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: [email protected] (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. International Immunopharmacology xxx (xxxx) xxx

1. Introduction reinforced by analyzing PL administration in epidermoid carcinoma of

the larynx (Hep-2) cells and human umbilical vein endothelial cells
Lung cancer is among the leading causes of mortality worldwide [58] HUVEC) in which PL modulated the expression of genes involved in
and can be classified into two subtypes: non-small cell lung cancer inflammatory processes [24]. In another investigation by our research
(NSCLC) and small cell lung cancer (SCLC) [62]. NSCLC represents 85% group on an experimental model of respiratory inflammation, PL treat­
of lung cancer cases and is divided into adenocarcinoma, squamous cell ment recovered lung tissues, increased the expression of the anti-
carcinoma (epidermoid) and large cell carcinoma [46]. inflammatory protein annexin-A1 (AnxA1) while reduced cyclo­
Tumor cells in the lung cancer microenvironment, chemokines and oxygenase (COX-2), nuclear factor kappa B (NF-kB) and neutrophil
inflammatory mediators as cyclooxygenase (COX-2), hypoxia-inducible elastase expressions, controlling the inflammation induced by cigarette
factor (HIF)-α, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-α, smoke [55].
and interleukin (IL)-1β, secreted by interstitial cells, interact and acti­ However, there are still few reports of the actions of PL in inflam­
vate pro-inflammatory signaling and cell growth pathways. This inter­ matory and neoplastic lung conditions. For this reason, in this study, we
action promotes the malignant biological behavior of lung cancer and sought to investigate the effect of PL administration on the treatment of
tumor progression [66,1]. Besides, alveolar type II (ATII) cells, that are precancerous inflammatory processes in the lung, induced by the
essential for lung homeostasis, also play a central role in acute and administration of benzopyrene in an experimental model, as a possible
chronic illness, being important for the pathogenesis of lung adenocar­ preventive therapeutic alternative in the treatment of lung cancers. To
cinoma as a progressive pre-neoplastic change [53]. verify the effectiveness of the treatment, physiological and histopatho­
Even before the appearance of the tumor, the existence of a chronic logical parameters, lymphocyte genotoxicity; inflammatory influx and
inflammatory process acting as a driving force for carcinogenesis is a levels of inflammatory mediators were studied.
well-established factor [25]. Among the critical causes associated with
inflammatory lung diseases, we can highlight tobacco, occupational 2. Materials and methods
exposure, diet, biological factors and environmental pollution [17,71].
Polycyclic aromatic hydrocarbons (PAHs) are chemical products that 2.1. Animals
contain at least two fused benzene rings, without heteroatoms and are
released into the environment by incomplete combustion of organic Male Balb/c mice (n = 30) (20–25 g body weight) were obtained
material, such as: coal, oil and organic polymers, and in urban areas, from the Didatic and Experimental Research Unit of the University
they are mainly launched by automobiles [68]. In this context, benzo­ Center Padre Albino (UNIFIPA) and kept in individual cages in a
pyrene stands out for being one of the main PAHs present in the envi­ controlled environment (24–25 ◦ C, 12 h light/dark cycle) with water
ronment and with a high proven carcinogenic capacity in humans [47], and food ad libitum.
therefore, used in vivo experimental models [23,26]. All experimental procedures were conducted according to the
Inflammatory changes triggered by intrapulmonary instillation of guidelines for biomedical research stated by the Brazilian Societies of
benzopyrene such as hyperplasia and formation of bronchus-associated Experimental Biology and approved by the Ethics Committees on Ani­
