Daidzein attenuates inflammation and exhibits antifibrotic effect against Bleomycin-induced pulmonary fibrosis in Wistar rats

Biomedicine & Preventive Nutrition 1 (2011) 236–244

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Original article

Daidzein attenuates inflammation and exhibits antifibrotic effect against Bleomycin-induced pulmonary fibrosis in Wistar rats Syamala Soumyakrishnan , Ganapasam Sudhandiran ∗ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu, India

a r t i c l e

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Article history: Received 16 September 2011 Accepted 28 September 2011 Keywords: Daidzein Bleomycin Collagen TNF-␣ Pulmonary fibrosis

a b s t r a c t Among the acute and chronic lung diseases, pulmonary fibrosis (PF) is the most fatal one because of its prognosis and treatment is so far an unsuccessful task. In this study, we have used the wellknown pulmonary fibrosis model, Bleomycin (BLM), to induce PF in Wistar rats. A single intratracheal instillation (3U/Kg BW) of BLM had been administered in fibrosis induced groups. The treatment drug daidzein (0.2 mg/KgBW) was administered by subcutaneous mode for 7 days. BLM administration reduces the bodyweight, antioxidant status (enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and non-enzymic antioxidants such as vitamin A, vitamin C, vitamin E and reduced glutathione) whereas, it increases the lung wet to dry ratio, hydroxyproline content, collagen deposition, lipid peroxidation and myeloperoxidase. Daidzein treatment improves the body weight, enzymic and non-enzymic antioxidant status and decreases the collagen deposition in lung as confirmed by Masson’s trichrome and Picro-sirius red staining. Daidzein treatment restored the lung architecture as revealed from histopathological and transmission electron microscopic studies. The levels of inflammatory cytokine Tumor necrosis factor(TNF-␣) was found to be increased in BLM-induced experimental group, whereas, on treatment with daidzein expression of TNF-␣ was found to be reduced. The results of the present study speculate that daidzein can be used as an agent against BLM-induced pulmonary fibrosis. © 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction Idiopathic pulmonary fibrosis (IPF) is an acute as well as one of the fatal human diseases for the reason that the deprived diagnosis and treatment of which is up to now an ineffective task [1–3]. The disease is the end result of several severe lung injuries, which is characterized by inflammatory reaction along with epithelial damage/activation followed by fibroblast/myofibroblast proliferation and extracellular matrix deposition [4]. The disease develops as a chronic inflammation, together with immune responses like tissue remodeling and repair processes occur consequently [5]. The pathological feature is characterized by the replacement of normal tissue by mesenchymal cells and extracellular matrix produced by these cells [6]. This is also mediated by the abnormal deposition of collagen in the lung [7]. The above two major hallmarks of fibrosis such as subepithelial myofibroblast/fibroblast foci and increased deposition of collagen with extracellular matrix results in the excess scar formation, thereby hardening the alveolar walls, which finally leads to the reduced ability to transport oxygen into the capillaries and the irreversible loss of total lung capac-

∗ Corresponding author. Tel.: +91 44 22202733; fax: +91 44 22352494. E-mail address: [email protected] (G. Sudhandiran). 2210-5239/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.bionut.2011.09.005

ity [8]. The major risk factors associated with pulmonary fibrosis include smoking, environmental exposure, gastroesophageal reflux disease, genetic factors, diabetes mellitus, infectious agents, and commonly prescribed drugs, such as bleomycin (BLM) [9]. BLM-induced lung fibrosis in rodents is the well-known animal model most frequently used to study human pulmonary fibrosis [10]. BLM (BLM) is an antibiotic complex produced by fermentation from Streptomyces verticillus, which also exhibits anticancer properties and is used as an antineoplastic agent to treat cancer in clinical chemotherapy [11,12]. This drug has been applied as cytostatic treatment of many malignant tumors, for instance, germ cell tumors, lymphomas, head, neck, and Kaposi’s sarcomas [12]. As the major side effect of BLM therapy, progression of pulmonary fibrosis occurs [11]. This limits its clinical use as an anticancer agent [13]. The reported mechanism of BLM is as follows: the BLM–iron complex reduces molecular oxygen to superoxide and hydroxyl radicals which can attack DNA, causing strand cleavage and this can induce lipid peroxidation, carbohydrate oxidation, alteration in lung prostaglandin synthesis and degradation, and increase in lung collagen synthesis [14]. Pulmonary fibrosis development is associated with the influx of activated inflammatory cells within lung parenchyma, the inflammatory cells produce reactive oxygen species (ROS) which may be the key contributory agents to the pathogenesis of BLM associated lung injury. These activated

