ORIGINAL PAPER

The therapeutic effects of anti-oxidant and anti-inflammatorydrug quercetin on aspiration-induced lung injury in rats

Mehmet Ziya Yilmaz • Aygul Guzel • Aysun Caglar Torun • Ali Okuyucu •

Osman Salis • Rifat Karli • Ayhan Gacar • Tolga Guvenc • Sule Paksu •

Volkan Urey • Naci Murat • Hasan Alacam

Received: 16 July 2013 / Accepted: 20 September 2013 / Published online: 13 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Aspiration pneumonitis refers to acute chemical

lung injury caused by aspiration of sterile gastric contents.

The aim of this study was to evaluate the role of quercetin

(QC) in acid aspiration-induced lung injury in rats. Twenty-

eight female Sprague–Dawley rats were used and divided

into the following groups (n = 7): sham (aspirated normal

saline, S), hydrochloric acid (aspirated HCl), S plus treat-

ment with QC (S ? QC), and HCl plus treatment with QC

(HCl ? QC). After aspiration, the treatment groups received

QC 60 mg/kg/day intraperitoneally once a day for 7 days. As

a result of acid aspiration, an increase was observed in the

levels of serum clara cell protein-16 (CC-16) and advanced

oxidation protein products, whereas there was a decrease in

serum thiobarbituric acid-reactive substances, superoxide

dismutase (SOD), and catalase levels. There was a signifi-

cant decrease in peribronchial inflammatory cell infiltration,

alveolar septal infiltration, alveolar edema, and alveolar

exudate scores, except in the alveolar histiocytes in the

HCl ? QC group. The expression of nitric oxide synthase,

which increased after aspiration in the HCl group, showed a

M. Z. Yilmaz (&) � A. C. Torun

Department of Pedodontia, Faculty of Dentistry,

Ondokuz Mayis University, Samsun, Turkey

e-mail: drziyilmaz@gmail.com

A. C. Torun

e-mail: aysunct@hotmail.com

A. Guzel

Department of Chest Disease, Faculty of Medicine,

Ondokuz Mayis University, Samsun, Turkey

e-mail: aygul.guzel@yahoo.com

A. Okuyucu � H. Alacam

Department of Medical Biochemistry, Faculty of Medicine,

Ondokuz Mayis University, Samsun, Turkey

e-mail: okuyucuali@hotmail.com

H. Alacam

e-mail: hasanalacam@hotmail.com

O. Salis

Samsun Highschool of Health, Ondokuz Mayis University,

Samsun, Turkey

e-mail: dr.osmansalis@hotmail.com

R. Karli

Department of Otorhinolaryngology, Faculty of Medicine,

Ondokuz Mayis University, Samsun, Turkey

e-mail: rifatkarli@yahoo.com

A. Gacar � T. Guvenc

Department of Pathology, Faculty of Veterinary Medicine,

Ondokuz Mayis University, Samsun, Turkey

e-mail: ayhangacar@omu.edu.tr

T. Guvenc

e-mail: tguvenc@omu.edu.tr

S. Paksu

Department of Pediatrics, Faculty of Medicine,

Ondokuz Mayis University, Samsun, Turkey

e-mail: karadenizsule@hotmail.com

V. Urey

Cizre State Hospital, Sırnak, Turkey

e-mail: volkanurey@hotmail.com

N. Murat

Industrial Engineering, Faculty of Engineering,

Ondokuz Mayis University, Samsun, Turkey

e-mail: nacimurat@omu.edu.tr

123

J Mol Hist (2014) 45:195–203

DOI 10.1007/s10735-013-9542-3

statistically significant decrease after the QC treatment.

After the treatment with QC, an increase in the serum SOD

level was observed, whereas a significant decrease was

determined in the serum CC-16 level relative to that of the

aspiration group (HCl). The antioxidant QC is effective in

the treatment of lung injury following acid aspiration and can

be used as a serum CC-16 biomarker in predicting the

severity of oxidative lung injury.

Keywords Lung injury � Aspiration pneumonitis �Quercetin � iNOS � CC-16

Introduction

Acute lung injury may occur due to primary lung diseases,

such as pneumonia and chronic obstructive pulmonary

disease, as well as secondary causes other than lung dis-

eases. Sepsis, aspiration of gastric contents, massive

transfusion, and blunt chest trauma are the most common

causes of secondary lung injury (Chu et al. 2004).

