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Protective effect of Berberis vulgaris fruit extract against Paraquat-induced pulmonary fibrosis in rats
Biomedicine & Pharmacotherapy 81 (2016) 329–336
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Protective effect of Berberis vulgaris fruit extract against Paraquat-induced pulmonary fibrosis in rats Seyed Ali Javad-Mousavia , Ali Asghar Hemmatib , Saeed Mehrzadic , Azam Hosseinzadehc , Gholamreza Houshmandb , Mohammad Reza Rashidi Nooshabadib , Mehrnaz Mehrabanid , Mehdi Goudarzib,e,* a
Department of Internal Medicine, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran c Department of Pharmacology, Iran University of Medical Sciences, Tehran, Iran d Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran e Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2015 Received in revised form 9 April 2016 Accepted 11 April 2016
Background: Pulmonary fibrosis induced by paraquat (PQ) has caused a large number of human fatalities all over the world, especially in Asian region. The main potential mechanism of PQ toxicity has been thought to be mediated by ROS. The present study was designed to evaluate the efficacy of the Berberis vulgaris fruit extract (BVFE) against PQ—induced pulmonary fibrosis in rats. Methods: Forty male rats were randomly divided into five experimental groups each containing eight rats. Groups 1 and 2, served as a negative and positive control and received a single dose of intratracheal instillation of saline and PQ (20 mg/kg), respectively. Groups 3–5 were treated with different doses of BVFE (100, 200, 400 mg/kg/day, orally) 1 week before the PQ injection and continued for 3 weeks. The rats were sacrificed 21 days after PQ. Malondialdehyde (MDA), Hydroxyproline, inflammatory and fibrogenic cytokine tumor necrosis factor (TNF)-a, interleukin (IL)-6 and transforming growth factor (TGF)-b1 in lung tissue were determined. Presence of fibrosis, inflammatory cells, connective tissue and collagen deposition in lung were evaluated microscopically by hematoxylin and eosin (H&E) staining. Dried extract was standardized by amount of berberine by HPTLC methods by silica gel plate. Results: The results showed that PQ could significantly increase the lung MDA, hydroxyproline, TNF-a, IL6 and TGF-b1 levels. BVFE ameliorated the biochemical and histological lung alterations induced by PQ. Conclusions: The present study indicates the hydroalcolic extract of Berberis vulgaris fruit has beneficial effects in rat pulmonary fibrosis induced by PQ in a dose-dependent manner, possibly by anti-oxidant and anti- inflammatory properties, which might be due to its berberine alkaloid content. ã 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Pulmonary fibrosis Paraquat Berberis vulgaris Oxidative stress Inflammation Rats
1. Introduction Pulmonary fibrosis is one of the main interstitial or diffuse parenchyma, lung diseases is characterized by a numerical expansion of fibroblasts that they synthesize excessive amounts of extracellular matrix, including collagen. Pulmonary fibrosis may result from a variety of acute and chronic diseases, including the idiopathic interstitial pneumonias, chronic inflammatory processes (sarcoidosis, Wegener’s granulomatosis), infections,
* Corresponding author at: Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. E-mail address: [email protected] (M. Goudarzi). http://dx.doi.org/10.1016/j.biopha.2016.04.027 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
environmental agents, exposure to ionizing radiation and certain medications [1,2]. Although current therapies for fibrotic lung diseases are ineffective and lung transplantation is a viable option for patients with end-stage pulmonary fibrosis, control and prevention of inflammatory events might delay progress of the fibrotic events. Although the precise mechanisms that drive the numeric expansion of fibroblasts and collagen accumulation in pulmonary fibrosis remain incompletely understood, tissue fibrosis is often viewed as the aberrant wound healing following sequential lung injuries. Inflammation and immune mechanisms, oxidative stress and oxidative signaling, and procoagulant mechanisms may be responsible for the combination of altered lung fibroblasts, loss of alveolar epithelial cells, and excessive accumulation of extracellular matrix (ECM) that can initiate the
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tissue damages and result in pulmonary fibrosis [3–5]. Many xenobiotics, including paraquat (PQ) [6], butyrated hydroxytoluene [7] and Amiodarone [8] and bleomycin [9–11] are capableof producing lung fibrosis by stimulating the overproduction of ROS, such as superoxide (O2), hydrogen peroxide (H2O2), peroxinitrite (ONOO), and hydroxyl radical (HO) that they are major mediators of lung inflammatory processes [12]. Among them, PQ (1, 10 dimethyl-4, 40 -bipyridinium dichloride) is an effective and nonselective quaternary nitrogen herbicide, which is widely used in many countries for broadleaf weed control. However, acute PQ poisoning by accidental and/or voluntary ingestion of commercial liquid formulations of PQ has caused a large number of human fatalities, especially in Asian region. Although PQ has toxic effects on various organs, including liver, kidney, heart and central nervous system, the lung is the primary target organ in PQ poisoning and death mostly occurs due to lung damage and pulmonary fibrosis. PQ is mainly accumulated in the lung through the process by which active polyamine is transported into the Clara cells and