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reactive oxygen species create breaks in DNA strand and lipid peroxidation [15]. At the start, BLM induces lung inflammation, which is followed by a progressive destruction of the normal lung architecture and the lesions formed as a result of this are associated with biochemical and functional changes that at least fairly bear a resemblance to those of human pulmonary fibrosis [16]. Experimental BLM-induced lung fibrosis completes within 28 days of the last stimulus [17]. Single intratracheal administration of BLM can cause pulmonary fibrosis in experimental rats [10]. Soy isoflavones are a major group of natural compounds collectively termed as isoflavonic phytoestrogens. Among them, the three major isoflavones in soybeans are Genistein, Daidzein and Glycetein have received much attention as dietary components to promote better health [18]. Daidzein is one of the most abundant isoflavanoid and phenolic compounds found in human diet as well as animal feedstuffs [19]. Daidzein possesses enormous health promoting physiological and pharmacological effects related to lower cholesterol, cancer prevention, reduced risk of cardiovascular disease, beneficial effects on bone, neuroprotective effect in experimental stroke and improves endothelial dysfunction [20–25]. In addition, in vivo studies on antidiabetic effects of daidzein have been previously reported [26]. In this study, for the first time, protective effect of daidzein against BLM-induced pulmonary fibrosis in experimental rats was investigated. 2. Materials and methods 2.1. Chemicals BLM sulfate, Daidzein and Dimethyl sulfoxide (DMSO) were purchased from Sigma chemicals, St. Louis, USA. All other chemicals used were of analytical grade.

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near normal values in BLM-induced rats after 28 d of experimental study. Hence, the dose of 0.20 mg/kg was chosen for the study. DMSO (0.01%) used in this study, did not educe any noticeable changes in the parameters performed, and hence DMSO was chosen as a vehicle. 2.5. Experimental groups The animals were divided into the following four groups (six rats in each group): • group-1: control rats were given 0.01% DMSO; • group-2: BLM was induced in rats via single intratracheal injection at a dosage of 3U/Kg W; • group-3: BLM-induced rats treated with daidzein (0.2 mg/Kg BW) in 0.1 ml 0.01% DMSO by subcutaneous injection for 7 days; • group-4: normal rats were given daidzein at a dosage of 0.2 mg/Kg BW in 0.1 ml 0.01% DMSO by subcutaneous injection for 7 days. 2.6. Preparation of lung tissue homogenate After the experimental period, the animals were sacrificed and the lung tissues were excised. After sacrificing the animals, the lungs were immediately excised and weighed. They were dried at 50 ◦ C for 72 h, and weighed again. The wet dry (W/D) lung weight ratio was calculated as an indicator of pulmonary edema. A small portion of the lung tissue was used for histopathological analysis. The remaining tissues were homogenized in 0.1 M Tris-HCl buffer (pH 7.4) and were used for biochemical measurements. 2.7. Biochemical parameters

Wistar male albino rats weighing between 200 and 240 g were used in this study. The rats were maintained in individual cages and acclimatized for a period of 7 d before the experiment was conducted. The animals were fed with commercial pellet diet (Hindustan lever Ltd., Bangalore, India) and were given free access to water. The experiments involved with animals were conducted according to the ethical norms approved by the Ministry of social justices and empowerment, Government of India and Institutional animal ethics committee guidelines (IAEC No. 01/052/09). Care was taken to minimize animal suffering.

Total protein content in the tissue homogenate was measured by the method of Lowry et al. [28]. The activity of superoxide dismutase (SOD) was assayed by the method described by Misra and Fridovich [29]. The activity of catalase (CAT) was assessed by Takahara et al. [30] and glutathione peroxidase (GPx) by Rotruck et al. [31]. Glutathione was measured according to the method of Ellman [32]. Vitamin C was estimated by the method described by Omaye et al. [33]. Vitamin E was measured according to the method of Desai [34]. Vitamin A was estimated by the method of Bayfield and Cole [35]. LPO was assessed by the measuring the level of malondialdehyde (MDA) following the method of Ohkawa et al. [36]. Lung MPO activity was estimated by Wei and Frenkel [37].

