The Efficiency of Proanthocyanidin in an Experimental Pulmonary Fibrosis Model: Comparison with Taurine

The Efficiency of Proanthocyanidin in an ExperimentalPulmonary Fibrosis Model: Comparison with Taurine

Yetkin Agackiran,1 Husamettin Gul,2 Ersin Gunay,3,8 Nalan Akyurek,4 Leyla Memis,4

Sibel Gunay,5 Yusuf Sinan Sirin,6 and Tayfun Ide7

Abstract—Pulmonary fibrosis (PF) is a progressive fatal disorder. Bleomycin (BLM) is a widelyused chemotherapeutic agent causing PF. Numerous agents have been investigated to prevent theprogression of PF so far, but there is still a need to find more efficacious agents. Proanthocyanidin(PA) is a strong antioxidant, the main ingredient of grape seed extract. Since PA is ready for use inpractice, we aimed to compare the preventive effect of PA in comparison with taurine (Tau) inBLM-induced PF. Forty Wistar male albino rats were used in the study and were divided into fourgroups: group 1, control; group 2, BLM-induced PF group; group 3, BLM-induced PF and treatedwith PA group; and group 4, BLM-induced PF and treated with Tau group. Treatments were begun10 days before and continued 21 days after BLM injection. PA and Tau effectively inhibited infl-ammation, edema, severity of fibrosis, fibrosis extension, inflammatory cell accumulation, iNOSstaining, and hydroxyproline level as well (p<0.05). Total histological scores of the PA group weresimilar to the control group; Tau was significantly higher than the control group but lower than theBLM group (p<0.05). We believe that PA could be a new treatment choice for PF, but furtherstudies need to be conducted to verify the findings of the current study.

KEY WORDS: bleomycin; proanthocyanidin; pulmonary fibrosis; iNOS; hydroxyproline; inflammation.

INTRODUCTION

Pulmonary fibrosis (PF) is a consequence of severaltypes of lung diseases which leads to respiratory failurewithin a few years after diagnosis [1]. Cytokines andreactive oxygen species (ROS) has been thought to be

responsible for the pathogenesis of PF [2]. Although theROS was shown to be essential to the development ofPF [3, 4], the exact pathophysiological mechanism forthe lung injury still remains unknown [5].

Bleomycin (BLM) is an antibiotic which has beenused in cancer chemotherapy. PF is one of the mostimportant side effects of BLM. BLM itself producessuperoxide and hydroxyl radicals by Fenton’s reactionand causes DNA damage [6]. BLM-induced lung injuryhas become an experimental model in animals forinterstitial pneumonitis and PF [7].

Despite the large number of compounds investigat-ed experimentally, none of these drugs have been able toqualify for clinical use, either due to lack of beneficialoutcome or significant adverse effects [7]. Therefore, itis advantageous to investigate new agents to prevent thedevelopment of lung fibrosis.

Proanthocyanidin (PA) is a biologically activepolyphenolic bioflavonoid produced by various plants.It is the main ingredient of grape seed extracts which hasstrong natural antioxidant effect with little cytotoxicbehavior [8]. Usually, PA has been taken as an enriched

These authors contributed equally to this work.

1 Department of Pathology, Ataturk Chest Diseases and ThoracicSurgery Training and Research Hospital, Kecioren, Ankara, Turkey

2 Department of Pharmacology, Gulhane Military Medical Academy,Ankara, Turkey

3 Department of Chest Diseases, School of Medicine, Afyon KocatepeUniversity, 03200 Afyonkarahisar, Turkey

4 Department of Pathology, Faculty of Medicine, Gazi University,Ankara, Turkey

5 Chest Diseases Clinic, Igdir State Hospital, Igdir, Turkey6 Department of Surgery, Faculty of Veterinary Medicine, Mehmet AkifErsoy University, Burdur, Turkey

7 Research and Developmental Institute, Gulhane Military MedicalAcademy, Ankara, Turkey

8 To whom correspondence should be addressed at Department ofChest Diseases, School of Medicine, Afyon Kocatepe University,03200 Afyonkarahisar, Turkey. E-mail: [email protected]

0360-3997/12/0400-1402/0 # 2012 Springer Science+Business Media, LLC

Inflammation, Vol. 35, No. 4, August 2012 (# 2012)DOI: 10.1007/s10753-012-9453-6

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grape seed extract in traditional herbal usage. It has beenproven to display protective effect against oxidativestress in numerous in vivo and in vitro studies. It has thescavenging effect of ROS [9], inhibitory effect ofischemia–reperfusion injury [10], and anti-inflammatoryand inhibitory effect of pesticide-induced oxidativestress [11]. In addition to its antioxidant effect, vasodi-lator [12], antithrombotic [13], cardioprotective [9, 14],and anticancer [15] effects have also been reported. Toour knowledge, despite its potential beneficial effects,PA has not been investigated in BLM-induced fibrosis.