Aspiration pneumonitis (AP; caused by aspiration of

gastric contents), which leads to fatal complications such as

acute respiratory distress syndrome (ARDS), is a serious

clinical situation that clinicians frequently encounter in

anesthesia and intensive care practices. Suppressed mental

status, loss of protective airway reflexes, nausea developing

spontaneously or after drug use, and stimulation of the

airway and gastrointestinal tract are the most common

causes of aspiration (Toouli et al. 2002; Gunaydin et al.

2012). The content, pH, and volume of the aspirated fluid

are among the factors that determine the severity of lung

injury occurring after aspiration. Among the gastric con-

tents, hydrochloric acid (HCl) has the most important

impact on lung injury (Beck-Schimmer and Bonvini 2011).

The pathophysiology of lung injury caused by gastric

acid aspiration remains unclear. It is crucial to understand

this process and put forward treatment options for lung

injury (Hassan et al. 2012; Marik and Kaplan 2003). Lung

atelectasis, peribronchial hemorrhage, alveolar edema,

obstruction of small airways causing the loss of surfactant,

neutrophil migration into the alveolar area, alveolar

necrosis, and ventilation-perfusion disorder, which devel-

ops as a result of hyaline membrane formation and intra-

pulmonary shunts, are among the pathological changes

caused by acid aspiration (Wong and Briars 2002; Francis

et al. 2009).

In the case of ARDS, a common inflammatory process

begins in the lung tissue after aspiration. In this process,

increased levels of free radicals and decreased levels of

antioxidants, such as superoxide dismutase (SOD), catalase

(CAT), glutathione peroxidase (GSH-Px), and glutathione

S-transferase, are considered to be the main causes of the

damage. The increase in free radicals during inflammation

causes lipid peroxidation and protein destruction in the

lung (Kalpana and Menon 2004). In this inflammation

process, thiobarbituric acid-reactive substances (TBARS)

are a marker of lipid peroxidation damage. Advanced

oxidation protein products (AOPP) serve as oxidative

markers of damage at the protein level (Wong and Briars

2002; Francis et al. 2009). The production of proinflam-

matory nitric oxide (NO), which is synthesized by induc-

ible NO synthase (iNOS), is also increased in lung damage

(Hauser et al. 2005).

Clara cell protein-16 (CC-16) is released from noncili-

ary tracheobronchial epithelial cells. This protein plays a

role in the immunosuppressive and antioxidant properties

of lung tissue. In particular, it prevents the degradation of

surfactant phospholipids and inhibits interferon synthesis.

Thus, CC-16 protects the respiratory tract against oxidative

stress (Krakowiak et al. 2013; Broeckaert and Bernard

2000). Studies have reported that CC-16 may serve as a

potential biomarker of the progression of many lung-spe-

cific diseases (Alacam et al. 2013; Dickens and Lomas

2012). AP is associated with significant oxidative damage,

and CC-16 may be an important biochemical parameter for

the diagnosis of lung disease due to AP.

Quercetin flavonol (3,5,7,30,40-pentahydroxyl flavone) is

a polyphenolic flavonoid with antioxidant, anti-inflamma-

tory, anti-ischemic, antiperoxidative, and antiapoptotic

features. Studies have suggested that QC treatment sig-

nificantly reduces tissue damage, primarily due to its anti-

inflammatory properties, and that it protects against tissue

damage by influencing lipid peroxidation and increasing

the effectiveness of the antioxidant defense system (Jin

et al. 2012; Muthukumaran et al. 2008). QC is found in

natural food sources, such as fruit and vegetables, and acts

as an antioxidant through the prevention of OH ion for-

mation via Fe ?2 and Cu ?2 in the body (Muthukumaran

et al. 2008; Bond 2002). The therapeutic efficacy of QC has

been tested in many diseases, with significant results

achieved in many cases (Jin et al. 2012; Yousef et al.

2010). Oxidative stress and acute inflammation are at the

forefront of the pathogenesis of AP. QC may be effective

against this disease. Therefore, this study investigated the

antioxidant, anti-inflammatory, and antiperoxidative prop-

erties of QC and its ability to prevent lung injury caused by

AP.

