The effects of α-tocopherol on oxidative damage and serum levels of Clara cell protein 16 in aspiration pneumonitis induced by bile acids

Abstract

Our aim in this study is to examine the effects of α-tocopherol (AT) on rats with aspiration pneumonitis induced with bile acids (BAs). The animals were divided in to four groups, namely saline group (n = 7), saline + AT group (n = 7), BA group (n = 7), and BA + AT group (n = 7). Saline and BA groups aspirated intratracheally with 1 ml/kg saline and 1 ml/kg bile acids, respectively. AT was given at 20 mg/kg/day dosage for 7 days to the groups. AT group was given 20 mg/kg/day AT for 7 days. Malondialdehyde (MDA), Clara cell protein 16 (CC-16), catalase (CAT), superoxide dismutase (SOD), as well as peribronchial inflammatory cell infiltration, alveolar septal infiltration, alveolar edema, alveolar exudate, alveolar histiocytes, and necrosis were evaluated. The CAT activity of the BA group was significantly lower than the saline group. In the BA + AT group, there was a significant increase in SOD and CAT activities when compared with that of the BA group. The CC-16 and MDA contents in the BA group were significantly higher than in the saline group. The CC-16 and MDA levels of the BA + AT group were significantly lower than BA group. Histopathologic changes were seen in BA group, and there was a significant decrease in the BA + AT group. In conclusion, AT might be beneficial in the treatment of aspiration pneumonitis induced by BAs because AT decreased oxidative damage and resulted in a decrease in CC-16 levels.

Keywords

Aspiration pneumonitis α-tocopherol bile acids Clara cell protein 16 malondialdehyde

Introduction

The entry of materials from oropharynx or gastrointestinal tract into the inferior of vocal cords and lower respiratory tract is called as the pulmonary aspiration. While microorganisms in the material aspirated lead to aspiration pneumonia, chemical agents lead to aspiration pneumonitis.^1,2 The severity of pulmonary damage after experiencing an aspiration pneumonitis may progress from a light subclinic picture to acute lung injury or acute respiratory distress syndrome.^3,4

The damage to lung tissue through aspirated material depends on the content and volume of the material aspirated and the accompanying bacterial contamination. Depending on these factors, aspiration pneumonitis has different aspiration syndromes with different clinic presentation, pathogenesis, and treatment strategies.⁵ Aspiration pneumonitis induced by bile acids (BAs), which may develop from bile reflux or severe bilious vomiting, is one of these syndromes. Aspiration pneumonitis induced with bile acids may occur with malrotation, volvulus duodenal stenosis or atresia, jejunoileal meconium diseases, intestinal atresia, Hirschsprung disease, bacterial enteritis, and duodenogastroesophageal reflux.⁶

Aspiration pneumonitis is histopathologically characterized by the acute accumulation of protein-rich pulmonary fluid and neutrophil infiltration in the alveolar space. Neutrophils pass through the endothelium and epithelium and then migrate to the alveolar area where they cause the secretion of cytotoxic proinflammatory materials, proteolytic enzymes, reactive oxygen and nitrogen species (ROS and RNS, respectively), cationic proteins, lipid mediators, and inflammatory cytokines.^3,7

–10 The disruption of the epithelial barrier due to the secreted ROS, cytokines, and mediators disturbs the transepithelial fluid transport and inhibits the reabsorption of alveolar edema and, as a result, mainly destruction of lung surfactant and biochemical and tissue damage occur.^11

–14 Although more studies are in progress, it is not entirely known how the lung damage in aspiration pneumonitis occurs.

The ROS generated in the physiological states of the body equalizes enzymatic and nonenzymatic antioxidant defense systems and protects cells against damage.^15,16 When ROS oxidizes fatty acids, which results in the formation of malondialdehyde (MDA). This compound can be used as an indicator of oxidative stress load.^17,18 Protein carbonyl (PC) is generated after ROS attacks the amino acids in proteins.¹⁹ Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) are the main antioxidant enzymes in the body, which provide protection from the ROS.^19
–21 Some publications indicate that the oxidant/antioxidant balance in lung injuries changes in favor of oxidative stress.²² In the literature, some markers have been found to indicate lung injury other than those that indicate oxidative damage.²³ Clara cell protein 16 (CC-16) is one of those markers.²⁴

CC-16 is produced by nonciliated Clara cells, and its main function is to protect lungs against oxidative stress, inflammation, and carcinogenesis.^25,26 Its amount increases after smoking,²⁷ while it decreases in steroid use.²⁸ Besides, when looked at the literature, it is also seen that the amount increases in lung damage.²⁹

Our aim in this study is to examine the histopathologic and biochemical effects of α-tocopherol (AT; vitamin E, one of the antioxidant vitamins) on rats with aspiration pneumonitis induced by BAs.

