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

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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.
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 inlt 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.
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... TNF-α and IL-6 via various signaling cascades directly and indirectly. The study revealed that neem leaf extract modulates the release of these pro-inflammatory cytokines in bronchoalveolar lavage fluid [124] of Chronic obstructive pulmonary disease (COPD) [125] and therefore it could be promising targets for COVID-19 mediated pneumonia. Additionally, one of the active compounds known as nimbolide present in leaf extract also inhibits the cytokine storm and protects endotoxin-induced acute respiratory distress by interfering with toll-like receptor 4 and inhibiting the TNF-α mediated NF-κB pathway. ...
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Pandemic coronavirus disease-2019 (COVID-19) is an infectious disease caused by the newly discovered virus “Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2)”. Considering the present scenario of COVID-19 outbreak and its impact on humankind, holistic remedies with respect to herbal medicine validated from ethnopharmacological rationale are now targeting approaches globally as a preventive care against SARS-CoV-2. Aim: This review is primarily focused on to deliver a concise fact of the coronaviridae family, pathophysiology, mechanism of action, ethnopharmacological validated Indian herbs for inhibiting the virus with possible targets. Experimental procedure: In this study, science mapping tool Bibliometrix R-package was used to perform bibliometric analysis and building data matrices for keywords co-occurrence investigation, country-wise scientific production; collaboration between the countries worldwide, co-word analysis on topic “keywords associated with SARS-CoV-2 and medicinal plants”. Results and Conclusion: Our findings is to deliver a concise knowledge about the coronaviridae family, pathophysiology, possible targets for managing the SARS-CoV-2, in addition to potential medicinal plants and their phytoconstituents against COVID-19. Target-specific inflammatory pathways due to post infection of SARS e.g. NLRP3, p38-MAPK, Metallopeptidase Domain 17; endocytosis pathways e.g. Clathrin, HMGB1 pathways are primarily highlighted along with relevant interleukins and cytokines, which directly/indirectly triggering to immune system and play a significant role. Based on selective pathways and potential lead, the outcome of our elaborated study put forward selected Indian medicinal plants that hold a very high probability as preventive care in this global crisis.
... TNF-α and IL-6 via various signaling cascades directly and indirectly. The study revealed that neem leaf extract modulates the release of these pro-inflammatory cytokines in bronchoalveolar lavage fluid [124] of Chronic obstructive pulmonary disease (COPD) [125] and therefore it could be promising targets for COVID-19 mediated pneumonia. Additionally, one of the active compounds known as nimbolide present in leaf extract also inhibits the cytokine storm and protects endotoxin-induced acute respiratory distress by interfering with toll-like receptor 4 and inhibiting the TNF-α mediated NF-κB pathway. ...
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This article is primarily reviewed and delivered a concise fact of the coronaviridae family, pathophysiology, mechanism of action, ethnopharmacological validated Indian herbs for inhibiting the virus with possible targets. Science mapping tool Bibliometrix R-package was used to understand bibliometric analysis on keywords associated with SARS-CoV-2 and medicinal plants. The outcome of our elaborated study put forward that the number of medicinal plants such as Andrographis paniculata, Azadirachta indica, Curcuma longa, Glycyrrhiza glabra, Ocimum sanactum, Tinospora cordifolia and Withania somnifera etc and their derivative phytoconstituents act on various pathways e.g. NLRP3, p38-MAPK, Metallopeptidase Domain 17; endocytosis pathways, HMGB1 triggers immune system directly/ indirectly against SARS-CoV-2/COVID-19 and could be the potential candidates as preventive remedies.
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The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has been a healthcare disaster because of the unique and distinct characteristics of the pathogen, the easy and rapid transmission of the virus from humans to humans, the challenges in diagnosis and confirmation of the disease and the inability to invent and distribute safe and effective drugs or vaccines worldwide that would work against all the variants of coronavirus. Bangladesh, despite being a third-world country with limited health resources, has not been one of the worst-hit countries in the world but has still suffered with the loss of nearly eleven thousand people. Traditional and herbal remedies have become popular in this sub-continent since long ago and used for the treatment and management of different diseases including infectious disease. In this review, we have summarized the reports of immunostimulating, anti-inflammatory, antiviral, and respiratory distress syndrome improving activities of prospective indigenous plants of Bangladesh that may be recommended for use as complementary and alternative medicine or may be potential sources for the discovery and development of anti-COVID-19 medicaments. Thus, the review will be beneficial for the researchers, complementary and alternative medicines or herbal medicine manufacturers, formulators to find out and manage the potential herbal/nutraceutical/medicinal agents for the preparation of complementary and alternative medicines, as well as to the scientist for further research for the discovery and development of therapeutics/new drugs for the prevention and treatment of COVID-19 as well as other viral infections.
