Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2012, Article ID 410232, 9 pages
Responsesina Rat Modelof Smoke-InducedLungInflammation
Jing Bai,Shi-LinQiu,Xiao-NingZhong, Qiu-PingHuang,Zhi-YiHe,
Jian-QuanZhang, Guang-NanLiu,Mei-HuaLi,andJing-Min Deng
Department of Respiratory Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning 530000, China
Correspondence should be addressed to Xiao-Ning Zhong, email@example.com
Received 17 January 2012; Revised 1 April 2012; Accepted 1 April 2012
Academic Editor: Kazuhito Asano
Copyright © 2012 Jing Bai et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Heavy smoking can induce airway inflammation and emphysema. Macrolides can modulate inflammation and effector T-cell res-
ponse in the lungs. However, there is no information on whether erythromycin can modulate regulatory T-cell (Treg) response.
This study is aimed at examining the impact of erythromycin on Treg response in the lungs in a rat model of smoking-induced
emphysema. Male Wistar rats were exposed to normal air or cigarette smoking daily for 12 weeks and treated by gavage with
100mg/kg of erythromycin or saline daily beginning at the forth week for nine weeks. The lung inflammation and the numbers of
inflammatory infiltrates in bronchoalveolar lavage fluid (BALF) were characterized. The frequency, the number of Tregs, and
the levels of Foxp3 expression in the lungs and IL-8, IL-35, and TNF-α in BALF were determined by flow cytometry, RT-PCR and
in the BALF and lung damages but increased the numbers of CD4+Foxp3+Tregs and the levels of Foxp3 transcription in the lungs,
associated with the inhibition of smoking-induced inflammation in the lungs of rats.
most prevalent illnesses worldwide and is estimated as the
third leading causeof mortality in 2020 .COPD is charac-
terised by airflow limitation that is poorly reversible. The
pathogenesis of COPD is usually progressive and associated
with an abnormal inflammatory response in the lungs, par-
ticularly in response to noxious particles or gases, such as
cigarette smoke . Recently, COPD-associated inflamma-
tion is thought to be an autoimmune response induced
by smoking or pathogenic microbials that activate lympho-
cytes and antigen-presenting cells . Previous studies have
shown that Th1 cells are predominantly associated with the
development of emphysematous lungs, leading to the prog-
smoke is associated with Th1 immunity remain unclear [4–
CD4+CD25+Foxp3+regulatory T cells (Tregs) are crucial
regulators of the maintenance of peripheral immunologic
tolerance, and Tregs can suppress effectors Th1, Th2, and
Th17 responses, inflammation, and autoimmune responses
[8, 9]. Tregs can secrete IL-35, which inhibits inflammatory
ciated with the development of many Th1-mediated chronic
inflammation and autoimmune disorders, including type 1
diabetes, multiple sclerosis, atherosclerosis, and rheumatoid
arthritis [11–14]. Interestingly, decreased numbers of Tregs
were detected in the lungs of subjects with emphysema
, suggesting that Tregs participate in the regulation of
emphysema-related inflammation in the lungs. However,
little is known on what therapeutic strategies could increase
the number of Tregs and IL-35 responses in the lungs of
subjects with emphysema-related inflammation. Currently,
anti-inflammatory steroids have been often used for the
treatment of COPD patients with acute exacerbation, but the
therapeutic efficacy of steroids is limited [16, 17]. Therefore,
discovery of new therapeutic reagents will be of great sig-
nificance in the management of patients with COPD.
2Mediators of Inflammation
Erythromycin is a 14-membered ring macrolide antibi-
otic and has been prescribed for the treatment of various
respiratory infections. Erythromycin can inhibit mitogen-
stimulated human T-cell proliferation and cytokine produc-
tion, which are associated with inhibition of the MAPK and
NF-κB activation [18, 19]. Furthermore, erythromycin can
ameliorate chronic inflammation in various animal models
[20, 21]. In addition, long-term treatment with low doses
of a 14-membered ring macrolide is beneficial for patients
with airway inflammatory diseases, such as diffuse panbron-
chiolitis (DPB) , cystic fibrosis [23, 24], bronchiectasis
, and bronchial asthma [26, 27]. Our previous study has
ber of smoking-induced airway inflammatory infiltrates and
airway remodelling in the lungs of rodents . However,
little is known on whether treatment with erythromycin
could modulate Treg and IL-35 responses in the lungs.
