Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Wu Shangjie, Duan Shu, Zhao Shuiping, Cai Ying, Chen Ping, Fang, Fang Xiang

Description

Objective To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549). Methods A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E 2 (PGE 2 ) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively. Results LPS increased the expression of COX-2 mRNA and production of PGE 2 in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE 2 . There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE 2 (r = 0.947, P < 0.05). Conclusion Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE 2 in cultured A549 cells.

Sujets

• PTGS2
• Lipopolysaccharide
• Atorvastatin
• Prostaglandin E2

Informations

• Publié par: biomed
• Publié le: 01 janvier 2005
• Nombre de lectures: 9
• Langue: English

Extrait

BioMed CentralRespiratory Research
Open AccessResearch
Atorvastatin reduces lipopolysaccharide-induced expression of
cyclooxygenase-2 in human pulmonary epithelial cells
1 1 1 2 2ShangJie Wu* , Shu Duan , ShuiPing Zhao , Ying Cai , Ping Chen and
3Xiang Fang
1Address: Division of Cardiovascular Disease, Department of Internal Medicine, The Second Xiangya Hospital, Center Southern University,
2Changsha, Hunan, China, Division of Respiratory Disease, Department of Internal Medicine, The Second Xiangya Hospital, Center Southern
3University, Changsha, Hunan, China and Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA
Email: ShangJie Wu* - [e-mail]; Shu Duan - [e-mail]; ShuiPing Zhao - [e-mail];
Ying Cai - [e-mail]; Ping Chen - [e-mail]; Xiang Fang - [e-mail]
* Corresponding author
Published: 03 April 2005 Received: 20 November 2004
Accepted: 03 April 2005
Respiratory Research 2005, 6:27 doi:10.1186/1465-9921-6-27
This article is available from: http://respiratory-research.com/content/6/1/27
© 2005 Wu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cyclooxygenase-2LipopolysaccharideAtorvastatinProstaglandin E Human pulmonary epithelial cell
2
Abstract
Objective: To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in
human pulmonary epithelial cells (A549).
Methods: A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the
presence or absence of atorvastatin. After incubation, the medium was collected and the amount
of prostaglandin E (PGE ) was measured by enzyme-linked immunosorbent assay (ELISA). The cells2 2
were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot
respectively.
Results: LPS increased the expression of COX-2 mRNA and production of PGE in a dose- and2
time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited
by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced
production of PGE . There was a positive correlation between reduced of COX-2 mRNA and2
decreased of PGE (r = 0.947, P < 0.05).2
Conclusion: Atorvastatin down-regulates LPS-induced expression of the COX-2 and
consequently inhibits production of PGE in cultured A549 cells.2
thesis of pro-inflammatory PGs such as PGE . Increased1. Introduction 2
Human pulmonary epithelial cell is one of major sources expression of COX-2 and production of PGE2 have been
of productive inflammatory biomediators, such as pros- found in pulmonary inflammatory disorders [2].
taglandin E (PGE ), interleukin-6 (IL-6), in respiratory2 2
inflammatory diseases [1]. Cyclooxygenase-2 (COX-2) is Statins is a class of compounds that decreases cholesterol
an inducible enzyme that is expressed in response to synthesis via inhibition of 3-hydroxy-3methylglutaryl
inflammatory cytokines, and it is responsible for the syn- coenzyme A (HMG-CoA) reductase. Recently, anti-
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(page number not for citation purposes)Respiratory Research 2005, 6:27 http://respiratory-research.com/content/6/1/27
inflammatory effects of statins have been described [3]. which yielded products of 342 bp (50 sec at 55°C for
For example, Atorvastatin reduces expression of the COX- annealing of the primers, 35 cycles), and CYCLOPHILIN
2 in cultured vascular smooth muscle cells [4]. However, A: forward: 5'-ATG GTC AAC CCC ACC GTG TTC TTC G-
it is not clear whether Atorvastatin also affects COX-2 3' and reverse: 5'-CGT GTG AAG TCA CCA CCC TGA CAC
expression in human pulmonary epithelial cells. Because A-3', which yielded products of 206 bp (50 sec at 55°C for
of importance of COX-2 in inflammatory respiratory dis- annealing primers, 38 cycles). The DNA products from
eases, we tested the effects of Atorvastatin on lipopolysac- RT-PCR reactions were analyzed on a 4% polyacrylamide-
charide (LPS)-induced expression of COX-2 in cultured urea gel in the same buffer. The polyacrylamide gels were
human pulmonary epithelial cells. dried and scanned using the ImageQuant densitometer
