Protective effects of neem (Azadirachta indica A. Juss.) leaf extract against cigarette smoke- and lipopolysaccharide-induced pulmonary inflammation

Abstract

Neem (Azadirachta indica A. Juss.) leaf has been reported to exert anti-inflammatory, antibacterial and antioxidant effects. The purpose of this study was to investigate the protective effects of neem leaf extract (NLE) against cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced pulmonary inflammation. Treatment with NLE significantly attenuated the infiltration of inflammatory cells, such as neutrophils and macrophages in bronchoalveolar lavage fluid (BALF). NLE also reduced the production of reactive oxygen species and the activity of neutrophil elastase in BALF. Moreover, NLE attenuated the release of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in BALF. NLE inhibited the recruitment of inflammatory cells and the expression of monocyte chemoattractant protein-1 (MCP-1) in the lungs of mice with CS- and LPS-induced pulmonary inflammation. NLE also decreased the expression of inducible nitric oxide synthase (iNOS) in the lungs of the mice CS- and LPS-induced pulmonary inflammation. Furthermore, treatment with NLE significantly attenuated the activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the lungs mice exposed to CS and LPS. NLE also inhibited the phosphorylation of nuclear factor (NF)-κB and inhibitor of NF-κB (IκB) in the lungs of mice expose to CS and LPS. These findings thus suggest that NLE has potential for use in the treatment of chronic obstructive pulmonary disease.

Full text

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE
Abstract. Neem (Azadirachta indica A. Juss.) leaf has
been reported to exert anti-inflammatory, antibacterial and
antioxidant effects. The purpose of this study was to investigate
the protective effects of neem leaf extract (NLE) against cigarett e
smoke (CS)- and lipopolysaccharide (LPS)-induced pulmonary
inflammation. Treatment with NLE significantly attenuated
the inltration of inammatory cells, such as neutrophils and
macrophages in bronchoalveolar lavage uid (BALF). NLE
also reduced the production of reactive oxygen species and
the activity of neutrophil elastase in BALF. Moreover, NLE
attenuated the release of pro-inf lammatory cytokines, such
as tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in
BALF. NLE inhibite d the recr uit ment of inamm atory cells and
the expression of monocyte chemoattractant protein-1 (MCP-1)
in the lungs of mice with CS- and LPS-induced pulmonary
inammation. NLE also decreased the expression of inducible
nitric oxide synthase (iNOS) in the lungs of the mice CS- and
LPS-induced pulmonary inammation. Furthermore, treatment
with NLE signicantly attenuated the activation of extracellular
signal-regulated kinase (ERK) and c-Jun N-terminal
kinase (JNK) in the lungs mice exposed to CS and LPS. NLE
also inhibited the phosphorylation of nuclear factor (NF)-κB
and inhibitor of NF-κB (IκB) in the lungs of mice expose to CS
and LPS. These ndings thus suggest that NLE has potential for
use in the treatment of chronic obstructive pulmonary disease.
Introduction
Chronic obstructive pulmonary disease (COPD) is a chronic
airway disease that leads to difculties breathing (1), and is
characterized by chronic inflammation of the respiratory
tract with increased numbers of inflammatory cells and
molecules (2). The worldwide incidence, prevalence and
mortality of COPD are increasing (3). Cigarette smoke (CS) is
a complex mixture of chemicals generated from the burning of
tobacco (4), and is the main cause of COPD (5). CS affects the
recruitment of inammatory cells, including neutrophils into
the lungs and is associated with chronic inammation of the
airways and a decline in lung function (6).
Neutrophils are the host defense inammatory cells that are
rapidly recruited to sites of infection (7). However, neutrophilic
inammation is the major cause of pulmonary inammation
in COPD pathophysiology (8). Activated neutrophils produce
several cytotoxic mediators, including reactive oxygen
species (ROS) and neutrophil elastase (NE), which aggravate
pulmonary inammation and emphysema (9). The increased
production of ROS accelerates the development of COPD through
the activation of mitogen-activated protein kinases (MAPKs)
and nuclear factor-κB (NF-κB) (10). NE activity is increased in
the lungs affected by COPD, which enhances the destruction
of alveolar structure (11). Tumor necrosis factor-α (TNF-α)
is a central inf lammatory cytokine that is associated with
Protective effects of neem (Azadirachta indica A. Juss.) leaf extract
against cigarette smoke- and lipopolysaccharide-induced
pulmonary inammation
JAE-WON LEE1, HYUNG WON RYU1, SO-YEON PARK1, HYUN AH PARK1,2,
OK-KYOUNG KWON1, HEUNG JOO YUK1, KRISHNA K. SHRESTHA3, MINWOO PARK4, JUNG HEE KIM1,
SANGWOO LEE5, SEI-RYANG OH1 and KYUNG-SEOP AHN1
1Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology,
Chungju-si, Chungbuk 363-883; 2College of Pharmacy, Chungnam National University, Daejeon 305-764,
Republic of Korea; 3Ethnobotanical Society of Nepal (ESON), Central Department of Botany, Tribhuvan University,
Kathmandu 44618, Nepal; 4SciTech Korea, Gangbuk-gu, Seoul 142-705; 5International Biological Material Research Center,
Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
Received March 30, 2016; Accepted September 25, 2017
DO I: 10. 3892/ ijm m.2017.3178
Correspondence to: Dr Kyung-Seop Ahn, Natural Medicine Research
Center, Korea Research Institute of Bioscience and Biotechnology,
30 Yeongudanji-ro, Cheongwon-gu, Chungju-si, Chungbuk 363-883,
Republic of Korea
E-mail: ksahn@kribb.re.kr
Abbreviations: COPD, chronic obstructive pulmonary disease;
CS, cigarette smoke; LPS, lipopolysaccharide; BALF, broncho-
alveolar lavage fluid; ROS, reactive oxygen species; NE, neutrophil
elastase; NLE, neem leaf extract; TNF-α, tumor necrosis factor-α;
IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1;
iNOS, inducible nitric oxide synthase; MAPKs, mitogen-activated
protein kinases; NF-κB, nuclear factor-κB; IκB, inhibitor of NF-κB
Key wo rds: neem, chronic obstructive pulmonary disease, cigarette
smoke, lipopolysaccharide, neutrophil, mitogen-activated protein
kinases, nuclear factor-κB

LEE et al: ANTI-I NFLAMMATORY EFFECTS OF NLE AGAINST CS- AND LPS-INDUCED PULMONARY INFLAMM ATION
2
many immune-mediated diseases, including COPD (12). It
is well known that the constitutive overexpression of TNF-α
affects the recruitment of inflammatory cells and promotes
emphysema in the lungs of animals (13). Interleukin (IL)-6 is
a pro-inammatory cytokine that plays a pivotal role in the
pathogenesis of COPD by modulating pulmonary function (14).