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Tuberculosis is a highly contagious infectious disease triggered by Mycobacterium tuberculosis, which is widely spread by aerosol. The major site of infection is usually the lungs however the disease can attack any extra-pulmonary site as well, which is further diagnosis by necrotizing granulomatous inflammation. World Health Organization reported almost 8.9–10 million people are suffering from tuberculosis in 2019, including 56% men and 32% women, and 12% children. Multidrug-resistant tuberculosis (MDR-TB) is a medical condition in which Mycobacterium tuberculosis strains resistant to at least isoniazid and rifampicin. In-vitro studies suggest that several bioactive compounds and their synthetic derivatives obtained from plants, fungi, and marine organism possesses antimycobacterial affinity. Phenolic compounds such as dihydrocubebin, hinokinin, ethoxycubebin possess the antimycobacterial activity. Mycobacterial cell envelope antagonists have been shown to obstruct the synthesis of mycolic acids, arabinogalactan, and peptidoglycan, essential components of the mycobacterial cell wall. The paramount antituberculous drugs hamper the development of mycolic acids or the aid mechanism which links them to the cell membrane. Medicines targeting RNA synthesis encompass those that restrict the assembly of bacterial DNA-dependent RNA polymerases, that are indispensable enzymes for RNA synthesis. Various molecular pathways for the target to cure tuberculosis entail the targets of M. tuberculosis cell wall synthesis, energy metabolism, folate metabolism, DNA replication, and RNA synthesis. Interestingly, in preserving the health of patients diagnosed with tuberculosis, medicinal plants have tremendous advantages with limited side effects as compare to the standard drugs.
Chapter
Pulmonary diseases have been identified as one of the major diseases that are responsible for higher level of mortality globally. The application of nanotechnology has been identified as a sustainable technology that could be utilized for successful delivery of biologically active constitutes available in the newly synthesized nanodrug. Several reports have also established the uses of nanocarriers in drug delivery to the lungs. The use of nanoparticle for drug delivery ensures systematic drug release and enhanced drug bioavailability. This increases drug effectiveness and reduces toxicity. Hence, this chapter intends to provide a comprehensive information on several medicinal plants that could be applied for the management of pulmonary diseases including chronic obstructive pulmonary disease, lung cancer, and lower respiratory tract infections. Detailed information were also provided on typical examples of nanodrug that could be applied for effective management of pulmonary diseases.
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Chung-pae (CP) inhalation therapy is a method frequently used in Korea to treat lung disease, especially chronic obstructive pulmonary disease (COPD). This study investigated the effects of CP inhalation on a COPD animal model. C57BL/6 mice received porcine pancreatic elastase (PPE) and lipopolysaccharide (LPS) alternately three times for 3 weeks to induce COPD. Then, CP (5 or 20 mg/kg) was administered every 2 h after the final LPS administration. The effect of CP was evaluated by bronchoalveolar lavage (BAL) fluid analysis, histological analysis of lung tissue, and reverse transcription polymerase chain reaction analysis of mRNA of interleukin- (IL-) 1β, tumor necrosis factor- (TNF-) α, IL-6, and tumor growth factor- (TGF-) β. Intratracheal CP administration reduced the number of leukocytes and neutrophils in BAL fluid, inhibited the histological appearance of lung damage, and decreased the mRNA levels of the proinflammatory cytokines IL-1β, TNF-α, IL-6, and TGF-β. Intratracheal CP administration effectively decreased the chronic inflammation and pathological changes in a PPE- and LPS-induced COPD mouse model. Therefore, we suggest that CP is a promising strategy for COPD.
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Objective: This study aimed to evaluate the therapeutic effects of potato extract (PE) on cigarette smoke (CS)-induced chronic obstructive pulmonary disease (COPD). Methods: PE was first prepared by frozen centrifugation, and its amino acid composition was detected. Toxicity of PE was analyzed by changes in morphology, behavior, routine blood indexes, and biochemical criteria of mice. Then, the COPD rat model was established by CS exposure, and PE, doxofylline, and prednisolone acetate were used to treat these rats. After 45 days of treatment, the morphology and behavior of rats were recorded. In addition, the histopathology of lung tissue was evaluated by chest x-ray and hematoxylin and eosin staining. The expression of interleukine-10 (IL-10), tumor necrosis factor-α (TNF-α), and granulocyte colony-stimulating factor (G-CSF) was detected in serum and lung tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Results: Various amino acids were identified in PE, and no toxicity was exhibited in mice. The CS-induced COPD rat model was successfully established, which exhibited significant thickened and disordered lung markings on 90% of the rats. After administering doxofylline and prednisolone acetate, inflammation symptoms were improved. However, side effects such as emaciation, weakness, and loosening of teeth appeared. In the PE group, obviously improved histopathology was observed in lung tissues. Meanwhile, it was revealed that PE could increase the expression of IL-10 and reduce the expression of TNF-α and G-CSF in COPD rats, and doxofylline and prednisolone acetate also elicited similar results. Conclusion: Our study suggests PE might be effective in the treatment of CS-induced COPD by inhibiting inflammation.
