Biological Trace Element Research

, Volume 151, Issue 1, pp 50–58

Comparative Analysis of the Protective Effects of Caffeic Acid Phenethyl Ester (CAPE) on Pulmonary Contusion Lung Oxidative Stress and Serum Copper and Zinc Levels in Experimental Rat Model

Authors

    • Department of Thoracic SurgeryIstanbul Medeniyet University Medical School
    • Department of Thoracic SurgeryIstanbul Medeniyet University Medical School and Süreyyapaşa Göğüs Hastalıkları ve Göğüs Cerrahisi Eğitim ve Araştırma Hastanesi
  • Okan Solak
    • Department of Thoracic SurgeryAfyon Kocatepe University Medical School
  • Cagatay Tezel
    • Department of Thoracic SurgerySureyyapasa Chest Diseases and Thoracic Surgery Teaching Hospital
  • Rana Sırmalı
    • Department of BiochemistryDışkapı Yıldırım Beyazıd Research Hospital
  • Zeynep Ginis
    • Department of BiochemistryDışkapı Yıldırım Beyazıd Research Hospital
  • Dilek Atik
    • Department of Emergency MedicineDışkapı Yıldırım Beyazıd Research Hospital
  • Yetkin Agackıran
    • Department of PathologyAtaturk Chest Diseases and Thoracic Surgery Research Hospital
  • Halis Koylu
    • Department of PhysiologySüleyman Demirel University Medical School
  • Namık Delibas
    • Department of BiochemistryBozok University Medical School
Article

DOI: 10.1007/s12011-012-9505-7

Cite this article as:
Sırmalı, M., Solak, O., Tezel, C. et al. Biol Trace Elem Res (2013) 151: 50. doi:10.1007/s12011-012-9505-7

Abstract

The aim of this study was to investigate the effects of caffeic acid phenethyl ester (CAPE) in the lungs by biochemical and histopathological analyses in an experimental isolated lung contusion model. Eighty-one male Sprague–Dawley rats were used. The animals were divided randomly into four groups: group 1 (n = 9) was defined as without contusion and without CAPE injection. Group 2 (n = 9) was defined as CAPE 10 μmol/kg injection without lung contusion. Group 3 (n = 36) was defined as contusion without CAPE-administrated group which consisted of four subgroups that were created according to analysis between days 0, 1, 2, and 3. Group 4 (n = 27) was defined as CAPE 10 μmol/kg administrated after contusion group divided into three subgroups according to analysis on days 1, 2, and 3. CAPE 10 μmol/kg was injected intraperitoneally 30 min after trauma and on days 1 and 2. Blood samples were obtained to measure catalase (CAT) and superoxide dismutase (SOD) activities and level of malondialdehyde (MDA) and for blood gas analysis. Trace elements such as zinc and copper were measured in serum. The lung tissue was also removed for histopathological examination. Isolated lung contusion increased serum and tissue SOD and CAT activities and MDA levels (p < 0.05). Both serum and tissue SOD, MDA, and CAT levels on day 3 were lower in group 4 compared to group 3 (p < 0.05). Further, the levels of SOD, MDA, and CAT in group 4 were similar compared to group 1 (p > 0.05). CAPE also had a significant beneficial effect on blood gases (p < 0.05). Both serum zinc and copper levels were (p < 0.05) influenced by the administration of CAPE. Histopathological examination revealed lower scores in group 4 compared to group 3 (p < 0.05) and no significant differences compared to group 1 (p > 0.05). CAPE appears to be effective in protecting against severe oxidative stress and tissue damage caused by pulmonary contusion in an experimental setting. Therefore, we conclude that administration of CAPE may be used for a variety of conditions associated with pulmonary contusion. Clinical use of CAPE may have the advantage of prevention of pulmonary contusion.

Keywords

Caffeic acid phenethyl esterThoracic traumaPulmonary contusionOxidative stressCopperZinc

Introduction

Lung contusion affects 17–25 % of adult blunt trauma patients and is the leading cause of death from blunt thoracic injury. The pathophysiology of pulmonary contusion and blunt chest trauma includes inflammation, increased alveolocapillary permeability and pulmonary edema, ventilation/perfusion mismatching, increased intrapulmonary shunting, and a loss of compliance [1, 2]. Systemic inflammatory response is characterized by an increase in reactive oxygen species (ROS) [3].