Materials and methods

The study was conducted upon the approval of Animal Experiments Local Ethics Committee, Ondokuz Mayis University (approval number: HADYEK 2011/45, approval date: August 25, 2011). Experimental animals were obtained from Ondokuz Mayis University Research and Application Center of Laboratory Animals. The surgical procedures, administration of drugs, and sample collection in this study were performed in the same laboratory.

Experimental animals

Sprague Dawley female rats (n = 28) weighing between 240 and 260 g were used in the study. The ambient temperature of the animal cages was maintained at 20–24°C and the moisture at 50–55%. The rats were kept in a 12-h dark and 12-h light environment, and they were given water and food ad libitum during the study. The animals were fasted the night before the surgical operation.

Study groups

There were four groups of animals used in the study. The first group (saline group) contained rats (n = 7) that were aspirated intratracheally with 1 ml/kg of 0.9% saline and given olive oil once a day for 7 days by oral gavage. The second group (saline + AT) contained rats (n = 7) that were aspirated intratracheally with 1 ml/kg of 0.9% saline and given 20 mg/kg/day³⁰ AT dissolved in olive oil once a day for 7 days by oral gavage. The third group (BA group) consisted of rats (n = 7) that were aspirated intratracheally with a 1-ml/kg solution containing 10 mg/kg taurocholic acid and 5 mg/kg taurochenodeoxycholate (BAs) and were given olive oil for 7 days by oral gavage. The fourth group (BA + AT group) consisted of rats (n = 7) that were aspirated intratracheally with a 1-ml/kg solution containing 10 mg/kg taurocholic acid and 5 mg/kg taurochenodeoxycholate and were given 20 mg/kg/day AT dissolved in olive oil once a day for 7 days by oral gavage.

Surgical procedures

Prior to the operation, 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine were administered to the rats and the rats were left to ventilate spontaneously. The front part of the neck of the rats was shaved and they were placed in the supine position. Lidocaine hydrochloride with epinephrine was administered at the very midpoint of the thyroid cartilage by the sternal notch and a horizontal incision was made. Cutaneous and subcutaneous fatty tissues were dissected. Strap muscles were retracted laterally to reach the trachea. The solution containing taurocholic acid and taurochenodeoxycholate was administered into the lumen of the trachea between the second and third tracheal rings by an insulin syringe. The cutaneous cut was then sutured.

Sample collection

The rats were killed at the end of day 7 and the blood and lung tissue samples were collected. The blood was centrifuged at 1000g for 10 min to obtain serum and plasma samples separately, which were stored at −80°C until processed for the estimation of biochemical parameters. Lung tissue samples were kept in 10% formalin at room temperature until they were processed.

Biochemical parameters

The samples were allowed to come to room temperature before further processing.

MDA quantitation

MDA content was assayed from the plasma samples.³¹ This was carried out by adding 2.5 ml of 8.3% trichloroacetic acid to 0.5 ml of plasma samples and then incubating the samples at 90°C for 15 min. The samples were water-cooled after incubation and centrifuged at 3000g and 4°C for 20 min. Then 1 ml of 0.225% thiobarbituric acid (TBA) was added to 2 ml of each supernatant, and the mixtures were incubated at 90°C for 15 min. After incubation, they were water-cooled and the absorbance was read at 532 nm. The MDA content was measured through the extinction coefficient (1.56 × 10⁵ per cm/M) of the TBA-MDA complex, and the results were presented in micromoles per deciliter.

Determination of CAT activity

The CAT activity of the plasma samples was determined by the Aebi et al. method.³² CAT was allowed to break down 18% of hydrogen peroxide in 50 mM phosphate buffer (pH 7). The activity was monitored at 240 nm, and the results were presented in kat/dl.