alveolar type I and II epithelial cells [13–16]. The mechanisms of PQ cytotoxicity have not been fully explained. The main potential mechanism of PQ toxicity has been thought to be mediated by ROS produced by the enzymatic one-electron reduction of PQ, which generates O2 anions and other free radicals that interact with membrane lipids leading to cell death and lung tissue damage. Since there are no specific antidotes or effective treatment for PQ, the mortality rate of PQ poisoning has remained high [17,18]. Berberis vulgaris is a shrub belongs to the Berberidaceae family. It grows in central and southern Europe, northwest Africa, and western Asia. Dried fruit of Berberis vulgaris known as Zereshk or Sereshk in Iran, which used as condiment and cultivated in the south of Khorasan province [19]. Different parts of this plant (root, bark, leaf and fruit) have extensively been used in traditional medicine for the treatment and prevention of various diseases including discomforts of cardiovascular, respiratory, kidneys, urinary and gastrointestinal tract, skin and infectious diseases [20]. Recent studies have demonstrated that Berberis vulgaris extracts possess a plethora of biological activities including antiarrhythmic, antibacterial, anticholinergic, antihistaminic, antihypertensive, anti-inflammatory, antinociceptive, vasodilatory, anti-tumor effects [19–24]. It seems that these effects are due to its content of bioactive compounds such as are isoquinoline alkaloids such as berbamine, palmatine, and particularly berberine. A number of studies have suggested that Berberis species has antioxidant and free radical scavenger activity [25–28]. The present investigation was carried out with the objective of evaluating the protective effects of Berberis vulgaris fruit extract (BVFE) against PQ-induced pulmonary fibrosis in rats. 2. Materials and methods 2.1. Chemicals Berberine hydrochloride, Trichloro acetic acid (TCA), Thiobarbituric acid (TBA), Bovine Serum Albumin (BSA), Hydroxyproline (HP), chloramine T and p-dimethylaminobenzaldehyde and Bradford reagent were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO), USA. PQ was purchased from Afrashimi Co. (Iran). All chemicals and reagents used were analytical grade. 2.2. Animals Forty male Sprague-Dawley strain rats, 8 weeks old, weighting 200 25 g were obtained animal house and research center of Jundishapur University of Medical Sciences, Ahvaz, Iran. Rats were kept in polypropylene cages at a controlled condition of
temperature (25 2 C) with a 12 h light: 12 h dark cycle. The animals were given standard rat chow and drinking water ad libitum. All experimental procedures were conducted according to the ethical standards and protocols approved by the Committee of Animal Experimentation of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 2.3. Extract preparation Berberis vulgaris fruit (BVF) were purchased in Ahvaz, southwest of Iran. Fruit were identified at department of pharmacognosy, School of Pharmacy, Ahvaz, Iran. The fruit (500 g) was soaked in a 70% aqueous ethanol solution in a large container for 3 days with occasional shaking. The extract was filtered through a clean cotton cloth and then was dried by using a rotary evaporator at 40 C. 2.4. HPTLC assay Dried extract was standardized by amount of berberine by HPTLC (Camag, Switzerland) methods by silica gel plate (20 10 cm 60 F 254 E MERCK KGaA co). Mobile phase n.propanol, water, formic acid (80:10:4). Standard of berberine (Sigma chemical Co, USA) was prepared in seven dilutions and calibrate for detection. 2.5. Experimental design The animals were randomly divided into five experimental groups each containing eight rats. Groups 1 and 2, served as a negative and positive control and received a single dose of intratracheal instillation of saline and PQ (20 mg/kg), respectively. Groups 3–5 were treated with different doses of BVFE (100, 200, 400 mg/kg/day, orally) 1 week before the PQ injection and continued for 3 weeks. The rats were sacrificed 21 days after PQ. 2.6. Tissue collection At the end of the treatment course, all rats were weighed and killed with a lethal dose (120 mg/kg, i.p.) of sodium pentobarbitone. After mid-line sternotomy, whole lung (include both lobes) was dissected out, separated from other tissues, and washed free of blood with ice-cold saline, then the whole lung weight was recorded and placed in a sterile plastic petri dish. One part of the right lung was fixed in formalin for histological examination, and the remaining lung tissues were immediately removed and washed in normal saline solution and frozen in liquid nitrogen. 2.7. Body weight and lung index In the course of the experiment, the body weight of rats was measured every 7 days. After sacrifice, the lung index was expressed as the ratio of wet lung weight (mg) to body weight (g). 2.8. Hydroxyproline (HP) assay The HP content of the lung tissue was determined using a colorimetric assay described else ware [29]. Briefly, 100 mg samples were homogenized in 6 molar HCl and hydrolyzed for five hours at 130 C. The pH was adjusted to 6.5–7.0 with NaOH, and the sample volume was adjusted to 30 ml with distilled water. The sample solution (1.0 ml) was mixed with 1.0 ml of chloramine T solution (0.05 mol/L), and then the mixture was incubated at room temperature for 20 min. When 1.0 ml of 20% dimethyl benzaldehyde solution was added, the mixture was incubated at 60 C for 20 min. The absorbance of each sample at 550 nm was measured. The results were calculated as mg HP per g wet lung weight using HP standards.