lymphoid tissue (BALT) are characteristic in the formation of neoplasms mal Use at UNIFIPA (Certificate n◦ 10/18), Brazil. The animals were
[54,51]. Research has shown that substances with anti-inflammatory divided into 3 groups: sham (vehicle – 10% DMSO), induced by ben­
activity can reduce the incidence of lung cancer [4,66]. In this sce­ zopyrene without treatment (BaP) and benzopyrene-induced and
nario, natural bioactive compounds (NBCs) such as terpenes and flavo­ treated with piperlongumin (BaP/PL) (n = 10/group).
noids that have many properties such as reducing inflammation and The experiments were designed to minimize the number of animals
stimulating the immune system [6], may represent a possibility for used and their suffering during the execution of the protocols. For this
preventive treatment for lung cancer. reason, PL control group was not included as the safety of PL has already
Among NBCs we highlight piperlongumin (PL) (piperlongumin, 5,6- been demonstrated even at higher concentrations (10 mg/kg) [39]. All
di-hidro-1-[(2E)-1-oxo-3 (3,4,5-trimetoxifenil)-2-propenil]-2(1H) pir­ animals were daily evaluated by the institution’s veterinarian.
idinona), the biologically active component of the Piper species
(Piperaceae) with great economic and medicinal importance [5] and
2.2. Benzopyrene exposure and PL treatment protocol
different pharmacological actions [6,61]. Several studies have shown
that PL has antitumor, antifungal, anxiolytic, antidepressant, cytotoxic,
For the benzopyrene exposure, two groups of animals were anes­
antibacterial, antiplatelet and anti-inflammatory properties, among
thetized intraperitoneally (ip) with 0.2 ml/100 g of ketamine and 0.05
others [28,5,31,52,16,60,24].
ml/100 g of xilasine and administered ip with 0.2 ml of the benzopyrene
Raj and collaborators [52] reported that PL induces apoptosis by
solution (Sigma – B1760) (100 mg/kg) diluted in dimethylsulfoxide
interfering with homeostatic regulators, redox and reactive oxygen
(DMSO) 10% [69].
species (ROS), such as glutathione S-transferase pi 1 (GSTP1) and
The therapeutic efficacy of PL (Sigma - SML 0221), against inflam­
carbonyl reductase (CBR1). The antitumor activity of PL involves cell
matory and pre-carcinogenic processes caused by exposure to benzo­
cycle arrest, activation of caspases and mitogen-activated protein ki­
pyrene, was evaluated in one of the induced groups, from the eighth
nases (MAPKs), deregulation of anti-apoptotic proteins such as Bcl-2 and
week post-induction, through the ip administration of PL (2.0 mg/Kg,
nuclear transcription factor kB (NF-kB) [34,15,36]. In breast cancer
diluted in DMSO 10%), 3× a week, on alternate days, [52,55], for 5
cells, PL reduced the expression of Bcl-2 and stopped the cell cycle by
weeks.
destabilizing the tubulin microtubules [42]. PL has also been shown to
The animals of all groups were euthanized after 12 weeks post-
be a potent inhibitor of NF-kB activation induced by carcinogens and
induction by excessive dose of anesthetic.
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 2.3. Physiological evaluations of plethysmography and weight
to reduce the mechanisms of resistance to chemotherapy. Jyothi and
collaborators [31] evaluated the concomitant treatment of PL with All animals were weekly weighed. At the end of the experiment, the
diferuloylmethane, an anti-inflammatory and anticancer agent. This animals were evaluated for plethysmography by measuring breathing
treatment increased the cytotoxicity induced by PL and the antitumor capacity, lung ventilation, frequency and inspired air volume by a spe­
activity of the chemotherapeutic agent. The potential of PL as a thera­ cific device and adapted for use (PowerLab, AD Instruments-Gas
peutic immunomodulator for cancer prevention and progression was Analyzer, Sydney, Australia).