Determination of total SOD activity

Total SOD activity was assayed from serum samples using the method established by Sun et al.³³ and modified by Durak et al.³⁴ This method is based on the principle that the superoxide radical generated by the xanthine/xanthine oxidase system reduces nitroblue tetrazolium (NBT) to form the blue–purple color. The SOD enzyme in the medium breaks down these superoxide radicals and prevents NBT from becoming colored. The samples were read at 560 nm, and the results were presented in units per milliliter.

Determination of PC content

The PC content of the serum samples was determined based on the principle that carbonyl groups in proteins react with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone.³⁵ The absorbance of the 2,4-dinitrophenylhydrazone formed was measured at 365 nm, and the results were presented in nanomoles per milliliter.

Clara cell protein 16

CC-16 content was assayed from the serum samples using enzyme-linked immunosorbent assay method in accordance with the manufacturer’s directions (USCN Life Science Incorporation, E90857Ra, China). The samples were pipetted into the CC-16-specific plates coated with monoclonal antibodies and then incubated. After incubation, biotin-conjugated secondary polyclonal antibodies were added to the sample wells. The plates were incubated and then washed. Avidin-conjugated horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine were added after washing. Sulfuric acid was added in the end to stop the reaction. The absorbance of the color formed was measured at 450 nm, and the results were presented in picograms per milliliter.

Histopathologic examination of lung tissues

Lung specimens were separately fixed in a neutral formalin solution with 10% buffer for 48 h. Then they were dehydrated in alcohol and embedded in paraffin blocks. Sections of 5 mm were cut, deparaffinized, and then stained with hematoxylin–eosin. The lung tissue samples were examined by a standard light microscope. Peribronchial inflammatory cell infiltration (PICI), alveolar septal infiltration (ASI), alveolar edema (AED), alveolar exudate (AEx), alveolar histiocytes (AH), and necrosis (NR) parameters were evaluated by modifying the four-point scale system of Takil et al.³⁶ (Table 1).

Table 1.

Pathologic scale used for the examination of lung tissues

	0	1	2	3
PICI	No	Prominent germinal centers of lymphoid follicles	Infiltration between lymphoid follicles	Confluent band-like form
ASI	No	Minimal	Moderate	Severe, impending of lumen
AED	No	Focal	In multiple alveoli	Widespread, involving lobules
AEx	No	Focal	In multiple alveoli	Prominent, widespread
AHI	No	Scattered in a few alveoli	Forming clusters in alveolar spaces	Filling the alveolar spaces
NR	No	Focal, few necrotic cells	Multifocal, small areas	Larger areas

^aPICI: peribronchial inflammatory cell infiltration; ASI: alveolar septal infiltration; AED: alveolar edema; AEx: alveolar exudate; AHI: alveolar histiocytes; NR: necrosis.

Statistical method

All statistical analyses were performed with SPSS statistical software (SPSS for Windows, version 15.0), and the results were presented as median, minimum value, and maximum value. The Mann-Whitney U test was performed to compare the groups’ biochemical and histopathologic parameters. The differences between the groups were considered significant if p < 0.05. The graphics used for the histopathologic results were made with GraphPad Prism 5.04.

Results

Biochemical findings

MDA, PC, and CC-16 contents as well as CAT and SOD activities were taken into account to understand the level of lung injury caused by BAs and to determine whether AT treatment was effective.

No significant statistical difference was determined between the MDA levels of the saline and saline + AT groups (p = 0.895). The MDA levels of the BA group were significantly higher than those of the saline group (p = 0.002). The MDA levels of the BA + AT group were significantly lower than those of the BA group (p = 0.001). According to the PC results, no significant differences were found among the groups, although it was slightly decreased in the groups where AT was administered. These results are shown in Table 2.

Table 2.

Serum CC-16, MDA, PC, SOD, and CAT contents^a

	Saline	Saline + AT	BA	BA + AT
CC-16 (pg/ml)	88.17 (52,95–170.95)	95.77 (61.78–140.59)	101.32 (92.55–57.53)^b	92.55 (70.59–23.17)^c
MDA (µM/dl)	32 (28–37)	32 (26–35)	41 (40–43)^b	31 (30–32)^c
PC (nM/ml)	785.67 (581.45–78.55)	771.71 (717.82–819.27)	805.31 (709.09–87.27)	703.64 (544.36–88.73)
SOD (U/ml)	4.94 (4.51–5.14)	4.99 (2.9–6.71)	4.94 (4.12–5.33)	5.82 (5.37–6.82)^c
CAT (k/dl)	1.09 (0.73–1.47)	1.13 (0.55–1.97)	0.37 (0.19–0.40)^b	1.57 (0.82–3.6)^c

BA: bile acid; AT: α-tocopherol; CC-16: Clara cell protein 16; MDA: malondialdehyde; PC: protein carbonyl content; SOD: superoxide dismutase; CAT: catalase.