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2.9. Lipid peroxidation assay The lipid peroxidation was expressed by measuring the amounts of MDA via the TBA color reaction by the method described by Buege and Aust [30]. Briefly, 0.5 ml of homogenate (prepared with 0.1 M Tris–HCl buffer (pH 7.4) at 4 C) was mixed with 2.5 ml of TCA (10%, w/v), the samples were centrifuged at 3000 rpm for 10 min and 2 ml of each sample supernatant was transferred to a test tube containing 1 ml of TBA solution (0.67%, w/v). The mixture was kept in boiling water for 10 min, forming a pink color solution. The mixture was then cooled immediately and the absorbance was measured at 532 nm by spectrophotometer. The concentration of MDA was calculated based on the absorbance coefficient of the TBA–MDA complex (e = 1.56 105 cm1 M1). 2.10. Measurement of TNF-a, IL-6, and TGF-b1 levels The samples were homogenized in Tris–HCl buffer (pH = 7.4) containing protease inhibitors (trypsin and other serine and cysteine proteases). All homogenized samples were centrifuged (20,000g, 4 C) in a refrigerated centrifuge for 20 min and the supernatant was taken and frozen at 80 C. Supernatant samples were thawed and analyzed for murine cytokine TNF-a, IL-6, and TGF-b1 levels using specific ELISA kits (eBioscience). For the expression of results of certain parameters, the protein content was estimated using bovine serum albumin as the standard protein. Cytokine concentrations in the samples were expressed as pg cytokine/mg of protein.
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changes in each histological section of the lung was assessed as the mean score of severity from observing microscopic fields. Twenty five fields in each rat lung section were analyzed. After examination of the whole fields of the section, the mean of the scores from all fields was considered as the fibrotic score. The entire lung section was reviewed at a magnification of 400. A score ranging from zero (normal lung) to eight (total fibrosis) was assigned. The mean score of all fields was taken as a fibrosis score of that lung section. Criteria for grading pulmonary fibrosis were as follows: A grade 0 normal lung; 1 = minimal fibrosis thickening of alveolar or bronchial walls; 2–3 = moderate thickening of the walls without obvious damage to the lung architecture; 4–5 = increased fibrosis with definite damage to lung architecture and formation of fibrotic bands or small fibrotic mass; 6–7 = severe distortion of structure and large fibrotic areas; “honeycomb lung” was placed in this category and grade 8 indicates a total fibrotic change of the field. The mean score of all fields was taken as the fibrosis score of that lung section [31]. 2.12. Statistical analysis The results are reported as mean SD. The statistical analyses were performed using one-way analysis of variance (ANOVA) by SPSS (v.20). Group differences were calculated by post hoc analysis using Tukey test. For all tests, differences with values of P < 0.05 were considered significant. 3. Results
2.11. Histological studies 3.1. HPTLC results Rat lungs on day 21 after PQ administration were inflated with a buffered 10% formalin solution for 24 h and embedded in paraffin. Sections (3 mm) were stained with hematoxylin and eosin for histological examination. The Ashcroft score was used for semiquantitative assessment of fibrotic changes. The severity of fibrotic
Determination amount of berberine in Berberis vulgaris hydroalcoholic extract (BVHE)showed many ingredients in BVHE that amount of berberine was measured as 0.763 percent of dried extract contains berberine (Fig. 1).
Fig. 1. HPTLC of amount of berberine. HPTLC chromatogram scanned at UV 254 nm. Regression via height: Polynomial Y = 276.6 + 10.03 X+(0.0476) X2 r = 0.93988, sdv = 17.32 Regression via area: Polynomial Y = 4189 + 337.4 X +(1.129) X2, r = 0.99275, sdv = 10.14.