Materials and methods

The experimental protocol and all animal procedures

were approved by the Experimental Animal Studies

Ethics Committee of Ondokuz Mayis University, Sam-

sun, Turkey.

196 J Mol Hist (2014) 45:195–203

123

Animals and experimental protocol

All animals were provided by the Experimental Research

Center of the medical faculty of Ondokuz Mayis Univer-

sity. Twenty-eight female Sprague–Dawley rats weighing

235–275 g were used in the experiments. They were kept

under standard experimental laboratory conditions (tem-

perature: 24 �C; dark/light cycle: 12/12 h; free access to

food and water; relative humidity: 60 %).

The experimental rats were randomly divided into the

following groups (n = 7): sham (aspirated saline; S group),

hydrochloric acid aspirated (HCl 0.1 M, pH 1.25; HCl

group), saline plus treatment with QC (S ? QC group), and

HCl plus treatment with QC (HCl ? QC group). The rats

were anesthetized with an intraperitoneal (i.p.) injection of

ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg)

and allowed to breathe spontaneously during the surgical

procedure. They were placed in a supine position. The

tracheal rings were revealed through a neck incision, and a

direct puncture with a 21-gauge needle was performed 2–4

tracheal rings below the larynx. Saline and HCl were

injected into the larynx via a 21-gauge syringe at a volume

of 1 ml/kg after the tracheal incision. The incision was

repaired with a 6-0 ethilon suture. All the experimental rats

were observed until they recovered from anesthesia.

After aspiration, the S ? QC and HCl ? QC groups

received 60 mg/kg/day i.p. injection of QC (Sigma

Chemical Co., St. Louis, MO, USA) for 7 days. At the end

of the seventh day, all the experimental rats were sacrificed

with an i.p. injection of ketamine hydrochloride. After the

surgical procedure, samples of the right lung (anterior lobe,

median lobe, posterior lobe, and post-caval lobe) and the

left lung (upper left lobe and lower left lobe) were exam-

ined. Lung lobe samples were allocated for histopatholo-

gical and immunohistochemical investigations, and at least

eight areas per lung section were analyzed. The average of

the scores from the lung sections examined determined the

final score in each category for each individual animal.

Histopathological studies

Midsagittal slices prepared from lung tissue samples were

fixed in 10 % buffered formalin for 24–72 h and embedded

in paraffin following routine procedures. Sections 5-lm

thick were prepared from the blocks and stained with

hematoxylin-eosin for routine microscopic examination.

Pathologists blinded to the study and the control groups

then examined all the microscopic slides by evaluating at

least eight randomly selected microscopic high-power

fields from each tissue sample. All the tissue slides were

scored according to the degree of peribronchial inflam-

matory cell infiltration (PICI), alveolar septal infiltration

(ASI), alveolar edema (AED), alveolar exudate (AEX), and

alveolar histiocyte (AHI) formation, using the 4-point scale

developed by Takil et al. (2003; Table 1).

Immunohistochemistry procedure

The lung tissue samples were fixed in 10 % neutral buf-

fered formalin and embedded in paraffin. All the samples

were sectioned at a thickness of 5 lm. The streptavidin–

biotin-peroxidase complex technique (Histostain Plus Kit,

Zymed, cat. no. 85-8943, CA, USA) was used for the

immunohistochemical study. Endogenous peroxidase

activity was removed by incubation with 2 % H2O2 in

methanol for 30 min at room temperature. Rabbit poly-

clonal anti-iNOS antibody (1/250, Abcam, cat. no. ab3523,

UK) was used as the primary antibody. Aminoethylcarba-

zole was used as the chromogen in H2O2 for 10 min, which

was controlled by visual observation under a microscope.

The sections were counterstained with Mayer’s hematox-

ylin for 1 min and rinsed with tap water. Subsequently, the

sections were mounted with an aqueous mounting medium.

Immunohistochemical iNOS staining of the lung tissue

slides was evaluated semi-quantitatively according to the

intensity of the differences between each experimental

group. The staining intensity of iNOS was recorded as faint

(-/?), mild (?), moderate (??), and strong (???).