(Gel Doc 2000, BioRad Co).
2. Methods
2.1 Materials 2.5 Western Blot analysis
Human pulmonary epithelial cell line (A549) was pur- After incubation, A549 cells were washed twice in phos-
chased from American Type Culture Collection (ATCC). phate-buffered saline, lysed in 200 µl lysis buffer (20 mM
Medium DMEM, trypsin, fetal bovine serum (FBS) and Tris/HCL, pH 7.5, 20 mM HECAMEG, 1 mM benzami-
LPS were purchased from Sigma-Aldrich. ECL chemilumi- dine). Protein content was determined by a microbicin-
nescence reagents, COX-2 polyclonal antibody were pur- choninic acid assay (Pierce) with bovine serum albumin
chased from Cayman Chemical Co. Anti-rabbit IgG, as standard. Western blot analysis was performed as
horseradish peroxidase linked whole antibody was described previously [6]. Briefly, the protein was sepa-
obtained from Amersham LIFE SCIENCE. HECAMEG was rated by electrophoresis on a 10% polyacrylamide gel at
from Vegatec (Villejuif, France). Trizol and electrophore- 180 V for 45 min. After transfer to nitrocellulose, the
sis reagents were from Promag Co. Atorvastatin was a gift membrane was blocked, incubated with a specific rabbit
from Beijing Honghui Medicine Co. polyclonal antibody against COX-2 (1:1000). The blots
were then incubated with a horseradish peroxidase-conju-
2.2 Cell culture gated donkey anti-rabbit antibody (1:5000). Antibody
A549 cells were grown in DEME medium supplemented labeling was detected by enhanced chemiluminescence.
with 5% FBS, 100 u/ml penicillin, 100 u/ml streptomycin, The films were scanned using an Arcus II Agfa scanner,
and 50 µg/L amphotericin B. Cells were sub-cultured into and densitometric analysis was performed using Sigma
six-well plates and maintained until sub-confluence. The Gel software.
medium was then replaced by a serum-free culture
2.6 Statistical analysismedium for 24 h prior to the addition of LPS and/or other
reagents. The cells were then incubated with various con- Statistical analysis was performed with SPSS analysis
centrations of LPS for 9 h, or 10 µg LPS for different times. (SPSS10.0 Software). PGE and RT-PCR data are presented2
For atorvastatin experiments, the cells were incubated in as mean ± S.D., and the differences between the multiple
the serum-free medium containing 10 µg LPS in the pres- treatment groups were analyzed by the one-way ANOVA,
ence or absence of different concentrations of atorvastatin LSD test. Data were correlated by nonparametric Spear-
for 9 h. man's rank method. Probability values of 0.05 or less were
considered to be statistically significant.
2.3 PGE assay2
After incubation, the medium was collected for measure- 3. Results
ment of PGE . PGE was determined by enzyme-linked 3.1 Dose- and time-dependent effects of LPS on COX-2 2 2
immunosorbent assays (ELISA, Shanghai Sun Biomedical mRNA expression and PGE2 production
Co. CV <10%). To determine the concentration dependent effect of LPS,
A549 cells were incubated with various concentrations of
2.4 RNA extraction, reverse transcription-polymerase LPS for 9 h. RT-PCR analysis indicated that LPS increased
chain reaction (RT-PCR) the expression of COX-2 mRNA in a concentration-
COX-2 mRNA was measured by RT-PCR as previously dependent manner (Figure 1A, top). Concentrations as
described [5]. Briefly, total RNA from different experimen- low as 5 µg/ml LPS were effective in inducing expression
tal conditions was obtained by Trizol method (Life tech- of the COX-2 mRNA. To determine the time-dependent
nologies) and the concentration of RNA was determined effect, A549 cells were incubated with LPS (10 µg/ml) for
by an absorbance at 260 nm. For RT-PCR, 100 ng of RNA different times. The expression of COX-2 mRNA was
from different experimental conditions was applied to the increased by LPS as early as 6 h, the earliest time point
access RT-PCR System. The following primers were used tested. It reached a maximum induction by 9 h of incuba-
for COX-2: forward: 5'-AAG CTG GGA AGC CTT CTC TA- tion, and remained stable for at least 12 h, the longest
3' and reverse: 5'-TTT CCA TCC TTG AAA AGG CGC-3', time tested (Figure 1B, middle). LPS also increased the
Page 2 of 6
(page number not for citation purposes)COX-2 OD /CYCL A OD
COX-2 OD /CYCL A OD
PGE (pg/ml)
2
PGE (pg/ml)
2
Respiratory Research 2005, 6:27 http://respiratory-research.com/content/6/1/27
A: * *
LPS 0 5 10 15( g/ml)
0.6
COX-2 (342bp) 0.4
CYCLOPHILIN
0.2
(206bp)
0.0
LPS 5 10 15 ( g/ml)
B:
LPS 10 g/ml * *
M c 69 12h 0.8
0.6
0.4
COX-2 (342bp)
CYCLOPHILIN 0.2
(206bp)
0.0
6h 9h 12h
LPS 10 g/ml
C:
* #
#*
60.0 60.0*
#
40.0 40.0
20.0 20.0
0.0 0.0
C 6h 9h 12hLPS 0 5 10 15 ( g/ml)
LPS 10 g/ml
A: Figure 1Dose-dependent effect of LPS on COX-2 mRNA expression
A: Dose-dependent effect of LPS on COX-2 mRNA expression. A5