^aThe results are presented as the median (minimum–maximum values).

^b p < 0.05 compared with saline group.

^c p < 0.05 compared with BA group.

The CAT and SOD activities of the saline group were not different from the saline + AT group (p = 0.798 and p = 0.701, respectively). The CAT activity of the BA group was significantly lower than that of the saline group (p = 0.002), but there was no significant difference between the SOD activities of both the groups (p = 0.562). These results indicate that the CAT activity, which is protective against damage in the group where BA was administered alone, was suppressed. In the BA + AT group, there was a significant increase in both SOD and CAT activities compared with that of the BA group (p = 0.002 and p = 0.002, respectively; Table 2). These results indicate that AT treatment increases the antioxidant capacity and prevents damages.

For the CC-16 contents, there was no significant difference between the saline group and the saline + AT group (p = 0.198). The CC-16 content in the BA group was significantly higher than that of the saline group (p = 0.012). The CC-16 levels of the BA + AT group were significantly lower than that of the BA group (p = 0.046). These results are shown in Table 2.

Histopathologic findings

There were no significant differences between the saline group and the saline + AT group relating to PICI, ASI, AED, AEx, AH, or NR. When the saline group was compared with the BA group, PICI, ASI, AED, AEx, AH, and NR were significantly higher in the BA group (p = 0.001, p = 0.003, p = 0.001, p = 0.023, p = 0.001, and p = 0.023, respectively). When the BA + AT group was compared with the BA group, PICI, ASI, AED, AEx, AH, and NR were significantly lower in the BA + AT group (p = 0.002, p = 0.032, 0.001, p = 0.023, p = 0.001, and p = 0.023 respectively). These results are shown in Figures 1 and 2.

Figure 1.

Histopathologic results of the study groups to which saline, saline + AT, BA, and BA + AT were administered. The results are presented in median values. BA: bile acids; AT: α-tocopherol; PICI: peribronchial inflammatory cell infiltration; ASI: alveolar septal infiltration; AED: alveolar edema; AEx: alveolar exudate; AH: alveolar histiocytes; NR: necrosis. ^a p < 0.05 compared with saline group; ^b p < 0.05 compared with BA group.

Figure 2.

Images of histopathologic section taken from the study groups: (a) saline group (H&E, ×40), (b) saline + AT group (H&E, ×40), (c) BA group (H&E, ×40), and (d) BA + AT group (H&E, ×40). Relatively standard lung parenchyma was observed in the saline and saline + AT group. In the BA group, an explicit PICI, interstitial inflammation, necrotic exudate in the lumen of the bronchial and significant inflammatory cell infiltration in interalveolar area were observed. PICI is very slight in the BA + AT group when compared with BA group. BA: bile acid; AT: α-tocopherol; H&E: hematoxylin–eosin; A: alveolar spaces; B: bronchiole; V: vascular structures; stars: peribronchial area; PICI: peribronchial inflammatory cell infiltration.

Discussion

One of the most important points in this study is that the aspiration pneumonitis was created by BAs. In the literature, a large number of studies examine aspiration pneumonitis caused by stomach acid and food particles.^37
–39 However, the damage caused by the aspiration of BAs associated with bile reflux or bilious vomiting has not been sufficiently studied. This study has helped to fill this gap to some extent.

MDA is one of the main markers that indicate oxidative stress in the body. MDA is created as a result of environmental ROS attacking lipids.¹⁸ In the study by Sahin et al., a significant increase in MDA levels was found in the aspiration pneumonitis created by enteral formula, and the hydrochloric acid and MDA levels were reduced in all groups after hyperbaric oxygen treatment. In the same study, activities of antioxidant enzymes SOD and GSH-Px were reduced in the untreated groups and increased in the treated groups.³⁹ Similarly, our study showed an increase in the MDA levels, but no significant change in the PC levels was observed, in the group where aspiration pneumonitis was created by BAs. In the same group, there was a significant decrease in CAT activity but no change in SOD activities, which is contrary to the results of Sahin et al. From these results, we can say that ROS resulted from the damage caused by BAs oxidizing lipids rather than the proteins in the medium, leading to a reduced antioxidant capacity of the medium. In the group where BAs and AT were administered, there was a significant decrease in MDA levels and a significant increase in CAT and SOD activities compared with the group not treated. These results indicate that AT treatment reduces oxidative damage in the medium, prevents lipid peroxidation, and increases antioxidant capacity.