Caffeic acid phenethyl ester (CAPE) is an antioxidant flavonoid. CAPE is the active component of the propolis purified from the hives of honeybees. It has antiviral, anti-inflammatory, antioxidant, and immunomodulatory properties [4, 5]. It has been shown that CAPE suppresses lipid peroxidation, inhibits lipoxygenase activities and tumor promotion, lipid peroxidation [6], lipoxygenase activities [7], protein tyrosine kinase [8], and ornithine decarboxylase [9]. It can completely block the in vitro production of ROS in human neutrophils and the xanthine/xanthine oxidase system at a concentration of 10 μM [10].

Various organs prevent the damaging effect of ROS by enzymatic and nonenzymatic antioxidant defense systems. The first line of defense that the body has against superoxide free radicals is the enzyme known as superoxide dismutase (SOD), which is considered to be the most effective antioxidant. SOD is of paramount importance for antioxidant system, protecting the cells, especially proteins synthesized in the human body from superoxide toxicity. In the process of removing superoxide free radicals, SOD rarely acts alone. It requires the enzyme called catalase (CAT) to remove hydrogen peroxide molecules, which are by-products of the reactions catalyzed by SOD. CAT not only removes hydrogen peroxide from our tissues, but also prevents cell damage via inhibition of formation of other, more toxic free radicals. Malondialdehyde (MDA) is a major end product of free radical reaction on membrane fatty acids. Although the cell is endowed with several antioxidant systems to limit the extent of lipid peroxidation, under certain conditions, protective mechanisms can be overwhelmed, leading to elevated tissue levels of peroxidation products.

Zinc (Zn) affects multiple aspects of the immune system [11, 12]. Zinc is essential for normal development and function of cell-mediating innate immunity, neutrophils, and natural killer cells. Macrophages are also affected by zinc deficiency. Phagocytosis, intracellular killing, and cytokine production are all affected by Zn deficiency. Zn deficiency adversely affects the secretion and functions of cytokines, the basic messengers of the immune system. Zn functions as an antioxidant and stabilizes membranes. Zn decreases ROS by several mechanisms. Zn is an inhibitor of NADPH oxidase, is required for SOD, and induces metallothionein, which is very effective in decreasing hydroxyl radical (·OH). ROS activates NF-κB, which in turn activates growth factors and antiapoptotic molecules, resulting in cancer cell proliferation. NF-κB activation also induces the generation of inflammatory cytokines and adhesion molecules. One mechanism by which Zn reduces inflammatory cytokine production involves the Zn-induced upregulation of a Zn finger protein, A20, which inhibits NF-κB activation via TRAF pathway. Zn, thus, not only functions as an antioxidant but also as an anti-inflammatory agent [11].

Copper (Cu) is an integral part of many important enzymes involved in a number of vital biological processes. Although normally bound to proteins, Cu may be released and become free to catalyze the formation of highly reactive hydroxyl radicals. Data obtained from in vitro and cell culture studies are largely supportive of Cu's capacity to initiate oxidative damage and interfere with important cellular events. Oxidative damage has been linked to chronic Cu overload and/or exposure to excessive Cu caused by accidents, occupational hazards, and environmental contamination. Additionally, Cu-induced oxidative damage has been implicated in disorders associated with abnormal Cu metabolism and neurodegenerative changes [13].

We herein developed an experimental rat model for isolated lung contusion that does not involve any significant associated injury to the heart and upper abdominal organs. To our knowledge, none of the studies could demonstrate both the direct and the indirect protective effects of CAPE on serum levels of essential elements and antioxidant enzyme activities after isolated lung contusion. This study was designed to examine the protective efficacy of CAPE on isolated lung contusion model by assessing the activity of CAPE on enzymatic and nonenzymatic antioxidant defense systems.

Material and Method

Animal Care and Lung Contusion Model

Eighty-one male Sprague–Dawley rats were used in the study. Rats weighing 276–312 g were housed in a temperature-controlled room (20–25 °C; 55–60 % humidity) on a 12-h light/dark cycle, fed a standard rat pellet food, and had ad libitum access to water. All rats were acclimatized for at least a week before the operation to allow them to adjust to the laboratory environment. The experiments were performed in accordance with “Guide for the Care and Use of Laboratory Animals, DHEW Publication No. (NIH) 85–23, 1985” and approved by the Ethical Committee of Ankara Numune Training and Research Center.