In this study, we evaluated the impact of treatment with
erythromycin for nine weeks on cigarette smoking-induced
inflammation in a rat model of emphysema. Our findings
smoking-induced airway inflammation and emphysema but
also increased Treg infiltrates and IL-35 production in the
lungs of rats.
2.1. Animals and Treatments. Male Wistar rats at 12 weeks of
age were obtained from the Animal Research Center of
Guangxi Medical University. The animals were housed indi-
vidually in standard laboratory cages with free access to
standard food and tap water ad libitum. The experimental
protocols were established, according to the guidelines of
Research Care Committee of Guangxi Medical University.
Individual rats (n = 40) were exposed either to room
air (control) or to cigarette smoke, as described previously
. Briefly, groups of rats (n = 20 per group) were exposed
to tobacco smoke with 20 cigarettes (Nanning Jiatianxia
unfiltered cigarettes: 12mg of tar and 0.9mg of nicotine) in a
per week for 12 consecutive weeks. As a result, an optimal
ratio of smoking to air at 1:6 was obtained and the levels of
oxygen exposed by the rats were kept at a 21 ± 1%, which is
similar to atmospheric oxygen concentrations. The rats tol-
erated the cigarette smoke without evidence of toxicity (the
levels of serum carboxyhemoglobin in rats were at ∼10%,
and no weight loss in the rats was observed). The levels
of serum carboxyhemoglobin in the smoking rats (n = 20)
were 8.3 ±1.4%, as compared with 1.0 ±0.2% in the control
rats (n = 20), which were similar to the concentrations of
blood carboxyhemoglobin of human smokers .
Three weeks after exposure to cigarette smoke, the rats
were randomized and treated by gavage with 100mg/kg/d of
erythromycin (Meichuang Pharmaceuticals, Dailian, China)
in saline (1mL) or saline alone daily for nine weeks, respec-
tively. We used this dose based on our previous findings
to show that treatment with 100mg/kg/d of erythromycin
inhibits smoke-related lung inflammation without obvious
adverse effect . The rats that exposed to regular air were
randomized and treated with erythromycin or saline in the
same manner. Accordingly, there were four groups of rats
(n = 10 per group). The normal group of rats were exposed
to regular air and treated with saline (group N); the smoking
group of rats were exposed to smoking air for 12 weeks and
treated with saline (group S); the erythromycin group of
rats were exposed to smoking air for 12 weeks and treated
with erythromycin (group E); the control group of rats were
exposed to regular air and treated with erythromycin (group
One day after the last smoking, animals were injected
intraperitoneally with 20mg/kg pentobarbital and subjected
to a thoracotomy. Their left lungs were lavaged through an
intratracheal cannula three times with 2mL of cold saline,
and the bronchoalveolar lavage fluid (BALF) samples were
collected. The left lungs were used for the preparation of
single cell suspension. The lower lobes of their right lungs
were fixed in 10% formalin for pathological examination.
2.2. Histology. The fixed lower lobes of the right lungs were
embedded in paraffin, and the midsagittal sections of the
lungs were stained with hematoxylin and eosin (H&E), fol-
lowed by examining under a light microscope. Three non-
consecutive lung sections from each animal and three non-
overlapping random fields from each section were examined
for the quantification of lung damages. Alveolar airspace
enlargement was assessed by the mean linear intercept (MLI)
by two independent individuals in a blinded manner, as des-
cribed previously . Briefly, multiple digital images of
histological sections were systematically captured at 100 ×
magnification. Images were overlaid with a 10 × 10 grid
(1mm2), and the MLI was established from every second
image (i.e., in a checkerboard fashion, averaging six images
for each rat). The distribution of the MLI values of all the
digital photographs was assessed using frequency distribu-
tion analysis and characterized using a Gaussian model.
lected BALF samples from the left lung tissues were cen-
trifuged, and their supernatants were stored at −80◦C for
ELISA analysis. The pelleted cells were resuspended in PBS
and a portion of the cells (1 × 105cells) was subjected to
cytospin centrifugation on glass slides and fixed with me-
thanol, followed by staining with May-Gr¨ unwald-Giemsa
solution, and a differential cell count was performed under a
2.4. Measurement of IL-8, IL-35, and TNF-α in BALF. The
concentrations of IL-8, IL-35, and TNF-α in BALF were
measured with a multiplex-enzyme-linked immunosorbent
assay (ELISA) system, according to the manufacturers’ ins-
tructions (Lincoplex Systems, St Charles, MO, USA).