Monocyte chemoattractant protein-1 (MCP-1) is one of the key
chemokines that contributes to the recruitment of inammatory
cells, such as neutrophils (15) and macrophages (16). MCP-1
levels are significantly increased in patients with COPD
compared with non-smokers (17). Inducible nitric oxide
synthase (iNOS) expression is induced by neutrophils and
macrophages in response to pro-inammatory stimuli (18,19)
and is known to have anti-inflammatory activity (20,21).
However, the continuous expression of iNOS is associated
with pulmonary inammation and emphysema (22). Recently,
it has also been reported that iNOS expression is higher in
the lungs of patients with COPD than non-smokers (23). The
MAPK signaling pathway promotes the inammatory response
by enhancing inammatory gene transcription (6,24). NF-κB
is a central transcription factor that plays an important role in
the expression of inammatory genes, such as iNOS, TNF-α
and IL-6 (25). CS has been shown to affect the activation of
MAPKs (26) and NF-κB (27).
Neem (Azadirachta indica A. Juss.) belonging to the family,
Meliaceae is an evergreen tree, cultivated in various parts of the
Indian subcontinent (28). The neem leaf has been reported to
exhibit various pharmacological activities, including anti-inam-
matory (29), antioxidant (30,31), antimicrobial (32) and antiviral
properties (33). Active constituents of the neem leaf include
nimbin, nimbidine, isomeldenin, β-sitosterol and quercetin (34).
Quercetin (35), β-sitoserol (36) and nimbidine (37) have been
shown to exert anti-inammatory effects. These effects are due
to the inhibition of pro-inammatory molecules, such as TNF-α,
iNOS and NF-κB. Recently, neem leaf extract (NLE) has been
reported to protect against endotoxemia in mice exposed to lipo-
polysaccharide (LPS) (38). However, to date, at least to the best
of our knowledge, the protective effects of NLE have not been
demonstrated in CS- and LPS-induced pulmonary inamma-
tion. Thus, the aim of this study was to investigate the protective
effects of NLE against cigarette smoke (CS)- and lipopolysac-
charide (LPS)-induced pulmonary inammation.
Materials and methods
Preparation of NLE. Neem leaf was collected from ward no. 11,
Hetauda, Nepal (latitude 27˚27'11.7'', longitude 85˚00'11.1'' and
531 m above sea level), and identied by Mr. M.R. Poudeyal of
the Ethnobotanical Society of Nepal (ESON). Voucher speci-
mens recorded as KRIB 0059759 and 760 have been deposited
in the herbarium of the Korea Research Institute of Bioscence
and Biotechnolgy (KRIB). After drying and grinding the
leaves of neem, the powder (52 g) was added to 100 liters of
methanol. The extraction was carried out using the method of
repercolation at room temperature. The extract was ltered and
concentrated by a rotavapor under reduced pressure, thereby
obtaining 2.99 g of neem methanolic extract. In the following
experiment, the neem leaves were dissolved in dimethyl sulf-
oxide (DMSO) at a concent ration of 20 mg/m l, and then diluted
to various concentrations prior to use.
Model of CS- and LPS-induced pulmonary inammation. CS-
and LPS-induced pulmonary inammation was induced using
a modication of the procedure described by Lee et al (6).
Briey, a total of 30 C57BL/6 mice (6 weeks old; weight, 20 g;
n=6/group) were whole-body exposed to room fresh air or CS
of 7 cigarettes for 50 min a day for 9 days. CS was generated
by 3R4F research cigarettes (Tobacco and Health Research
Institute, University of Kentucky, Lexington, KY, USA). LPS
was instilled intrana sally on day 8 (5 µg dissolved in 50 µl
distilled water). The mice were randomly divided into 5 groups
as follows: the normal control (NC), the CS + LPS (CS with
intranasal LPS instillation) group, the ROF (CS with intranasal
LPS instillation) + roumilast [10 mg/kg, per os (p.o)] group, and
the NLE 10 or 20 (CS with intranasal LPS instillation) + NLE
(10 or 20 mg/kg, p.o) groups. All the animal experiments were
approved by the Institutional Animal Care and Use Committee
of t h e Kore a Re s ea r ch I n stit u t e of Bio s c ienc e and Biot e chn o l o g y
and performed in compliance with the National Institutes of
Health Guidelines for the Care and Use of Laboratory Animals
and National Animal Welfare Law of Korea.
Measurement of inflammatory cells in bronchoalveolar
lavage uid (BALF). BALF collection was performed using
the method of Shin et al (5). In brief, the mice were admin-
istered an intraperitoneal injection of a pentobarbital (50 mg/
kg; Hanlim Pharm, Co., Seoul, Korea) 24 h after the final
challenge, and a tracheostomy was performed. To obtain the
BALF, ice-cold phosphate-buffered saline (PBS) (0.7 ml) was
infused into the lung and withdrawn via tracheal cannulation
twice (total volume, 1.4 ml). To determine differential cell
counts, 100 µl of BALF were centrifuged at 1,500 rpm for
5 min and the number of neutrophils and macrophages was
counted using Diff-Quik® staining reagent according to the
manufacturer's instructions (IMEB Inc., Deereld, IL, USA).
Measurement of ROS and NE in BALF. The effects of NLE on
the production of ROS were determined using 2',7'-dichlorou-
orescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO,
USA). Briey, the in a m mator y cells were isolate d from BALF
and incubated with 20 µM DCFH-DA for 10 min at 37˚C. The
level of intracellular ROS was then determined using a uores-
cence microscope at 488 nm excitation and 525 nm emission (8).
The activity of NE was examined using N-succinyl-(Ala)3-p-
nitroanilide (Sigma-Aldrich) in 37˚C for 90 min, according to
the protocol described by Sakuma et al (39).
Measurement of the level of pro-inammatory cytokines in
BA L F. The levels of pro-inammatory cytokines (TNF-α and
IL-6) in BALF were determined using ELISA according to the
manufacturer's instructions (R&D Systems, Shanghai, China).
The absorbance was measured at 450 nm using a microplate
reader (Molecular Devices, Sunnyvale, CA, USA), as previ-
ously described (4).
