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From the Department of Anesthesiology, Second Affiliated Hospital, Wenzhou Medical College, Wenzhou, China; *Departments of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; and Departments of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Address correspondence to Sheng-Wei Jin, MD, PhD, Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhon University of Science and Technology, 1277 Jiefang Rd., Wuhan, Hubei Province, People’s Republic of China, 430022. Address e-mail to jinshengwei{at}sohu.com.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: All the animals were randomly assigned to one of six groups (n = 6 per group). In the sham-vehicle group, mice were treated with 0.9% saline 60 min after they were challenged with saline. The sham-ATL group was identical to the sham-vehicle group except that ATL (0.7 mg/kg, IV) was administered, and the sham-ZnPP group was identical to the sham-vehicle group except that Zinc protoporphyrin IX (ZnPP, 25 mg/kg IV) was administered. In the LPS-vehicle group, mice were treated with vehicle 60 min after they were challenged with LPS. The LPS-ATL group was identical to the LPS-vehicle group but received ATL. The ZnPP-ATL-LPS group was identical to the LPS-ATL group, but ZnPP was administered 30 min before ATL.
RESULTS: Inhalation of LPS increased inflammatory cell counts, tumor necrosis factor-
, and protein concentration in bronchoalveolar lavage fluid and also induced lung histological injury and edema. Posttreatment with ATL inhibited tumor necrosis factor-, nitric oxide, and malondialdehyde production, with the outcome of decreased pulmonary edema, lipid peroxidation, and the infiltration of neutrophils in lung tissues. In addition, ATL promoted the formation of heme oxygenase-1 in the lung tissues. Heme oxygenase-1 activity was also increased in the lung tissues after ATL stimulation. The beneficial effects of ATL were abolished by ZnPP.
CONCLUSIONS: This study demonstrates that posttreatment with ATL significantly reduces LPS-induced acute lung injury in mice.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism, which leads to the generation of biliverdin, free iron, and carbon monoxide (4–6). Three mammalian HO isoforms have been identified, one of which (HO-1) is a stress-responsive protein induced by various oxidative agents. HO-1 has been implicated in the cytoprotective defense response against oxidative injury (6). Studies have shown that HO-1 and its product, carbon monoxide, can reduce tissue edema, leukocyte adhesion and migration, and production of cytokines (7,8). The HO-1 pathway also mediates the antiinflammatory effect of interleukin (IL)-10 in mice (9). The expression of HO-1 has been shown to be increased in lipopolysaccharide (LPS)-induced ALI (10), and this up-regulated pulmonary expression of HO-1 is now considered an important part of the host antioxidative response during ALI (11–13).
Lipoxins (LX) and aspirin-triggered lipoxin A4 (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes that elicit distinct antiinflammatory and proresolution bioactions, including the inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, the repression of proinflammatory cytokine production, and the inhibition of cell proliferation and migration (14,15). These two series have emerged as founding members of the first class of lipid/chemical mediators that are ‘‘switched on" in the resolution phase of an inflammatory response and can function as "braking signals" in inflammation (15,16). Acetylation of cyclooxygenase (COX) by aspirin results in the biosynthesis of the ATL (17). Such ATL as well as synthetic analogs of ATL resist enzymatic degradation, 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 aspirin’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). ATLs and several synthetic ATL analogs have already been demonstrated to have impressive antiinflammatory activity in experimental renal ischemia reperfusion injury (23), zymosan A-induced peritonitis (19), allergen-mediated pulmonary inflammation (24), and dextran sodium sulfate-induced colitis (25). However, whether ATL can reduce pulmonary inflammation and lung injury induced by LPS and, if so, what the underlying mechanisms are, remain unclear.