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Objective: Cigarette smoking is the main cause of chronic obstructive pulmonary disease (COPD) and may modulate the immune response of exposed individuals. Mast cell function can be altered by cigarette smoking, but the role of smoking in COPD remains poorly understood. The current study aimed to explore the role of cigarette smoke extract (CSE)-treated mast cells in COPD pathogenesis. Methods: Cytokine and chemokine expression as well as degranulation of bone marrow-derived mast cells (BMMCs) were detected in cells exposed to immunoglobulin E (IgE) and various doses of CSE. Adoptive transfer of CSE-treated BMMCs into C57BL/6J mice was performed, and macrophage infiltration and polarization were evaluated by fluorescence-activated cell sorting (FACS). Furthermore, a coculture system of BMMCs and macrophages was established to examine macrophage phenotype transition. The role of protease serine member S31 (Prss31) was also investigated in the co-culture system and in COPD mice. Results: CSE exposure suppressed cytokine expression and degranulation in BMMCs, but promoted the expressions of chemokines and Prss31. Adoptive transfer of CSE-treated BMMCs induced macrophage infiltration and M2 polarization in the mouse lung. Moreover, CSE-treated BMMCs triggered macrophage M2 polarization via Prss31 secretion. Recombinant Prss31 was shown to activate interleukin (IL)-13/IL-13Rα/Signal transducers and activators of transcription (Stat) 6 signaling in macrophages. Additionally, a positive correlation was found between Prss31 expression and the number of M2 macrophages in COPD mice. Conclusion: In conclusion, CSE-treated mast cells may induce macrophage infiltration and M2 polarization via Prss31 expression, and potentially contribute to COPD progression.
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The antler of Sika deer (Cervus nippon Temminck) has been used a natural medicine in Korea, China and Japan, and a monoacetyldiaglyceride (1-palmitoyl-2-linoleoyl-3-acetylglycerol, PLAG) was found in the antler of Sika deer as a constituent for immunomodulation. In this study, we investigated protective effects of EC-18 (a synthetic copy of PLAG) on inflammatory responses using a cigarette smoke with lipopolysaccharide (LPS)-induced airway inflammation model. Mice were exposed to cigarette smoke for 1 h per day for 3 days. Ten micrograms of LPS dissolved in 50 μL of PBS was administered intra nasally 1 h after the final cigarette smoke exposure. EC-18 was administered by oral gavage at doses of 30 and 60 mg/kg for 3 days. EC-18 significantly reduced the number of neutrophils, reactive oxygen species production, cytokines and elastase activity in bronchoalveolar lavage fluid (BALF) compared with the cigarette smoke and LPS induced mice. Histologically, EC-18 attenuated airway inflammation with a reduction in myeloperoxidase expression in lung tissue. Additionally, EC-18 inhibited the phosphorylation of NF-κB and IκB induced by cigarette smoke and LPS exposure. Our results show that EC-18 effectively suppresses neutrophilic inflammation induced by cigarette smoke and LPS exposure. In conclusion, this study suggests that EC-18 has therapeutic potential for the treatment of chronic obstructive pulmonary disease.
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The p66Shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction and might play a role in cigarette smoke (CS)-induced airway epithelial cell injury. CS can induce an excessive amount of reactive oxygen species (ROS) generation, which can cause mitochondrial depolarization and injury through the oxidative stress-mediated Serine36 phosphorylation of p66Shc. The excessive production of ROS can trigger an inflammatory response and mucin hypersecretion by enhancing the transcriptional activity of pro-inflammatory cytokines and mucin genes. Therefore, we speculate that p66Shc plays an essential role in airway epithelial cell injury and the process of mucin generation in CS-induced chronic inflammatory airway diseases. Our present study focuses on the role of p66Shc in ROS generation, and on the resulting mitochondrial dysfunction, inflammatory response and mucus hypersecretion in CS-stimulated human bronchial epithelial cells (16HBE). We found that CS disturbed the mitochondrial function by increasing the level of phosphorylated p66Shc in these cells and that the effects were significantly reduced by silencing p66Shc. Conversely, the ectopic overexpression of wild-type p66Shc enhanced these effects. We also found that high levels of ROS inhibited FOXO3a transcriptional activity, which led to NF-κB activation. Subsequently, activated NF-κB promoted pro-inflammatory cytokine production and mucin hypersecretion. Thus, manipulating p66Shc might offer a new therapeutic modality with which to treat chronic inflammatory airway diseases.