Immunostaining was evaluated in at least eight randomly

selected areas per lung section, using two sections from

each animal at 4009 magnification. The final score cal-

culated in each category for each individual rat was the

mean of the scores from the lung sections examined.

Table 1 The 4-point scale used for the histopathological assessment

0 1 2 3

PICI No Prominent germinal

centers of

lymphoid follicles

Infiltration

between

lymphoid

follicles

Confluent

bandlike

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

PICI peribronchial inflammatory cell infiltration, ASI alveolar septal

infiltration, AED alveolar edema, AEX alveolar exudate, AHI alveolar

histiocytes

J Mol Hist (2014) 45:195–203 197

123

Biochemical procedure

Blood samples taken from the rats were placed in antico-

agulant-free tubes and left for 30 min to coagulate prop-

erly. They were centrifuged at 3,000 rpm for 10 min. The

resulting serum samples were kept frozen at 80 �C until

evaluation.

Measurement of serum levels of clara cell protein-16

The concentrations of CC-16 were assessed using a sand-

wich enzyme immunoassay according to the manufac-

turer’s instructions (USCN Life Science Inc., Wuhan,

People’s Republic of China). The plate was precoated with

an antibody specific to CC-16. Samples were added to the

wells with a biotin-conjugated antibody specific to CC-16.

Next, avidin conjugated to horseradish peroxidase was

added to the wells and incubated. After a tetramethyl-

benzidine substrate solution was added, the enzyme-sub-

strate reaction was terminated by adding a sulfuric acid

solution, and the color change was measured photometri-

cally at 450 nm. The CC-16 concentration in the samples

was determined by comparing the optical density of the

samples to the standard curve. The results are presented in

pg/mL.

Measurement of serum levels of advanced oxidation

protein products

The serum levels of the AOPP were assessed according to

the manufacturer’s instructions (Immundiagnostik AG,

Bensheim, Germany). The test is based on spectroscopic

analysis of modified proteins at 340 nm. The results are

presented in lmol/L.

Measurement of serum levels of thiobarbituric

acid-reactive substances

The concentrations of TBARS were assessed according to

the manufacturer’s instructions (Cayman Chemical Com-

pany, cat. no. 10009055, USA). The test is based on the

formation of malondialdehyde-thiobarbituric acid (MDA-

TBA) adduct by the reaction of MDA and TBA under high

temperatures (90–100 �C) and acidic conditions. The

MDA-TBA adduct was then measured colorimetrically at

530–540 nm. The results are presented in lmol/L.

Measurement of serum levels of superoxide dismutase

The serum levels of SOD were assessed according to the

manufacturer’s instructions (Cayman Chemical Company,

cat. no. 706002). The test uses tetrazolium salt to detect

superoxide radicals generated by xanthine oxidase and

hypoxanthine. One unit of SOD is defined as the amount of

enzyme needed to exhibit 50 % dismutation of the super-

oxide radical. The results are presented in U/mL.

Measurement of serum levels of catalase

The serum levels of CAT were assessed according to the

manufacturer’s instructions (Cayman Chemical Company,

cat. no. 707002). The test is based on the reaction of the

enzyme with methanol in the presence of an optimal con-

centration of H2O2. The formaldehyde produced was

measured colorimetrically with 4-amino-3-hydrazino-5-

mercapto-1,2,4-triazole as the chromogen. This chromogen

specifically forms a bicyclic heterocycle with aldehydes,

which changes from colorless to a purple color upon oxi-

dation. The resulting color was measured at 532 nm. The

results are presented in nmol/min/mL.

Statistical methods

Data acquired from this study were analyzed with the SPSS

15.0 package software. The measurements obtained were

expressed in mean (±) standard deviation (SD) form. As all

the measurements did not comply with a normal distribu-

tion, all the values were compared with Mann–Whitney

U tests. The level of statistical significance was accepted as

p \ 0.05.

Results

Biochemical findings

The CC-16 serum levels were significantly higher in the

HCl group than in the S group (p \ 0.01; Table 2). The

serum levels of TBARS and CAT were lower in the HCl

group than in the S group. Additionally, the AOPP serum

levels were higher in the HCl group than in the S group.