In the present study, we examined the effects of a stable analog of aspirin-triggered ATL namely 15-epi-16-parafluoro-phenoxy-lipoxin A4, on LPS-induced ALI in mice. Additionally, to gain a better understanding of the mechanisms of action of ATL, we also investigated its effects on the protein expression and activity of HO-1, intrapulmonary nitric oxide (NO), malondialdehyde (MDA) and tumor necrosis factor (TNF)- production, and the immunohistochemical localization of HO-1 in the lungs. Finally, we investigated whether Zinc protoporphyrin IX (ZnPP), an inhibitor of HO, attenuates the protective effects of ATL.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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LPS-Induced ALI
All animals were anesthetized with pentobarbital (70 mg/kg ip). The induction of ALI in a mouse model by LPS instillation was described previously (26,27). Briefly, the mice were exposed to 0.3 mg of LPS (Escherichia coli serotype O55:B5, Sigma, St. Louis, MO) per mL in 0.9% saline or to 0.9% saline by aerosolization for 20 min under a laminar flow hood by using a flow rate of 2 L/min. At 8 h after inhalation of LPS or 0.9% saline, the mice were killed for collection of bronchoalveolar lavage fluid (BALF) and lungs, because dramatic differences in pulmonary inflammation were observed for this time (26). The mice were randomly allocated into the following groups. In the sham-vehicle group, the mice were treated with 0.9% saline (vehicle for ATL, 5 mL/kg, IV) 60 min after they were challenged with saline, (n = 6). The sham-ATL group was identical to the sham-vehicle group except that ATL (0.7 mg/kg, IV, 5-epi-16-parafluoro-phenoxy-Lipoxin A4, Berlex Inc., USA) was administered instead of vehicle (n = 6). The sham-ZnPP group was identical to the sham-vehicle group except that ZnPP (25 mg/kg IV, Porphyrin, Logan, UT) was administered instead of vehicle (n = 6). In the LPS-vehicle group, the mice were treated with vehicle 60 min after they were challenged with LPS (n = 6). The LPS-ATL group was identical to the LPS-vehicle group, but received ATL (0.7 mg/kg, IV) instead of vehicle (n = 6). The ZnPP-ATL-LPS group was identical to the LPS-ATL group, but ZnPP (25 mg/kg IV) was administered 30 min before ATL (n = 6). In our preliminary experiments, we found 0.1 mg/kg ATL did not affect the inflammatory response in mice challenged with LPS. However, 0.4 mg/kg ATL could inhibit the inflammatory response to LPS, and reached the maximal effect at 0.7 mg/kg (data not shown), so we chose 0.7 mg/kg ATL in our experiments. ATL was delivered as a bolus injection into the tail vein in 100 µL of 0.9% saline. During the experiment, the mice breathed spontaneously without any mechanical ventilatory support or supplemental oxygen.
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-µm pore-size filter and then used immediately or stored at –80°C for measurement of TNF- concentration 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).
Tissue Samples Collection
Mouse lungs were excised, and the right lung lower lobe was fixed in 10% (w/v) neutral buffered formalin (pH 7.0) until processing in paraffin wax. The rest of the lung was cut off, snap frozen in liquid nitrogen, and stored at –80°C until subsequent analysis.
Lung Morphology
Mouse lungs were fixed overnight at room temperature. The lungs were embedded in paraffin, and 5-µm sections were cut and stained with hematoxylin and eosin for histological analysis.
Lung Wet Weight to Dry Weight Ratio
To quantify the magnitude of pulmonary edema, we evaluated the wet weight to dry weight (W/D) ratio of the lung. Portions of the harvested wet left lungs were weighed, then placed in an oven for 24 h at 80°C, and weighed when dry. The W/D ratio was then calculated.
Measurement of Myeloperoxidase Activity
As an index of neutrophil infiltration, tissue-associated myeloperoxidase (MPO) activity was determined by a modification of the method of Gray et al. (28). Briefly, frozen lung tissues were thawed and homogenized in a phosphate buffer (20 mM, pH 7.4). After centrifugation at 30 000g for 30 min, the pellet was resuspended in another potassium phosphate buffer (50 mM, pH 6.0) with 0.5% hexadecyltrimethyl ammonium bromide. Samples were centrifuged at 20,000g for 15 min at 4°C, and supernatants were saved. MPO activity in the lungs was assayed by measuring absorbance changes spectrophotometrically at 460 nm, using 0.167 mg/mL O-dianisidine hydrochloride and 0.0005% hydrogen peroxide. Results were expressed as units MPO per gram of wet tissue.