2.3. Induction of pulmonary fibrosis

2.8. Histolopathological analysis

Pulmonary fibrosis was induced in rats by single intratracheal injection of BLM as previously reported in our laboratory [27]. For induction of pulmonary fibrosis, male wistar rats (n = 6) received a single dose of 3 U/kg BW. BLM sulfate (Sigma, St. Louis, USA) was dissolved in 0.3 ml of 0.9% NaCl solution by intratracheal instillation on day 1 of the experimental period.

The lung tissue samples were fixed in 10% buffered formalin, routinely processed and embedded in paraffin. Three-micrometerthick sections were placed on slides and stained with hematoxylin and eosin (H&E).

2.4. Experimental design

Excess collagen deposition is the major hallmark of pulmonary fibrosis, which is formed from the non-proteinogenic aminoacid precursor, hydroxy proline. Hydroxyproline content present in the lung tissue homogenate of control and experimental groups was estimated by the method of Neuman and Logan [38]. Lung tissue collagen content was examined under the polarization microscopy by Picrosirius red staining, which is the specific staining for collagen [39]. Sirius red was dissolved in saturated picric acid solution (0.1%) and was used as the staining solution. Tissue sections with three-micrometer thickness were stained with sirius red to identify collagen fibers under polarized microscope (Olympus BX50) as

2.2. Animals

A pilot study was conducted with five different doses of daidzein (0.10, 0.20, 0.30, 0.40 mg/kg body weight) dissolved in 0.01% of DMSO administered subcutaneously in order to determine the optimum dosage. Daidzein was administered 6 h after BLM induction for seven days subcutaneously, during the 28 days of experimental period. It was observed that daidzein treatment at a dose of 0.20 mg/kg body weight, significantly (p < 0.05) altered hydroxyproline level in lung tissue homogenate, serum activities of alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) to

2.9. Collagen specific studies

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described by Chen et al. [40]. Areas of collagen deposition appeared yellow/orange color when visualized under a polarized microscope. Lung tissues from control and experimental groups of rats were collected and fixed with 3.6% buffered formaldehyde. After overnight fixation, lungs were embedded in paraffin. Five-micrometer-thick sections were stained with Masson’s trichrome stain.

2.12. Statistical methods

2.10. Analysis of TNF-˛ expression by immunohistochemistry

3. Results

Paraffin embedded tissue sections of 3 micrometer thickness were rehydrated first in xylene and then in graded ethanol solutions. Then the slides were blocked with 5% BSA in Tris Buffered Saline (TBS) for 2 h. The sections were then immunostained with primary antibody goat polyclonal IgG TNF-␣ (Santacruz Biotech, USA) at a concentration of 1 ␮g/ml with 5% BSA in TBS and incubated overnight at 4 ◦ C. After washing the slides thrice with TBS, the sections were then incubated with HRP conjugate secondary antibody goat polyclonal IgG (Bangalore Genei, India), diluted 1:2000 with 5% BSA in TBS and incubated for 2 h at room temperature. Sections were then washed with TBS and incubated for 5–10 min in a solution of 0.02% diaminobenzidine containing 0.01% hydrogen peroxide. Counterstaining was performed using hematoxylin, and the slides were visualized under a light microscope.