However, these differences were not statistically significant

(p [ 0.05, p [ 0.05, and p [ 0.05, respectively; Table 2).

The SOD serum levels were significantly lower in the HCl

group than in the S group (p \ 0.01).

The QC Treatment decreased the CC-16 serum levels in

the HCl ? QC group compared to the HCl group

(p \ 0.05). The SOD serum levels increased in the treated

groups compared to the untreated groups (p = 0.001;

Table 2). The QC treatment decreased the serum levels of

AOPP and TBARS in the HCl ? QC group compared to

the HCl group, but they were not statistically significant

(p [ 0.05 and p [ 0.05, respectively). The CAT serum

levels did not significantly increase in the HCl ? QC

group compared to the HCl group (p [ 0.05; Table 2).

198 J Mol Hist (2014) 45:195–203

123

Histopathological results

The PICI, ASI, AED, and AEX scores increased in the HCl

group compared to the S group (p \ 0.01, p = 0.001,

p \ 0.05, and p \ 0.01, respectively). However, AHI scores

increased in the HCl group compared to the S group,

although this finding was not statistically significant

(p [ 0.05; Figs. 1, 2). There was a significant decrease in the

PICI, ASI, AED, and AEX scores in the HCl ? QC group

compared to the HCl group (p \ 0.01, p \ 0.01, p \ 0.05,

and p \ 0.05, respectively; Figs. 1, 2), but the decrease in

the AHI score was not significant (p [ 0.05). None of the

histopathological scores were statistically significant in the S

group compared to the S ? QC treatment group (p [ 0.05).

Immunohistochemical results

Immunohistochemical iNOS staining was evaluated semi-

quantitatively according to the intensity and the proportion of

the stained cells. The iNOS-positive cell numbers were sig-

nificantly higher in the HCl group (median 4?) than in the S

group (median ?; p \ 0.05; Fig. 3; Table 3). The iNOS

expression was significantly lower in the HCl ? QC group

(median ?) compared to the HCl group (median 4?;

p \ 0.05; Fig. 3; Table 3). However, after the QC treatment,

there was no statistically significant difference in the expres-

sion of iNOS in the S ? QC group (median 1?) compared to

the S group (median -/?; p [ 0.05; Fig. 3; Table 3).

Discussion

The study showed a significant reduction in the expression

of iNOS and in the histopathological scores for PICI, ASI,

AED, and AEX after the QC treatment. The QC treatment

was also effective in reducing the serum level of CC-16

and resulted in a statistically significant increase in the

serum level of the antioxidant SOD.

Acute lung damage can occur for direct or indirect

reasons, and it may include many structural changes,

including inflammation, proliferation, and fibrosis. Sepsis,

pneumonia, aspiration of gastric contents, emergency

transfusion, and trauma are the most common causes.

Irrespective of the cause, the lack of distinction among

pathological changes is noteworthy. Many studies have

attributed these pathological changes to an increase in

epithelial and vascular permeability, endothelial damage,

acute inflammation, and coagulation disorders (Toouli

et al. 2002; Wong and Briars 2002; Francis et al. 2009).

AP is a frequent cause of ARDS. Suppressed mental sta-

tus, loss of protective airway reflexes, nausea developing

spontaneously or after drug use, and stimulation of the air-

ways and the gastrointestinal tract are the most common

causes of aspiration (Gunaydin et al. 2012). The HCl com-

ponent of the gastric contents is the most common cause of

lung damage in aspiration. Acid aspiration causes alveolar

necrosis due to obstruction of the small airways and venti-

lation-perfusion imbalance. This gives rise to hypoxia in the

early period as a result of intrapulmonary shunts and leads to

ARDS by causing atelectasis, alveolar edema, and surfactant

loss in the lung. Atelectasis, peribronchial hemorrhage,

pulmonary edema, bronchial epithelial cell injury, hyaline

membrane formation, and neutrophil migration into the

alveolar space are among the pathological changes occurring

in the lung tissue (Wong and Briars 2002; Francis et al.