Western blot analysis. Lung tissues were homogenized using
a homogenizer with a lysis buffer (Intron Biotechnology, Inc.,
Seoul, Korea). Protein samples were denatured and resolved
on 10% SDS-polyacrylamide gels and transferred onto a
nitrocellulose membrane. The membrane was incubated
with blocking solution for 1 h. Specific antibodies against

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 3
MCP-1 (1;1,000; ab25124; Abcam, Cambridge, MA,
USA), iNOS (1;1,000; ADI-905-431; Enzo Life Sciences,
Farmingdale, NY, USA), p-ERK (1:1,000; #9101; Cell
Signaling Technology, Inc., Danvers, MA, USA), ERK
(1:1,000; sc-154; Santa Cruz Biotechnology, Santa Cruz, CA,
USA), p-J N K (1:1,000; KAP-SA011; Enz o Life Sciences), JNK
(1:1,000; sc-474; Santa Cruz Biotechnology), p-p38 (1:1,000;
ADI-KAP-MA022; Enzo Life Sciences), p-38 (1:1,000;
sc-7149; Santa Cruz Biotechnology), p-p65 (1:1,000; #3033;
Cell Signaling Technology, Inc.), p65 (1:1,000; sc-372; Santa
Cruz Biotechnology), p-inhibitor of NF-κB (IκB; 1:1,000;
sc-371; Santa Cruz Biotechnology) and β-actin (1;2,500;
#4967; Cell Signaling Technology, Inc.) were incubated over-
night at 4˚C with 5% skim milk. The membranes were washed
in Tris-buffered saline with Tween 20 (TBST) and incubated
with the Peroxidase-AfniPure goat anti-mouse IgG (H+L)
(1:2,000; 115-035-003; Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA, USA) and the Peroxidase-
AfniPure goat anti-rabbit IgG (H+L) (1:2,000; 111-035-003;
Jackson ImmunoResearch Laboratories, Inc.) for 2 h at room
temperature. The blots were washed 3 times with TBST, and
then developed with an enhanced chemiluminescence (ECL)
kit (Amersham Biosciences, Piscataway, NJ, USA).
Histological analysis. After the BALF samples were collected,
lung tissues were xed in 10% (v/v) neutral-buffered formalin
solution. For histological examination, the lung tissues were
embedded in parafn, sectioned at 4 µm thickness, and stained
with a hematoxylin and eosin (H&E) solution (Sigma-Aldrich)
to estimate the inammatory response.
Statistical analysis. All values shown in the figures are
expressed as the means ± SD obtained from at least 3 indepen-
dent experiments. Statistical signicance was carried out using
a two-tailed Student's t-test. A p-value <0.05 was considered to
indicate a statistically signicant difference.
Results
NLE inhibits the inltration of inammatory cells in the BALF
of mice with CS- and LPS-induced pulmonary inammation.
Given the fact that the inltration of inammatory cells, such as
neutrophils and macrophages is increased in the BALF of mice
with CS- and LPS-induced pulmonary inflammation (9), we
investigated whether NLE inhibits the inltration of neutrophils
and macrophages in BALF. As shown in Fig. 1, we observed that
increased numbers of neutrophils and macrophages were detected
in the BALF of mice in the CS and LPS group compared with
those in the normal control group. However, treatment with NLE
signicantly attenuated the numbers of neutrophils and macro-
phages in BALF, compared with the CS and LPS group in a
concentration-dependent manner (Fig. 1). The effect of 20 mg/kg
NLE was similar to that of treatment with 10 mg/kg ROF.
NLE attenuates the production of ROS and NE in BALF.
It is well known that ROS production and NE activity are
Figure 1. Effect of neem leaf extract (NLE) on the inltration of neutrophils
and macrophages in the bronchoalveolar lavage uid (BALF) of mice with
cigarette smoke (CS)- and lipopolysacchar ide (LPS)-induced pulmona ry
inammation. (A and B) The BALF differential cell count was determined
using the Diff-Quick® staining reagent (x400 magnication). The values are
expressed as means ± SD (n=6 mice per g roup). NC, normal control mice with
PBS only; CS + LPS, cigarette smoke (CS) and lipopolysaccharide (LPS);
ROF, roumilast (10 mg/kg) + CS and LPS; NLE 10 or 20, NLE (10 or 20 mg/
kg) + CS and LPS. #p<0.01 indicates a statistically signicant difference from
the normal control group. *p<0.05 and **p<0.01 indicate statistically signicant
differences compared to t he CS and LPS group.
Figure 2. Effect of neem leaf extract (NLE) on the production of reactive
oxygen species (ROS) and neutrophil elastase (NE) in bronchoalveolar lavage
uid (BALF). (A) ROS production and (B) NE activity. Data are expressed
as the means ± SD. #p<0.01 indicates a statistically signicant difference
from the normal control group. *p<0.05 and **p<0.01 indicate statistically
signicant differences compared to the cigarette smoke (CS) and lipopolysac-
charide (LPS) group.

LEE et al: ANTI-I NFLAMMATORY EFFECTS OF NLE AGAINST CS- AND LPS-INDUCED PULMONARY INFLAMM ATION
4
increased in the BALF of mice with CS- and LPS-induced
pulmonary inammation (5,6). Thus, in this study, the levels of
ROS and NE were examined in the BALF of mice with CS and
LPS-induced pulmonary inammation. As shown in Fig. 2,
the levels of ROS and NE were signicantly increased in the
CS and LPS group. However, treatment with NLE signicantly
decreased the levels of ROS and NE (Fig. 2). In particular,
treatment with 20 mg/kg NLE more effectively attenuated the
levels of those molecules compared with 10 mg/kg ROF.
NLE decreases the levels of TNF-α and IL- 6 in BALF. The
increased release of TNF-α and IL-6 in BALF is one of the
major characteristics of COPD (5). Thus, to determine whether
NLE affects the release of pro-inflammatory cytokines in
Figure 4. Effect of neem leaf extract (NLE) on the inltration of inammatory cells in lungs of mice with cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced
pulmonar y inam mation. (A) Peribronchial lesion (x400 magnication): (a) negative control, (b) CS + LPS, (c) roumilast, (d) NLE 10 mg/kg and (e) NLE 20 mg/
kg. (B) Quantitative analysis of airway inammation in lung tissue stained with H&E solution. (C) Monocyte chemoattractant protein-1 (MCP-1) expression was
detected by western blot analysis. NC, normal control mice with PBS only; CS + LPS, cigarette smoke (CS) and lipopolysaccharide (LPS); ROF, roumilast (10 mg/
kg) + CS and LPS; NLE 10, NLE (10 mg/kg) + CS and LPS; NLE 20, NLE (20 mg /kg) + CS an d LPS. Data are expressed as the mean s ± SD. #p<0.01 indicates a sta-
tistically signicant difference from the ormal control group. *p<0.05 and **p<0.01 indicate statistically signicant differences compared with the CS and LPS group.
Figure 3. Effect of neem leaf extract (NLE) on the levels of pro-inammatory cytokines, such as tumor necrosis factor-α (T NF-α) and interleukin-6 (IL- 6)
in bronchoalveolar lavage uid (BALF). (A) The levels of TNF-α and (B) IL-6 were measured by ELISA. The absorbance was measured at 450 nm using a
microplate reader. Data are expressed as the means ± SD. #p<0.01 indicates a statistically signicant differencefrom the normal control group. **p<0.01 indicate
statistically signicant differences compa red to the cigarette smoke (CS) and lipopolysaccharide (LPS) group.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 5
BALF, the levels of TNF-α and IL-6 were examined by ELISA.