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3.2. Effects of BVFE and PQ on lung indices The lung index (wet lung weight/body weight) in the PQ group was significantly increased compared with the control group on day 28 (p < 0.001). However, the rats received BVFE (200 and 400 mg/kg) significantly lower lung index compared with the PQ group (p < 0.01 and p < 0.001, respectively) (Fig. 2). 3.3. Effects of BVFE and PQ on HP content of lung tissue HP is the main component of collagen protein in the body. We measured the HP content as a marker of fibrosis in the lung tissue. As shown in Fig. 3, the HP content in lung of the PQ-treated mice significantly increased compared with the control group (p < 0.001). Administration of BVFE (200 and 400 mg/kg) significantly reduced the content of HP in lung tissue (p < 0.05 and p < 0.001, respectively). These findings were consistent with the histological results. 3.4. Effects of BVFE and PQ on MDA levels Results showed that a significant rise in MDA level in lung of rats exposed to PQ which is an index of lipid peroxidation, when compared with the control group (p < 0.001). As shown in Fig. 4, the decrease in MDA level was observed in pretreated rats by BVFE (200 and 400 mg/kg) (p < 0.001).
Fig. 3. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on hydroxyproline content in the lungs of rats with pulmonary fibrosis. Each column represents mean SEM. ### P < 0.001 compared to the control group; *p < 0.05, ***p < 0.001 compared to the PQ group.
3.5. Effect of BVFE and PQ on the levels of TNF-a, IL-6 and TGF-b1 in lung tissues To evaluate the effect of BVFE on PQ-induced lung injury, we measured the levels of important proinflammatory and profibrotic cytokines TNF-a, IL-6 and TGF-b1 in lung tissues (Fig. 5). Administration of PQ caused significant elevation of tissue levels of TNF-a, IL-6 and TGF-b1 as compared with those in the control group. Treatment of rats with BVFE in three doses of 100,200 and 50 mg/kg significantly reduced PQ-induced production of TNF-a, IL-6 and TGF-b1 compared to the PQ group in a dose-dependent manner. 3.6. The light microscopic findings Photomicrographic evaluation of pulmonary fibrosis by infiltration of fibroblast, inflammatory cells and extracellular matrix
Fig. 4. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on MDA in the lungs of rats with pulmonary fibrosis. Each column represents mean SEM. ## P < 0.01, ###P < 0.001 compared to the control group; ***p < 0.001 compared to the PQ group.
showed grade 0 for control and grade 8 more prominent for PQ group. The rats pretreated by Berberis vulgaris showed grade 6– 7 for the dose of 100 mg/kg of Berberis vulgaris and grade 4–5 more evident for the lung photomicrographs in the dose of 200 and 400 mg/kg of BVFE (Fig. 6). 4. Discussion Fig. 2. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on Lung Index of rats with pulmonary fibrosis. Each column represents mean S.E.M. # P < 0.05, ###P < 0.001 compared to the control group; **p < 0.01, *** p < 0.001 compared to the PQ group.
Acute respiratory distress syndrome (ARDS) is the major cause of death after PQ poisoning, which clinical course of it divided into three phases: (1) an early inflammatory phase characterized by
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Fig. 5. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on PQ-induced production of proinflammatory cytokines TNF-a, IL-6 and TGF-b1 in lung tissues of rats. Each column represents mean SEM. # P < 0.05, ###P < 0.001 compared to the control group; *P < 0.05, **P < 0.01, ***p < 0.001 compared to the PQ group.
damage and destruction of alveolar epithelial, pulmonary edema, and infiltration of inflammatory cells, (2) proliferative phase with a process of pneumocyte and fibroblast proliferation, and (3) phase of collagen deposition and massive pulmonary interstitial fibrosis (PIF) [32,33]. It is clear that inflammation response is a major component in the pathogenesis of interstitial lung fibrosis and inflammatory cytokines, particularly TNF-a, TGF-b1, interferon (IFN)-c, platelet-derived growth factor (PDGF), interleukin (IL)-1, and IL-6 are found to be involved in the initiation and development of this process [34,35]. TGF-b1 is a multifunction cytokine, which not only stimulates the proliferation and differentiation of fibroblasts, synthesizes extracellular matrix, suppresses collagen degradation, enhances collagen deposition but also promotes inflammatory responses by releasing related inflammatory cells during the development of lung fibrosis [36]. It is well known that abundant oxygen free radicals such as HO, O2, H2O2, nitrite oxide (NO) and ONOO may participate in sophisticated process of PQ toxicity [18,37,38]. PQ is metabolized by several enzyme systems (NADPH-cytochrome P450 reductase, xanthine oxidase, NADHubiquinone oxidoreductase and nitric oxide synthase), which leads to generation of PQ mono-cation radical (PQ+) that Inside the cell, it is rapidly reoxidized by oxygen to PQ2+ with the concomitant production of O2. O2 anion could be transformed by various reactions, for example SOD can catalyze the dismutation of the highly reactive O2 anion to O2 and to the less reactive species H2O2. H2O2 may then be reduced to HO in the