Measurement of MDA
The MDA levels, an indicator of lipid peroxidation (29), were determined using a commercial kit (Jiancheng Bioengineering Institute, Nanjing, China). The principle of the method is the spectrophotometric measurement of the color generated by the reaction of thiobarbituric acid with MDA. For this purpose, frozen lung tissues were weighed and homogenized (1:10, w/v) in 0.1 M phosphate buffer (pH 7.4) in an ice bath. The homogenate was centrifuged at 3000g for 20 min at 4°C. MDA levels in the supernatants were measured strictly following the recommendations of the manufacturer. The protein content of the supernatant was determined by BCA Protein Assay Kit (Pierce) and the concentration of MDA was expressed as nanomoles per milligram of protein.
Measurement of Nitrite and Nitrate Concentration
Nitrite/nitrate production, an indicator of NO synthesis, was measured in the lung homogenates with a NO assay kit (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s instruction.
Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from tissue samples using Trizol reagent (Invitrogen, Life Technologies, NY) according to the manufacturer’s protocol. First-strand complementary DNA (cDNA) was synthesized using oligo-dT primer and the AMV RT (Promega, Madison, WI). The reaction was incubated at 42°C for 60 min in a thermocycler. The cDNA samples were then incubated at 85°C for 6 min to stop the reaction. For amplification of TNF- cDNA, the sequences of primers were 5'-GGC AGG TCT ACT TTG GAG TCA TTG C-3' (sense) and 5'-ACA TTC GAG GCT CCA GTG AAT TCG G-3' (antisense). The primers used for amplification of ß-actin cDNA as an internal standard were 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3' (sense) and 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3' (antisense). The PCR products of TNF- and ß-actin were 300 and 349 base pairs (bp) in length, respectively.
cDNA was amplified in 25-µL reactions. PCR reactions were initiated at 94°C for 5 min, followed by 34 cycles of amplification (denaturation at 94°C for 1 min, annealing at 58°C for 30 s, and extension at 72°C for 30 s) with a final primer extension at 72°C for 7 min. The PCR products were separated on 2% agarose gel and stained with ethidium bromide. The intensity of each TNF- mRNA band was quantified by densitometry using a gel documentation and analysis system (GDS8000, Ultra-Violet Products, Cambridge, UK) and normalized to values for ß-actin.
Protein Extraction and Western Blotting
For Western blot analysis, the total proteins were prepared from lung tissues as previously described (1). Samples (50 µg protein) were mixed with sample buffer, separated by 12% SDS-PAGE and electroblotted to a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature with blocking solution [5% nonfat milk in Tris buffered saline with Tween 20 (TBST)]. Blots were then incubated overnight 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 peroxidase conjugated secondary antibody for 1 h at room temperature. Immunoreactive proteins were visualized with the use of enhanced chemiluminescence detection (Amersham Biosciences, Piscataway, NJ) and exposed to radiograph film (Fuji Hyperfilm).
HO Activity Assay
The frozen sample of the right lung was homogenized in 4 mL of solution containing 50 mM Tris (pH 7.4), 250 mM sucrose and a mixture of proteases inhibitors. The homogenate was centrifuged for 20 min at 10,000g and at 4°C. The supernatant was used for measuring HO activity. Protein concentration in supernatants was determined by using the BCA Protein Assay Kit (Pierce). The reaction mixture consisted of 200 µL of lung supernatant, 50 µL of liver cytosol, 20 µL of 1 mM heme b solution, 200 µL of 2.75 mM ß-NADPH solution, and 530 µL of 2 mM MgCl2 100 mM phosphate buffer (pH 7.4). The samples were incubated in a 37°C water bath in the dark for 1 h. The reaction was stopped by placement on ice. An NADPH-free reaction mixture provided a baseline against which the measured concentrations were compared. The absorbency of the samples was measured by UV/Visible spectrophotometer Ultrospec 2000 (Pharmacia Biotech) at 464 nm and 530 nm. The amount of bilirubin formed was calculated from the difference in absorbance at 464 nm and 530 nm. The values are expressed as picomoles of bilirubin formed per milligram of protein per hour.