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

2.4. Biochemical blood assays UK) for 12 h. Then, incubated with the biotinylated secondary antibody
and immersed in a streptavidin peroxidase conjugate complex. The
Blood was collected by cardiac puncture in heparinized syringes and diaminobenzidine substrate (DAB) was used for staining (DAB Kit,
separated in aliquots for the analysis of hemoglobin by a commercial Kit Invitrogen) and, afterwards, the sections were counterstained with He­
(LAB test, Minas Gerais, Brazil) and reading on the spectrophotometer. matoxylin. Proteins were quantified by densitometry (arbitrary units
from 0 to 255) [55] in the image analyzer (Leica Image Analysis soft­
2.5. Comet assay 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
For the Comet Assay, two slides per animal were prepared using a sample.
mixture of agarose (1.5% in PBS) boiled for one minute, cooled and
solidified three times. Then, the slides were bathed and placed under 2.8. Evaluation of anti-inflammatory protein annexin A1 by Western
refrigeration for later use. One aliquot of 10 µl of blood was added to blotting
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 Lung fragments were macerated in liquid nitrogen and 650 µl of the
water bath together with 10 µl of saline/blood solution and this mixture protease inhibitor solution (Protease Inhibitor Cocktail Set I, Millipore
was placed on the slides, covered with coverslips and taken to the Corporation, USA) and phosphatases (PhosphoSafe, Novagen, Millipore
refrigerator by at least thirty minutes to solidify the Low Melting Corporation, USA) were added, according to the manufacturer’s in­
agarose. Then, the coverslips were removed and the slides were placed structions. The material was incubated for 20 min, at 4 ◦ C, under con­
in a glass vat to be immersed in pH 10 lysis solution (2.5 M NaCl, 100 nM stant agitation, and then centrifuged at 14,000 rpm, for 10 min, at 4 ◦ C,
EDTA, 10 mM Tris, 35 mM Lauryl) at 4◦C for one hour in the refrigerator with the supernatants collected and immediately frozen at – 80 ◦ C.
and at room temperature shelter of light. Afterwards, the slides were The Bradford assay (Biorad, Hemel Hempsted, UK) was performed to
placed in the pH13 electrophoresis buffer solution (200 mM EDTA, 10 N determine the protein concentration. An aliquot was mixed (1:1) with
NaOH) for about 30 min. Then, the slides were neutralized and washed 2× “loading buffer” (125 mM Tris base, pH 6.8, containing 10% mer­
for 3x of five minutes with 5 ml of neutralization solution pH 7.5 (0.4 M captoethanol, 4% sodium dodecyl sulfate, 20% glycerol, 0.1 bromo­
Tris). Subsequently, the slides were immersed in 100% ethanol for 10 phenol blue %) and denatured in water at 100 ◦ C for 5 min. The samples
min for fixation. After drying, 100 µl of solution for staining with (20 mg of protein per well) and molecular weight markers were sepa­
ethidium bromide was added and the slides were covered with a rated on SDS-PAGE gel (10% sodium dodecyl sulfate − 10% poly­
coverslip for analysis under a fluorescence microscope (Fluo3, Bel). acrylamide gel) and subsequently transferred to a nitrocellulose
The cells were classified according to tail length: class 0, undamaged, membrane. The AnxA1 protein was detected using the primary anti-
without tail; class 1: with short tail, smaller than the diameter of the AnxA1 antibody (1:1000) (Life Technologies). The signal was ampli­
nucleus; class 2, with a tail corresponding to one or two times the fied using secondary anti-rabbit IgG HRP conjugated antibody (Prom­
diameter of the nucleus; class 3, tail length greater than twice the ega) and the reaction product visualized with DAB staining (DAB Kit,
diameter of the nucleus [5]. Invitrogen). The α-Tubulin antibody (1:500, Sigma) and β-actin (1:500,
Fifty lymphocytes per group were counted and DNA damage (tail Sigma) were used as a controls. The protein expressions in the mem­
length, percentage of DNA damage and tail moment) was determined branes were quantified by densitometry in the image analyzer (Leica
using the CometScore software version 1.5 [33]. Image Analysis software).