presence of iron via the Fenton reaction. PQ also directly or indirectly induces NOS (NO synthase)-mediated NO production and NO may react with O2 to generate ONOO, a very strong oxidant and a nitrating agent, which react with several biomolecules leading to enormous implications in biological process. It is well known that reactive oxygen species can activate nuclear factor kappa B (NF-kB) that in normal condition it is bound to an inhibitory protein (IkBa). The active form of NF-kappa B rapidly translocates into the nucleus where it binds to specific sequences of DNA called response elements (RE) and induces target genes involved in inflammation, such as inflammatory enzymes, cytokines and chemokines leading to platelet adhesion and aggregation, fibrogenesis and attraction of inflammatory cells. Free radicals directly or indirectly damage membrane components of the cells and it is one of the production of reactive oxygen species, which monitored by measuring MDA level. It is shown that PQ causes increased contents of MDA, an index of lipid peroxidation, and decreased activities of SOD and GSH-PX, three oxidant stress markers that are considered to reflect the injured levels of oxygen radicals [39–44]. The results of our study were consistent with these reports. Our data confirmed that all rats received a single dose of intratracheal PQ (20 mg/kg)
presented a typical pattern of pulmonary fibrosis, which was correlated with alveolar thickening associated with fibroblasts and myofibroblasts proliferation that this histopathological examination was accompanied by an increase in lung weight, MDA level and hydroxyproline as an index of the amount of collagen. However, our data showed that the BVHE induced a significant decrease in lung weight, MDA and collagen amounts in a dosedependent manner in rats pulmonary fibrosis induced by PQ. Since ancient times, the extracts of various Berberidaceae (Berberis aquifolium, Berberis vulgaris and Berberis aristata) have been administered as a valuable natural resource of traditional remedy for the treatment of chronic inflammatory disorders while Berberis vulgaris consumption has been approved by FDA due to its great amount of phenolic and flavonoids compounds, which have significant antioxidant and cytoprotective activities [21,44–46]. These components were found to possess significant reductive ability and radicals scavenging activity and are able to absorb and neutralize free radicals, quench singlet and triplet oxygen, or decompose peroxides. In previous studies it was shown that Berberis vulgaris extract is able to increase CAT, SOD and GPX activities and is able to increase GSH content probably by stimulation of g-glutamyl cycle and/or enzymatic regeneration of GSH from oxidized glutathione [47,48]. Also, El-Wahab et al. have shown that Berberis vulgaris extract is a potent inhibitor for hepatocytes' lipid peroxidation induced by Fe2+ and H2O2 and can reduce the generation of NO in a concentration dependent manner [49]. In this way its ability to scavenge free radicals and reduce NO production leads to inhibition of ONOO generation. It was shown Berberis vulgaris ethanolic extract induced apoptosis in MCF-7 cells that it might be due to its antioxidant and free radical scavenging activities [50]. Minaiyan et al. investigated the protective effect of Berberis vulgaris fruit extract (orally and rectally) on acute colitis induced by acetic acid and they saw that Berberis vulgaris fruit extract, which administered orally, was effective to reduce total colitis index and they suggested that this effect may be attributed to the its anthocyanin constituents or may be mediated by its berberine alkaloid content, which could be attributed to this alkaloid possible potential in activation of mechanism that resembles glucocorticoides and/or raising blood corticosterone levels [51]. In another study, Majeed et al. evaluated gastro protective activity of B. vulgaris seed powder. They saw B. vulgaris was efficacious to reduce aspirin induced gastro toxicity in male adult albino mice. They suggested this effect of B. vulgaris mainly is due to its antioxidant potential [52]. Also, it has shown that Berberis vulgaris has anti-inflammatory effect that it may be attributed to the presence of phenolic compounds in Berberis vulgaris extract [53].
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Fig. 6. The figures are representatives of the lungs from animals in each treatment group. Lung tissues were stained 21 days after administration of PQ in saline with H&E. Appearance of rat lungs in different groups: (A) control (B) PQ (C) PQ treated with 100 mg/kg Berberis vulgaris fruit extract (D) PQ treated with 200 mg/kg Berberis vulgaris fruit extract and (E) PQ treated with 400 mg/kg Berberis vulgaris fruit extract. Lung parenchyma of control is well preserved and intact. Extensive interstitial infiltration and fibrosis showing in group (B). A marked prevention of the PQ-induced histological changes was seen in the rat lungs in groups (D) and (E).
Hermenean et al. showed that Berberis vulgaris can reduce infiltration of inflammatory cells induced by carbon tetrachloride (CCl4) in the liver and can protect hepatocytes against liver injuries induced by CCl4 [54].