2.5. Lung Cell Preparation. A single-cell suspension of whole
left lung tissue was prepared by combined procedures of
Mediators of Inflammation3
mechanical fragmentation, enzymatic digestion, and centri-
fugation, as described in previous studies [5, 15]. The pre-
pared lung cells were used for flow cytometry analyses.
Briefly, lungs were flushed via the right ventricle with 10mL
of warm (37◦C) HBSS (calcium and magnesium free) con-
taining 5% fetal bovine serum (FBS, Sigma, Beijing, China),
100U/mL of penicillin, and 100μg/mL of streptomycin
(Gibco BRL). The lungs were then cut into small pieces
(∼2mm in diameter) and digested with 150U/mL of colla-
genase (Worthington Biochemical, Freehold, NJ, USA) in
HBSS with being shaken at 37◦C for 1h. Using a plunger
from a 5-mL syringe, the lung pieces were triturated
through a mess of 100μM into HBSS, and the resulting cell
suspension was filtered through nylon mesh. The cells were
washed twice, and mononuclear cells were isolated using
density centrifugation in 30% percoll (Pharmacia, Uppsala,
Sweden). The total numbers of cells were counted. The col-
lected leukocytes (1×106cells) were used for flow cytometry
analysis and the remaining cells were used for the extraction
of total RNA for RT-PCR analysis.
2.6. Flow Cytometry. The collected cells (1 × 106) from indi-
vidual rats were stained with PE-Cy5-conjugated anti-CD4
(clone: OX-35) or its isotype control (BD Pharmingen, San
and stained with PE-conjugated anti-Foxp3 (clone: FJK16s)
or its isotype control (eBioscience, Wembley, UK) at 4◦C for
another 40 minutes. The frequency and the number of Tregs
were determined by flow cytometry on a FACSCalibur (BD
PharMingen) and analysed by FCS Express software.
2.7. RNA Isolation and RT-PCR. Total RNA was extracted
from the lung cells of individual rats with TRIzol reagent,
according to the manufacturers’ instructions (Invitrogen,
Carlsbad, CA, USA). The quality and quantity of total RNA
were analysed by a spectrophotometer. The RNA samples
were reversely transcribed into cDNA using a reverse trans-
cription kit (Finn-zymes, Espoo, Finland) and oligo (dT)
primers. The relative levels of Foxp3 mRNA transcripts to
control β-actin in individual samples were characterized by
quantitative RT-PCR using SYBR Green on a LightCycler
(iCycler IQ, BioRad, USA) and the specific primers. The
sequences of primers were forward 5?-GGAGATTAC-
TGCCCTGGCTCCTA-3?, and reverse 5?-GACTCATCG-
TACTCCTGCTTGCTG-3?for β-actin and forward 5?-TGA-
GCTGGCTGCAATTCTGG-3?and reverse 5?-ATCTAGCTG-
CTCTGCATGAGGTGA-3?for Foxp3. The PCR amplifica-
tions were performed in triplicate at 95◦C for 30sec and
subjected to 40 cycles of 95◦C for 5sec and 60◦C for 30sec.
The values of Foxp3 mRNA transcripts in each sample were
normalized to that of β-actin and the relative levels of Foxp3
mRNA transcripts were calculated.
2.8. Statistical Analysis. Data are expressed as means ± SD.
Differences among groups were analysed using the analysis
of variance (ANOVA) and post hoc Student’s t-test, the
plicable using statistical package SPSS 11.0 (SPSS, Chicago,
IL, USA). The association between two variants was analyzed
using Spearman’s rank method. A P value of <0.05 was con-
sidered statistically significant.
3.1. Treatment with Erythromycin Reduces the Smoking-In-
duced Lung Damages in Rats. Following smoking for 12
weeks and treatment with erythromycin for 9 weeks, the
lung tissue sections of the different groups of rats were
stained with H&E and subjected to quantitative analysis of
the lung airspace (Figure 1). We observed the enlargement
of air spaces and many inflammatory infiltrates in the lungs
was no significant difference in the MLI values between the
N and C groups of rats. In contrast, the MLI values in the S
and E group of rats were significantly greater than that in
the N and C groups of rats (P < 0.05), demonstrating that
long-term heavy smoking-induced lung emphysema in rats.