Table 1 denotes the effect of daidzein on the body weight of BLM and daidzein administered groups of rats. In BLM-induced group, because of severe tissue damage caused by free radicals, a marked reduction of body weight was observed. However, upon treatment with daidzein body weight was improved marginally near to normal control groups of rats. W/D ratio is an indicator for pulmonary edema, BLM-induced group of rats show a higher value of w/d ratio as compared with control group of rats, whereas in daidzein treated group of rats, w/d ratio was found to be lowered. Table 2 demonstrates the activities of SOD, CAT, GPx, GR, GSH, vitamin A, vitamin E and vitamin C in lung tissue homogenate of control and experimental group of rats. In BLM-induced groups, a significant depletion in the levels of SOD, CAT, GPx, GR, GSH, vitamin A, vitamin E and vitamin C were observed. However, upon treatment with daidzein, these levels were altered and reached closer to normal. The levels of oxidative stress parameters such as LPO and MPO are presented in Table 3. The result of this study shows the increased level of LPO in BLM-administered group, which might be due to tissue injury and damage. These levels are significantly lowered in daidzein treated group of rats. Daidzein alone group of rats exhibits similar value as that of control group. Similarly, the neutrophil infiltration indicator, MPO levels were also increased in BLM administered experimental group of rats. However, a remarkable descend in the levels of MPO was observed in daidzein group of rats as compared to BLM injured group of rats. Fig. 1 illustrates the histology of lung sections of control and experimental groups of rats. BLM administered animals (Fig. 1B)

The data was evaluated by using SPSS/11.5 software. Hypothesis testing method included one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. p < 0.05 was considered to indicate statistical significance. All the results were expressed as mean S.D. for six rats in each group.

2.11. Ultrastructural studies using transmission electron microscopy Upper and lower portions in lobes of right lungs of control and experimental groups of rats were randomly selected and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 18 h. The lungs were dissected into small pieces and postfixed for 1.5 h in 1% osmium tetroxide dissolved in 0.1 M phosphate buffer (pH 7.4), then dehydrated through a series of graded ethanol solutions and embedded in araldite (epoxy resin). Ultrathin sections were cut, stained with uranyl acetate and lead nitrate, mounted on copper grids and examined under a transmission electron microscope.

Table 1 Effect of Daidzein on body weight and Wet to Dry (W/D) ratio of control and experimental groups of rats. Parameters

Control

BLM

BLM + Daidzein

Daidzein alone

Initial body weight (g) Final body weight (g) W/D ratio

215.50 ± 3.72 229.83 ± 7.27 4.05 ± 0.47

218.66 ± 2.80 206.33 ± 11.80a 5.14 ± 0.68a

219.50 ± 3.50 225.50 ± 12.38b 4.55 ± 0.23b

215.33 ± 3.14 229.66 ± 6.91 3.92. ± 0.47c ns

Values are given as mean ± s.d for groups of six rats each. Values are given statistically significant at *p < 0.05, ns: non significant. a BLM-induced vs. control. b BLM + daidzein vs. BLM-induced. c Daidzein alone vs. control. Table 2 Enzymic and non-enzymic activities in Lungs of control and experimental groups of rats. Parameters

Control

SOD CAT GPx GR GSH Vitamin A Vitamin E Vitamin C

10.25 48.20 6.31 2.02 22.59 1.88 2.94 2.07

± ± ± ± ± ± ± ±

BLM 2.83 7.07 0.76 0.47 3.41 0.55 0.42 0.49

6.51 35.03 5.06 1.12 16.47 0.70 2.31 1.23

BLM + daidzen ± ± ± ± ± ± ± ±

0.72a * 8.60a * 0.51a * 0.40a * 3.04a * 0.53a * 0.22a * 0.15a *

8.55 43.89 6.10 1.79 20.27 1.57 2.61 1.74

± ± ± ± ± ± ± ±

0.46b * 2.74b * 0.29b * 0.21b * 1.39b * 0.52b * 0.18b * 0.13b *

Daidzein alone 10.61 50.29 6.24 1.99 21.61 1.85 3.10 2.19

± ± ± ± ± ± ± ±

1.21c 3.83c 0.41c 0.40c 2.70c 0.56c 0.23c 0.45c

ns ns ns ns ns ns ns ns

Values are given as mean ± s.d for groups of six rats each. Values are given statistically significant at *p < 0.05, ns: non significant. Activities are expressed as SOD-50% inhibition of adrenaline auto oxidation/min, CAT-␮M H2 O2 consumed/mg protein/min, GPx-␮g GSH utilized/mg protein/minute, GR-nmol NADPH oxidized/ mg protein/min, GSHmg/100g tissue, Vit A-␮g/mg protein, Vit E-␮g/mg protein, Vit C-␮g/mg protein. a BLM-induced vs. control. b BLM + daidzein vs. BLM-induced. c Daidzein alone vs. control.