2.6. Quantitative analyses of bronchoalveolar lavage (BAL) 2.9. Cytokine levels

To obtain BAL, at the end of the experiment the animals had a can­ In all groups, the cytokines IL-1β, IL-17 and TNF-α were quantified in
nulated trachea and a clamped right lung. The left lung was washed 3 the pulmonary macerate supernatant, using commercially available
times with PBS and the liquid obtained was centrifuged for 10 min at ELISA immunological test kits (Sigma) and according to the manufac­
1,500 rpm. The supernatant was stored at − 70 ◦ C for subsequent mea­ turer’s specification.
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 2.10. Statistical analysis
flammatory cells in a Neubauer camera (values as number of cells ×
103/ml). Data collected from the experiments were analyzed using GraphPad
Prism® version 6. software (GraphPad Software, Inc.). The results ob­
2.7. Histopathological and immunohistochemical analysis tained were previously submitted to descriptive analysis and determi­
nation of normality using the Shapiro-Wilk normality test. As the
After BAL collection, the right lung was removed, fixed in 4% samples showed normal distribution, Analysis of Variance (ANOVA) was
formaldehyde and processed for inclusion in paraffin. 4 µm sections used, followed by the Bonferroni test. All values obtained were
were used for histopathological analysis after staining with expressed as mean ± S.E.M. and P values less than 0.05 were considered
hematoxylin-eosin (HE), under the Leica microscope (DM500). To statistically significant.
perform the inflammatory score, 10 different slides of each animal were
analyzed according the categories: Peribronchial inflammation and 3. Results
perivascular inflammation and graded as follows: 0, no inflammatory
cells; 1, occasional cuffing with inflammatory cells; 2, most bronchi or 3.1. Increased pulmonary frequency, volume and ventilation parameters
vessels surrounded by a thin layer (1–5) of inflammatory cells; 3, most were attenuated after treatment with PL
bronchi or vessels surrounded by a thick layer (>5 cells) [39].
Immunohistochemical studies were used to evaluate the expressions The lung volume (Fig. 1A), respiratory frequency (Fig. 1B) and pul­
of AnxA1, Bcl-2, COX-2 and NF-kB. Lung sections were processed for monary ventilation (Fig. 1C) were increased in the benzopyrene-
antigenic recovery with citrate buffer pH 6.0, blockade of the endoge­ induced-group without treatment (BaP) compared to the sham group
nous peroxidase activity and incubated with the primary polyclonal (p < 0.001) but a significant reduction in these pulmonary parameters
antibodies rabbit:anti AnxA1 (1:1000) (Invitrogen), anti-Bcl − 2 (1: occurred in animals treated with PL (BaP/PL) (p < 0.001) (Lung volume:
150), anti-COX-2 (1: 500) and anti-NF-kB (1: 1000) (Abcam, Cambrigde, Sham 2.356 ± 0.120; BaP 7.505 ± 0.065; BaP/PL 2.411 ± 0.194;