Taurine (Tau), 2-aminoethanesulfonic acid, is anormal constituent of the human diet and is present inmost mammalian tissues and cells. It is a powerfulantioxidant [16]. It has been shown to be protectiveagainst BLM-induced lung injury [17]. Tau has alsobeen found beneficial in preventing experimental re-expansion pulmonary edema [18]. We have used Tau asa reference to compare the effects of PA.

The aim of this study is to investigate the potentialpreventive effects of PA in BLM-induced lung fibrosis.Lung damage was assessed by histological score andlung hydroxyproline level, a marker of collagendeposition.

MATERIALS AND METHODS

Reagents

Bleomycin sulfate was used in its commercial form,lyophilized flacon, from Onko&Koçsel (Istanbul, Tur-key). Tau was purchased from Sigma Chemical Co.(Ankara, Turkey). PA that was derived from grape seedswas obtained from General Nutrition Centers, Inc.(Pittsburgh, PA, USA). According to gas chromatogra-phy–mass spectrophotometry measurements by themanufacturer, this PA compound basically consists of30 % catechin and epicatechin and 25 % procyanidin,with the remaining 45 % made up of a variety of otherflavonoids. Other chemicals and reagents were fromstandard commercial sources.

Animals and Fibrosis Induction

Male Wistar albino rats (Breeding Colony ofResearch Center, Gulhane Military Medical School,Ankara Turkey) weighing 210–240 g were used. Ratswere maintained in a 12-h light–dark cycle (lights onfrom 0800 hours) at a constant ambient temperature

(25±2 °C) with normal rat chow and water availablead libitum during the study.

This study was approved by the ExperimentalAnimal Ethics Committee of Gulhane Research Institute,and the National Institute of Health’s Guide for the Careand Use of Laboratory Animals was followed.

To produce PF, all animals received a singlesublethal dose of BLM via direct intratracheal injectionwith cervical cut-down with isoflurane anesthesia.Control animals received saline with the same protocol.Three weeks after intratracheal injection, animals werekilled by a lethal dose of pentobarbital and exsanguina-tion was performed.

Experimental Groups

Animals were divided into four groups of ten rats:saline (control group), BLM, BLM + PA, BLM + Tau.BLM was given with a single intratracheal injection andinstillations at a dose of 7.5 mg/kg in a final volume of1 mL of distilled water similar to a previous method[19]. Tau 5 % (w/v) was given with drinking water andPA was given orally by gavage on a daily basis at100 mg/kg dose. All treatments started 10 days beforeand continued 21 days after the intratracheal injection ofBLM.

Histological Assessment

The lungs of rats were fixed in 10 % neutralformaldehyde and embedded in paraffin. Serial 5-μmsections were used through the middle of the lobeincluding the hilum to the peripheral region stained withhematoxylin–eosin (H&E) and Masson’s trichromestain. In addition, two sections from the right and leftlungs were separated for iNOS immunohistochemicalexamination. Pathologic grading was performed by twoexperienced pathologists for the extent and severity ofinflammation and fibrosis in lung parenchyma. Six lungsections from each animal were systematically scannedusing a ×10 objective and each successive field wasscored using the following grading scheme: grade 0 fornormal tissue, grades 1–4 for presence of inflammationand fibrosis. The severity of lesions was graded as 1(mild), 2 (moderate), 3 (severe), and 4 (severe inflam-mation accompanied by total distortion of structure). Theextent of lesions was graded as 1 (<10 % of the slide), 2(10–40 %), 3 (40–70 %), and 4 (>70 % of tissueaffected) [20]. Fields predominantly occupied by por-tions of large bronchi or vessels were not counted. Thepattern of distribution of the lesions was defined as

1403Proanthocyanidin in Bleomycin-Induced Pulmonary Fibrosis

multifocal and/or diffused, with or without affection ofthe subpleural zone. Edema was scored in a progressivemanner as normal (grade 0), perivascular (grade 1),interstitial (grade 2), intra-alveolar (grade 3), andorganized (grade 4). Infiltration of inflammatory cellswas graded 0–4, relating to their increasing presence inthe interstitial, peribronchiolar, and intra-alveolar spacesby counting each cell type in ten random fields.Infiltration of inflammatory cells (macrophages, neutro-phils, and lymphocytes) through the lung tissue wasgraded between 0 and 4, relating to their increasingpresence in the interstitial, peribronchiolar, and intra-alveolar spaces by each cell in ten random fields (grade0, normal; grade 1, mild; grade 2, moderate; grade 3,severe; grade 4, severe infiltration accompanied by totaldistortion of lung architecture).