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Table 3 Assessment of Lipid peroxidation and Myeloperoxidase activities in the lung tissues of control and experimental groups of rats. Parameters

Control

BLM

BLM + Daidzein

Daidzein alone

LPO MPO

2.71 ± 0.45 65.85 ± 4.34

4.44 ± 1.39a * 77.03 ± 8.06a *

2.94 ± 0.50b * 64.99 ± 2.26b *

2.72 ± 0.27c ns 65.58 ± 5.17c ns

Values are given as mean ± s.d for groups of 6 rats each. Values are given statistically significant at *p < 0.05, ns: non significant. Level of LPO expressed as nmol MDA formed/min/mg protein and MPO activity of lung tissue expressed in terms of mU/g of wet tissue/min. a BLM-induced vs. control. b BLM + daidzein vs. BLM-induced c Daidzein alone vs. control.

Fig. 1. Lung histology of control and experimental groups of rats stained with Hematoxylin & Eosin. Histopathological analysis of lung sections of control and experimental groups of rats observed in a light microscope at magnification 40×. A. Control rats showing normal lung histology without any pathological deformities. B. BLM-induced group displaying collapse of alveolar spaces and large number of leukocytes accumulated in alveolar walls. C. Less accumulation of leukocytes with clear alveolar spaces and corresponds to Daidzein treated group of rats. D. Daidzein alone treated group of rats shows normal lung histology.

show distorted architecture such as areas of increased alveolar thickening, leukocytes accumulation in alveolar walls and increased fibrosis, whereas in daidzein treated group (Fig. 1C) shows these conditions are prominently reduced as compared to BLM-induced group (Fig. 1B). The level of hydroxyproline in control and experimental groups of rats is depicted in Table 4. In BLM-induced group an increased level of hydroxyproline was observed, whereas upon treatment with daidzein restored the above level to closer to normal. Figs. 2 and 3 shows the Picrosirius red and the Masson’s trichrome staining respectively. Due to tissue injury, excess collagen has been deposited, as evident in Figs. 2B and 3B. However, daidzein group of rats exhibited predominant reduction of collagen deposition in the lung tissues Figs. 2C and 3C. Daidzein alone administered group represented in Figs. 2D and 3D which shows a similar pattern as observed in control group. The involvement of TNF-␣ in pulmonary fibrosis was studied by immunohistochemical staining analysis as depicted in Fig. 4. We have noticed that the expression of TNF-␣ was moderately higher in BLM-induced groups while daidzein administration caused a

markable reduction of TNF-␣ activity. Control groups showed a small amount of positive expression of TNF-␣. The expression of TNF-␣ in daidzein alone group shows negligible expression (Fig. 4D). The ultrastructural changes in BLM-induced group of rats shows bulging of mitochondria, distortion alveolar epithelium, ballooning

Table 4 Hydroxyproline content in the lung tissues of control and experimental groups of rats. Experimental groups

Hydroxyproline (mg/g dried tissue)

Control BLM BLM + Daidzein Daidzein alone

9.01 12.24 10.01 9.24

± ± ± ±

1.48 2.02a * 0.83b * 1.24c ns

Values are given as mean ± s.d for groups of six rats each. Values are given statistically significant at *p < 0.05, ns: non significant. a BLM-induced vs. control. b BLM + daidzein vs. BLM-induced. c Daidzein alone vs. control.

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Fig. 2. Assessment of collagen architecture by Picro-sirius red staining in control and experimental groups of rats. Tissue sections were stained with Picric acid and Sirius red (Direct red 80). Collagen was in yellow/orange colour when viewed under a polarized microscope at 20× magnification. A. Control section showing scarcely deposited collagen. B. BLM-induced group shows increased collagen deposition. C. Section of Daidzein treated group of rats shows comparatively less collagen deposition to BLM-induced group. D. Daidzein alone treated group shows normal lung histology.

Fig. 3. Assessment of collagen architecture by Masson’s trichrome Staining in control and experimental groups of rats. Masson’s trichrome staining analysis of lung sections of control and experimental groups of rats a light microscope at magnification 10×. A. Control rats showing normal lung histology. B. BLM-induced group showing collapse of alveolar spaces and dense deposition of collagen around the alveolar walls. C. Collagen accumulation is less corresponds to BLM and Daidzein treated rats. D. Daidzein alone treated group shows normal lung histology.