Groups

Rats were randomly divided into four groups as follows:
  • Group 1 (n = 9): without contusion and without CAPE injection

  • Group 2 (n = 36): intraperitoneal injection of CAPE (10 μmol/kg) without lung contusion

  • Group 3 (n = 27): contusion without CAPE injection; This group was further divided into four subgroups according to the time of analysis on days 0, 1, 2, and 3.

  • Group 4 (n = 9): CAPE (10 μmol/kg) administrated 30 min after contusion; This group was further divided into three subgroups according to the time of analysis on days 1, 2, and 3. CAPE (10 μmol/kg) was administrated intraperitoneally on days 1 and 2.

Anesthesia and Trauma Model

Animals were anesthetized with intramuscular injection of 100/10 mg/kg of ketamine/xylazine. Trauma model: Rats fasted for 12 h before the process. An apparatus was constructed, which comprised of a pipe through which a specific weight was allowed to free fall and a stand to prevent the impact of weight to dissipate to the head and abdominal space of the rat. This also allowed a space around the pipe to protect the sternum and heart. Energy of the impact (E) was calculated by the formula E = mgh where m is the mass (kilogram), g is the gravitational acceleration (9.8 m/s2), and h is the height where the weight is dropped (meter friction force was ignored). Blunt thoracic trauma of 1.78 J force was applied to rats, except groups 1 and 2 [14].

Biochemical Analysis

Under a semi-sterile condition, midsternotomy was performed in all animals at the end of the procedure. The procedure was performed 30 min after CAPE administration in group 4. Five-milliliter blood sample was drawn from the ascending aorta for biochemical assays. Blood samples were centrifuged at 1,500×g for 15 min and serum was separated. Serum samples were stored in a freezer at −20 °C and lung tissues were stored in a freezer at −80 °C until biochemical analysis. Samples were studied for 2 weeks. Determinations of SOD and CAT enzyme activities and MDA levels were performed on samples of the right lungs of the rats in the four experimental groups.

Determination of Catalase Activity

The tissue was homogenized (Qiagen, Switzerland) at 16,000 rpm on ice in 5–10 ml cold buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA) per gram tissue. It was centrifuged at 10,000×g for 15 min at 4 °C. The supernatant was removed for assay and stored on ice. It was stored at −80 °C until the time of analysis. CAT level was measured in the supernatant and serum. CAT activity was determined using a commercial CAT assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). Results were expressed as nanomole per minute per milligram protein wet tissue for lung tissue and nanomole per minute per milliter for serum.

The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. Purpald specifically forms a bicyclic heterocycle with aldehydes, which, upon oxidation, changes from colorless to a purple color. The catalase activity is measured at 540 nm using a plate reader (EPOCH, ABD).

Determination of Superoxide Dismutase Activity

The lung tissue was homogenized (Qiagen, Switzerland) at 16,000 rpm on ice in 5–10 ml cold buffer 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose per gram tissue. It was centrifuged at 1,500×g for 5 min at 4 °C. The supernatant was removed for assay and stored on ice. It was stored at −80 °C until the time of analysis. SOD activity was measured in the supernatant and serum by a commercial SOD assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). The results were expressed as units per milligram protein wet tissue for lung tissue and units per milliliter for serum.

SOD assay kit utilizes tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The reactions were initiated by adding xanthine oxidase, incubating for 20 min at room temperature, and reading the absorbance at 440 nm using a plate reader (EPOCH, ABD).

Determination of Malondialdehyde Level

The tissue was homogenized (Qiagen, Switzerland) at 16,000 rpm on ice in 1 ml of PBS, pH 7.4. The homogenate was centrifuged at 5,000×g for 5 min at 4 °C. The supernatant was removed for assay and it was stored at −80 °C until the time of analysis. MDA levels were determined using a commercial ELISA kit (Cusabio Biotech Co., People's Republic of China). MDA level was measured in the supernatant and serum. Results were expressed as picomoles per milligram protein wet tissue for lung tissue and picomoles per milliliter for serum.

This assay employs the quantitative sandwich enzyme immunoassay technique. The enzyme–substrate reaction is terminated by the addition of a sulfuric acid solution and color change is measured at 450 nm using a plate reader (EPOCH, ABD).