Interestingly, the MLI values in the E groups of rats were
significantly less than that in the S group of rats although
they remained greater than that in controls. In addition,
treatment with erythromycin mitigated smoke-induced his-
vious observation . These data indicated that treatment
with erythromycin significantly diminished smoking-related
emphysema in the lungs of rats.
3.2. Treatment with Erythromycin Modulates the Smoking-
Induced Inflammatory Infiltrates in BALF. To quantify the
airway inflammation response, we evaluated the numbers
of inflammatory infiltrates in BALF and found significantly
increased numbers of total infiltrates, particularly macro-
phages, lymphocytes, and neutrophils in the BALF from the
smoking rats, as compared with that in the N and C groups
of rats (P < 0.05, Figure 2). In contrast, the total numbers
of inflammatory infiltrates, macrophages, lymphocytes, and
neutrophils in the BALF from the erythromycin-treated
smoking rats were reduced significantly, as compared with
those in the smoking rats without erythromycin treatment.
In addition, treatment with erythromycin did not cause
obvious adverse effect in rats, consistent with our previous
findings . These data demonstrated that treatment
with erythromycin significantly mitigated smoking-induced
inflammatory cell infiltration in the lungs of rats.
3.3. Treatment with Erythromycin Alters the Levels of TNF-α
and IL-8 in BALF. Analysis of the concentrations of TNF-
α and IL-8 in the BALF indicated that significantly higher
levels of TNF-α and IL-8 were detected in BLAF from the
smoking rats, as compared with that in the N and C groups
of rats (Figure 3). Furthermore, the levels of TNF-α and IL-
8 in BALF from the smoking rats that had been treated
with erythromycin were significantly lower than that in the
smoking rats without erythromycin treatment. Apparently,
proinflammatory cytokine production in the lungs.
4 Mediators of Inflammation
Figure 1: Treatment with erythromycin protects against the smoking-induced emphysema in rats. The lung tissue sections from different
groupsofratsweresubjected toH&Estaining,andthealveolar airspaceenlargement was assessedusingMLIbytwoindependentindividuals
in a blinded manner. Data are representative images or expressed as mean value ± SD of each group of rats (n = 10) from five separate
experiments. (a) Morphological changes in the lungs of rats (magnification × 100). (b) Quantitative analysis of alveolar airspace. Group N:
rats exposed to regular air without any special treatment; Group C: rats exposed to regular air and were treated with erythromycin; Group S:
rats exposed to smoking air and were treated with saline; Group E: rats exposed to smoking air and were treated with erythromycin daily for
nine weeks beginning at the forth weeks smoking.∗P < 0.05.
3.4. Treatment with Erythromycin Alters the Numbers of Tregs
in the Lungs of Rats. Flow cytometry analysis revealed that
the frequency and the number of Tregs in the lung paren-
chyma of smoking rats were significantly lower than that of
theNandCgroupsofcontrolrats(P < 0.01,Figure 4),while
the frequency and the number of Tregs in the erythromycin-
treated group of rats were higher than that of the S group
of rats (P < 0.05). A similar pattern of the relative levels of
of rats. Apparently, treatment with erythromycin mitigated
heavy smoking-induced reduction in the numbers of Tregs
in the lungs of rats.
3.5. Treatment with Erythromycin Alters the Levels of IL-35 in
BALF. IL-35 is an inhibitory cytokine and is predominantly
secreted by Tregs. Next, we determined the levels of IL-35 in
BALF from different groups of rats. The concentrations of
IL-35 in the BALF from the S group of rats were significantly
lower than that in the N and C groups of control rats
(Figure 5). Interestingly, the levels of IL-35 in the BALF from
E group of rats were similar to that in the N and C groups of
rats and were significantly higher than that in the S group of
rats. Apparently, treatment with erythromycin increased the
levels of IL-35 responses in the lungs of rats.
COPD and emphysema are common destructive inflamma-
tory diseases that are leading causes of mortality worldwide.
