. Quantitative analyses of bronchoalveolar lavage (BAL)
To obtain BAL, at the end of the experiment the animals had a can­
nulated trachea and a clamped right lung. The left lung was washed 3
times with PBS and the liquid obtained was centrifuged for 10 min at
1,500 rpm. The supernatant was stored at − 70 ◦ C for subsequent mea­
surement of cytokines and the pellet was resuspended in 500 µl of PBS
and 10 µl aliquots were stained in Turk (1:10) for quantification of in­
flammatory cells in a Neubauer camera (values as number of cells ×
103/ml).
2.7. Histopathological and immunohistochemical analysis
After BAL collection, the right lung was removed, fixed in 4%
formaldehyde and processed for inclusion in paraffin. 4 µm sections
were used for histopathological analysis after staining with
hematoxylin-eosin (HE), under the Leica microscope (DM500). To
perform the inflammatory score, 10 different slides of each animal were
analyzed according the categories: Peribronchial inflammation and
perivascular inflammation and graded as follows: 0, no inflammatory
cells; 1, occasional cuffing with inflammatory cells; 2, most bronchi or
vessels surrounded by a thin layer (1–5) of inflammatory cells; 3, most
bronchi or vessels surrounded by a thick layer (>5 cells) [39].
Immunohistochemical studies were used to evaluate the expressions
of AnxA1, Bcl-2, COX-2 and NF-kB. Lung sections were processed for
antigenic recovery with citrate buffer pH 6.0, blockade of the endoge­
nous peroxidase activity and incubated with the primary polyclonal
antibodies rabbit:anti AnxA1 (1:1000) (Invitrogen), anti-Bcl − 2 (1:
150), anti-COX-2 (1: 500) and anti-NF-kB (1: 1000) (Abcam, Cambrigde,
3
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UK) for 12 h. Then, incubated with the biotinylated secondary antibody
and immersed in a streptavidin peroxidase conjugate complex. The
diaminobenzidine substrate (DAB) was used for staining (DAB Kit,
Invitrogen) and, afterwards, the sections were counterstained with He­
matoxylin. Proteins were quantified by densitometry (arbitrary units
from 0 to 255) [55] in the image analyzer (Leica Image Analysis soft­
ware), by marking 20 points on the cells present in the alveoli, in five
fields photographed with a 40x objective and selected at random in each
sample.
2.8. Evaluation of anti-inflammatory protein annexin A1 by Western
blotting
Lung fragments were macerated in liquid nitrogen and 650 µl of the
protease inhibitor solution (Protease Inhibitor Cocktail Set I, Millipore
Corporation, USA) and phosphatases (PhosphoSafe, Novagen, Millipore
Corporation, USA) were added, according to the manufacturer’s in­
structions. The material was incubated for 20 min, at 4 ◦ C, under con­
stant agitation, and then centrifuged at 14,000 rpm, for 10 min, at 4 ◦ C,
with the supernatants collected and immediately frozen at – 80 ◦ C.
The Bradford assay (Biorad, Hemel Hempsted, UK) was performed to
determine the protein concentration. An aliquot was mixed (1:1) with
2× “loading buffer” (125 mM Tris base, pH 6.8, containing 10% mer­
captoethanol, 4% sodium dodecyl sulfate, 20% glycerol, 0.1 bromo­
phenol blue %) and denatured in water at 100 ◦ C for 5 min. The samples
(20 mg of protein per well) and molecular weight markers were sepa­
rated on SDS-PAGE gel (10% sodium dodecyl sulfate − 10% poly­
acrylamide gel) and subsequently transferred to a nitrocellulose
membrane. The AnxA1 protein was detected using the primary anti-
AnxA1 antibody (1:1000) (Life Technologies). The signal was ampli­
fied using secondary anti-rabbit IgG HRP conjugated antibody (Prom­
ega) and the reaction product visualized with DAB staining (DAB Kit,
Invitrogen). The α-Tubulin antibody (1:500, Sigma) and β-actin (1:500,
Sigma) were used as a controls. The protein expressions in the mem­
branes were quantified by densitometry in the image analyzer (Leica
Image Analysis software).