Also, it was shown that in patients with non-alcoholic fatty liver disease, Berberis vulgaris extract can reduces liver enzymes, triglycerides and cholesterol [55]. The results obtained in the present study have shown that the hydroalcolic extract of Berberis vulgaris has beneficial effects in rat
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pulmonary fibrosis induced by PQ in a dose-dependent manner. However, future researches will be needed to clarify the mechanism(s) and the other active compound(s) involved in this effect. 5. Conclusion The results revealed that hydroalcoholic extract of Berberis vulgaris might be able to diminish the fibrogenic effects of PQ on lung. This effect of Berberis vulgaris can be attributed to active ingredients of the plant with anti-oxidant and anti-inflammatory properties. Conflict of interest There is no conflict of interest to be reported. Acknowledgment This paper financially supported by a grant of Student Research Committee of Ahvaz Jundishapur University of Medical Sciences. Grant Number (92s74). References [1] M.I. Schwarz, T.E. King, Interstitial Lung Dis., PMPH, USA, 2003. [2] M. Wilson, T. Wynn, Pulmonary fibrosis: pathogenesis, etiology and regulation, Mucosal Immunol. 2 (2) (2009) 103–121. [3] T.J. Gross, G.W. Hunninghake, Idiopathic pulmonary fibrosis, N. Engl. J. Med. 345 (7) (2001) 517–525. [4] C. Kroegel, B. Mock, U. Hengst, A. Reissig, Interferon-g-1b: therapeutic option in advanced idiopathic pulmonary fibrosis? Respiration 71 (6) (2004) 656–657. [5] N.W. Todd, I.G. Luzina, S.P. Atamas, Molecular and cellular mechanisms of pulmonary fibrosis, Fibrogenesis Tissue Rep. 5 (1) (2012) 11. [6] M.J. Khodayar, M. Kiani, A.A. Hemmati, A. Rezaie, M.R. Zerafatfard, M.R. Rashidi Nooshabadi, M. Goudarzi, The preventive effect of atorvastatin on paraquatinduced pulmonary fibrosis in the rats, Adv. Pharm. Bull. 4 (4) (2014) 345–349. [7] I. Adamson, D. Bowden, M. Cote, H. Witschi, Lung injury induced by butylated hydroxytoluene: cytodynamic and biochemical studies in mice, Lab. Investig. 36 (1) (1977) 26–32. [8] A. Hemmati, N. Zaeemzadeh, A. Arzi, T. Jalali, I. Rashidi, Protective effect of caffeic acid phenethyl ester (CAPE) on amiodarone-Induced pulmonary fibrosis in rat, Iran. J. Pharm. Res. (2011) 321–328. [9] I. Javadi, M. Rashidi Nooshabadi, M. Goudarzi, R. Roudbari, Protective effects of celery (Apium graveloens) seed extract on bleomycin-Induced pulmonary fibrosis in rats, J. Babol Univ. Med. Sci. 17 (2015) 70–76. [10] A.A. Hemmati, A. Rezaie, P. Darabpour, Preventive effects of pomegranate seed extract on bleomycin-induced pulmonary fibrosis in rat, Jundishapur, J. Nat. Pharm. Prod. 8 (2013) 76. [11] A.A. Hemmati, N. Aghel, Z. Nazari, B. Mohammadian, N. Hasanvand, Protective effect of grape seed extract against the fibrogenic effect of bleomycin in rat lung, Iran. J. Pharm. Sci. 2 (2006) 143–150. [12] V.L. Kinnula, J.D. Crapo, K.O. Raivio, Generation and disposal of reactive oxygen metabolites in the lung, Lab. Investig. 73 (1) (1995) 3. [13] H. Forman, T.K. Aldrich, M.A. Posner, A.B. Fisher, Differential paraquat uptake and redox kinetics of rat granular pneumocytes and alveolar macrophages, J. Pharmacol. Exp. Ther. 221 (2) (1982) 428–433. [14] H. Naito, M. Yamashita, Epidemiology of paraquat in Japan and a new safe formulation of paraquat, Hum. Exp. Toxicol. 6 (1) (1987) 87–88. [15] M.S. Rose, L.L. Smith, I. Wyatt, Evidence for energy-dependent accumulation of paraquat into rat lung, Nature (1974). [16] N.A. van der Wal, J.F. van Oirschot, A. van Dijk, J. Verhoef, B.S. Van Asbeck, Mechanism of protection of alveolar type II cells against paraquat-induced cytotoxicity by deferoxamine, Biochem. Pharmacol. 39 (11) (1990) 1665–1671. [17] D. Bonneh-Barkay, S.H. Reaney, W.J. Langston, D.A. Di Monte, Redox cycling of the herbicide paraquat in microglial cultures, Mol. Brain Res. 134 (1) (2005) 52–56. [18] Z.E. Suntres, Role of antioxidants in paraquat toxicity, Toxicology 180 (1) (2002) 65–77. [19] M. Fatehi, T.M. Saleh, Z. Fatehi-Hassanabad, K. Farrokhfal, M. Jafarzadeh, S. Davodi, A pharmacological study on Berberis vulgaris fruit extract, J. Ethnopharmacol. 102 (1) (2005) 46–52. [20] M. Imanshahidi, H. Hosseinzadeh, Pharmacological and therapeutic effects of Berberis vulgaris and its active constituent, berberine, Phytother. Res. 22 (8) (2008) 999–1012. [21] N. Ivanovska, S. Philipov, Study on the anti-inflammatory action of Berberis vulgaris root extract, alkaloid fractions and pure alkaloids, Int. J. Immunopharmacol. 18 (10) (1996) 553–561.