Immunohistochemical Localization of HO-1
Tissue sections were prepared from paraffin-embedded lung sample. After deparaffinization, endogenous peroxidase was quenched with 3% hydrogen peroxide for 30 min. The sections were permeablized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 5% (v/v) normal goat serum in PBS for 20 min. Immunohistochemical staining was performed using indirect streptavidin-peroxidase technique (SP kit, Zymed, CA). The sections were incubated overnight with rabbit polyclonal anti-HO-1 antibody (Santa Cruz Biotechnology; 1:100 dilution) and followed by incubation with a biotin-conjugated secondary antibody for 30 min. Sections then were stained with diaminobenzidine and examined under light microscope. Negative controls were performed using normal goat serum instead of primary antibody.
Statistical Analysis
Data were expressed as mean ± sd. The statistical analysis was performed using SPSS 11.0 programs (SPSS, Chicago, IL). All data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons. We considered P < 0.05 as statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of ATL on LPS-Induced Pulmonary Edema and Microvascular Permeability
The W/D ratio and BALF total protein concentration increased significantly in the LPS-vehicle group as compared with the sham-vehicle group (P < 0.05, Fig. 2). This increase was significantly (P < 0.05) reduced in the LPS-ATL group. The inhibitory effect of ATL was completely reversed by pretreatment with 25 mg/kg ZnPP. The treatment of mice with ATL or ZnPP alone did not modify the lung edema formation and microvascular permeability (Fig. 2).
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Effects of ATL on Neutrophil Infiltration in the Lungs
In order to measure neutrophil infiltration in response to LPS, the total number of white blood cells (WBCs) and neutrophils in BALF and lung MPO activity, an index of neutrophil sequestration, were determined. Inhalation of LPS increased the total WBC and neutrophil counts in the BALF and the lung MPO activity at 8 h. This increase in total WBC and neutrophil counts and MPO activity was significantly reduced in the LPS-ATL group (P < 0.05, Figs. 3A–3C). However, the inhibitory effects of ATL on these variables were abolished by the pretreatment with ZnPP (Figs. 3A–3C). ATL or ZnPP treatment alone did not cause significant changes in total WBC and neutrophil counts in the BALF and the lung MPO activity from the sham-treated mice (Figs. 3A–3C).
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Effects of ATL on Lipid Peroxidation and NO Production in the Lung
Lung MDA levels increased markedly in the LPS-vehicle group compared with the sham-vehicle group (Fig. 4B), whereas the increase was significantly attenuated in the LPS-ATL group, as shown in Figure 4B. The LPS-ATL group also had decreased pulmonary production of NO, as indicated by lower lung nitrite/nitrate levels (Fig. 4A). However, the inhibitory effects of ATL on these variables were abolished by the pretreatment with ZnPP (Figs. 4A and 4B). ATL or ZnPP treatment alone did not cause significant changes in MDA levels and NO production in lung tissues from sham-treated mice (Figs. 4A and 4B).
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Effects of ATL on LPS-Induced Pulmonary TNF-
The expression of TNF- mRNA in the lungs was found to be markedly enhanced in the LPS-vehicle group (Fig. 5A). The enhancement of TNF- mRNA expression was greatly attenuated in the LPS-ATL group (Fig. 5A). A similar observation was made regarding the TNF- protein content in the BALF (Fig. 5B). The inhibitory effects of ATL on TNF- mRNA and protein expression were significantly blocked by pretreatment with ZnPP (Figs. 5A and 5B).