Immunohistochemistry

Immunohistochemical study was performed accord-ing to the ABC technique as described previously [21].The positive immunostaining of iNOS was scored in asemiquantitative manner in order to determine thedifferences between control and treatment groups. Thescoring procedure of positive iNOS staining was asfollows: weak (±), mild (+), moderate (++), strong (+++),and very strong (++++). The analysis was performed inat least ten randomly selected microscopic high-powerfields from each lung section, in two sections fromeach animal at ×400 magnification. The final scoredetermined in each category for each individual animalwas the average of the scores from the sections of thelung examined.

Hydroxyproline Measurement

Lung hydroxyproline content was measured asoutlined previously [22]. Briefly, samples were homog-enized and then hydrolyzed in 6 N HCl for 18 h at 110°C. The hydrolysate was then neutralized with 2.5 MNaOH. Aliquots (2 mL) were analyzed for hydroxypro-line content after the addition of 1 mL of chloramine T,1 mL of perchloric acid, and 1 mL of dimethylamino-benzaldehyde. Samples were read in a spectrophotometerfor absorbance at 550 nm. Results are expressed asmilligrams of hydroxyproline per lung.

Statistical Analysis of Results

Statistical analyses were performed using SPSSstatistical software (SPSS for Windows, version 15.0).

Biochemical data were expressed as the mean ± standarddeviation. Statistical analysis of histopathological scoreswere carried out by analysis of variance (ANOVA)followed by appropriate post hoc tests including Bon-ferroni correction and unpaired t test. Significance wasaccepted when p<0.05.

RESULTS

BLM-treated rats showed severe fibrosis, edema,and large numbers of inflammatory cells, while PA- andTau-treated rats showed less inflammation (Fig. 1). PA-and Tau-treated rats had significantly lower histopatho-logical scores than the BLM group (p<0.05). PA moreefficiently suppressed inflammation (Fig. 2), edema(Fig. 3), severity of fibrosis (Fig. 4), fibrosis extension(Fig. 5), macrophage, neutrophil, and lymphocyteaccumulation (Fig. 6a, c), and iNOS staining (Figs. 7and 8) than the Tau group. BLM caused an increase iniNOS immunohistochemical staining in the lung of rats.PA and Tau treatment decreased iNOS staining signifi-cantly (p<0.05), but the degree of iNOS staining in bothgroups remained higher than the controls (p<0.05).Whereas lung tissue changes showed that the totalhistopathologic scores of PA were similar to the controlgroup, the Tau group was significantly different from thecontrol group (p<0.05). The BLM group had signifi-cantly higher total histopathologic scores than others(p<0.05) (Fig. 9).

Lung hydroxyproline content significantly in-creased in the BLM group compared with either salinecontrol group or treatment group (p<0.05). Hydroxy-proline content was 4.78±0.34, 3.81±0.15, 3.02±0.31,and 2.32±0.22 μg lung−1 in BLM-, Tau-, PA-, andsaline-treated rats, respectively. Tau and PA significantlydecreased the augmented collagen deposition in BLM-exposed rats (p<0.05), but their levels were higherthan the controls (p<0.05). The decreasing order ofhydroxyproline was consistent with the histopathologicalscores.

DISCUSSION

PF is a chronic disease for which there is noeffective treatment. BLM-induced PF is reported in2–40% of cases, this ratio increases with increasingdoses [23].

1404 Agackiran, Gul, Gunay, Akyurek, Memis, Gunay, Sirin, and Ide

To the best of our knowledge, this is the first animalstudy that demonstrated the preventive effect of PA onBLM-induced PF. In this study, we found that PA could

effectively prevent BLM-induced lung fibrosis in rats.Additionally, BLM caused an increase in content of thelung hydroxyproline level; PA and Tau treatments

Fig. 1. Histopathological examination of lung tissues. a Normal (H&E), b BLM (H&E), c BLM (Masson’s trichrome), d PA (H&E), and e Tau (H&E)groups showed normal rat lung tissue morphology, severe inflammatory cell infiltration, prominent interstitial and peribronchial fibrosis, mild infla-mmatory cell infiltration, mild–moderate inflammatory cell infiltration, and moderate–severe inflammatory cell infiltration, respectively (×100).