Blood Gas Analysis

Blood pH, pO2, pCO2, and HCO3 levels were determined on Siemens–Advia Rapid Lab. 1200 blood gas analyzer.

Protein Measurements

Protein measurements were made in homogenate and supernatant according to the method explained elsewhere by Lowry et al. [15].

Measurement of Serum Zinc and Copper Levels

Blood samples were centrifuged at 2,465×g for 10 min and the serum samples were aliquoted and were stored at −20 °C for serum zinc and copper measurements. Before the analyses, the stored samples were taken into room temperature and diluted with deionized water (1:10). The serum levels of zinc and copper were analyzed by atomic absorption spectrophotometer (Shimadzu-AA6501F, Japan) at 213,9 and 324,8 nm, respectively. Calibration was made using rat serum-based standards with known Zn and Cu concentration. Quality assurance was ensured using certified reference serum as a quality control.

Histopathological Analysis

Lung tissues were fixed in 10 % formalin, dehydrated in graded concentrations of ethanol, cleared in xylene, and embedded in paraffin. At least eight tissue sections in 5-μm thickness were obtained; they were then stained with hematoxylin–eosin and examined by a pathologist in a blinded manner. All histopathological changes were detailed in each lung tissue, including intra-alveolar hemorrhage, alveolar edema, disruption and congestion, and leukocyte infiltration. Alveolar edema and congestion were scored on a scale from 0 to 3 where 0 = absence of pathology (<5 % of maximum pathology), 1 = mild (<10 %), 2 = moderate (15–20 %), and 3 = severe (20–25 %). Leukocyte infiltration was evaluated to determine the severity of inflammation that resulted from contusion. Each section was divided into ten subsections, and leukocytic infiltration was examined in each of subsections at a magnification of ×400 with the following scale: 0, no extravascular leukocytes; 1, <10 leukocytes; 2, 10–45 leukocytes; and 3, >45 leukocytes. An average of the numbers was used for comparison [16].

Statistical Analysis

Data were analyzed by SPSS 15.0, which is a commercially available statistical software package. Distributions of the groups were analyzed with one sample Kolmogorov–Smirnov test. Biochemical results showed normal distribution and one-way ANOVA test was performed and post hoc multiple comparisons were done with LSD. Histopathological results were analyzed by Kruskal–Wallis and Mann–Whitney U tests. Results were presented as mean ± SD. P values less than 0.05 were regarded as statistically significant.

Results

We found that on day 0, isolated lung contusion resulted in elevated serum and tissue SOD, CAT activities, and MDA level (p < 0.05) in group 3 when compared to group 1. There were no differences in serum or tissue SOD, CAT activities, and MDA level between groups 1 and 2 (p > 0.05). On day 1, no difference was detected in serum or tissue SOD, CAT activities, and MDA level between groups 3 and 4 (p > 0.05). However, serum and tissue SOD, CAT activities, and MDA level were elevated (p < 0.05) in groups 3 and 4 compared to group 1. On day 2 of the experiment, serum and tissue SOD, CAT activities, and MDA level were increased (p < 0.05) in group 3 when compared to group 4. Further, levels of serum SOD, CAT activities, and MDA level in group 4 were higher (p < 0.05) than group 1. However, no significant differences were found between groups 4 and 1 in levels of tissue MDA level and SOD, CAT activities (p > 0.05). On the following day, same results in serum and tissue SOD, CAT activities, and MDA level between groups 3 and 4 were obtained. In contrast to the previous day, no significant difference was detected in serum and tissue SOD, CAT activities, and MDA levels between groups 4 and 1 (p > 0.05; Table 1).
Table 1

Tissue and serum malondialdehyde (MDA) levels, superoxide dismutase (SOD), and catalase (CAT) activities in pulmonary contusion injury in rats (n = 9 and mean ± SD)

 

MDA (tissue) (pmol/mg protein)

SOD (tissue) (U/mg protein)

CAT (tissue) (nmol/min/mg protein)

MDA (serum) (pmol/ml)

SOD (serum) (U/ml)

CAT (serum) (nmol/min/ml)

