Effects of lycopene on the model of oleic acid-induced acute lung injury

Giriş: Bu çalışmada, akciğer hasarı modelinde likopenin koruyucu etkisinin araştırılması amaçlandı. Materyal ve Metod: Çalışmaya 28 adet Wistar rat alındı. Kontrol grubuna (n= 7) serum fizyolojik + etanol (9/1) infüzyonu uygulandı. Oleik asit (OA) grubuna (n= 7), OA (100 mg/kg) tek doz intravenöz olarak uygulandı. Mısır yağı + OA grubuna (n= 7), beş hafta mısır yağı (1 mL/gün) gavajla verildi. Likopen + OA grubuna (n= 7), beş hafta likopen gavajla verildi ve beşinci haftanın sonunda OA (100 mg/kg) uygulandı. OA verildikten dört saat sonra kan ve akciğer doku örnekleri alındı. Malondialdehid, süperoksit dismutaz, glutatyon peroksidaz ve doku katalaz enzim aktivite düzeyleri ölçüldü. Bulgular: Kontrole göre OA ile mısır yağı + OA gruplarında artmış olan serum ve akciğer doku malondialdehid düzeyi, likopen + OA grubunda kontrol değeri düzeyinde idi (p< 0.05). Serum ve doku süperoksit dismutaz ve glutatyon peroksidaz enzim aktivitesi kontrole yakın değerler veya hafif artışlar görülürken, likopen + OA grubunda diğer gruplara göre belirgin artış mevcuttu (p< 0.05). Kontrol grubunun histopatolojik değerlendirmesi normalken, OA ve mısır yağı + OA gruplarında perivasküler, alveoler ödem, hemoraji, belirgin nötrofil infiltrasyonu, alveoler yapılarda destrüksiyon saptandı. Likopen + OA grubunda daha az nötrofilik infiltrasyon, perivasküler ve alveoler ödem izlendi. Sonuç: Likopenden zengin diyet akciğer hasarının önlenmesinde önemli role sahip olabilir. Anahtar Kelimeler: Akut akciğer hasarı, rat model, likopen.


INTRODUCTION
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), is a syndrome characterized by the acute onset, hypoxemia refractory to oxygen therapy, increased lung microvascular permeability and diffuse alveolar damage (1).ALI/ARDS in which lung injury and cellular elements (neutrophils, macrophages/monocytes, lymphocytes, platelets) and humoral components (complement system, cytokines, coagulation/fibrinolysis system, kinin system, lipid mediators, oxidants, proteases, nitric oxide, growth factors, neuropeptides) that arise as a result of activation of the system plays an important role in response mediator.ALI/ARDS in patients with early stage taken in the bronchoalveolar lavage (BAL) fluid increased neutrophils and neutrophil products have been identified.Neutrophiles cause endotelial and epitelial cell damage by excreting free radicals, inflammatory mediators, proteases (elastase, collagenase, reactive oxygen species), cytokines like tumor necrosis factor alfa (TNF-α) (2).Oxidant-mediated tissue injury of ALI/ARDS has an important role in pathogenesis.DNA, protein and lipid oxidation causes the formation of free radicals in lung injury are involved.The release of proteolytic enzymes and production of toxic oxygen radicals are the main factors in tissue damage.Plasma antioxidant levels decreased in patients with ARDS.Free oxygen radicals and neutrophile streams are thought to consume the total antioxidant capacity of lungs (3,4).
Oleic acid (OA)-induced model of lung injury is frequently used in investigation of novel ALI/ARDS treatment methods (5).ALI after the implementation of OA, decreased serum total antioxidant capacity (6).
Caratenoids found in fruits and vegetables, have protective effects against oxidative damages as well as activate enzymes which prevent oxidants harmful effects, stimulate the immune system, and cell proliferation (7).Lycopene the most important member of the carotene group, consisting of only carbon and hydrogen atoms contain carotenoids.Lycopene is a carotenoid which can not be synthesized but can be stored in human body (8).In a study, lycopene have been shown to reduce the risk of lung cancer when daily and regularly consumed (9).In experimentally induced gastric cancers, lycopene has been showed to decrease the lipid peroxidation by increasing the antioxidant capacity (10).
Many pharmacological agents in the treatment of ARDS have been tried but it has still a high mortality rate.If ALI, which is accepted as a beginning form of ARDS, may be prevented or may be treated, mortality rate will be decreased.In this OA induced lung injury model, we aimed to investigate the protective effects of lycopene in the oxidant-antioxidant systems.

Study Design
The local ethics committee of Firat University has approved the experimental protocol.Twenty eight female Wistar rats (140-160 g) were received from Experimental Research Unit of Firat University, Faculty of Medicine.Wistar rats were seperated into four groups as follows: Control group (n= 7): Animals of control group were fed in standard rat chow for five weeks, and applied PBS + ethanol (9/1) infusion on the last day of examination.
OA group (n= 7): ALI was performed by a single intravenous injection of 100 mg/kg OA (cis-9-octadecenoic acid; Sigma-Aldrich Germany) on the last day of examination after feeding in standard rat chow for five weeks.The suspension comprised 25 mg/mL pure OA suspended in ethanol, then 0.9% NaCL added to the suspension (ethanol/NaCl= 1/9) (6).
OA + corn oil group (n= 7): One mL of corn oil was given daily to this group by gavage for five weeks.At the end of the 5 th week, a single dose of 100 mg/kg OA was administered.
Lycopene + OA group (n= 7): Twenty mg/kg/day Lycopene (Lycopene 10% FS; Roche redivivo) in the corn oil was given by gavage to this group for five weeks.At the end of the 5 th week, a single dose of 100 mg/kg OA was administered.