2.9. Cytokine levels
In all groups, the cytokines IL-1β, IL-17 and TNF-α were quantified in
the pulmonary macerate supernatant, using commercially available
ELISA immunological test kits (Sigma) and according to the manufac­
turer’s specification.
2.10. Statistical analysis
Data collected from the experiments were analyzed using GraphPad
Prism® version 6. software (GraphPad Software, Inc.). The results ob­
tained were previously submitted to descriptive analysis and determi­
nation of normality using the Shapiro-Wilk normality test. As the
samples showed normal distribution, Analysis of Variance (ANOVA) was
used, followed by the Bonferroni test. All values obtained were
expressed as mean ± S.E.M. and P values less than 0.05 were considered
statistically significant.
3. Results
3.1. Increased pulmonary frequency, volume and ventilation parameters
were attenuated after treatment with PL
The lung volume (Fig. 1A), respiratory frequency (Fig. 1B) and pul­
monary ventilation (Fig. 1C) were increased in the benzopyrene-
induced-group without treatment (BaP) compared to the sham group
(p < 0.001) but a significant reduction in these pulmonary parameters
occurred in animals treated with PL (BaP/PL) (p < 0.001) (Lung volume:
Sham 2.356 ± 0.120; BaP 7.505 ± 0.065; BaP/PL 2.411 ± 0.194;
T.E.B. Ashino et al. International Immunopharmacology xxx (xxxx) xxx

Fig. 1. Physiological analyses (pleth­

ysmography and weight) hemoglobin
levels- Capacity of the volume of
inspired air (A). Pulmonary frequency
assessment (B), pulmonary ventilation
capacity (C) per minute. Initial and final
weighing in grammes. I: Initial. F: Final.
(D). Biochemical analysis of hemoglobin
in whole blood per by g/dL (E). Sham
group, induced by untreated benzopyr­
ene (BaP) and induced and treated with
piperlongumin (BaP/PL) animals. Re­
sults presented as mean ± S.E.M. (n =
10/group), *** p < 0.001 vs sham
group; ** p < 0.01 vs sham group; ###
p < 0.001 vs BaP group, ## p < 0.01 vs
BaP group.

Respiratory frequency: Sham 157.9 ± 1.908; BaP 190.5 ± 0.712; BaP/PL moment, p < 0,05) (Fig. 2B).
154.5 ± 1.274; Lung ventilation: Sham 374.3 ± 22.65; BaP 1439 ±
7.976; BaP/PL 371.2 ± 31.56). 3.4. PL preserved lung histological organization and reduced the influx of
Weighing assessments did not show significant differences among inflammatory cells in BAL and lungs
groups (Fig. 1D).
Histopathological analyses showed normal pulmonary structures in
3.2. PL decreased hemoglobin levels after benzopyrene exposure the sham group (Fig. 3A and B) with terminal bronchioles of simple
cubic epithelium, well-defined alveolar ducts, alveolar sacs and alveoli
Dosages of hemoglobin showed an increase in the blood of animals as well thin intra-alveolar septa. Differently, important changes
exposed to benzopyrene without treatment (BaP) (13.94 ± 0.26; p < occurred in the pulmonary architecture by administration of benzo­
0.01) compared to the sham group (12.19 ± 0.36) as well as, reduction pyrene. Diffuse congestion, regions of fibrosis and foci of hyperplasia
in the concentrations of this protein in the animals treated with PL (BaP/ involving alveoli and terminal bronchioles consisting of cuboid cells,
PL) (11.47 ± 0.68p < 0.01) compared to the untreated (BaP) (Fig. 1E). with dense chromatin and cell and nuclear atypia were observed in
animals induced without treatment (BaP), (Fig. 3C and D). Neutrophils
3.3. PL reduced DNA damage were also observed in the hyperplasia regions. However, in the animals
treated with PL (BaP/PL), preservation of the pulmonary architecture
The averages of the comet assay parameters used to measure DNA occurred, maintaining the histological characteristics similar to the non-
damage in the three study groups are shown in Table 1. The measured induced animals (Sham) (Fig. 3E and F). Animals BaP exhibited a severe
parameters (tail length, DNA damage and tail moment) indicated pattern of lung inflammation (p < 0,001) that was mitigated by PL
greater DNA damage in the BaP group (Fig. 2C) compared to the sham administration (p < 0,001) (Fig. 3G). All histopathological findings were
group (Fig. 2A). There was a significant reduction in these damages after confirmed by evaluation of a pathologist that performed.