2009). In the present study, AP was induced by HCl, which is

the most important component in gastric aspiration. In the

histopathological examination, there was a significant

Table 2 Evaluation of serum CC-16, AOPP, TBARS, SOD, and CAT levels in the study and treatment groups

Study groups Treatment groups

Sham (S) HCl S ? QC HCl ? QC

CC-16 (pg/mL) 37.60 ± 6.22 62.28 ± 20.08a 33.04 ± 6.53 41.09 ± 4.80d

AOPP (lmol/L) 42.13 ± 11.49 57.61 ± 0.11 36.36 ± 9.33 44.92 ± 23.99

TBARS (lmol/mL) 16.92 ± 2.50 14.14 ± 2.17 11.18 ± 1.86c 12.29 ± 3.57

SOD (U/mL) 1.08 ± 0.15 0.39 ± 0.39b 1.19 ± 0.10 1.14 ± 0.08e

CAT (nmol/min/mL) 72.19 ± 22.93 50.51 ± 18.05 111.77 ± 82.90 57.86 ± 40.35

All serum levels are presented as mean ± SD. Sham (S); Normal saline aspirated, HCl; Hydrochloric acid aspirated, S ? QC; S group treated

with quercetin, HCl ? QC; HCl group treated with quercetin, CC-16; Clara cell protein-16, AOPP; Advanced oxidation protein products,

TBARS; Thiobarbituric acid-reactive substances, SOD; Superoxide dismutase, CAT; Catalasea p \ 0.01 compared to sham groupb p \ 0.01 compared to sham groupc p = 0.001 compared to sham groupd p \ 0.05 compared to HCl and HCl ? QC groupse p = 0.001 compared to HCl and HCl ? QC groups

J Mol Hist (2014) 45:195–203 199

123

increase in PICI, ASI, AED, and AEX in the HCl aspiration

group compared with the S group. The immunochemical

analysis also revealed a significant increase in iNOS

expression in the HCl group when compared with the S

group, with inflammation in the lung tissue.

Many studies have reported the powerful proinflamma-

tory effect of NO, which aggravates tissue damage caused by

inflammation (Hauser et al. 2005; Song et al. 2013). Studies

have demonstrated that the inhibition of NO synthase by

iNOS inhibitors is effective in preventing such tissue damage

(Victor et al. 2004; Sakaguchi and Furusawa 2006). The

current study confirms the results published in the literature.

After treatment with QC, the immunohistochemical analysis

showed a significant decrease in iNOS expression, which had

increased after aspiration in the acid aspiration group. In a

study using gastrointestinal decontamination agents to treat

Fig. 1 Evaluation of all groups according to the histopathological

scores. PICI peribronchial inflammatory cell infiltration, ASI alveolar

septal infiltration, AED alveolar edema, AEX alveolar exudate, AHI

alveolar histiocytes; Sham (S); Normal saline aspirated, HCl;

Hydrochloric acid aspirated, S ? QC; S group treated with quercetin,

HCl ? QC; HCl group treated with quercetin

200 J Mol Hist (2014) 45:195–203

123

acute lung injury, S-methylisothiourea, which is an iNOS

inhibitor, was found to be effective in preventing lung

damage (Guzel et al. 2012).

Life-threatening complications caused by gastric acid

aspiration, such as pneumonia, ARDS, airway obstruction,

pleural effusion, pneumothorax, and bronchopleural fistula,

and the associated pathological changes by AP have

increased the importance of studies to identify treatment

strategies (Choi et al. 2006). Therefore, in the present

study, we investigated the efficacy of QC treatment in rats

exposed to acid aspiration to determine whether it might be

useful in treating lung pneumonitis due to its anti-inflam-

matory (Nair et al. 2006) and antioxidant (Ray and Husain

2002) properties. It resulted in significant improvements, as

shown by the results of the histopathological and immu-

nohistochemical analyses noted above.

Lung tissue inflammation begins with the formation of

free oxygen radicals (FORs). It then progresses with the

increase in and activation of neutrophils and the release of

large amounts of FORs from neutrophils in the region. Some

studies have suggested that FORs play a critical role in the

etiology of inflammatory diseases (Kalpana and Menon

2004). Free radicals cause a change in membrane perme-

ability or deterioration in the integrity of the membrane by

lipid peroxidation in the mitochondria and cell membrane

(Piwowar et al. 2007). MDA is the most important product of

lipid peroxidation. Free and bound MDA can be determined

by measuring levels of TBARS (Zhou et al. 2012). AOPP, a

cross-linked protein that contains ditrozin, is another serum

biomarker of the oxidative modification of proteins (Nayyar

et al. 2012). In our research, TBARS and AOPP were studied

to demonstrate the oxidative load of lung damage. No sig-

nificant difference was seen in the levels of AOPP in

response to the QC treatment, but the amounts of TBARS

decreased significantly.