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Effects of ATL on LPS-Induced Pulmonary HO-1
In the sham-vehicle group and Sham-ZnPP group, the lung expression of HO-1 protein was almost absent. However, the expression of HO-1 protein was found to be enhanced in the LPS-vehicle group and markedly enhanced in the LPS-ATL group, Sham-ATL group and ZnPP-ATL-LPS group (Fig. 6A). A similar observation was made regarding lung HO-1 activity, but ZnPP treatment significantly reduced lung HO activity (Fig. 6B). Immunohistochemical analysis of lung sections obtained from mice in the sham-vehicle group and LPS-vehicle group revealed weak staining for HO-1 (Figs. 7A and 7B), intense positive staining for HO-1 was observed in the LPS-ATL group, and the ZnPP-ATL-LPS group (Figs. 7C and 7D).
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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 markedly increased when compared with that of mice receiving LPS alone (80% versus 13% survival; P < 0.001).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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B suppressed neutrophils apoptosis in vivo, so it is likely to inhibit inflammatory resolution (33). Recently, resolution of acute inflammation was shown to be an active, rather than a passive, process endogenous chemical mediators or autacoids play key roles in controlling inflammation and its programmed resolution (34). Among them, LX and ATL evoke bioactions in a range of physiologic and pathophysiologic processes and serve as endogenous lipid/chemical mediators that stop neutrophilic infiltration and initiate resolution (14). Strategies that hasten the resolution of the illness may ultimately be as important as those that attenuate inflammatory lung injury (2). Specifically in ALI, resolution is characterized by clearance of polymorphonuclear neutrophils (PMN) from the lung, restoration of epithelial barrier function and vascular permeability, and gradual resolution of fibrosis (35). It has been suggested that LX/ATL are involved in the resolution of inflammation by promoting nonphlogistic phagocytosis of apoptotic PMN by macrophages in vitro (16,36) and in vivo (37). LX also promotes gradual resolution of fibrosis in lung (38). HO-1 is one of the original endogenous factors identified as playing a crucial role in the resolution of acute pleuritis (39), with current studies indicating that HO-1 is another mediator of resolution (32). Against this background, our purpose was to evaluate if ATL has protective action against LPS-induced ALI and the role of HO-1 when given therapeutically; that is, when the disease has already been induced, because it is more similar to the clinical situation.
Our data clearly demonstrated that a synthetic ATL exerts potent antiinflammatory effects in mouse lung after LPS challenge. Posttreatment with ATL inhibited TNF-, NO and MDA production, with the outcome of decreased pulmonary edema, lipid peroxidation and the infiltration of neutrophils in lung tissue. Furthermore, we demonstrated that posttreatment of mice with ATL up-regulates the HO-1 protein expression and activity. Finally, our data provided evidence that the inhibitor of HO (ZnPP) is able to abolish the beneficial effects of ATL in vivo, suggesting that the HO-1 play an essential role in the antiinflammatory and pro-resolution effects of ATL.
Neutrophils are key players in the pathogenesis of ALI, releasing lipid and enzyme mediators and oxygen radicals (2,40). In the current study, posttreatment of mice with ATL attenuated the LPS-induced increase of neutrophil counts in BALF and MPO activity in lung, suggesting an inhibition of neutrophil infiltration. Moreover, this inhibition was associated with the protective effects of ATL as evaluated by morphologic changes, W/D ratio, and BALF total protein concentration. Consistent with our findings, similar results have shown that LX A4/ATL and their metabolically stable analogs inhibit recruitment of PMNs and protect against neutrophil-mediated tissue injury by attenuating their chemotaxis, adhesion and transmigration across vascular EC and epithelial cells, and by reducing production of chemokines that direct PMN trafficking (41–43). LX/ATL is also involved in the resolution of inflammation by promoting nonphlogistic phagocytosis of apoptotic PMN by macrophages in vitro (16,36) and in vivo (37). These findings, therefore, suggest that ATL attenuates lung neutrophil infiltration and stimulates clearance of apoptotic neutrophils to lead to ameliorated lung injury.