treatment with PL (tail length, p < 0,01, DNA damage, p < 0,001 and tail The quantification of leukocytes in BAL showed a significant increase
in lymphocytes (25.92 ± 2.46; p < 0.001) (Fig. 3H), monocytes (9.10 ±
0.64; p < 0.001) (Fig. 3I) and neutrophils (6.50 ± 0.29; p < 0.001)
Table 1
Comparison of the damage parameters by the comet test.
(Fig. 3J) in the BaP group compared to the sham (Lymphocytes: 9.78 ±
1.28; Monocytes: 3.75 ± 0.94; Neutrophils: 0.60 ± 0,40). The anti-
Groups/ Length of tail DNA damage (%) Tail moment
inflammatory action of PL was observed by reducing the number of
Parameters (μm) (μm)
lymphocytes (16.57 ± 2.44; p < 0.05), monocytes (5.20 ± 1.32; p <
Sham 275,5 ± 8,150 7,330 ± 0,4630 14,67 ± 2,093 0.05) and neutrophils (4.25 ± 0.75; p < 0.05) compared to untreated
BaP 4862 ± 1005*** 59,26 ± 2,540*** 3163 ± 776,0*
BaP/PL 944,6 ± 326,2## 27,51 ± 814,8 ± 694,5#
mice (BaP).
3,883***/###

BaP, group exposed to benzopyrene without treatment; BaP/PL, group exposed 3.5. PL decreased the expression of NF-kB and COX-2 and modulates the
to benzopyrene and treated with piperlongumin. Results presented as mean ± S. endogenous AnxA1 protein
E.M., * p < 0.05 and *** p < 0.001 vs sham group; #p < 0.05, ## p < 0.01 and
### p < 0.001 vs BaP group. Immunohistochemical analyses of the lungs of benzopyrene-induced

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T.E.B. Ashino et al. International Immunopharmacology xxx (xxxx) xxx

Fig. 2. Lymphocyte nuclei by comet assay - Class 0 cell, without apparent damage, Sham group (A), class 1 cell, where the length of the tail is less than the
diameter of the nucleus, group induced by benzopyrene and treated with PL (BaP/PL) (B) and class 2 cell, with a tail corresponding to one or two times the diameter
of the nucleus, a group induced by untreated benzopyrene (BaP) (C). Bars: 10 μm.

Fig. 3. Histopathological analysis of the lung and BAL inflammatory influx quantification - Sham group with normal appearance (A) and thin intra-alveolar
septa (B). Group exposed to benzopyrene without treatment (C and D) with disorganization of pulmonary architecture and extensive regions of hyperplasia. Atypical
cells with dense chromatin (D). Treated group exposed to benzopyrene and treated with piperlongumin (BaP/PL) with preserved pulmonary architecture. Staining:
Hematoxylin-Eosin. Bars: 200 μm (A, C, E) and 50 μm (B, D, F). Inflammation score (G). Quantification of Lymphocytes (H), Monocytes (I) and Neutrophils (J), in a
Neubauer chamber, in sham groups, induced by untreated benzopyrene (BaP) and induced and treated with piperlongumin (BaP/PL). Results presented as mean ± S.
E.M. (n = 10), ** p < 0.01; *** p < 0.001 vs sham group; # p < 0.05 vs BaP group.

and untreated animals (BaP) showed increased expressions of the anti-
inflammatory protein AnxA1 (p < 0.01; Fig. 4A-D), proinflammatory
enzyme COX-2 (p < 0.001; Fig. 4E-H), anti-apoptotic protein Bcl-2 (p <
0.01; Fig. 4I-L) and nuclear transcription factor NF-kB (p < 0.001;
Fig. 4M-P) compared to the sham group.