Biochemical Analysis
Four hours after OA infusion, rats were decapitated under intramuscular 80 mg/kg ketamine anesthesia, according to the ethics guidelines, and blood samples were collected for biochemical analysis.Blood samples were centrifuged at 3500 rpm for 10 minutes, separated serum samples transferred to eppendorf tubes and stored at -80°C until analysis.Right lungs were removed, wrapped separately in aluminum foil, frozen in dry ice and stored at -80°C until the preparation of tissue homogenate samples for the measurement of malondialdehyde (MDA) levels and catalase (CAT), glutathione-peroxidase (GSH-Px), superoxide dismutase (SOD) activities.
Frozen lung tissues dissolved, washed with isotonic NaCl solution and dried in room temperature with adsorbent paper.Also the wet weights of tissues were determined.Tissues were kept cold and sliced into small pieces with a bistoury and transferred to the glass tubes. 2 mL cold Tris-HCl buffer solution (pH 7.4; 0.2 M Tris-HCl buffer) was added to the tissues and this buffer used for all studies.Tissues were homogenized in 16.000 rpm for two minutes using Ultra Turrax T25 Basic (Germany) Homogenizer.The homogenization completed to three minutes by adding 4 mL buffer else.A portion of the homogenates were vortexed and transferred to eppendorf tubes.The homogenates centrifuged at 3500 g for 45 minutes at +4°C for preparing the supernatants.

Measurement of MDA levels:
Serum MDA levels were measured by the thiobarbituric acid (TBA) method, which was modified from methods of Satoh and Yagi (11,12).Peroxidation was measured as the production of MDA, which in combination with TBA forms a pink chromogen compound whose absorbance was measured spectrophotmetrically at 532 nm.Serum MDA results were expressed as nmol/mL.Lung tissue MDA levels were analyzed by the method of Ohkawa and expressed as nmol/mg protein (13).
Measurement of SOD activity levels: SOD activity in lung tissue and serum samples was measured according to the method of Sun et al. and modification of Durak et al. by determining the reduction of nitro blue tetrazolium (NBT) by superoxide anion produced with xanthine/xanthine oxidase system (14,15).One unit for SOD activity was expressed as the amount of protein that causes 50% inhibition in NBT reduction rate.Results were defined as units per milligram protein (U/mg protein).
Measurement of GSH-Px activity levels: GSH-Px activity in lung tissue and serum samples were measured according to the method of Paglia and Valentine, by monitoring the oxidation of reduced nicotinamide adenine dinucleotid phosphate (NADPH) at 340 nm (16).Enzyme units were defined as the number of micromoles of NADPH oxidised per minute.Results were defined as units per milligram protein (U/mg protein).

Measurement of CAT activity levels:
CAT activity in lung tissue samples were determined according to the method of Aebi by measuring the decomposition of hydrogen peroxide at 240 nm (17).And results were expressed as rate constant per second per milligram protein (k/mg protein).

Histologic Analysis of the Lung
Left lungs were fixed in 10% formaldehyde.After embedding in paraffin, the tissues were cut into 3 µm sections and stained with hematoxylin-eosin methods and assessed by light microscope (Olympus BX-50, Japan).
Histological apperance of groups were graded as follows (18);

Statistical Analysis
Data are expressed as mean ± SE.For statistical analysis, the non-parametric Kruskal-Wallis test was used.
Comparisons between groups were performed using the Mann-Whitney Rank Sum test.p value < 0.05 denotes the presence of a significant statistical difference.

RESULTS
Serum MDA levels of OA group was statistically higher than control group (p< 0.05), when we compared lycopene + OA group with OA group, we saw that serum MDA levels was significantly lower in lycopene + OA group (p< 0.01).Tissue MDA levels in control and lycopene + OA groups was similar, levels of control and lycopene + OA group was significantly lower than cornoil + OA group (p< 0.05).
Serum SOD levels was significantly higher in lycopene + OA group than control group and OA group (for both p< 0.01).Tissue SOD levels of OA group was significantly higher than control group (p< 0.01).Moreover, lycopene + OA group levels was significantly higher than other three groups (p< 0.01, p< 0.05, p< 0.01, respectively).
When evaluated serum GSH-Px levels of groups, although lycopene + OA group and cornoil + OA group levels was higher than control group, no significant difference was seen between groups.Tissue GSH-Px levels of lycopene + OA group was significantly higher than other three groups (p< 0.01, p< 0.01, p< 0.05, respectively).
Serum and tissue levels of MDA, SOD, GSH-Px, and tissue levels of CAT of all groups is shown on Table 1.
Histopathological examination of lung tissue were as follows; control group had a normal appearance (Figure 1), OA, cornoil + OA groups had perivascular edema, alveolar edema, hemorrage, prominent neutrophil infiltration, and destruction in alveolar structure (Figure 2,3).Lycopene + OA group had less neutrophilic infiltration, perivascular and alveolar edema, and alveolar structure was prevented (Figure 4).