The smoking-induced emphysema is thought to be an auto-
immune disease and is mediated predominantly by Th1 res-
ponses in the lung . In this study, we employed a rat
model of smoking-related airway inflammation and emphy-
sema to test the therapeutic effect of treatment with ery-
thromycin and the potential mechanisms. Our data showed
that treatment with erythromycin significantly reduced
smoking-induced lung inflammation and damages, consis-
with erythromycin increased the numbers of Tregs, accom-
panied by increased levels of inhibitory IL-35 in the lungs of
rats. The increased levels of IL-35 may contribute to the inhi-
bition of erythromycin on smoking-related inflammation.
Our novel findings extend previous observations and suggest
that erythromycin may be valuable for the intervention of
Mediators of Inflammation5
Total cells in BALF (×105/mL)
Macrophages in BALF (×105/mL)
Lymphocytes in BALF (×105/mL)
Neutrophils in BALF (×105/mL)
Figure 2: Treatment with erythromycin reduces the numbers of inflammatory infiltrates in the lungs of rats. BALF samples were collected
from individual rats and the cells were stained with May-Gr¨ unwald-Giemsa. The numbers of total inflammatory infiltrates, macrophages,
samples and mean values (lines) for each group (n = 10). Group N: rats exposed to regular air without any special treatment; Group C: rats
exposed to regular air and were treated with erythromycin; Group S: rats exposed to smoking air and were treated with saline; Group E: rats
exposed to smoking air and were treated with erythromycin daily for nine weeks beginning at the forth weeks smoking.∗P < 0.05.
airway inflammation by upregulating Treg responses in pa-
tients with COPD in the clinic.
Macrolideantibiotics havebeen usedforthetreatmentof
lung inflammation in patients with COPD in the clinic .
Previous studies have shown that macrolides, especially for
erythromycin, can modulate immune responses and inhibit
with COPD by its anti-inflammatory activities. In this study,
we employed a well-known cigarette-smoking-inuced rat
erythromycin on the airway inflammation and lung dam-
ages. We detected high values of MLI, great numbers of
inflammatory infiltrates, and high levels of TNF-α and IL-8
in the lungs of smoking rats, demonstrating that heavy
lungs of rats. Furthermore, we found that treatment with
erythromycin mitigated the smoking-induced emphysema
and reduced the numbers of inflammatory infiltrates and
levels of TNF-α and IL-8 in the lungs of rats. Our data were
consistent with a previous report that treatment with clar-
ithromycin for six months decreases airspace enlargement in
the smoke-induced emphysema in mice . Our findings
Heavy smoking can modulate the function of antigen-
presenting cells, which may induce T-cell autoimmunity
against the lungs and Th1 immunity has been thought to be
related to the pathogenic process of COPD [15, 34]. Micro-
bials, such as erythromycin, can modulate T-cell responses
and inhibit airway inflammation [23, 27, 35]. Notably, Tregs
are potent regulators of T-cell autoimmunity and inflam-
mation and IL-35 is predominantly produced by Tregs and
contributes to regulatory T-cell function [8, 9]. We found
6 Mediators of Inflammation
IL-8 in BALF (pg/mL)
TNF-α in BALF (pg/mL)
Figure 3: Treatment with erythromycin decreases the levels of TNF-α, IL-8 in the lungs of rats. The levels of TNF-α and IL-8 in BALF of
individual rats were analyzed by ELISA. Data shown are mean values of individual samples fromthree separate experiments and mean values
for each group of rats (n = 10). Group N: rats exposed to regular air without any special treatment; Group C: rats exposed to regular air and
were treated with erythromycin; Group S: rats exposed to smoking air and were treated with saline; and Group E: rats exposed to smoking
air and were treated with erythromycin daily for nine weeks beginning at the forth weeks smoking.∗P < 0.05,∗∗P < 0.01.
that treatment with erythromycin enhanced Treg responses,
which may contribute to the inhibition of airway inflamma-
tion. Evidentially, in comparison with that in the smoking
rats, treatment with erythromycin significantly increased the
frequency and the numbers of Treg infiltrates in the lungs.
Furthermore, treatment with erythromycin upregulated the
levels of Foxp3 mRNA transcripts in the lungs. In addition,
treatment with erythromycin increased the levels of IL-35
in the BALF, given that Tregs can inhibit pathogenic T-cell
responses and IL-35 is crucial for the function of Tregs .