Group 1

243 ± 19

161 ± 12

63 ± 11

88 ± 12

47 ± 13

31 ± 11

Group 2

249 ± 11

159 ± 14

69 ± 13

85 ± 11

45 ± 12

33 ± 14

Group 3

Day 0

723 ± 12

414 ± 19

308 ± 15

302 ± 24

286 ± 21

215 ± 16

Day 1

580 ± 22

418 ± 21

273 ± 32

294 ± 16

241 ± 19

204 ± 19

Day 2

556 ± 16

374 ± 18

214 ± 19

241 ± 21

201 ± 23

176 ± 24

Day 3

481 ± 23

349 ± 13

187 ± 21

249 ± 19

185 ± 17

172 ± 16

Group 4

Day 1

491 ± 23

396 ± 18

249 ± 23

281 ± 19

219 ± 24

193 ± 21

Day 2

284 ± 15

185 ± 26

79 ± 12

163 ± 16

103 ± 12

124 ± 11

Day 3

258 ± 17

172 ± 21

72 ± 13

92 ± 13

53 ± 13

39 ± 12

p values

Day 0, groups 1–2

n.s

n.s

n.s

n.s

n.s

n.s

Day 0, groups 3–1

0.0001

0.0001

0.0001

0.0001

0.0001

0.0001

Day 1, groups 3–4

n.s

n.s

n.s

n.s

n.s

n.s

Day 1, groups 4–1

0.004

0.003

0.004

0.004

0.002

0.003

Day 2, groups 3–4

0.003

0.002

0.001

0.001

0.003

0.001

Day 2, groups 4–1

n.s

n.s

n.s

0.004

0.004

0.003

Day 3, groups 3–4

0.001

0.001

0.002

0.001

0.002

0.001

Day 3, groups 4–1

n.s

n.s

n.s

n.s

n.s

n.s

No significant differences were found in blood gas analyses including pH, pO2, pCO2, and HCO3 values between groups 1 and 2 (p > 0.05). On day 0, pH, pO2, and HCO3 were lower and pCO2 was higher in group 3 than group 1, and difference was significant (p < 0.05). On day 1, no difference was found between groups 3 and 4 though the pH, pO2, and HCO3 levels were decreased and pCO2 was increased in both groups 3 and 4 in comparison to group 1 (p < 0.05). The next day, significant difference was observed between groups 3 and 4 (p < 0.05). On day 3, no difference was detected between groups 4 and 1 (p > 0.05; Table 2).
Table 2

Mean values and standard deviations of blood pH, pO2, pCO2, and HCO3 in pulmonary contusion injury in rats (n = 9 and mean ± SD)

 

pH

pO2

pCO2

HCO3

Group 1

7.3 ± 0.16

84.8 ± 1.6

32.3 ± 6.2

21.8 ± 2.6

Group 2

7.3 ± 0.18

84.6 ± 5.3

33.8 ± 2.1

21.3 ± 1.7

Group 3

Day 0

6.9 ± 0.03

46.4 ± 1.8

54.2 ± 4.3

15.4 ± 0.2

Day 1

7.0 ± 0.09

49.6 ± 2.1

51.5 ± 2.1

16.3 ± 2.1

Day 2

7.0 ± 0.13

50.1 ± 3.2

52.7 ± 6.5

16.1 ± 7.4

Day 3

7.1 ± 0.18

54.6 ± 0.7

45.1 ± 9.7

17.2 ± 4.8

Group 4

Day 1

7.0 ± 0.19

54.8 ± 1.3

51.3 ± 0.9

17.1 ± 2.1

Day 2

7.2 ± 0.06

68.8 ± 0.9

42.8 ± 3.8

19.2 ± 5.3

Day 3

7.3 ± 0.01

82.8 ± 0.9

31.9 ± 2.1

20.5 ± 3.6

p values

Day 0, groups 1–2

n.s

n.s

n.s

n.s

Day 0, groups 3–1

0.0001

0.0001

0.0001

0.0001

Day 1, groups 3–4

n.s

n.s

n.s

n.s

Day 1, groups 4–1

0.004

0.0001

0.0001

0.0001

Day 2, groups 3–4

0.003

0.0001

0.001

0.0001

Day 2, groups 4–1

n.s

n.s

0.009

0.001

Day 3, groups 3–4

0.001

0.003

0.001

0.001

Day 3, groups 4–1

n.s

n.s

n.s

n.s

Serum Zn and Cu levels were comparable in groups 1 and 2. Descriptive statistics of all diagnostic parameters are presented in Table 3. Serum level of Zn was decreased (p < 0.05) and this was influenced by the administration of CAPE (p < 0.05). There was a significant (p < 0.05) decrease in group 3 when compared to group 1 in Zn level on day 0. In the following days, there was a marked decrease in the level of Zn in untreated contusion group compared to CAPE-treated contusion group (p < 0.05). On the third day of the study, the serum Zn level was similar between groups 4 and 1 (p > 0.05).
Table 3