DISCUSSION
Histopathological changes in acute lung injury begins with increase in neutrophile leucocytes, and the formation of free oxygen radicals in lung cells.Lipid peroxidation is the main cause of the damage caused by free oxygen radicals in cells and tissues.Lipid peroxidation completed with converting of lipid peroxides to active aldehide and other carbonyl compounds.MDA, alcohol, etane, pentanes were some of the compounds pro-  Tissue CAT levels 0.08 ± 0.02* # 0.12 ± 0.01 † 0.12 ± 0.04 ‡ 0.20 ± 0.05 § (U/mg) duced at the end of the reaction.So, MDA is used as an indirect indicator of lipid peroxidation (19,20).
Plasma and BAL levels of MDA have been shown to increase significantly in experimental studies of acute lung injury (21,22).Septic lung injury models in rats showed the increase in lung tissue MDA levels which decreased after application of N-acetylsistein or methylene blue (18,23).Karahan et al. reported that lycopene decreased plasma and sera MDA levels that increased in experimental oxidative stress induced by cisplatin and gentamycine (24).Another study in which gastric carcinogenesis was induced by Nmethyl-N-nitro-N-nitrosoguanidin showed the increase in lipid peroxidation, and the investigators reported that lycopene decreased the lipid peroxidation products in blood (25).In an acute lung injury model in rats, Köksel et al. reported the increase in MDA levels in lung tissue, plasma and BAL, and decrease in these levels after applying an antioxidant, caffeic acid phenethyl ester (CAPE) (26).
In our study, higher sera and tissue MDA levels of group II and III proved the lung injury formation, and serum and tissue MDA levels of lycopene given group (group IV) even below the levels found in the control group to suggest that lycopene prevents lipid peroxidation.
It is known that in order to prevent the lipid peroxidation in ARDS patients, increase in total antioxidant capacity and decrease in glutation specific antioxidants occurs and this causes a spesific decrease in antioxidant defence of lung (27).Liu et al. determined a decrease in SOD enzyme activity at early stages of ARDS induced by oleic acide (28).In an other study conducted in ARDS patients, no changes were seen in SOD, GSH-Px enzyme activities but a little increase in CAT enzyme activities (3).In other studies carried out in ARDS patients and patients with sepsis found the increased serum levels of CAT, and SOD, decreased levels of glutation (29)(30)(31).It is also reported that decreases in GSH-     Px, SOD, and CAT enzyme activities in oxidative stress can be regulated by lycopene (25,32).
In our study, a little increase determined in tissue, and serum SOD, GSH-Px enzyme activities and tissue CAT activities in OA and corn oil + OA groups when compared with control group.However, significant increases were determined in lycopene + OA group when compared with other groups.Dose of lycopene intake in the studies shows heterogeneity.While 6.5 mg/day lycopene decreases lung cancer risk in nonsmoker women, the dose in nonsmoker men is 12 mg/day (9).30 mg lycopene per day is indicated for prevention of exercise-induced asthma (33).Although high levels of lycopene and other carotenoids in lungs provide an additional protection against to oxidative damage, no dose-dependent relationship between increased tomato consumption and reduced risk of lung cancer was found (34).Lower values of serum carotenoids were found in dead lung cancer patients and it is reported that carotenoid support slowed down the disease progression in lung cancer patients (35).The dose of lycopene as an antioxidant used in our study (20 mg/kg) may be accepted as an effective dose and there were no side effects reported for this dose.
Koksel et al. showed alveolar edema, congestion, neutrophil infiltration and damage in pulmonary structures in oleic acid lung injury model.These authors reported that pulmonary injury was decreased by giving antioxidant CAPE and NAC (6,26).In an experimental model of sepsis, Ozdulger et al. determined interstitiel edema, inflammatory cell infiltration and degeneration in pulmonary structure and also they reported edema, infiltration and pulmonary degeneration decreased with NAC therapy Liu and his colleagues (18,28) have created an ARDS model in rats with OA, and they reported that pulmonary interstitial edema and pulmonary hemorrhage were reduced when SOD was given before OA application.Gultekin and colleagues have created an experimental acute pancreatitis in rats and showed neutrophil infiltration, alveolar edema, enlargement and wall thickening in lungs when decreased by giving leptin (36).
In our study, we determined that corn oil + OA, and OA groups had intense neutrophil infiltration, prominent perivascular and alveolar edema, hemorrhage, and impaired alveolar structure consistent with the literature.Lycopene + OA group showed mild neutrophil infiltration, perivascular and alveolar edema, and preserved alveolar structure, suggests that lycopene may prevent the progression of lung damage.
As a result, lycopene rich diet has an important role in preventing damages in lungs that is open to oxidative stress, and we can say that extensive clinical studies will better explain the subject.