Posttreatment with Aspirin-Triggered Lipoxin A4 Analog

Endotoxin causes multiple organ dysfunction, in-
cluding acute lung injury (ALI) or its severe form,
acute respiratory distress syndrome (1,2). ALI is char-
acterized by an extensive neutrophil influx into the
lungs, the expression of proinflammatory mediators,
and damage of the lung epithelium and endothelium,
which results in pulmonary edema and the deteriora-
tion of gas exchange (2,3).mediators that are ''switched on" in the resolution
phase of an inflammatory response and can function
as "braking signals" in inflammation (15,16). Acetyla-
tion of cyclooxygenase (COX) by aspirin results in the
biosynthesis of the ATL (17). Such ATL as well as
synthetic analogs of ATL resist enzymatic degrada-
tion, and thus have longer-lasting antiinflammatory
bioactivity than the native eicosanoid (18,19). Thus,
they are useful tools and offer leads for developing
novel therapeutic interventions.
A recent study (20) reported that daily treatment
with small-dose aspirin triggers the formation of ATL
in healthy individuals, which may account for aspi-
rin's antiinflammatory actions in vivo. Aspirin also
induces HO-1 expression on endothelial cells (EC) in a
COX-independent manner, which offers protection
against prooxidant insults (21). More recently, it was
reported that an aspirin-triggered lipoxin A4 stable
analog, 15-epi-16-parafluoro-phenoxy-lipoxin A4 (ATL),
was able to induce endothelial HO-1 expression (22).
METHODS
Animals
Thirty-six pathogen-free male C57BL/6 mice wei-
ghing 20–25 g (purchased from the Laboratory Ani-
mal Section, Tongji Medical College, Wuhan, China)
were used for this study. The mice were fed a standard
diet and water ad libitum. Our university's Animal
Care and Scientific Committee approved the experi-
mental protocol.
BALF Collection
BALF was harvested as previously described
(26,27). Approximately 3.0 mL of BALF was obtained
from each mouse. One-hundred microliters of BALF
was centrifuged for 5 min at 400g by using a cytospin
on a Superfrost/Plus microscopic slide, and BALF
cells were stained by the Diff -Quick method (Fisher,
Chicago, IL). The rest of the BALF was passed through
a 0.22- m pore-size filter and then used immediately
or stored at 80°C for measurement of TNF- concen-
tration by ELISA kit (R&D Systems, Minneapolis,
MN), and the total protein concentration in recovered
BALF was determined by using the BCA Protein
Assay Kit (Pierce, Rockford, IL).
Protein Extraction and Western Blotting
For Western blot analysis, the total proteins were
prepared from lung tissues as previously described
(1). Samples (50 g protein) were mixed with sample
buffer, separated by 12% SDS-PAGE and electroblot-
ted to a nitrocellulose membrane. The membrane was
blocked for1hat room temperature with blocking
solution [5% nonfat milk in Tris buffered saline with
Tween 20 (TBST)]. Blots were then incubated over-
night at 4°C with primary anti-HO-1 antibody (1:100
dilution; Santa Cruz Biotechnology, Santa Cruz, CA).
Then, the membrane was washed in 5% nonfat milk in
TBST and was incubated with a horseradish peroxi-
dase conjugated secondary antibody for1hat room
temperature. Immunoreactive proteins were visual-
ized with the use of enhanced chemiluminescence
detection (Amersham Biosciences, Piscataway, NJ)
and exposed to radiograph film (Fuji Hyperfilm).
RESULTS
Effects of ATL on LPS-Induced ALI
(Histopathologic Evaluation)
The sham-vehicle group had normal pulmonary
histology (Fig. 1A). In contrast, the lung tissues from
the LPS-vehicle group were significantly damaged,
with alveolar disarray and severe inflammatory cell
infiltration (Fig. 1B). All indicated that there was ALI
Treatment effect of ATL on LPS-Induced ALI
Furthermore, we investigated in vivo the effects of
ATL on the survival rate after LPS challenge. Thirty
mice were randomized into a LPS-vehicle group (n
15) and LPS-ATL group (n 15). The survival rate of
mice in each group was monitored for 72 h. The
survival rate in the ATL treatment group was mark-
edly increased when compared with that of mice
receiving LPS alone (80% versus 13% survival; P
0.001).
Hecho por:
Willson A Mendoza C.
C.I: 16.959.604
CRF
FUENTE:http://www.anesthesia-analgesia.org/content/104/2/369.full.pdf