The anti-inflammatory and protective effect of PL (BaP/PL) was
observed by the significant reduction in the expression of COX-2 (p <
0.05), Bcl-2 (p < 0.01) and NF-kB (p < 0.001) in relation to untreated
animals (BaP). In contrast, the expression of the AnxA1 protein
remained high after treatment with PL. The specificity of the immuno­
staining was confirmed by the reaction controls for each antibody
(Fig. 4Q).
The expression of the AnxA1 protein was also verified in the pul­
monary macerate supernatant by means of Western blotting and the
results obtained corroborate the observations of immunohistochemistry
(Fig. 5A and D). Interestingly, treatment with PL markedly reduced the
expression of α-Tubulin (Fig. 5B and E).
3.6. Treatment with PL reduced the cytokine pro-inflammatory levels
Elevated levels of pro-inflammatory cytokines IL-1β (p < 0.001), IL-
17 (p < 0.05) and TNF-α (p < 0.01) were observed in the pulmonary

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T.E.B. Ashino et al. International Immunopharmacology xxx (xxxx) xxx

Fig. 4. AnxA1, COX-2, Bcl-2 and NF-kB expressions in the lung: Immunohistochemistry of AnxA1 (A, B, C), COX-2 (E, F, G), Bcl-2 (I, J, K) and NF-kB (M, N, O) in
sham groups, benzopyrene-induced and untreated (BaP) and benzopyrene-induced and treated with piperlongumin (BaP/PL). Densitometric analyses of AnxA1 (D),
COX-2 (H), Bcl-2 (L) and NF-kB (P). Results presented as mean ± S.E.M. (n = 10/group). ** p < 0.01 and *** p < 0.001 vs sham group. # p < 0.05; ## p < 0.01 e.
### p < 0.001 vs BaP group. Absence of immunostaining in the reaction control (Q). Counter staining: Hematoxylin. 50 μm bars.

Fig. 5. Quantification of protein AnxA1 - Expression AnxA1 by Western blotting (A). Reaction controls with α-tubulin (B) and β-actin (C), densitometric analysis of
AnxA1 (D), tubulin (E) and β-actin (F).

6
T.E.B. Ashino et al.
Fig. 6. Cytokine levels in lung macerate supernatant - IL-1β (A); IL-17 (B); TNF-α (C). Results presented as mean ± S.E.M. (n = 10/group). * p < 0.1, ** p < 0.01;
*** p < 0.001 vs Sham # p < 0.05; ## p < 0.01 vs exposed benzopyrene group without treatment.
supernatant of the BaP mice compared to the control (Fig. 6). The
administration of PL promoted a reduction in the levels of these pro-
inflammatory mediators (IL-1β and IL-17, p < 0.01 and TNF-α, p <
0.05) (Fig. 6A and C).
4. Discussion
In the search for new therapeutic strategies to prevent the progres­
sion of inflammatory lung diseases, we evaluated the therapeutic po­
tential of PL in a model of carcinogenesis induced by benzopyrene in
Balb/c mice. The obtained results indicated protective effects of PL
administration with maintenance of pulmonary parameters and hemo­
globin levels similar to sham group, preservation of pulmonary archi­
tecture, reduced DNA damage, decreased inflammatory influx and levels
of pro-inflammatory mediators.
Initially the physiological data of weighing and plethysmography
were analyzed. Although the weight of the animals did not show vari­
ation among the groups studied, our results showed an increase in
plethysmographic parameters (volume, frequency and pulmonary
ventilation) in the group induced by benzopyrene without treatment
(BaP) in comparison to Sham group, in addition to a significant reduc­
tion of the same parameters in animals induced by benzopyrene and
treated with PL (BaP/PL). Similar physiological results were observed in
mice exposed to cigarette smoke and treated with PL [55]. Full-body
plethysmography is an interesting resource that allows you to assess
the animal consciously and spontaneously, checking the volume of
inspired air, the frequency and the capacity of ventilation more reliable
and without the interferences related to anesthesia that the most inva­
sive methods need [37].