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Fig. 4. Immunohistochemical analysis of TNF-␣ expression in the lungs of control and experimental groups of rats at 10× magnification. A. Control rats showing normal histology. B. BLM-induced group of rats showing abundant TNF-␣ (indicated by arrows) expression. C. Daidzein treated group showing reduced TNF-␣ (brown colour) expression. D. Section of lung in daidzein alone group showing minimal TNF-␣ (indicated by arrows) expression. Quantification of TNF-␣ staining represents the number of stained (positive) cells per 10× field was averaged across 15 field for each rat section. Hypothesis testing method included one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test < 0.05 was considered to indicate statistical significance. Values are given as mean ± s.d. for groups of six rats each. Values are given statistically significant at *P < 0.05, ns: non significant, a BLM-induced vs. control; b BLM + daidzein vs. BLM-induced.

of endothelial cells, hemorrhage, increased collagen deposition and shrunken cell nucleus, which represents the other experimental models of lung damage. Daidzein treated to BLM-induced rats remarkably decreased all the above reflected ultrastructural changes. The improvement of these observable changes during daidzein administration shows its promising protective effect against BLM-induced pulmonary fibrosis (Fig. 5). 4. Discussion BLM, the antineoplastic agent can cause toxicity in generating inflammatory response and fibrosis in a short period time, and

this model is the well-established animal model for the study of human pulmonary fibrosis [41,42]. BLM causes severe lung damage by the up regulation of inflammatory cytokines, reduced lung volume, increased synthesis of hydroxyproline, accumulation of collagen in the lung airways and apoptosis of alveolar epithelial cells resulting in the respiratory failure and death [13,43]. The present study addresses the anti fibrotic effect of daidzein against BLM-induced pulmonary fibrosis in rats. BLM causes organ damage by various modes such as the depletion of antioxidant enzymes, activation of inflammatory cytokines, neutrophil recruitment, apoptosis in the alveolar epithelial cells [44,45]. Daidzein and its various health promoting effects such as free radical

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Fig. 5. Ultrastrutural studies using transmission electron microscopy in lung sections of control and experimental groups of rats. A. Lung tissues of control group of animals showing normal nuclei and cytoplasm (10,000×). B. Accumulation of collagen in BLM-induced group of rats (10,000×). C. Shrunken and dense abnormal nuclei in lungs of BLM-induced groups of rats (7,000×). D. Less endothelial injury in daidzein treated group of rats (10,000×). E. Type II epithelium irregularity is reduced in daidzein treated group of rats (10,000×). F. Normal architecture of nuclei is seen in daidzein alone groups of rats (15,000×).

scavenging, anti-inflammatory, chemo-preventive, anti-apoptotic has been reported earlier [46,47]. In this study, a marked reduction in the body weight was observed in the BLM-induced group of animals, which might be due to the progression of the fibrosis. BLM generates ROS through an iron dependent mechanism [48]. The ROS molecules are deleterious and damage the biomolecules such as protein, lipid and DNA, which leads to the loss of enzyme activity and membrane integrity [15]. It has been widely accepted that the anti-oxidant enzymes protects the cell from the toxic exogenous and endogenous compounds by

their free radical scavenging mechanism [49]. SOD is an abundant antioxidant enzyme found in the extracellular matrix of the lung, which protects the tissue from injury by catalyzing the dismutation of superoxide into oxygen and hydroperoxides [50]. Catalase is another antioxidant enzyme found in peroxisomes. This enzyme functions as the catalyst for the conversion of hydrogen peroxide, which formed previously by the dismutation of SOD, into water and molecular oxygen. Similarly, Gpx is also a powerful endogenous antioxidant enzyme, which contains the non-metallic element selenium. This enzyme protects the system from the harmful effects of