As shown in Fig. 3, treatment with NLE effectively inhibited the
release of these cytokines in BALF.
NLE reduces the recruitment of inflammatory cells and
the expression of MCP-1 in the lungs of mice with CS- and
LPS-induced pulmonary inammation. To examine whether
NLE affects the recruitment of inflammatory cells and the
expression of MCP-1 in the lungs of mice with CS- and
LPS-induced pulmonary inf lammation, the infiltration of
inammatory cells was determined by H&E staining. As shown
in Fig. 4A and B, the mice in the CS and LPS group exhibit ed an
increased inltration of inammatory cells. However, treatment
with NLE signicantly reduced the recruitment of inamma-
tory cells in a concentration-dependent manner. Consistent with
the decrease in inammatory cell recruitment, treatment with
NLE also signicantly decreased the expression of MCP-1 in
the lungs, suggesting that NLE attenuated the recruitment of
inammatory cells (Fig. 4C). Similar to the results shown above,
the effect of 20 mg/kg NLE was similar to that of treatment with
10 mg/kg ROF.
NLE inhibits the expression of iNOS in lungs of mice with CS-
and LPS-induced pulmonary inammation. As the increased
expression of iNOS induced by neutrophils (40) and macro-
phages (2) is an important in the pathologenesis of COPD, we
investigated whether NLE affects the level of iNOS in the lungs
of mice with CS- and LPS-induced pulmonary inammation.
As shown in Fig. 5, iNOS expression was increased in the lungs
of mice in the CS and LPS group. However, treatment with
NLE effectively inhibited the expression of iNOS, compared
with normal control mice.
NLE attenuates the activation of ERK and JNK in the lungs
of mice with CS- and LPS-induced pulmonary inammation.
MAPK activation plays an important role in the inammatory
response regulating the release of pro-inammatory cytokines
and mediators. Thus, we investigated whether NLE treatment
attenuates the activation of MAPKs in the lungs of mice with
CS- and LPS-induced pulmonary inflammation. As shown
in Fig. 6, the activation of MAPKs (ERK, JNK and p38) was
signicantly increased in the lungs of mice in the CS and LPS
group. However, treatment with NLE signicantly decreased
the activation of ERK and JNK in a concentration-dependent
manner (Fig. 6A and B). The inhibitory effect of 20 mg/kg
Figure 6. Effect of neem leaf extract ( NLE) on the activation of ERK and
JNK in lungs of mice. (A) The activation of ERK, (B) JNK and (C) p38 was
detected by western blot analysis. Data a re expressed as the means ± SD.
#p<0.01 indicates a statistically signicant difference from the normal con-
trol group. *p<0.05 and **p<0.01 indicate statistically signicant differences
compared to the cigarette smoke (CS) and lipopolysaccharide (LPS) group.
Figure 5. Effect of neem leaf extract (NLE) on the expression of inducible
nitric oxide synthase (iNOS) in lungs of mice. The expression of iNOS was
detected by western blot analysis. Data a re expressed as the means ± SD.
#p<0.01 indicates a statistically signicant difference from the normal control
group. *p<0.05 indicate statistically signicant differences compared to the
cigarette smoke (CS) and lipopolysaccharide (LPS) group.

LEE et al: ANTI-I NFLAMMATORY EFFECTS OF NLE AGAINST CS- AND LPS-INDUCED PULMONARY INFLAMM ATION
6
NLE on ERK and JNK activation was similar to that of treat-
ment with 10 mg/kg ROF. No signicant attenuation of p38
activation was observed with NLE (Fig. 6C).
NLE decreases the phosphorylation of NF-κB and IκB in
lungs of mice with CS- and LPS-induced pulmonary inam-
mation. NF-κB is activated by a number of stimuli, including
pro-inammatory mediators and LPS. In response to these
molecules, IκB is phosphorylated, ubiquitinated and degraded,
resulting in the phosphorylation and nuclear translocation of
NF-κB (20,41). In the present study, treatment with NLE inhib-
ited the phosphorylation of NF-κB and IκB in the lungs of mice
with CS- and LPS-induced pulmonary inammation (Fig. 7).
Discussion
In the present study, we examined the protective effects of NLE
against CS- and LPS-induced pulmonary inammation. NLE
sign i cantly in h i bited the inlt ra t i on of in a m m a t o r y cells, such
as neutrophils and macrophages in BALF. NLE also reduced
the production of ROS and NE, and decreased the release of
pr o -in a m mato r y cyto k ines in BAL F. NLE at t enu ated th e accu-
mulation of inammatory cells and the expression of MCP-1 in
the lungs of mice with CS- and LPS-induced pul monar y inam-
mation. Furthermore, NLE inhibited the expression of iNOS in
the lungs of mice with CS- and LPS-induced pul monar y inam-
mation. NLE also attenuated the activation of MAPKs (ERK
and JNK) and NF-κB in the lung tissue.
COPD is a global health epidemic the incidence of which is
increasing (42), and it is associated with a high risk of morbidity
and mortality (43). COPD is characterized by chronic airway
inflammation and mucus hypersecretion (44). It is also well
known that CS exposure and bacterial infection are associated
with the development of COPD (4,45,46). CS is the most impor-
tant risk factor that increases the recruitment of inammatory
cells in the lungs and the number of goblet cells in the small
airway (47). LPS is a major constituent of the Gram-negative
bacterial cell wall that stimulates the inammatory response (48).
The recruitment of inflammatory cells, such as neutrophils
and macrophages in the airways is a characteristic sign of
COPD (6,49,50). ROS production promote the inammatory
response in the lungs via the activation of transcription factors,
such as NF-κB and MAPK signal transduction pathways (10).
Increased ROS production induced by neutrophils has been
reported to promote the oxidation of proteins, DNA and lipids
which leads to lung damage (10,51). A number of studies have
reported that NE levels are increased in response to CS (52,53) or
CS and LPS (5,6), which increases the inammatory cell recruit-
ment, emphysema and the production of mucus in the lungs (54).
CS is the most important source of elevated levels of ROS and
NE in COPD (55). In the present study, treatment with NLE
signicantly inhibited inammatory cell inltration in BALF
and in the lungs of mice with CS- and LPS-induced pulmonary
inammation (Figs. 1 and 4A and B). NLE also attenuated the
production of ROS and the activity of NE (Fig. 2).
Pro-inammatory cytokines, including TNF-α and IL-6 play
an important role in the pathological processes of COPD (56).
TNF-α is a central cytokine that regulates inammation through
neutrophil recruitment and endothelial activation (57). IL-6 is
involved in the pathogenesis of lung diseases, such as COPD (58).