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Golkho, Using HPLC to determination the composition and antioxidant activity of Berberis vulgaris, Eur. J. Sci. Res. 29 (1) (2009) 47–54. [28] G. Motalleb, Evaluation of phenolic contentand total antioxidant activity in Berberis vulgaris fruit extracts, J. Biol. Sci. (2005). [29] G. Kesava Reddy, C.S. Enwemeka, A simplified method for the analysis of hydroxyproline in biological tissues, Clin. Biochem. 29 (3) (1996) 225–229. [30] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–310. [31] T. Ashcroft, J.M. Simpson, V. Timbrell, Simple method of estimating severity of pulmonary fibrosis on a numerical scale, J. Clin. Pathol. 41 (4) (1988) 467–470. [32] L.L. Smith, M.S. Rose, A comparison of the effects of paraquat and diquat on the water content of rat lung and the incorporation of thymidine into lung DNA, Toxicology 8 (2) (1977) 223–230. [33] G. Vijeyaratnam, B. 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Orhan, Some physicochemical characteristics, bioactive content and antioxidant characteristics of non-Sprayed barberry (Berberis vulgaris L.) fruits from Turkey, Erwerbs-Obstbau 56 (4) (2014) 123–129. [49] A.E.A. El-Wahab, D.A. Ghareeb, E.E. Sarhan, M.M. Abu-Serie, M.A. El Demellawy, In vitro biological assessment of berberis vulgaris and its active constituent, berberine: antioxidants, anti-acetylcholinesterase, anti-diabetic and anticancer effects, BMC Complement. Altern. Med. 13 (1) (2013) 218. [50] Z. Hoshyar, A.Zarban Mahboob, The antioxidant and chemical properties of Berberis vulgaris and its cytotoxic effect on human breast carcinoma cells, Cytotechnology (2015) 1–7. [51] M. Minaiyan, A. Ghannadi, P. Mahzouni, E. Jaffari-Shirazi, Comparative study of Berberis vulgaris fruit extract and berberine chloride effects on acetic acidinduced colitis in rats, Iran. J. Pharm. Res. (2011) 97–104. [52] W. Majeed, B. Aslam, I. Javed, T. Khaliq, F. Muhammad, A. Ali, A. 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Protective effect of Berberis vulgaris fruit extract against Paraquat-induced pulmonary fibrosis in rats Seyed Ali Javad-Mousavia , Ali Asghar Hemmatib , Saeed Mehrzadic , Azam Hosseinzadehc , Gholamreza Houshmandb , Mohammad Reza Rashidi Nooshabadib , Mehrnaz Mehrabanid , Mehdi Goudarzib,e,* a
Department of Internal Medicine, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran c Department of Pharmacology, Iran University of Medical Sciences, Tehran, Iran d Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran e Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2015 Received in revised form 9 April 2016 Accepted 11 April 2016
Background: Pulmonary fibrosis induced by paraquat (PQ) has caused a large number of human fatalities all over the world, especially in Asian region. The main potential mechanism of PQ toxicity has been thought to be mediated by ROS. The present study was designed to evaluate the efficacy of the Berberis vulgaris fruit extract (BVFE) against PQ—induced pulmonary fibrosis in rats. Methods: Forty male rats were randomly divided into five experimental groups each containing eight rats. Groups 1 and 2, served as a negative and positive control and received a single dose of intratracheal instillation of saline and PQ (20 mg/kg), respectively. Groups 3–5 were treated with different doses of BVFE (100, 200, 400 mg/kg/day, orally) 1 week before the PQ injection and continued for 3 weeks. The rats were sacrificed 21 days after PQ. Malondialdehyde (MDA), Hydroxyproline, inflammatory and fibrogenic cytokine tumor necrosis factor (TNF)-a, interleukin (IL)-6 and transforming growth factor (TGF)-b1 in lung tissue were determined. Presence of fibrosis, inflammatory cells, connective tissue and collagen deposition in lung were evaluated microscopically by hematoxylin and eosin (H&E) staining. Dried extract was standardized by amount of berberine by HPTLC methods by silica gel plate. Results: The results showed that PQ could significantly increase the lung MDA, hydroxyproline, TNF-a, IL6 and TGF-b1 levels. BVFE ameliorated the biochemical and histological lung alterations induced by PQ. Conclusions: The present study indicates the hydroalcolic extract of Berberis vulgaris fruit has beneficial effects in rat pulmonary fibrosis induced by PQ in a dose-dependent manner, possibly by anti-oxidant and anti- inflammatory properties, which might be due to its berberine alkaloid content. ã 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Pulmonary fibrosis Paraquat Berberis vulgaris Oxidative stress Inflammation Rats
1. Introduction Pulmonary fibrosis is one of the main interstitial or diffuse parenchyma, lung diseases is characterized by a numerical expansion of fibroblasts that they synthesize excessive amounts of extracellular matrix, including collagen. Pulmonary fibrosis may result from a variety of acute and chronic diseases, including the idiopathic interstitial pneumonias, chronic inflammatory processes (sarcoidosis, Wegener’s granulomatosis), infections,