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free radicals by reducing these into alcohols and water. GSH is the non-protein thiol, which protects the cellular system from various free radicals. In this study, a marked depletion of SOD, CAT, GPx, GSH and GR was observed which might be due to the tissue injury caused by the free radicals. In the daidzein treated group of rats, shows a significant increase of these parameters were observed, which can be due to the anti oxidant activity of daidzein. Vitamin A plays a vital role to tone down the oxidative stress mediated by lipid peroxidation and also recognized to repair the damaged tissues [51,52]. Vitamin E functions as the lipid-soluble antioxidant of biological membranes and lipoproteins, furthermore known to decrease collagen deposition and protect lungs from oxidative damage [53–55]. Vitamin C is another physiologically important antioxidant with various metabolic activities and exhibits protective effects on tissues from lipid peroxidation both in vivo and in vitro [56,57]. Vitamins A, E and C levels were depleted due to the oxidative stress generated upon BLM induction. These levels were regulated by daidzein treatment, suggesting allevation over oxidative stress. It is known that the depletion of antioxidant defenses and/or raise in free radical production deteriorates the prooxidantantioxidant balance, leading to oxidative stress-induced cell death [58]. In this study, significant rise of LPO level was observed in BLMinduced lung tissues. It has been reported that lipid peroxidation is known to agitate the integrity of cellular membranes, leading to the outflow of cytoplasmic enzymes [59]. Myeloperoxidase, a heme enzyme that is abundantly present in the cytoplasmic granules of neutrophils is an indicator of polymorpho nuclear leukocyte accumulation [60]. MPO is activity is linked to both influx of inflammatory cells and oxidative stress [61,62]. BLM-induced animals exhibited increased lipid peroxidation and MPO activity in the lung tissue when compared to the control animals. During the daidzein treatment, a reduction in LPO level and MPO activity were observed, that might be due to the antioxidant potential of the drug. In this study, we have observed marked structural distortion of the alveolar space with collapsed alveolae, interalveolar inflammation and thickened alveolar wall and abnormal collagen deposition in BLM-induced rats. The histological results of the present study clearly demonstrate that daidzein due to its antioxidant potency, exerts a significant attenuation of the extent and severity of the histological signs of tissue damage caused by BLM. BLM-induced group of rats produced significant increase in lung hydroxyproline content compared to control group of rats. Since, the aminoacid 4-hydroxyproline is the precursor for collagen, the estimation of the amino acid following acid digestion of collagen would be a good biochemical index of collagen content. This result is in accordance with previous findings, which demonstrated remarkable increment in lung hydroxyproline content as an index of collagen accumulation and deposition [63–66]. This finding was further confirmed by collagen specific studies using Masson’s trichrome staining and Picro-sirius red staining analysis of lung sections for collagen deposition. In picro-sirius red staining studies, BLM-induced group of rats show abnormal collagen deposition and accumulation in peribronchial and perialveolar tissues, which can be visualized under polarized light. Daidzein administered group portrayed the intensity of collagen deposition considerably reduced, which might be due to the anti-fibrotic effect of daidzein. TNF-␣, a potent pro-inflammatory cytokine, acts as one major molecule among the multifaceted networks of cellular and molecular interactions that regulate the fibrotic process [44]. In this study, a significant elevation in the TNF-␣ expression was observed in BLM-administered groups of animals. The tissue injury caused by BLM is found to be inflammation mediated and which might be due to the production of free radicals during BLM induction that possibly leads to activation of NF-␬B and increase in the synthesis

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of TNF-␣ [43,67]. Administration of daidzein substantially reduced the expression of TNF-␣. Transmission electron microscopic studies reveal the ultra structural changes in control and BLM-induced group of rats, where BLM-induced group exhibited the endothelial injury, hemorrhage, dense collagen fibers, ballooning of mitochondria, abnormal endoplasmic reticulum and either dense, shrunken or elongated nuclei. This data is in accordance with the previous reports with respect to the experimental models of pulmonary fibrosis [68]. However, treatment with daidzein to BLM-induced groups of rats remarkably reduced the above stated ultra structural changes. In conclusion, the results of this study demonstrate that daidzein possesses anti-fibrotic effect against BLM-induced pulmonary fibrosis in rats. Further studies are in progress to elucidate the mechanism by which daidzein protects lungs against fibrosis.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

Acknowledgements The authors thank Dr. Upendra Nongthomba, Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India for his help in utilizing polarization microscope. We also thank Mrs.Rita Rajan, Department of Gastrointestinal sciences, Christian Medical College, Vellore, India for her assistance in Transmission electron microscopic studies.

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