It has also been reported that exposure to CS increases macro-
phage accumulation that contributes to the development of
COPD by increasing the levels of IL-6 (59). Recently, TNF-α
and IL-6 were identied to be involved in CS- and LPS-induced
COPD (4,5,60). In the present study, NLE decreased the release
of TNF-α and IL-6 in BALF (Fig. 3). MCP-1 is a chemokine
that plays a key role in the migration of neutrophils and macro-
phages (15,16). Recently, the increased expression of MCP-1 was
detected in the lungs of mice exposed to CS (61). The present
data demonstrated benecial effects of NLE against the CS- and
LPS-induced expression of MCP-1 (Fig. 4C). iNOS has been
implicated in the pathophysiology of inammatory diseases,
including COPD (23), and the high expression of iNOS has been
reported to affect pulmonary inammation (62). It has also been
reported that the inhibition of iNOS exerts protective effects in
a wide variety of respiratory diseases (63). The present study
dem onstrated that N L E sig n ic a ntly sup p r e ssed the ex p r essio n of
iNOS in the lungs of mice with CS and LPS-induced pulmonary
inammation in a concentration-dependent manner (Fig. 5).
MAPKs have been reported to regulate pro-inammatory
molecules (64,65) and have been widely studied in pulmonary
inammation (4,5,66). MAPKs (ERK, JNK and p38) mediate
pro-inammatory gene transcription in response to cytokines
and LPS (5,67). CS leads to the activation of MAPKs (68-71).
It has also been reported that MAPK activation affects ROS
production in lungs affected by COPD (10). The present data
demonstrated that the activation of MAPKs (ERK, JNK
and p38) was induced by CS and LPS in the lungs of mice.
However, NLE treatment signicantly inhibited the activation
or ERK and JNK (Fig. 6A and B). No signicant inhibition of
p38 activation was observed with NLE treatment (Fig. 6C).
Figure 7. Effect of neem leaf extract (NLE) on the activation of nuclear
fac tor-κB (NF-κB) in lungs of mice. The phosphorylation of NF-κB and
inhibitor of NF-κB (IκB) was detected by western blot analysis. Data are
expressed as the means ± SD. #p<0.01 indicates a statistically signicant
difference f rom the normal control group. *p<0.05 and **p<0.01 indicate
statistically signicant differences compared to the cigarette smoke (CS) and
lipopolysaccharide (LPS) group.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 7
NF-κB is a key transcription factor in the inammatory
response, and is activated by numerous extracellular stimuli,
including pro-inflammatory cytokines, such as TNF-α and
IL-6 (10). The NF-κB-dependent production of these cyto-
kines affects the recruitment of inflammatory cells, such
as neutrophils and macrophages to lung tissue, causing lung
injury or emphysema (9,72,73) Therefore, NF-κB signaling is
considered to be an important therapeutic target for pulmonary
inammation induced by CS (74). In this study, NLE treatment
signicantly inhibited the elevated phosphorylation levels of
NF-κB and IκB induced by CS and LPS in lung tissue (Fig. 7).
The neem tree (Azadirachta indica A. Juss.; Meliaceae) is
indigenous to India, and now this tree is cultivated widely in
areas of the world (75). Azadirachta indica A. Juss has been
widely used as neem and has been used in medicine for over
2,0 00 years (76). Various parts of the neem tree have been used
in medicines and food, as well as as insecticides, and many
bioactive constituents, including limonoids (tetra-nortriterpe-
noids) have been isolated and identied (77). NLE has been
reported to possess antibacterial activity (78-80). It has also
been demonstrated that NLE induces apoptosis in the breast
cancer cells (81). Neem leaf fraction has been reported to
possess antioxidant properties (82,83). Recently, it has also
been shown that NLE protects LPS-induced endotoxemia (38).
However, to date, at least to the best of our knowledge, the
protective effects of NLE have not been investigated in CS- and
LPS-induced pulmonary inammation.
In conclusion, the present data demonstrated that NLE
signicantly inhibited the inltration of inammatory cells, such
as neutrophils and macrophages in the lungs of mice with CS-
and LPS-induced pulmonary inammation. NLE also attenuated
the production of inammatory mediators, including ROS, NE,
TNF-α and IL-6 in BALF. Furthermore, NLE decreased the
expression of MCP-1 and iNOS in the lungs of mice with CS-
and LPS-induced pulmonary inammation. NLE also inhibited
the activation of MAPKs (ERK and JNK) and NF-κB in the lungs
of mice. These results thus suggest that NLE may have potential
for use as a valuable therapeutic agent in the treatment of COPD.
Acknowledgements
This study was supported by a grant from the Ministry of
Science, ICT and Future Planning (FGC 1011534), Ministry
for Health and Welfare (HI14C1277) and the KRIBB Research
Initiative Program (KGM 1221713) of the Republic of Korea.
References
1. Lee H, Jung KH, Lee H, Park S, Choi W and Bae H: Casticin,
an active compound isolated from Vitex Fructus, ameliorates the
cigarette smoke-induced acute lung inammatory response in a
murine model. Int Immunopharmacol 28: 1097-1101, 2015.
2. Barnes PJ: Chronic obstructive pulmonary disease * 12: new
treatments for COPD. Thorax 58: 803-808, 2003.
3. Park YC, Jin M, Kim SH, Kim MH, Namgung U and Yeo Y:
Effects of inhalable microparticle of ower of Lonicera japonica
in a mous e mo d e l of CO P D. J Et h nopha r ma c o l 151: 123-1 3 0, 2014.
4. Bak JH, Lee SM and Lim HB: Safety assessment of mainstream
smoke of herbal cigarette. Toxicol Res 31: 41-48, 2015.
5. Shin IS, Shin NR, Park JW, Jeon CM, Hong JM, Kwon OK,
Kim JS, Lee IC, Kim JC, Oh SR, et al: Melatonin attenuates
neutrophil inflammation and mucus secretion in cigarette
smoke-induced chronic obstructive pulmonary diseases via the
suppression of Erk-Sp1 signaling. J Pineal Res 58: 50-60, 2015.
6. Lee JW, Shin NR, Park JW, Park SY, Kwon OK, Lee HS,
Hee Kim J, Lee HJ, Lee J, Zhang ZY, et al: Callicarpa japonica
Thunb. attenuates cigarette smoke-induced neutrophil inam-
mation and mucus secretion. J Ethnopharmacol 175: 1-8, 2015.
7. Kruger P, Saffarzadeh M, Weber AN, Rieber N, Radsak M,
von Bernuth H, Benarafa C, Roos D, Skokowa J and Hartl D:
Neutrophils: between host defence, immune modulation, and
tissue injury. PLoS Pathog 11: e1004651, 2015.
8. Shin IS, Ahn KS, Shin NR, Lee HJ, Ryu HW, Kim JW, Sohn KY,
Kim HJ, Han YH and Oh SR: Protective effect of EC-18, a
synthetic monoacetyldiglyceride on lung inflammation in a
murine model induced by cigarette smoke and lipopolysac-
charide. Int Immunopharmacol 30: 62-68, 2016.