The effects of CAPE administration on serum copper and zinc levels in pulmonary contusion injury in rats (n = 9 and mean ± SD)

 

Zinc (μg/dl)

Copper (μg/dl)

Group 1

151.5 ± 1.3

89.6 ± 2.4

Group 2

153.3 ± 0.7

87.9 ± 1.3

Group 3

Day 0

62.9 ± 5.1

162.2 ± 3.4

Day 1

69.5 ± 1.4

146.8 ± 4.4

Day 2

64.4 ± 1.7

139.1 ± 6.4

Day 3

76.7 ± 1.9

130.1 ± 0.4

Group 4

Day 1

73.9 ± 2.3

113.7 ± 5.4

Day 2

98.4 ± 0.7

98.3 ± 3.4

Day 3

145.2 ± 0.9

92.2 ± 1.4

p values

Day 0, groups 1–2

n.s

n.s

Day 0, groups 3–1

0.0001

0.0001

Day 1, groups 3–4

n.s

0.002

Day 1, groups 4–1

0.002

0.004

Day 2, groups 3–4

0.003

0.003

Day 2, groups 4–1

0.002

0.002

Day 3, groups 3–4

0.002

0.003

Day 3, groups 4–1

n.s

n.s

Serum Cu level was inversely correlated with Zn level after lung injury. Higher serum Cu level was shown after contusion (Table 3). The rise in the Cu level was blocked on the second day of CAPE administration, which was significant (p < 0.05) when group 4 was compared to group 3. On day 3, no difference was detected between groups 4 and 1 (p > 0.05).

Histopathological examination scores revealed no difference between groups 1 (Fig. 1) and 2 (p > 0.05). On day 0, the score was higher in group 3 (Fig. 2a, b) compared to group 1 (p < 0.05). Higher scores in group 4 (Fig. 3) than group 1 (p < 0.05) and in group 3 than group 4 (p < 0.05) were observed in the following 2 days. On day 3, score was still higher (p < 0.05) in group 3 than group 4, while the difference between group 4 (Fig. 4) and group 1 was not significant (p > 0.05) (Table 4).
https://static-content.springer.com/image/art%3A10.1007%2Fs12011-012-9505-7/MediaObjects/12011_2012_9505_Fig1_HTML.jpg
Fig. 1

Representative photomicrographs from lungs of group 1 rats, showing the normal lung histology (H&E; ×200)

https://static-content.springer.com/image/art%3A10.1007%2Fs12011-012-9505-7/MediaObjects/12011_2012_9505_Fig2_HTML.gif
Fig. 2

a Representative photomicrographs from lungs of group 3 of rats on day 0, showing extensive leukocyte infiltration (H&E; ×400). b Representative photomicrographs from lungs of group 3 of rats on day 0, showing extensive congestion, edema, and hemorrhage (H&E; ×200)

https://static-content.springer.com/image/art%3A10.1007%2Fs12011-012-9505-7/MediaObjects/12011_2012_9505_Fig3_HTML.jpg
Fig. 3

Representative photomicrographs from lungs of group 4 of rats on day 1. A moderate alleviation in contusion-induced histopathological changes is seen in the rat lung (H&E; ×200)

https://static-content.springer.com/image/art%3A10.1007%2Fs12011-012-9505-7/MediaObjects/12011_2012_9505_Fig4_HTML.jpg
Fig. 4

Representative photomicrographs from lungs of group 4 of rats on day 3. Marked alleviation in congestion, edema, hemorrhage, and leukocyte infiltration can be observed. (H&E; ×200)

Table 4

The values of pathologic score in pulmonary contusion injury in rats (n = 9 and mean ± SD)

 