Fig. 2. Degree of inflammation. Effects of intratracheal instillated bl-eomycin (BLM) on the development of inflammation; saline control(C), BLM, proanthocyanidin (PA), and taurine (Tau) groups. Y axisvalue represents the number of the rats, *p<0.05, significantly higherdegree the than C, PA, and Tau groups; ANOVA followed by Bonferronicorrection post hoc test.

Fig. 3. Degree of edema. Effects of BLM on the development ofedema. *p<0.05, significantly higher degree the than control (C),proanthocyanidin (PA), and taurine (Tau) groups; ANOVA followed byBonferroni correction post hoc test.


decreased hydroxyproline level, which is a marker ofcollagen deposition and an index of fibrosis.

A large number of compounds have been suggestedfor the treatment or inhibition of the progression of PF

experimentally, such as glucocorticoids [24], N-acetyl-cysteine [25], interferon-gamma [26], blockade of trans-forming growth factor-beta [27], L-carnitine, ginkgobiloba [28], interleukin (IL)-4, and IL-13 [29]. Cortico-steroids may have symptomatic relief, but they do notappear to inhibit the progression of fibrosis, and theirbeneficial effects remain in question [24]. Cytotoxicdrugs (cyclophosphamide, azathioprine, etc.) have notbeen shown to improve lung function and they haveharmful side effects.

There are some studies related to the protectiveeffect of Tau on BLM-, amiodarone-, and ozone-inducedPF [30–33]. The protective effects of Tau againstcytotoxicity and oxidative stress have been observed incells and tissues, both in vivo and in vitro [34–36]. In theprevious studies, it has been shown that Tau significantlyprevented BLM-induced lung fibrosis [31, 33, 37].Therefore, we choose Tau as a positive control agent tocompare its beneficial effect with PA. In our study,extension of PF and degree of inflammation werereduced by oral Tau and PA administration. Althoughthe dose of Tau was higher in the present study than inthe previous studies (5 % w/v) in drinking water [31,33], the preventive effect of PA was greater than Tau.Whereas the PA group was similar to the controls, theTau group had significantly higher histopathologicalscores than the controls.

PA has natural polyphenols which are safe, potent,and bioavailable free radical scavengers and antioxidantspossessing a broad spectrum of health benefits. Grapeseed extract is one of the good sources of PA. It acts as apowerful antioxidant helping the body neutralize freeradical damage, believed to play a role in tissuedeterioration and aging [14, 38]. Due to the chemicalproperties of PAs, the availability of the phenolichydrogens as hydrogen-donating radical scavengers andsinglet oxygen quenchers predicts their antioxidantactivity [39, 40]. In our study, PA effectively preventedBLM-induced lung fibrosis. The mechanism by whichPA limits fibrosis is unclear, but the most likely one is itsstrong antioxidant effect, since ROS was shown to beessential in the development of fibrosis [3, 4]. Themechanism of the antioxidant effect of PAs is based onthe inhibition of xanthine oxidase and binding of freeradicals [14, 41]. It has potent hydroxyl and other freeradical scavenging abilities that contribute to its strongcardioprotective effect, even more powerful than vita-mins C and E [14, 42].

On the other hand, Rodriguez et al. showed thatneither hydroxyl nor superoxide radicals contributed to

Fig. 4. Degree of severity of fibrosis. Effects of bleomycin (BLM) onthe severity of fibrosis. *p<0.05, significantly higher degree than thecontrol (C), proanthocyanidin (PA), and taurine (Tau) groups; ANOVAfollowed by Bonferroni correction post hoc test.

Fig. 5. Degree of fibrosis extension. Effects of bleomycin (BLM) onthe severity of fibrosis extension. *p<0.05, significantly different fromthe control (C), proanthocyanidin (PA), and taurine (Tau) groups; A-NOVA followed by Bonferroni correction post hoc test.