9. Song HH, Shin IS, Woo SY, Lee SU, Sung MH, Ryu HW,
Kim DY, Ahn KS, Lee HK, Lee D, et al: Piscroside C, a novel
iridoid glycoside isolated from Pseudolysimachion rotundum
va r. subinegrum suppresses airway inammation induced by
cigarette smoke. J Ethnopharmacol 170: 20-27, 2015.
10. Rahman I and Adcock IM: Oxidative stress and redox regulation
of lung inammation in COPD. Eur Respir J 28: 219-242, 2006.
11. Shapiro SD, Goldstein NM, Houghton AM, Kobayashi DK,
Kelley D and Belaaouaj A: Neutrophil elastase contributes to
cigarette smoke-induced emphysema in mice. Am J Pathol 163:
2329-2335, 2003.
12. Mukhopadhyay S, Hoidal JR and Mukherjee TK: Role of
TNFalpha in pulmonary pathophysiology. Respir Res 7:
125, 2006.
13. Lundblad LK, Thompson-Figueroa J, Leclair T, Sullivan MJ,
Poynter ME, Irvin CG and Bates JH: Tumor necrosis factor-alpha
overexpression in lung disease: a single cause behind a complex
phenotype. Am J Respir Crit Care Med 171: 1363-1370, 2005.
14. Rincon M and Irvin CG: Role of IL-6 in asthma and other inam-
matory pulmonary diseases. Int J Biol Sci 8: 1281-1290, 2012.
15. Wan MX, Wang Y, Liu Q, Schramm R and Thorlacius H: CC
chemokines induce P-selectin-dependent neutrophil rolling
and recruitment in vivo: intermediary role of mast cells. Br J
Pharmacol 138: 698-706, 2003.
16. Deshmane SL, Kremlev S, Amini S and Sawaya BE: Monocyte
chemoattractant protein-1 (MCP-1): an overview. J Interferon
Cytokine Res 29: 313-326, 2009.
17. Traves SL, Culpitt SV, Russell RE, Barnes PJ and Donnelly LE:
Increased levels of the chemokines GROalpha and MCP-1
in sputum samples from patients with COPD. Thorax 57:
590-595, 2002.
18. McNeill E, Crabtree MJ, Sahgal N, Patel J, Chuaiphichai S,
Iqbal AJ, Hale AB, Greaves DR and Channon KM: Regulation
of iNOS function and cellular redox state by macrophage Gch1
reveals specic requirements for tetrahydrobiopterin in NRF2
activation. Free Radic Biol Med 79: 206-216, 2015.
19. Webb JL, Polak JM and Evans TJ: Effect of adhesion on inducible
nitric oxide synthase (iNOS) production in purified human
neutrophils. Clin Exp Immunol 123: 42-48, 2001.
20. Lee JW, Bae CJ, Choi YJ, Kim SI, Kwon YS, Lee HJ, Kim SS and
Chun W: 3,4,5-Trihydroxycinnamic acid inhibits lipopolysac-
charide (LPS)-induced inammation by Nrf2 activation in vitro
and improves survival of mice in LPS-induced endotoxemia
model in vivo. Mol Cell Biochem 390: 143-153, 2014.
21. Marinovic MP, Morandi AC and Otton R: Green tea catechins
alone or in combination alter functional parameters of human
neutrophils via suppressing the activation of TLR-4/NFκB p65
signal pathway. Toxicol In Vitro 29: 1766-1778, 2015.
22. Roh GS, Yi CO, Cho YJ, Jeon BT, Nizamudtinova IT, Kim HJ,
Kim JH, Oh YM, Huh JW, Lee JH, et al: Anti-inammatory
effects of celecoxib in rat lungs with smoke-induced emphysema.
Am J Physiol Lung Cell Mol Physiol 299: L184-L191, 2010.
23. Jiang WT, Liu XS, Xu YJ, Ni W and Chen SX: Expression of
nitric oxide synthase isoenzyme in lung tissue of smokers with
and without chronic obstructive pulmonary disease. Chin Med J
(Engl) 128: 1584-1589, 2015.
24. Singh D: P38 inhibition in COPD; cautious optimism. Thorax 68:
705-706, 2013.
25. Oh YC, Jeong YH, Ha JH, Cho WK and Ma JY: Oryeongsan
inhibits LPS-induced production of inf lammatory mediators
via blockade of the NF-kappaB, MAPK pathways and leads to
HO-1 induction in macrophage cells. BMC Complement Altern
Med 14: 242, 2014.
26. Liang Z, Xie W, Wu R, Geng H, Zhao L, Xie C, Li X, Zhu M,
Zhu W, Zhu J, et al: Inhibition of tobacco smoke-induced bladder
MAPK activation and epithelial-mesenchymal transition in mice
by curcumin. Int J Clin Exp Pathol 8: 4503-4513, 2015.

LEE et al: ANTI-I NFLAMMATORY EFFECTS OF NLE AGAINST CS- AND LPS-INDUCED PULMONARY INFLAMM ATION
8
27. Li L, Sun J, Xu C, Zhang H, Wu J, Liu B and Dong J: Icariin
ameliorates cigarette smoke induced inammatory responses via
suppression of NF-κB and modulation of GR in vivo and in vitro.
PLoS One 9: e102345, 2014.
28. Mahfuzul Hoque MD, Bari ML, Inatsu Y, Juneja VK and
Kawamoto S: Antibacterial act ivity of guava (Psid ium guaj ava L.)
and Neem (Azadirachta indica A. Juss.) extracts against
foodborne pathogens and spoilage bacteria. Foodborne Pathog
Dis 4: 481-488, 2007.
29. Okpanyi SN and Ezeukwu GC: Anti-inammatory and anti-
pyretic activities of Azadirachta indica. Planta Med 41:
34-39, 1981.
30. Rao AD, Devi KN and Thyagaraju K: Isolation of antioxidant
principle from A zadirachta seed kernels: determination of its
role on plant lipoxygenases. J Enzyme Inhib 14: 85-96, 1998.
31. Yanpallewar SU, Sen S, Tapas S, Kumar M, Raju SS and
Acharya SB: Effect of Azadirachta indica on parac etamol-induced
hepatic damage in albino rats. Phytomedicine 10: 391-396, 2003.
32. Almas K: The antimicrobial effects of extracts of Azadi-
rachta indica (Neem) and Salvadora persica (Arak) chewing
sticks. Indian J Dent Res 10: 23-26, 1999.
33. Badam L, Joshi SP and Bedekar SS: ‘In vitro’ antiviral activity of
neem (Azadirachta indica. A. Juss) leaf extract against group B
coxsackieviruses. J Commun Dis 31: 79-90, 1999.
34. Siddiqui BS, Afshan F, Gulzar T and Hanif M: Tetra-
cyclic triterpenoids from the leaves of Azadirachta indica.
Phytochemistry 65: 2363-2367, 2004.