The smoking habit, an important cause in lung carcinogenesis [57],
is linked to the elevation of hemoglobin levels, which occurs due to the
high affinity of carbon monoxide, resulting from the burning of tobacco,
when binding to hemoglobin forming carboxyhemoglobin. This associ­
ation makes the binding site for the oxygen molecule unavailable,
decreasing its concentrations in the blood. Thus, to compensate for this
reduction, smokers [40] and rats exposed to cigarette smoke [50,55]
maintain increased hemoglobin level, as observed in benzopyrene-
induced-animals without treatment in our study. In contrast, after
treatment with PL, the hemoglobin measurement was similar to sham
group, indicating the protective role of this NBC, as also observed by
SantA ́ na and collaborators [55] in a model of pulmonary inflammation
induced by cigarette smoke. Similarly, in the model of carcinogenesis
induced by benzopyrene with capsaicin treatment, the restoration of
hemoglobin levels close to that of the control group was also observed
[2].
After verifying the reversion promoted by PL in the alterations
caused by benzopyrene in the pulmonary parameters and hemoglobin
levels, we evaluated the genotoxicity using the comet test. The analyses
showed that benzopyrene promoted damage to the lymphocyte DNA,
7
International Immunopharmacology xxx (xxxx) xxx
but the administration of PL partially reduced these changes. The comet
assay has been used to assess DNA damage by exposure to genotoxic
agents in the workplace [33,20] and in vitro [18]. Increased rates of
DNA damage were observed in pneumocytes II of rats exposed to ciga­
rette smoke for periods of 3 and 6 weeks, with a cumulative effect [11].
In addition, damage induced by benzopyrene was observed in cells of
the nematode Caenorhabditis elegans, in a dose-dependent manner [27].
In research related especially to the lung tumor, PL showed great
therapeutic potential by inhibiting cell proliferation in vitro and in vivo
studies in a NSCLC model, through different pathways such as Pi3K/Akt
[64,56,72], NF-kB [70] and as ROS inducer [32]. PL induced damage to
the DNA of chinese hamster pulmonary fibroblast cells, detected by
neutral and alkaline comet assay, in a dose-dependent manner [5].
Furthermore, the genotoxicity of PL was also indicated by the comet
assay in head and neck squamous cell carcinoma cells, in which DNA
damage and ROS levels were elevated after treatment with PL [22].
These authors suggest that treatment with PL selectively increases ROS
levels and induces cell death in cancer cells compared to normal ones.
Following this study, we analyzed the inflammatory cells in the
bronchoalveolar lavage and observed an increase in lymphocytes,
monocytes and neutrophils in animals exposed to benzopyrene. This
agent triggers inflammatory changes in the lung, such as hyperplasia
and formation of bronchus-associated lymphoid tissue (BALT) [54]. The
deleterious effects caused by the excessive oxidative stress that occurs
through the use of cigarettes also destabilizes the cytoskeleton of
phagocytic cells leading to deficiency in bacterial control and clearance
of apoptotic remains [7].
Studies show that in metastatic non-small cell lung cancer, the
inflammation index can be used to assess patients’ prognosis [29]. In­
filtrates of cells of the immune system, such as lymphocytes, neutrophils
and macrophages have important significance in clinical management,
and may contribute to a favorable prognosis or not depending on their
density and location [8]. Research on the benzopyrene-induced lung
carcinogenesis model [2,63] showed a reduction in lymphocytes and
monocytes, but an increase in neutrophils. In previous studies of our
research group, increased lymphocytes and macrophages in animals
exposed to cigarette smoke was also observed [49,50,35].
Even though BAL analysis, in some cases such as COVID-19 after
negative upper respiratory tract swabs, play a limited role, its investi­
gation is important to establish a different diagnosis of an infectious or
non-infectious nature [14]. In our study, the anti-inflammatory effect of
PL led to reduction of lymphocytes, monocytes and neutrophils in BAL,
which corroborates the positive data obtained in previous researches of
our group, in models of respiratory inflammation induced by exposure
to cigarette smoke. In those researches, BAL leukocytes influx was
mitigated by administering an herbal mixture [49] and PL [55]. A study
with another NBC, capsaicin, showed a reduction in leukocytes, espe­
cially neutrophils, in mice induced to benzopyrene lung carcinogenesis
[2].