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

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

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 but the administration of PL partially reduced these changes. The comet
administration of PL promoted a reduction in the levels of these pro- assay has been used to assess DNA damage by exposure to genotoxic
inflammatory mediators (IL-1β and IL-17, p < 0.01 and TNF-α, p < agents in the workplace [33,20] and in vitro [18]. Increased rates of
0.05) (Fig. 6A and C). 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].
4. Discussion In addition, damage induced by benzopyrene was observed in cells of
the nematode Caenorhabditis elegans, in a dose-dependent manner [27].
In the search for new therapeutic strategies to prevent the progres­ In research related especially to the lung tumor, PL showed great
sion of inflammatory lung diseases, we evaluated the therapeutic po­ therapeutic potential by inhibiting cell proliferation in vitro and in vivo
tential of PL in a model of carcinogenesis induced by benzopyrene in studies in a NSCLC model, through different pathways such as Pi3K/Akt
Balb/c mice. The obtained results indicated protective effects of PL [64,56,72], NF-kB [70] and as ROS inducer [32]. PL induced damage to
administration with maintenance of pulmonary parameters and hemo­ the DNA of chinese hamster pulmonary fibroblast cells, detected by
globin levels similar to sham group, preservation of pulmonary archi­ neutral and alkaline comet assay, in a dose-dependent manner [5].
tecture, reduced DNA damage, decreased inflammatory influx and levels Furthermore, the genotoxicity of PL was also indicated by the comet
of pro-inflammatory mediators. assay in head and neck squamous cell carcinoma cells, in which DNA
Initially the physiological data of weighing and plethysmography damage and ROS levels were elevated after treatment with PL [22].
were analyzed. Although the weight of the animals did not show vari­ These authors suggest that treatment with PL selectively increases ROS
ation among the groups studied, our results showed an increase in levels and induces cell death in cancer cells compared to normal ones.
plethysmographic parameters (volume, frequency and pulmonary Following this study, we analyzed the inflammatory cells in the
ventilation) in the group induced by benzopyrene without treatment bronchoalveolar lavage and observed an increase in lymphocytes,
(BaP) in comparison to Sham group, in addition to a significant reduc­ monocytes and neutrophils in animals exposed to benzopyrene. This
tion of the same parameters in animals induced by benzopyrene and agent triggers inflammatory changes in the lung, such as hyperplasia
treated with PL (BaP/PL). Similar physiological results were observed in and formation of bronchus-associated lymphoid tissue (BALT) [54]. The
mice exposed to cigarette smoke and treated with PL [55]. Full-body deleterious effects caused by the excessive oxidative stress that occurs
plethysmography is an interesting resource that allows you to assess through the use of cigarettes also destabilizes the cytoskeleton of
the animal consciously and spontaneously, checking the volume of phagocytic cells leading to deficiency in bacterial control and clearance
inspired air, the frequency and the capacity of ventilation more reliable of apoptotic remains [7].
and without the interferences related to anesthesia that the most inva­ Studies show that in metastatic non-small cell lung cancer, the
sive methods need [37]. inflammation index can be used to assess patients’ prognosis [29]. In­
The smoking habit, an important cause in lung carcinogenesis [57], filtrates of cells of the immune system, such as lymphocytes, neutrophils
is linked to the elevation of hemoglobin levels, which occurs due to the and macrophages have important significance in clinical management,
high affinity of carbon monoxide, resulting from the burning of tobacco, and may contribute to a favorable prognosis or not depending on their
when binding to hemoglobin forming carboxyhemoglobin. This associ­ density and location [8]. Research on the benzopyrene-induced lung
ation makes the binding site for the oxygen molecule unavailable, carcinogenesis model [2,63] showed a reduction in lymphocytes and
decreasing its concentrations in the blood. Thus, to compensate for this monocytes, but an increase in neutrophils. In previous studies of our
reduction, smokers [40] and rats exposed to cigarette smoke [50,55] research group, increased lymphocytes and macrophages in animals
maintain increased hemoglobin level, as observed in benzopyrene- exposed to cigarette smoke was also observed [49,50,35].
induced-animals without treatment in our study. In contrast, after Even though BAL analysis, in some cases such as COVID-19 after
treatment with PL, the hemoglobin measurement was similar to sham negative upper respiratory tract swabs, play a limited role, its investi­
group, indicating the protective role of this NBC, as also observed by gation is important to establish a different diagnosis of an infectious or
SantA ́ na and collaborators [55] in a model of pulmonary inflammation non-infectious nature [14]. In our study, the anti-inflammatory effect of
induced by cigarette smoke. Similarly, in the model of carcinogenesis PL led to reduction of lymphocytes, monocytes and neutrophils in BAL,
induced by benzopyrene with capsaicin treatment, the restoration of which corroborates the positive data obtained in previous researches of
hemoglobin levels close to that of the control group was also observed our group, in models of respiratory inflammation induced by exposure
[2]. to cigarette smoke. In those researches, BAL leukocytes influx was
After verifying the reversion promoted by PL in the alterations mitigated by administering an herbal mixture [49] and PL [55]. A study
caused by benzopyrene in the pulmonary parameters and hemoglobin with another NBC, capsaicin, showed a reduction in leukocytes, espe­
levels, we evaluated the genotoxicity using the comet test. The analyses cially neutrophils, in mice induced to benzopyrene lung carcinogenesis
showed that benzopyrene promoted damage to the lymphocyte DNA, [2].