A variety of enzymatic and nonenzymatic antioxidant

systems remove FORs from the body (Piwowar et al.

2007). The superoxide group, which causes extreme dam-

age to cells, is transformed into hydrogen peroxide and

oxygen by the cuprous enzyme, SOD. Then, H2O2, which

is less potent than molecules belonging to the superoxide

group, is nullified following transformation into products

with weak effects, which are exerted via enzymatic path-

ways, such as CAT in tissues, peroxidase, and GSH-Px

(Kovacic and Cooksy 2005; Di Filippo et al. 2006). In the

present study, the activity of SOD decreased with acid

aspiration and increased after treatment with QC. SOD

played a prominent role in eliminating the increase in the

Fig. 2 Histopathological evaluation of lung tissues. a S group, with

weak peribronchial inflammatory cell infiltration HE 94; b S ? QC

group, with weak edema formation around the peribronchial area

(arrow head) and no peribronchial inflammatory cell infiltration

HE 94; c HCl group, with severe peribronchial inflammatory cell

infiltration (star) HE 94; d HCl ? QC group, with prominent

germinal centers of lymph follicles (stars) HE 910

J Mol Hist (2014) 45:195–203 201

123

levels of FORs in the lungs. The activity of SOD was

decreased in the acid aspiration groups but increased fol-

lowing the treatment with QC. Levels of SOD were higher

in the cytosol, and CAT existed in the peroksizam.

Therefore, QC may have had no effect on CAT levels. In

addition, as SOD effectively removes superoxide radicals

from the environment, the amount of radicals may not have

reached a sufficiently high level to damage lipids and

proteins or to be detected in the serum. Therefore, levels of

AOPP and TBARS levels may not have increased.

Moreover, CC-16, which is released from nonciliary

clara cells and has anti-inflammatory and immunomodu-

latory effects, is known to protect the lungs and airways

against oxidative stress (Broeckaert and Bernard 2000).

Released from the airway epithelium, CC-16 is a biomarker

of lung epithelial injury (Benson et al. 2005; Lakind et al.

2007). In a study that induced lung injury by aspiration of

bile acids in rats, the serum amount of CC-16 increased in

the lung injury group and was reduced with a-tocopherol

treatment (Alacam et al. 2013). Similarly, in the present

study, the serum level of CC-16 was higher in the AP group

and lower in the treatment group.

As a result, both inflammation and oxidative stress

parameters were reduced after treatment with QC. The

levels of CC-16, which increased in response to epithelium

damage, were reduced by QC treatment. This suggests that

CC-16 can be used as a biomarker in damage assessment of

lung injury due to secondary causes. This study may mark

progress toward the clinical use of QC in treating lung

injury after acid aspiration.

References

Alacam H, Karlı R, Alıcı O, Avcıl B, Guzel A, Kozan A, Mertoglu C,

Murat N, Salıs O, Guzel A, Sahin M (2013) The effects of

Fig. 3 Immunohistochemical expression of iNOS in study and

treatment groups. a Sham group, with weak immunopositive reaction

of iNOS and inflammatory reaction; b S ? QC group, with interstitial

inflammatory cell infiltration and weak immunopositive reaction of

iNOS; c HCl group, with moderate inflammatory reaction and strong

iNOS positive reaction in the bronchial epithelium and interstitial

tissue; d HCl ? QC group, with minimal inflammatory reaction in the

interalveolar septum and mild immunopositive reaction of iNOS.

Immunoperoxidase technique, Harris hematoxyline counter staining,

AEC chromogen, 920

Table 3 Semiquantitative positive cell numbers for iNOS in lung

tissues for each group

Study groups Treatment groups

Sham (S) HCL S ? QC HCL ? QC

iNOS ? ???? -/? ?

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