35. Chang YC, Tsai MH, Sheu WH, Hsieh SC and Chiang AN: The
therapeutic potential and mechanisms of action of quercetin in
relation to lipopolysaccharide-induced sepsis in vitro and in vivo.
PLoS One 8: e80744, 2013.
36. Loizou S, Lekakis I, Chrousos GP and Moutsatsou P: Beta-sitosterol
exhibits anti-inammatory activity in human aortic endothelial
cells. Mol Nutr Food Res 54: 551-558, 2010.
37. Pillai NR and Santhakumari G: Anti-arthritic and anti-inam-
matory actions of nimbidin. Planta Med 43: 59-63, 1981.
38. Kim WH, Song HO, Jin CM, Hur JM, Lee HS, Jin HY, Kim SY
and Park H: The methanol extract of Azadirachta indica A. Juss
leaf protects mice against lethal endotoxemia and sepsis. Biomol
Ther (Seoul) 20: 96-103, 2012.
39. Sakuma T, Takahashi K, Ohya N, Usuda K, Handa M and
Abe T: ONO-5046 is a potent inhibitor of neutrophil elastase in
human pleural effusion after lobectomy. Eur J Pharmacol 353:
273-279, 1998.
40. Rytilä P, Rehn T, Ilumets H, Rouhos A, Sovijärvi A,
Myllärniemi M and Kinnula VL: Increased oxidative stress
in asymptomatic current chronic smokers and GOLD stage 0
COPD. Respir Res 7: 69, 2006.
41. Lee JW, Kwon JH, Lim MS, Lee HJ, Kim SS, Lim SY and Chun W:
3,4,5-Trihydroxycinnam ic acid increases heme-oxygenase-1 (HO-1)
and decreases macrophage infiltration in LPS-induced septic
kidney. Mol Cell Biochem 397: 109-116, 2014.
42. Barnes PJ: Chronic obstructive pulmonary disease: a growing
but neglected global epidemic. PLoS Med 4: e112, 2007.
43. Vestbo J, Hurd SS, Agustí AG, Jones PW, Vogelmeier C,
Anzueto A, Barnes PJ, Fabbri LM, Martinez FJ,
Nishimura M, et al: Global strategy for the diagnosis, mana-
gement, and prevention of chronic obstructive pulmonary
disease: GOLD executive summary. Am J Respir Crit Care
Med 187: 347-365, 2013.
44. Yang J, Yu HM, Zhou XD, Huang HP, Han Zh, Kolosov VP
and Perelman JM: Cigarette smoke induces mucin hyperse-
cretion and inammatory response through the p66shc adaptor
protein-mediated mechanism in human bronchial epithelial cells.
Mol Immunol 69: 86-98, 2016.
45. Shao MX, Nakanaga T and Nadel JA: Cigarette smoke
induces MUC5AC mucin overproduction via tumor necrosis
factor-alpha-converting enzyme in human airway epithelial
(NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 287:
L420-L 427, 2004.
46. Chung KF: Inflammatory mediators in chronic obstructive
pulmonary disease. Curr Drug Targets Inf lamm Allergy 4:
619-625, 2005.
47. Thorley AJ and Tetley TD: Pulmonary epithelium, cigarette
smoke, and chronic obstructive pulmonary disease. Int J Chron
Obstruct Pulmon Dis 2: 409-428, 2007.
48. Kwak HG and Lim HB: Inhibitory effects of Cnidium monnieri
fruit extract on pulmonary inammation in mice induced by
cigarette smoke condensate and lipopolysaccharide. Chin J Nat
Med 12: 641-647, 2014.
49. Baraldo S, Turato G, Badin C, Bazzan E, Beghé B, Zuin R,
Calabrese F, Casoni G, Maestrelli P, Papi A, et al: Neutrophilic
inltration within the airway smooth muscle in patients with
COPD. Thorax 59: 308-312, 2004.
50. Li H, Yang T, Ning Q, Li F, Chen T, Yao Y and Sun Z: Cigarette
smoke extract-treated mast cells promote alveolar macrophage
inltration and polarization in experimental chronic obstructive
pulmonary disease. Inhal Toxicol 27: 822-831, 2015.
51. Neofytou E, Tzortzaki EG, Chatziantoniou A and Siafakas NM:
DNA damage due to oxidative stress in chronic obstructive
pulmonary disease (COPD). Int J Mol Sci 13: 16853-16864, 2012.
52. Chan KH, Chan SC, Yeung SC, Man RY, Ip MS and Mak JC:
Inhibitory effect of Chinese green tea on cigarette smoke-induced
up-regulation of airway neutrophil elastase and matrix metal-
loproteinase-12 via antioxidant activity. Free Radic Res 46:
1123-1129, 2012.
53. Iizuka T, Ishii Y, Itoh K, Kiwamoto T, Kimura T, Matsuno Y,
Morishima Y, Hegab AE, Homma S, Nomura A, et al:
Nrf2-deficient mice are highly susceptible to cigarette
smoke-induced emphysema. Genes Cells 10: 1113-1125, 2005.
54. Bhowmik A, Chahal K, Austin G and Chakravorty I: Improving
mucociliar y clearance in chronic obstructive pulmonary disease.
Respir Med 103: 496-502, 2009.
55. van E eden SF and Sin DD: Oxidative stress in chronic obstructive
pulmonary disease: a lung and systemic process. Can Respir
J 20: 27-29, 2013.
56. Hwang JH, Lee BJ, Jung HJ, Kim KI, Choi JY, Joo M and
Jung SK: Effects of Chung-pae inhalation therapy on a mouse
model of chronic obstructive pulmonary disease. Evid Based
Complement Alternat Med 2015: 461295, 2015.
57. Lee E, Yun N, Jang YP and Kim J: Lilium lancifolium Thunb.
extract attenuates pulmonary inflammation and air space
enlargement in a cigarette smoke-exposed mouse model. J
Ethnopharmacol 149: 148-156, 2013.
58. Pedroza M, Schneider DJ, Karmouty-Quintana H, Coote J,
Shaw S, Corrigan R, Molina JG, Alcorn JL, Galas D,
Gelinas R, et al: Interleukin-6 contributes to inammation and
remodeling in a model of adenosine mediated lung injury. PLoS
One 6: e22667, 2011.
59. Fernandez-Real JM, Broch M, Vendrell J and Ricart W: Smoking,
fat mass and activation of the tumor necrosis factor-alpha
pathway. Int J Obes Relat Metab Disord 27: 1552-1556, 2003.
60. Xu GH, Shen J, Sun P, Yang ML, Zhao PW, Niu Y, Lu JK,
Wang ZQ, Gao C, Han X, et al: Anti-inammatory effects of
potato extract on a rat model of cigarette smoke-induced chronic
obstructive pulmonary disease. Food Nutr Res 59: 28879, 2015.