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

In the histopathological analyses, the lungs of untreated animals showed inflammation [41]. In addition, the increase in serum IL-17A in BALB/c
diffuse congestion, regions of fibrosis and foci of hyperplasia involving mice was associated with mycoplasma pathology characterized by the
alveoli and terminal bronchioles, consisting of cuboid cells, with dense presence of airway neutrophils, weight loss, development of macro­
chromatin as well as cellular and nuclear atypia. The group treated with PL scopic lung lesions and persistent infection, suggesting that increased IL-
showed similar characteristics to the sham group, with preservation of the 17A contributes to the severity of the disease during respiratory myco­
pulmonary histological structure and reduced inflammatory score. These plasma infection [43]. The IL-17A can also contribute to disease pro­
findings confirm not only the validity of the model of induction of chronic gression in patients with severe COPD [38].
inflammation by benzopyrene, with progressive alteration of the pulmonary Regarding to the treatment, other herbal medicines, in different
environment, leading to carcinogenesis, but also the efficacy of PL in the models, also reduced levels of inflammatory mediators. Ginko Biloba
proposed treatment. A similar disarrangement of pulmonary architecture extract inhibited the expression of IL-1β, IL-6, TNF-α and COX-2 in the
with the presence of hyperplasia was also observed by other authors in cochlea of rats after noise-induced hearing loss [13]. Dicentrine, an
models of benzopyrene-induced carcinogenesis for periods longer than 14 aporphine alkaloid found in the roots of Lindernis megaphylla, increased
weeks [64,26]. Inflammatory mediators, including cytokines, chemokines TNF-α-induced cell death in A549 lung cancer cells, reducing cell in­
and extracellular enzymes modulate molecular events in tumors induced by vasion due, at least in part, to the suppression of kB signaling pathways
benzopyrene [57]. Again, the treatment with PL showed a protective effect [45]. An herbal mixture (Arctium lappa, Plantago major, Mikania glom­
against the damage caused by benzopyrene and is in line with another study erata Spreng and Equisetum arvense) decreased IL-1β, IL-6, TNF-α and
that revealed important differences after treatment with PL in pulmonary MCP-1 plasma levels and lung expression of AnxA1 and NF-kB in a COPD
inflammation induced by cigarette smoke, evidenced by the reduction of model [49]. In a benzopyrene-induced precancerous lung lesion model
cellular influx in bronchoalveolar lavage and lung tissue, preservation of in mice, the treatment with Phyllanthus emblica L, an Asian medicinal
alveolar structures and attenuation of proteases activity [55]. plant, attenuated levels of macrophage inflammatory protein (MIP-2),
To deepen the analysis, we proceeded to study the expression of anti TNF-α, IL-6, IL-1β, COX-2 and HIF-α in lung tissue [65]. Moreover,
and pro inflammatory proteins, anti-apoptotic and nuclear transcription treatment of neoplastic cells with PL alone or with AnxA1 mimetic
factor. The results indicated the inhibition of COX-2, Bcl-2 and NF-kB peptide reduced cell proliferation and viability and modulated the
expressions with PL administration, while AnxA1 expression remained expression of MCP-1, IL-8 and genes involved in inflammatory processes
high. The effects of the pro-apoptotic molecular activity of PL include [24].
the low expression of Bcl-2 [48], supporting our findings. Han and Finally, an interesting result obtained by the Western blotting tech­
collaborators [21] evaluated the action of PL in several cell lines induced nique was the intense reduction of the α-Tubulin protein with the
by different stimuli, including cigarettes. These authors showed that PL administration of PL. Meegan and collaborators [42] indicated PL as a
regulated the expression of COX-2 through the inhibition of NF-kB and microtubule destabilizing agent with antiproliferative effects, which