BLM-induced DNA damage, but Fe(II) and O2 werenecessary considering that other ROS such as ·OH mightplay a role in BLM-induced lung fibrosis [43]. Oligonol,a type of PA, prior to the administration of ferric–nitrilotriacetic complex (a Fenton chemistry model),administration significantly reduced the extent of lipidperoxidation in mice [44]. This suggests that inhibitionof Fenton’s reaction could contribute to the beneficialeffects of PA. In addition, BLM can cause the generationof acrolein in vivo [45]. Acrolein is a strong pulmonaryirritant and causes DNA damage and protein synthesis ata low concentration more strongly than H2O2 and nearlyequal to that of ·OH. Resveratrol, a polyphenoliccompound found in grapes with a similar action to PA,showed a protective effect against the damage caused byacrolein exposure followed by hydrogen peroxidetreatment in human retinal pigmental epithelial cells[46]. This suggests that PA might also prevent acrolein-induced damage in BLM-induced lung fibrosis.

Besides its free radical scavenging and antioxidantactivity, PA has also phospholipase A2, cyclooxygenase,and lipoxygenase inhibitory activity which may contrib-ute to its beneficial effects [40, 47]. It has been recently

Fig. 6. Degree of inflammatory cell accumulation. Effects of bleomy-cin (BLM) on the severity of fibrosis extension: a macrophage, b ne-utrophil, and c lymphocyte. *p<0.05, significantly higher than thecontrol (C), proanthocyanidin (PA), and taurine (Tau) groups; ANOVAfollowed by Bonferroni correction post hoc test.

�

Fig. 7. Degree of iNOS immunostaining. Effects of bleomycin (BLM)on iNOS immunostaining. *p<0.05, significantly higher than the con-trol (C), proanthocyanidin (PA), and taurine (Tau) groups; ANOVA f-ollowed by Bonferroni correction post hoc test.


shown that PA could inhibit the expression of metal-lothionein genes in human HepG2 cells. Metallothioneinis important in the regulation of heavy metals, i.e., Znand Cu homeostasis. It is plausible that this metal-regulating effect may also contribute to the beneficialeffect of PA [48]. However, the exact mechanism of itspreventive effect on BLM-induced lung fibrosis shouldbe further investigated.

Our results suggest that there is a significantreduction in the activity of iNOS in lung tissue in thePA and Tau treatment groups in parallel with thedecrease of inflammatory cell infiltration, severity offibrosis, and edema. Saleh et al. reported increasedproduction of NO and peroxynitrite in patients withidiopathic PF [49]. Gurujeyalakshmi et al. reportedincreased NO levels in bronchoalveolar lavage fluid ofBLM-treated mice [33]. Kalayarasan et al. reported thatBLM could increase the expression of iNOS which inturn stimulates NF-kappa B in rats [50]. Yildirim et al.found that BLM could decrease the activities ofantioxidant enzymes and increase the NO levels in rats[51]. However, iNOS-derived NO production might beeither cytotoxic or cytoprotective, depending on theexperimental and clinical conditions [52]. Germano etal. showed that the upregulation of iNOS might inhibitalveolar macrophage proliferation and protect from

Fig. 8. Immunohistochemical expression of iNOS. Immunohistochemical expression of iNOS in lung tissues from a control, b BLM, c PA, and d Taugroups which revealed slightly positive iNOS immunoreactivity, strongly positive iNOS immunoreactivity, weak mildly positive iNOS immunorea-ctivity, and mildly positive iNOS immunoreactivity, respectively (×200).

Fig. 9. Total histopathological scores of the groups. Total histopath-ological scores including inflammation, edema, severity of fibrosis, fi-brosis extension, inflammatory cell accumulation, and iNOSimmunostaining: a control (C), b proanthocyanidin (PA), c taurine (T-au), and d bleomycin (BLM) groups. *p<0.05, different from the BLMgroup; **p<0.05, different from the control and BLM groups; ***p<0.05, different from the control, PA, and Tau groups (ANOVA followedby Bonferroni correction post hoc test).


BLM-induced lung injury [53]. It could be speculatedthat iNOS may increase as a result of increasing ROSand other inflammatory mediators to limit their deterio-rating effects further, but in the presence of antioxidantand anti-inflammatory agents which scavenge ROS andreduce inflammatory mediators, so iNOS might not beactivated so much.

CONCLUSIONS

In conclusion, our results indicate that PA exhibits aprotective effect against BLM-induced PF in rats. Since PA-rich grape seed extract is commonly used in herbal practice,our findings support the use of PA as a potential protectiveagent in BLM-induced PF. However, further studies need tobe conducted to verify the findings of the current study.

ACKNOWLEDGMENTS

The authors would like to thank Patricia B. Uptonfor the critical reading of the manuscript.

Conflict of Interest. The authors declare that there areno conflicts of interest.

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The Efficiency of Proanthocyanidin in an Experimental Pulmonary Fibrosis Model: Comparison with Taurine