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Ethnopharmacological relevance: There are ethnopharmacological reports supporting the use of neem (Azadirachta indica A. Juss) leaf against bacterial and worm infections. However there is a lack of studies about its effect on bacterial biofilm formation and Schistosoma mansoni worms. This study reports the in vitro effects of neem leaf ethanolic extract (Neem EE) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and planktonic aggregation formation, and against S. mansoni worms. Materials and methods: Quantification of the Azadirachtin (AZA), thought to be one of their main compounds related to biological effects, was performed. The effect of sub-inhibitory concentrations of Neem EE on biofilm formation and planktonic aggregates of S. aureus was tested using the crystal violet dye method and atomic force microscopy (AFM) analysis, respectively. Changes in S. mansoni motor activity and death of worms were analyzed in vitro after exposition to the extract. Treated schistosomes were also examined using confocal laser scanning microscopy. Results: It was observed the presence of AZA in the extract (0.14±0.02mg/L). Testing Neem EE sub-inhibitory concentrations, a significant biofilm adherence inhibition from 62.5µg/mL for a sensitive S. aureus and 125µg/mL for two MRSA strains was observed. AFM images revealed that as the Neem EE concentration increases (from 250 to 1000µg/mL) decreased ability of a chosen MRSA strain to form large aggregates. In relation of anti-schistosoma assay, the extract caused 100% mortality of female worms at a concentration of 50µg/mL at 72h of incubation, while 300µg/mL at 24hours of incubation was required to achieve 100% mortality of male worms. The extract also caused significant motor activity reduction in S. mansoni. For instance, at 96h of incubation with 100µg/mL, 80% of the worms presented significant motor activity reduction. By the confocal microscopy analysis, the dorsal surface of the tegument of worms exposed to 300µg/mL (male) and 100µg/mL (female) of the extract showed severe morphological changes after 24h of treatment. Conclusions: Neem leaf ethanolic extract presented inhibitory effect on MRSA biofilm and planktonic aggregation formation, and anthelmintic activity against S. mansoni worms.
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Callicarpa japonica Thunb. (CJT) is traditionally used as an herbal remedy for the treatment of inflammatory diseases in Korea, China, and Japan. In this study, we evaluated the effects of Callicarpa japonica Thunb. (CJT) on the development of COPD using a Cigarette smoke (CS)-induced murine model and cigarette smoke condensate (CSC)-stimulated H292 cells, human pulmonary mucoepidermoid cell line. Callicarpa japonica Thunb. was isolated from the leaves and stem of C. japonica. The methanol extract profile was obtained by UPLC Q-TOF-MS analysis. In in vivo experiment, the mice received 1h of cigarette smoke for 10 days. Callicarpa japonica Thunb. was administered to mice by oral gavage 1h before cigarette smoke exposure for 10 days. In in vitro experiment, we evaluated the effect of Callicarpa japonica Thunb. on the expression of MUC5AC and proinflammatory cytokines in H292 cells stimulated with CSC. CJT treatment effectively suppressed the infiltration of neutrophils, and decreased the production of ROS and the activity of neutrophil elastase in the bronchoalveolar lavage fluid (BALF) induced by CS. CJT also significantly attenuated production of proinflammatory cytokines such as IL-6 and TNF-α in the BALF, and reduced the infiltration of inflammatory cells and the production of mucus in lung tissue induced by CS. In in vitro experiments, CJT decreased the expression of MUC5AC and proinflammatory cytokines in CSC-stimulated H292 cells. Furthermore, CJT attenuated the phosphorylation of ERK induced by CSC in H292 cells. Taken together, CJT effectively reduced the neutrophil airway inflammation and mucus secretion induced by CS in murine model, and inhibited the expression of MUC5AC in CSC-stimulated H292 human lung cell line. These findings suggest that CJT has a therapeutic potential for the treatment of COPD. Copyright © 2015. Published by Elsevier Ireland Ltd.
Article
Tobacco smoke (TS) has been shown to cause bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial pathophysiological process in cancer development. MAPK pathways play central roles in tumorigenesis including EMT process. Curcumin is a promising chemopreventive agent for several types of cancers. In the present study we investigated the effects of TS on MAPK pathway activation and EMT alterations in the bladder of mice, and the preventive effect of curcumin was further examined. Results showed that exposure of mice to TS for 12 weeks resulted in activation of ERK1/2, JNK, p38 and ERK5 MAPK pathways as well as AP-1 proteins in bladder. TS reduced mRNA and protein expression levels of epithelial markers E-cadherin and ZO-1, while mRNA and protein expression levels of the mesenchymal markers vimentin and N-cadherin were increased. Curcumin treatment effectively attenuated TS-triggered activation of ERK1/2, JNK and p38 MAPK pathways, AP-1 proteins and EMT alterations in bladder tissue. These results suggest the protective effects of curcumin in TS-induced MAPK activation and EMT, thus providing new insights into the chemoprevention of TS-associated bladder cancer.