reduced the production of IL-6 in a dose-dependent manner. Son et al. provides evidence that PL affects the polymerization of tubulin. In
[59] showed that PL inhibits the activation of NF-kB in atherosclerotic another study by our group the results also suggested an inhibitory effect
lesions. Also, PL mitigated the symptoms and activation of macrophages of PL on tubulin expression [24]. This effect of PL is important for
and T cells by inhibiting NF-κB signaling in an experimental autoim­ controlling the cell proliferation of cancer cells.
mune encephalomyelitis [19]. Regarding lung cancer, PL induced In view of the above, our results are consistent with the literature
apoptosis and suppressed the DNA binding activity of NF-κB in NSCLC data and confirm the therapeutic potential of PL as a modulatory com­
cells and also inhibited tumor growth of NSCLC in vivo, dose depen­ pound in precancerous inflammatory processes promoted by benzo­
dently [70]. pyrene in lung, through the regulation of different proteins and chemical
In addition, an investigation carried out by our research group also mediators.
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 5. Conclusions
rette smoke [55]. AnxA1 overexpression was assessed in bronchial
epithelial cells treated with benzopyrene [10]. This study indicated that Together, our results show the efficiency of the PL in maintaining the
AnxA1 has a protective effect on bronchial injury induced by benzo­ pulmonary parameters (ventilation, frequency and volume) and hemo­
pyrene, with inhibition of apoptosis by regulating the expressions of Bcl- globin levels similar to the sham group. PL also promoted the reduction
2, Bax and cyclin D1. Previous studies showed increased expression of of DNA damage and influx of inflammatory cells (lymphocytes, mono­
AnxA1, COX-2 and NF-kB in rats and mice exposed to cigarette smoke cytes and neutrophils) into BAL, besides helping to preserve the pul­
[35,49,50,55]. monary architecture. Furthermore, the administration of PL maintained
Exposure to benzopyrene induced increased levels of IL-1β, TNF-α the levels of AnxA1 increased, reduced the expressions of COX-2, Bcl-2
and ROS production, through the activation of NF-kB. Activation of this and NF-kB as well as of the cytokines IL-1β, IL-17 and TNF-α. Therefore,
pathway stimulates cell proliferation and differentiation, inhibits our data indicate the potential of PL for future anti-inflammatory and
apoptosis and stimulates inflammation and angiogenesis [30,57]. In anti-tumor strategies in pulmonary disorders.
addition, the mediators involved recruit and activate leukocytes, mainly
neutrophils, to the site of inflammation, which creates a microenvi­ Funding
ronment that promotes the even more pronounced release of inflam­
matory mediators [44,57]. This work was supported by Grants from Fundação de Amparo à
In view of the results obtained, we complemented the analysis of Pesquisa do Estado de São Paulo - FAPESP (2015/03359-5 to APG),
chemical mediators by measuring pro-inflammatory cytokines. The Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq
studies indicated increased levels of IL-1β, IL-17 and TNF-α in the su­ (404190/2016-2 to APG) and Centro Universitário Padre Albino (UNI­
pernatants of the lung macerate of animals induced by benzopyrene and FIPA) research programme.
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, Declaration of Competing Interest
numerous macrophages and high levels of IL-1β, COX-2, TGF-α in the
bronchoalveolar lavage [67]. Other studies indicated that IL-17 acts The authors declare that they have no known competing financial
synergistically with NF-kB activators such as TNF-α and IL-1β and that interests or personal relationships that could have appeared to influence
IL-17 can promote tumor development through chronic tissue the work reported in this paper.

8
T.E.B. Ashino et al. International Immunopharmacology xxx (xxxx) xxx

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