T.E.B. Ashino et al.
In the histopathological analyses, the lungs of untreated animals showed
diffuse congestion, regions of fibrosis and foci of hyperplasia involving
alveoli and terminal bronchioles, consisting of cuboid cells, with dense
chromatin as well as cellular and nuclear atypia. The group treated with PL
showed similar characteristics to the sham group, with preservation of the
pulmonary histological structure and reduced inflammatory score. These
findings confirm not only the validity of the model of induction of chronic
inflammation by benzopyrene, with progressive alteration of the pulmonary
environment, leading to carcinogenesis, but also the efficacy of PL in the
proposed treatment. A similar disarrangement of pulmonary architecture
with the presence of hyperplasia was also observed by other authors in
models of benzopyrene-induced carcinogenesis for periods longer than 14
weeks [64,26]. Inflammatory mediators, including cytokines, chemokines
and extracellular enzymes modulate molecular events in tumors induced by
benzopyrene [57]. Again, the treatment with PL showed a protective effect
against the damage caused by benzopyrene and is in line with another study
that revealed important differences after treatment with PL in pulmonary
inflammation induced by cigarette smoke, evidenced by the reduction of
cellular influx in bronchoalveolar lavage and lung tissue, preservation of
alveolar structures and attenuation of proteases activity [55].
To deepen the analysis, we proceeded to study the expression of anti
and pro inflammatory proteins, anti-apoptotic and nuclear transcription
factor. The results indicated the inhibition of COX-2, Bcl-2 and NF-kB
expressions with PL administration, while AnxA1 expression remained
high. The effects of the pro-apoptotic molecular activity of PL include
the low expression of Bcl-2 [48], supporting our findings. Han and
collaborators [21] evaluated the action of PL in several cell lines induced
by different stimuli, including cigarettes. These authors showed that PL
regulated the expression of COX-2 through the inhibition of NF-kB and
reduced the production of IL-6 in a dose-dependent manner. Son et al.
[59] showed that PL inhibits the activation of NF-kB in atherosclerotic
lesions. Also, PL mitigated the symptoms and activation of macrophages
and T cells by inhibiting NF-κB signaling in an experimental autoim­
mune encephalomyelitis [19]. Regarding lung cancer, PL induced
apoptosis and suppressed the DNA binding activity of NF-κB in NSCLC
cells and also inhibited tumor growth of NSCLC in vivo, dose depen­
dently [70].
In addition, an investigation carried out by our research group also
showed a reduction in NF-kB and COX-2 and an increase in AnxA1 after
treatment with PL in lung inflammation induced by exposure to ciga­
rette smoke [55]. AnxA1 overexpression was assessed in bronchial
epithelial cells treated with benzopyrene [10]. This study indicated that
AnxA1 has a protective effect on bronchial injury induced by benzo­
pyrene, with inhibition of apoptosis by regulating the expressions of Bcl-
2, Bax and cyclin D1. Previous studies showed increased expression of
AnxA1, COX-2 and NF-kB in rats and mice exposed to cigarette smoke
[35,49,50,55].
Exposure to benzopyrene induced increased levels of IL-1β, TNF-α
and ROS production, through the activation of NF-kB. Activation of this
pathway stimulates cell proliferation and differentiation, inhibits
apoptosis and stimulates inflammation and angiogenesis [30,57]. In
addition, the mediators involved recruit and activate leukocytes, mainly
neutrophils, to the site of inflammation, which creates a microenvi­
ronment that promotes the even more pronounced release of inflam­
matory mediators [44,57].