Alveolar edema and congestion

Leukocyte infiltration

Group 1

0.00 ± 0.00

0.00 ± 0.00

Group 2

0.00 ± 0.00

0.00 ± 0.00

Group 3

Day 0

2.55 ± 0.45

2.72 ± 0.28

Day 1

2.41 ± 0.11

2.53 ± 0.21

Day 2

2.32 ± 0.16

2.49 ± 0.14

Day 3

2.14 ± 0.22

2.02 ± 0.42

Group 4

Day 1

1.74 ± 0.12

1.54 ± 0.31

Day 2

1.22 ± 0.24

1.04 ± 0.35

Day 3

0.32 ± 0.16

0.28 ± 0.27

p values

Day 0, groups 1–2

n.s

n.s

Day 0, groups 3–1

0.0001

0.0001

Day 1, groups 3–4

0.003

0.002

Day 1, groups 4–1

0.003

0.003

Day 2, groups 3–4

0.002

0.002

Day 2, groups 4–1

0.004

0.001

Day 3, groups 3–4

0.003

0.002

Day 3, groups 4–1

n.s

n.s

Discussion

Pulmonary contusion occurs in 30–75 % of major thoracic traumas. It is a severe injury associated with high mortality, which is about 11–22 % after isolated severe contusion [1]. Pulmonary contusion is characterized by intra-alveolar hemorrhage, alveolar rupture, disconnection of alveoli from bronchioles, and interstitial edema of the lungs after trauma. Pulmonary contusion induced by blunt chest trauma leads to an early inflammatory process which is clearly associated with activation of the oxidant–antioxidant cascade [1, 2].

It has been suggested that the main causative factors after pulmonary contusion are ROS such as superoxide, hydrogen peroxide, peroxynitrites, and hydroxyl radicals, which are generated also in normal physiological conditions in the human body. These ROS are capable of initiating and promoting oxidative damage in the form of lipid peroxidation. The antioxidant serves as a defensive factor against free radicals in the body. Enzymes such as superoxide dismutase, catalase, and glutathione peroxidase fight against oxidation and act as a protective mechanism [1, 2, 11].

Potential agent selected for this study was CAPE, which is an active component of honeybee propolis and it is known to have powerful antimicrobial, anti-inflammatory, antineoplastic, and anti-oxidizing effects [49]. Previous studies demonstrated that CAPE is beneficial in decreasing free oxygen radicals and preventing consumption of free radical scavenging enzymes, acting similarly as these antioxidant enzymes [17, 18]. It has been shown that CAPE suppresses lipid peroxidation and inhibits lipoxygenase activities and tumor promotion [69]. The anti-inflammatory properties of CAPE have been attributed to suppression of prostaglandin and leukotriene synthesis [19, 20].

We hypothesized that CAPE would effectively protect tissue from pulmonary contusion by its antioxidant and anti-inflammatory effects. Therefore, we investigated the effects of CAPE on the activities of MDA, SOD, and CAT and on the levels of essential elements such Zn and Cu, changes in blood gases and markers of inflammation in tissue specimens after isolated lung contusion. To our knowledge, this is the first study to explore these effects of CAPE in an attempt to prevent oxidative stress after isolated pulmonary contusion. Our biochemical and histological results demonstrated that CAPE could reduce the damage to the rat of pulmonary contusion-dependent lung injury.

SOD and CAT are antioxidant enzymes and MDA is a major end product of free radical reaction. Therefore, if SOD and CAT will increase, MDA level is expected to decrease or vice versa. However, the results of the present study showed that SOD, CAT activities, and MDA level were increased after contusion and reduced to normal levels after CAPE administration. Thus, we hypothesized that free radical activity is augmented after contusion. The increased levels of free radicals intensify the MDA production. Simultaneously, SOD and CAT activities are increased due to increased expression and/or increased activation. CAPE decreases both SOD and CAT activities and MDA levels. It appears that CAPE not only acts directly on enzymatic activity but also, and especially, blocks the production of free oxygen radicals.

Zn is an intracellular signaling molecule and it plays an important role in cell-mediated immune functions and oxidative stress. These unique properties of zinc may have significant therapeutic benefits in several diseases in humans. In many diseases, concurrent zinc deficiency may complicate the clinical features, affect adversely immunological status, increase oxidative stress, and increase the generation of inflammatory cytokines [11]. Result of the present study showed that contusion itself reduced serum Zn levels. CAPE exhibited positive effect on Zn level after contusion and interrupted the decline in Zn level.