Among many possible mediators for ALI, NO, reactive oxygen species, and proinflammatory cytokines have been implicated in the pathogenesis of sepsis-associated lung injury. Some evidence indicates that large amounts of NO produced by inducible NO synthase (iNOS) react rapidly with the superoxide radical to form peroxynitrite (ONOO–), which has been found in the early stages of endotoxin shock and is proposed to play an important role in the pathogenesis of sepsis-associated lung injury and acute respiratory distress syndrome (44). TNF- is an important mediator for sepsis and concomitant lung injury. In conjunction with IL-1ß, TNF- activates the inflammatory cascade by inciting the production of several cytokines and chemokines, and by enhancing endothelial adhesion molecules expression on vascular EC that promotes neutrophil adherence to these cells (45). To explore the underlying mechanisms by which ATL achieves its beneficial effects, we investigated its influence on pulmonary NO, MDA and TNF- production. We demonstrated here that posttreatment of mice with ATL markedly induced pulmonary HO-1 protein expression and activity. Moreover, posttreatment of mice with ATL markedly attenuated the LPS-induced increase in pulmonary TNF- and NO, MDA level and the administration of ZnPP blocked the effect. These findings, therefore, suggest that the activities of HO-1 play an important role in inhibiting of TNF-, NO, and MDA production, leading to a mitigation of neutrophilic lung inflammation.
Excessive NO release from the iNOS has been suggested to play a crucial role in the development of endotoxin-induced ALI (46). We have recently reported (1,47) that acute endotoxemia produces a large amount of NO through the upregulation of the expression for iNOS mRNA. We also found lung generation of ONOO–, which is detected as nitrotyrosine residues. It was reported that intracellular NO signal was markedly enhanced in LX A4/ATL-treated neutrophils (48). However, in our study, ATL decreased the production of NO in LPS-induced ALI. Thus, it depends on different cell level, animal models and the dose of LPS. Several studies have demonstrated that HO-1 inhibited iNOS expression in LPS-stimulated RAW264.7 macrophages (49) and HO-1 mediate the inhibitory effect of IL-10 on LPS-induced iNOS expression (9). We demonstrated here that ATL treatment prevents the production of NO and induces expression of HO-1 in the lungs of LPS-treated mice. We propose that the protective effect of ATL against lung damage may be, in part, due to the inhibition of iNOS expression through HO-1.
LX A4/ATL analogs have been reported to markedly attenuate ONOO– formation in human neutrophils (48). This was thought to inhibit the superoxide production, because LX A4/ATL analogs lead to accumulation of the polyisoprenoid presqualane diphosphate, which inhibits phospholipase D directly, and thereby blocks assembly of NADPH oxidase (50). Decreased superoxide production is consistent with a shift in the superoxide–NO ratio, resulting in decreased ONOO– formation. Here, we propose that ATL may also decrease ONOO– formation by markedly inducing the HO-1 protein expression and activity to scavenge superoxide and NO production. Reductions in ONOO– formation were associated with the attenuation of nuclear accumulation of transcription factors NF-B and AP-1 (48), which act in concert to induce IL-8, TNF- gene transcription. HO-1 also has been reported to suppress LPS-activated TNF- production in macrophages (9). These findings, therefore, suggest the essential role of HO-1 in the antiinflammatory function of ATL.
In summary, this study demonstrates that posttreatment with ATL, attenuates ALI induced by LPS in mice. In addition, we also demonstrate that ATL induces HO-1, HO antagonist, ZnPP, and counteracts the protective effect of ATL. Thus, the mechanism of the salutary effect of ATL is dependent on the HO-1. The expression of HO-1, in turn, results in a reduction of the production of NO, MDA, TNF- as well as neutrophil infiltration in the lung. Our findings reveal a novel mechanism for the antiinflammatory and pro-resolution activity of ATL. LX, ATL, and its analogs may provide new opportunities to design ‘‘resolution targeted" therapies with high degree of precision in controlling LPS-induced ALI.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supported by National Natural Science Foundation of China Grant Nos. 30200704, 30570726.
Reprints will not be available from the author.
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P < 0.05 vs. LPS-vehicle group; #P < 0.05 vs. LPS-ATL group.