In view of the results obtained, we complemented the analysis of
chemical mediators by measuring pro-inflammatory cytokines. The
studies indicated increased levels of IL-1β, IL-17 and TNF-α in the su­
pernatants of the lung macerate of animals induced by benzopyrene and
the reduction of these levels by the administration of PL, reinforcing the
previous analyses. Investigations with rats exposed to environmental
pollution showed increased expression of IL-1β in the lung parenchyma,
numerous macrophages and high levels of IL-1β, COX-2, TGF-α in the
bronchoalveolar lavage [67]. Other studies indicated that IL-17 acts
synergistically with NF-kB activators such as TNF-α and IL-1β and that
IL-17 can promote tumor development through chronic tissue
8
International Immunopharmacology xxx (xxxx) xxx
inflammation [41]. In addition, the increase in serum IL-17A in BALB/c
mice was associated with mycoplasma pathology characterized by the
presence of airway neutrophils, weight loss, development of macro­
scopic lung lesions and persistent infection, suggesting that increased IL-
17A contributes to the severity of the disease during respiratory myco­
plasma infection [43]. The IL-17A can also contribute to disease pro­
gression in patients with severe COPD [38].
Regarding to the treatment, other herbal medicines, in different
models, also reduced levels of inflammatory mediators. Ginko Biloba
extract inhibited the expression of IL-1β, IL-6, TNF-α and COX-2 in the
cochlea of rats after noise-induced hearing loss [13]. Dicentrine, an
aporphine alkaloid found in the roots of Lindernis megaphylla, increased
TNF-α-induced cell death in A549 lung cancer cells, reducing cell in­
vasion due, at least in part, to the suppression of kB signaling pathways
[45]. An herbal mixture (Arctium lappa, Plantago major, Mikania glom­
erata Spreng and Equisetum arvense) decreased IL-1β, IL-6, TNF-α and
MCP-1 plasma levels and lung expression of AnxA1 and NF-kB in a COPD
model [49]. In a benzopyrene-induced precancerous lung lesion model
in mice, the treatment with Phyllanthus emblica L, an Asian medicinal
plant, attenuated levels of macrophage inflammatory protein (MIP-2),
TNF-α, IL-6, IL-1β, COX-2 and HIF-α in lung tissue [65]. Moreover,
treatment of neoplastic cells with PL alone or with AnxA1 mimetic
peptide reduced cell proliferation and viability and modulated the
expression of MCP-1, IL-8 and genes involved in inflammatory processes
[24].
Finally, an interesting result obtained by the Western blotting tech­
nique was the intense reduction of the α-Tubulin protein with the
administration of PL. Meegan and collaborators [42] indicated PL as a
microtubule destabilizing agent with antiproliferative effects, which
provides evidence that PL affects the polymerization of tubulin. In
another study by our group the results also suggested an inhibitory effect
of PL on tubulin expression [24]. This effect of PL is important for
controlling the cell proliferation of cancer cells.
In view of the above, our results are consistent with the literature
data and confirm the therapeutic potential of PL as a modulatory com­
pound in precancerous inflammatory processes promoted by benzo­
pyrene in lung, through the regulation of different proteins and chemical
mediators.
5. Conclusions
Together, our results show the efficiency of the PL in maintaining the
pulmonary parameters (ventilation, frequency and volume) and hemo­
globin levels similar to the sham group. PL also promoted the reduction
of DNA damage and influx of inflammatory cells (lymphocytes, mono­
cytes and neutrophils) into BAL, besides helping to preserve the pul­
monary architecture. Furthermore, the administration of PL maintained
the levels of AnxA1 increased, reduced the expressions of COX-2, Bcl-2
and NF-kB as well as of the cytokines IL-1β, IL-17 and TNF-α. Therefore,
our data indicate the potential of PL for future anti-inflammatory and
anti-tumor strategies in pulmonary disorders.
Funding
This work was supported by Grants from Fundação de Amparo à
Pesquisa do Estado de São Paulo - FAPESP (2015/03359-5 to APG),
Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq
(404190/2016-2 to APG) and Centro Universitário Padre Albino (UNI­
FIPA) research programme.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
T.E.B. Ashino et al.
Acknowledgements
To Dr Daniel Henrique Gonçalves for his assistance in pathological
evaluations and to Dr Rodrigo Berguio Vidotti for veterinary support.
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