Cu ions are considered multifunctional, participating in a broad spectrum of intracellular processes under normal and pathologic conditions. Cu complexes show a diverse in vitro biological activity, ranging from antibacterial and anti-inflammatory to cytostatic and enzyme inhibitory [13, 21]. The present study found that, unlike the CAPE–Zn interaction, Cu levels were increased after contusion, and CAPE reduced the Cu level after contusion and brought the level to that of the control group.

The data on the critical role of neutrophils in the etiology of pulmonary endothelial damage are convincing. For example, morphological data revealed deposition and aggregation of neutrophils in the pulmonary vasculature in animals with acute lung injury [22]. Clinical data are partially based on bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome (ARDS). Neutrophil elastase and neutrophil-derived oxidants in the lavage fluid have been demonstrated [22]. The release of elastase and the generation of oxidants by neutrophils can lead to severe pulmonary injury. Neutrophils derived from the pulmonary artery blood in critical patients with ARDS appear to be in a functionally and metabolically activated state compared to critical patients without ARDS [22, 23]. During ARDS, neutrophils are present in high concentrations and are in a metabolically active state so they can release proteases and oxygen metabolites that are toxic to the lung. As mentioned above, pulmonary contusion is associated with a leukocyte-mediated secondary inflammatory response leading to capillary leak and protein extravasation. Early after contusion, designated as day 0, enzyme levels gradually increased in the contusion group compared to the control group in the present study. Further, due to alveolar hemorrhage, congestion, and edema, leukocytic infiltration begins in the alveolar space [3]. The present study, in agreement with others, showed a significant decrease in leukocytic infiltration scores with the use of CAPE in contused rats [2124].

Koltuksauz et al. [24] designed an experimental study in order to evaluate the effects of CAPE on ischemia–reperfusion injury in rat intestine. The study showed that prophylactic administration of CAPE in ischemic condition prevented reperfusion injuries by eliminating oxygen radicals and inhibiting polymorphonuclear leukocyte infiltration. Histopathological changes vary in the contused lung. When histopathological changes in lung parenchyma were assessed in our study, there was, as predicted, a statistically significant difference between the contusion and non-contusion groups. Moreover, this experimental study confirmed that histopathological score, which defines leukocyte concentration, was lower in CAPE-administrated contusion group than in the contusion group without CAPE administration on days 1 and 2. On day 3, the score was still higher in contusion group without CAPE than CAPE-administered contusion group. On the other hand, no difference was found between CAPE-administrated contusion group and the control group, suggesting that CAPE can diminish the potential effect of isolated lung contusion by lowering the leukocytic infiltration.

The increase in antioxidant enzymes in ischemic tissues is a defense mechanism against oxidative stress. Marked decrease in antioxidant enzymes after reperfusion means that these enzymes, which are proteins, were degraded in combating with oxidant attack developed during reperfusion. The effect of CAPE on these enzymes is not clear; however, the increase in the activities of both enzymes in CAPE-treated ischemia group is less than that in the saline-injected ischemia group [25]. This suggests that CAPE acts in parallel with SOD enzyme and diminishes formation of free oxygen radicals. This action may be explained with the inhibition of polymorphonucleated leukocyte infiltration by CAPE in the ischemic tissue.

Blunt thoracic trauma can lead to direct pulmonary vascular damage, resulting in impaired alveolar perfusion. This ultimately ends with ventilation–perfusion mismatch and worsening of gas exchange, as shown by Turut et al. [26] in a rat contusion model. With regard to the arterial oxygenation of contused rats, blood pH, pO2, and HCO3 measurements were decreased while pCO2 was increased in the contusion group compared to the control group in the very early stages of the present study. We detected statistically significant reduction in acidosis, beginning from the second day in CAPE administration after contusion group. The difference was so prominent that on day 3, due to the curative effect of CAPE, no difference in blood gas levels was detected in contused CAPE-administrated group and the control group.

Blood gases recovered in the CAPE group. SOD, CAT activities, and MDA level were lower in group 4 than in group 3. Histopathological scores were found to be lower in group 4.

In conclusion, CAPE seems to be effective in protecting against the severe oxidative stress and tissue damage caused by pulmonary contusion in an experimental setting. Therefore, we conclude that, in order to decrease pulmonary contusion injury, administration of CAPE may be a promising agent for a variety of conditions associated with pulmonary contusion and acute administration of CAPE would be helpful in clinical practice. Although the exact mechanisms remain to be elucidated, CAPE could be an effective agent.

Copyright information

© Springer Science+Business Media, LLC 2012