Although the increased Treg responses in the lungs by treat-
ment with erythromycin were moderate the significantly
reduced inflammation suggests that marginal effect of ery-
thromycin on increasing Treg response in the lung may be
sufficient in suppressing smoking-related inflammation. We
understand that our data did not demonstrate that the in-
creased Treg responses were responsible for the inhibition of
smoke-related lung inflammation. We are interested in fur-
ther investigation of whether adoptive transfer of Tregs or
inactivation of Tregscouldmodulate smoke-induced inflam-
change the effect of treatment with erythromycin on smoke-
induced lung damage in rats.
While there is clear evidence that treatment with macro-
lide antibiotics inhibits effector T-cell proliferation and cyto-
kine production there currently is little information on
how macrolide antibiotics modulate T-cell immunity. Ery-
thromycin may modulate the components of gut microbiota
and promote the development of Tregs. Indeed, the compo-
nents of gut microbiota are crucial for the development of
Tregs in rodents. Furthermore, a previous study has shown
that Roxithromycin inhibits chemokine-induced chemotaxis
of Th1 and Th2 cells but does not affect regulatory T-cell
migration . Erythromycin may act, like Roxithromycin,
and inhibit the migration of effector T cells, but not Tregs,
leading to relative increase in the numbers of Tregs in the
lungs of rats. In addition, erythromycin has been shown
to downregulate dendritic cell function and cytokine pro-
duction, particularly for LPS-stimulated dendritic cell mat-
uration and activation . However, treatment with ery-
thromycin does not affect peptidoglycan-induced dendritic
cell activation . It is possible that erythromycin may
modulate dendritic cell function toward to promoting Treg
development. Indeed, we found that treatment with ery-
thromycin upregulated Foxp3 transcription and IL-35 pro-
duction. Given that IL-35 has been shown to promote Treg
proliferation the increased levels of IL-35 may feedback en-
hance Treg responses in the lungs of rats. We are interested in
further investigating the mechanisms underlying the role of
erythromycin in regulating Treg responses.
In summary, treatment of COPD currently remains a sig-
nificant challenge, and pharmacological understanding of
drugs for the treatment of COPD is crucial for the control
of disease progression. Our data indicated that treatment
with erythromycin significantly reduced smoking-related
35 responses in the lungs of rats. Therefore, our findings may
provide new insights into understanding the pharmacologi-
cal action of erythromycin in the management of COPD in
Mediators of Inflammation7
The frequency of tregs
The number of tregs
The relative levels of Foxp3
Figure 4: Treatment with erythromycin modulates the frequency and the number of Treg and Foxp3 transcription in the lungs of rats. The
frequency of Tregs, the number of Tregs (b), and the relative levels of Foxp3 mRNA transcripts to β-actin in the lungs (c) were analyzed by
flow cytometry (a) and RT-PCR, respectively. The isolated lung cells were stained with anti-CD4 and anti-Foxp3 and subjected to flow cyto-
metry analysis. Data are expressed as mean numbers of individual samples and mean values (lines) of each group or the mean ± SD of the
relative levels of Foxp3 mRNA transcripts of each group (n = 10 per group) of rats from three separate experiments. Group N: rats exposed
to regular air without any special treatment; Group C: rats exposed to regular air and were treated with erythromycin; Group S: rats exposed
to smoking air and were treated with saline; and Group E: rats exposed to smoking air and were treated with erythromycin daily for nine
weeks beginning at the forth weeks smoking.∗P < 0.05,∗∗P < 0.01.
8Mediators of Inflammation
IL-35 in BALF (pg/mL)
Figure5:Treatmentwitherythromycin increases thelevels ofIL-35
in the lungs of rats. The levels of IL-35 in BALF of individual rats
were analyzed by ELISA. Data shown are mean values of individual
samples from three separate experiments and mean values (lines)
of each group of rats (n = 10). Group N: rats exposed to regular
air without any special treatment; Group C: rats exposed to regular
air and were treated with erythromycin; Group S: rats exposed to
to smoking air and were treated with erythromycin daily for nine
weeksbeginningattheforthweekssmoking.∗P < 0.05,∗∗P < 0.01.
The authors would like to thank Dr. Hui Chen (the Fifth
Affiliated Hospital of Guangxi Medical University) for her
excellent assistance in statistical analyses. This study was
supported by a grant from the National Nature Science
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