61. Chen X, Guan XJ, Peng XH, Cui ZL, Luan CY and Guo XJ:
Acetylation of lysine 9 on histone H3 is associated with increased
pro-inammatory cytokine release in a cigarette smoke-induced
rat model through HDAC1 depression. Inflamm Res 64:
513-526, 2015.
62. Zhao Y, Cui A, Wang F, Wang XJ, Chen X, Jin ML and
Huang KW: Characteristics of pulmonary inflammation in
combined pulmonary fibrosis and emphysema. Chin Med J
(Engl) 125: 3015-3021, 2012.
63. Hesslinger C, Strub A, Boer R, Ulrich WR, Lehner MD and
Braun C: Inhibition of inducible nitric oxide synthase in respi-
ratory diseases. Biochem Soc Trans 37: 886-891, 2009.
64. Li D, Xu D, Wang T, Shen Y, Guo S, Zhang X, Guo L, Li X, Liu L
and Wen F: Silymarin attenuates airway inammation induced
by cigarette smoke in mice. Inammation 38: 871-878, 2015.
65. Ng DS, Liao W, Tan WS, Chan TK, Loh XY and Wong WS:
Anti-malarial drug artesunate protects against cigarette
smoke-induced lung injury in mice. Phytomedicine 21:
1638 -16 4 4, 2014.
66. Ma WJ, Sun YH, Jiang JX, Dong XW, Zhou JY and Xie QM:
Epoxyeicosatrienoic acids attenuate cigarette smoke
extract-induced interleukin-8 production in bronchial epithelial
cells. Prostaglandins Leukot Essent Fatty Acids 94: 13-19, 2015.
67. Coskun M, Olsen J, Seidelin JB and Nielsen OH: MAP kinases in
inam matory bowel disease. Cli n Ch im Acta 412: 513-520, 2011.
68. Hoshino S, Yoshida M, Inoue K, Yano Y, Yanagita M, Mawatar i H,
Yamane H, Kijima T, Kumagai T, Osaki T, et al: Cigarette smoke
extract induces endothelial cell injury via JNK pathway. Biochem
Biophys Res Commun 329: 58-63, 2005.
69. Xu X, Balsiger R, Tyrrell J, Boyaka PN, Tarran R and
Cormet-Boyaka E: Cigarette smoke exposure reveals a novel role
for the MEK/ERK1/2 MAPK pathway in regulation of CFTR.
Biochim Biophys Acta 1850: 1224-1232, 2015.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9
70. Marumo S, Hoshino Y, Kiyokawa H, Tanabe N, Sato A, Ogawa E,
Muro S, Hirai T and Mishima M: p38 mitogen-activated protein
kinase determines the susceptibility to cigarette smoke-induced
emphysema in mice. BMC Pulm Med 14: 79, 2014.
71. Shen N, Gong T, Wang JD, Meng FL, Qiao L, Yang RL, Xue B,
Pan FY, Zhou XJ, Chen HQ, et al: Cigarette smoke-induced
pulmonary inflammatory responses are mediated by
EG R-1/G GPPS/ MA PK sig na l i n g. Am J Pa t h o l 178: 110-118, 2 011.
72. Metcalfe HJ, Lea S, Hughes D, Khalaf R, Abbott-Banner K and
Singh D: Effects of cigarette smoke on toll-like receptor (TLR)
activation of chronic obstructive pulmonary disease (COPD)
macrophages. Clin Exp Immunol 176: 461-472, 2014.
73. Zhao Y, Xu Y, Li Y, Xu W, Luo F, Wang B, Pang Y, Xiang Q,
Zhou J, Wang X, et al: NF-κB-mediated inammation leading to
EMT via miR-200c is involved in cell transformation induced by
cigarette smoke extract. Toxicol Sci 135: 265-276, 2013.
74. Edwards MR, Bartlett NW, Clarke D, Birrell M, Belvisi M and
Johnston SL: Targeting the NF-kappaB pathway in asthma and
chronic obstructive pulmonary disease. Pharmacol Ther 121:
1-13, 2009.
75. Akihisa T, Noto T, Takahashi A, Fujita Y, Banno N, Tokuda H,
Koike K, Suzuki T, Yasukawa K and Kimura Y: Melanogenesis
inhibitory, anti-inammatory, and chemopreventive effects of
limonoids from the seeds of Azadirachta indicia A. Juss. (neem).
J Oleo Sci 58: 581-594, 2009.
76. Faccin-Galhardi LC, Yamamoto KA, Ray S, Ray B,
Carvalho Linhares RE and Nozawa C: The in vitro antiviral
property of Azadirachta indica polysaccharides for poliovirus. J
Ethnopharmacol 142: 86-90, 2012.
77. Akihisa T, Takahashi A, Kikuchi T, Takagi M, Watanabe K,
Fukatsu M, Fujita Y, Banno N, Tokuda H and Yasukawa K: The
melanogenesis-inhibitory, anti-inammatory, and chemopreventive
effects of limonoids in n-hexane extract of Azadirachta indica A.
Juss. (neem) seeds. J Oleo Sci 60: 53-59, 2011.
78. Agyare C, Spiegler V, Sarkodie H, Asase A, Liebau E and
Hensel A: An ethnopharmacological survey and in vitro conr-
mation of the ethnopharmacological use of medicinal plants as
anthelmintic remedies in the Ashanti region, in the central part
of Ghana. J Ethnopharmacol 158: 255-263, 2014.
79. Quelemes PV, Perfeito ML, Guimarães MA, dos Santos RC,
Lima DF, Nascimento C, Silva MP, Soares MJ, Ropke CD,
Eaton P, et al: Effect of neem (Azadirachta indica A. Juss) leaf
extract on resistant Staphylococcus aureus biolm formation
and Schistosoma mansoni worms. J Ethnopharmacol 175:
287-294, 2015.
80. Sharma J, Gairola S, Sharma YP and Gaur RD: Ethnomedicinal
plants used to treat skin diseases by Tharu community of district
Udham Singh Nagar, Uttarakhand, India. J Ethnopharmacol 158:
140 -206, 2014.
81. Othman F, Motalleb G, Lam Tsuey Peng S, Rahmat A, Basri R
and Pei Pei C: Effect of neem leaf extract (Azadirachta indica)
on c-Myc oncogene expression in 4T1 breast cancer cells of
BALB/c mice. Cell J 14: 53-60, 2012.
82. Manikandan P, Anandan R and Nagini S: Evaluation of Azadi-
rachta indica leaf fractions for in vitro antioxidant potential
and protective effects against H2O2-induced oxidative damage
to pBR322 DNA and red blood cells. J Agric Food Chem 57:
6990-6996, 2009.
83. Sithisarn P, Supabphol R and Gritsanapan W: Comparison of
free radical scavenging activity of Siamese neem tree (Azadi-
rachta indica A. Juss var. siamensis Valeton) leaf extracts
prepared by different methods of extraction. Med Princ Pract 15:
219-222, 2006.