In lung pathology studies, CC-16 levels were found to be lower in some publications⁴⁰ and higher in other publications.⁴¹ In the study performed by Kucejko et al.,⁴¹ an increase in serum CC-16 levels was found in patients with sarcoidosis and idiopathic pulmonary fibrosis. CC-16 levels were increased in sputum, bronchioalveolar lavage fluid (BALF), and nasal lavage in smokers compared with nonsmokers.^26,40 However, other results indicated a decrease in BALF in lung diseases.^42,43 In our study, an increase in CC-16 levels was seen in the group where BAs were administered alone and the levels reduced after treatment with AT. This increase shows that CC-16 mixes with blood depending on the damage in the Clara cells. The damage decreases after the administration of AT, and the decrease in damage ensures that CC-16 stays in the cell.

There are a large number of studies on aspiration pneumonitis performed using different agents in experimental animals. Regardless of the material aspirated, in aspiration pneumonitis, patchy-shaped areas are observed due to the accumulation of neutrophils. There are decreases in alveolar hemorrhaging, intra-alveolar and interstitial edema, and alveolar fluid clearing.⁴⁴ In addition, there is damage in alveolar–capillary barrier regardless of etiology. In response to the damage in the barrier, gas exchange gets impaired and respiratory failure is developed.^3,45,46

In lung injuries, polymorphonuclear leukocytes pass through the endothelium by way of adhesion molecules and accumulate on lung tissues. A wide range of molecules play a role in helping the neutrophils hold onto lung capillaries, diffuse into lung tissues, and stay in the tissue for a long time. Inflammation markers, such as C5a and IL-8, play quite an important role in neutrophils that immigrate into the lungs.⁴⁷ Proteolytic enzymes, ROS/RNS, cytokines, and chemokines secreted by neutrophils cause damage to endothelial cells and alveoli, while elastase secreted by neutrophils results in endothelial cell death.⁴⁸

It is not entirely understood how BAs cause damage in the cells. However, the studies indicate that BAs, due to their detergent-like characteristics, increase solubility of lipids in membrane structures.^49,50 Furthermore, due to their lipophilic characteristics, they easily pass through the cell membrane, break the organization of the cell membranes, and cause damage in subcellular cell functions.^50,51 As can be seen, BA itself and the inflammation caused indirectly by it result in severe damage in the lungs.

In parallel with the results of both Takil et al.³⁶ and Sahin et al.,³⁹ our study showed a significant increase in PICI, ASI, AED, AExD, AH, and NR in the group where aspiration pneumonitis is created by BAs when compared with the control group. In addition, there was a significant decrease in these parameters in the groups treated with BAs and AT. The results show that AT is effective in decreasing the tissue damage induced by BAs.

AT is a natural antioxidant found in low concentrations in the cell membrane. It inhibits lipid peroxidation in the cell membrane because of its ability to catch ROS.⁵² In the study carried out by Rocksen et al., AT administered 1 h after the intraperitoneal administration of lipopolysaccharide prevented neutrophils from accumulating in the alveoli and, therefore, avoided cytotoxic damage to the epithelium and endothelium.⁵³ AT decreases the number of adhesion molecules in leucocytes and endothelial cells and prevents neutrophils from sticking to the endothelium and accumulating in alveolar area.^54,55 In another study of hypoxic lung injury, the administration of AT inhibited lipid peroxidation and ensured that the membrane phospholipid content remained at the same level as the control group.⁵⁶ AT also regulates the cellular and humoral immune system. It inhibits protein kinase C and prevents proinflammatory signal pathways.^54,57 In our study, it caused both biochemical and histopathologic recoveries in lung injuries. AT probably made use of some the abovementioned mechanisms during recovery.

In conclusion, our results indicate that AT might be beneficial in the treatment of aspiration pneumonitis created by BAs, because the administration of AT decreased oxidative damage in the medium and resulted in a decrease in CC-16 levels.

Footnotes

Conflict of Interest

The authors declared no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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