* Corresponding author at: Department of Pharmacology and Toxicology, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. E-mail address: [email protected] (M. Goudarzi). http://dx.doi.org/10.1016/j.biopha.2016.04.027 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
environmental agents, exposure to ionizing radiation and certain medications [1,2]. Although current therapies for fibrotic lung diseases are ineffective and lung transplantation is a viable option for patients with end-stage pulmonary fibrosis, control and prevention of inflammatory events might delay progress of the fibrotic events. Although the precise mechanisms that drive the numeric expansion of fibroblasts and collagen accumulation in pulmonary fibrosis remain incompletely understood, tissue fibrosis is often viewed as the aberrant wound healing following sequential lung injuries. Inflammation and immune mechanisms, oxidative stress and oxidative signaling, and procoagulant mechanisms may be responsible for the combination of altered lung fibroblasts, loss of alveolar epithelial cells, and excessive accumulation of extracellular matrix (ECM) that can initiate the
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tissue damages and result in pulmonary fibrosis [3–5]. Many xenobiotics, including paraquat (PQ) [6], butyrated hydroxytoluene [7] and Amiodarone [8] and bleomycin [9–11] are capableof producing lung fibrosis by stimulating the overproduction of ROS, such as superoxide (O2), hydrogen peroxide (H2O2), peroxinitrite (ONOO), and hydroxyl radical (HO) that they are major mediators of lung inflammatory processes [12]. Among them, PQ (1, 10 dimethyl-4, 40 -bipyridinium dichloride) is an effective and nonselective quaternary nitrogen herbicide, which is widely used in many countries for broadleaf weed control. However, acute PQ poisoning by accidental and/or voluntary ingestion of commercial liquid formulations of PQ has caused a large number of human fatalities, especially in Asian region. Although PQ has toxic effects on various organs, including liver, kidney, heart and central nervous system, the lung is the primary target organ in PQ poisoning and death mostly occurs due to lung damage and pulmonary fibrosis. PQ is mainly accumulated in the lung through the process by which active polyamine is transported into the Clara cells and alveolar type I and II epithelial cells [13–16]. The mechanisms of PQ cytotoxicity have not been fully explained. The main potential mechanism of PQ toxicity has been thought to be mediated by ROS produced by the enzymatic one-electron reduction of PQ, which generates O2 anions and other free radicals that interact with membrane lipids leading to cell death and lung tissue damage. Since there are no specific antidotes or effective treatment for PQ, the mortality rate of PQ poisoning has remained high [17,18]. Berberis vulgaris is a shrub belongs to the Berberidaceae family. It grows in central and southern Europe, northwest Africa, and western Asia. Dried fruit of Berberis vulgaris known as Zereshk or Sereshk in Iran, which used as condiment and cultivated in the south of Khorasan province [19]. Different parts of this plant (root, bark, leaf and fruit) have extensively been used in traditional medicine for the treatment and prevention of various diseases including discomforts of cardiovascular, respiratory, kidneys, urinary and gastrointestinal tract, skin and infectious diseases [20]. Recent studies have demonstrated that Berberis vulgaris extracts possess a plethora of biological activities including antiarrhythmic, antibacterial, anticholinergic, antihistaminic, antihypertensive, anti-inflammatory, antinociceptive, vasodilatory, anti-tumor effects [19–24]. It seems that these effects are due to its content of bioactive compounds such as are isoquinoline alkaloids such as berbamine, palmatine, and particularly berberine. A number of studies have suggested that Berberis species has antioxidant and free radical scavenger activity [25–28]. The present investigation was carried out with the objective of evaluating the protective effects of Berberis vulgaris fruit extract (BVFE) against PQ-induced pulmonary fibrosis in rats. 2. Materials and methods 2.1. Chemicals Berberine hydrochloride, Trichloro acetic acid (TCA), Thiobarbituric acid (TBA), Bovine Serum Albumin (BSA), Hydroxyproline (HP), chloramine T and p-dimethylaminobenzaldehyde and Bradford reagent were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO), USA. PQ was purchased from Afrashimi Co. (Iran). All chemicals and reagents used were analytical grade. 2.2. Animals Forty male Sprague-Dawley strain rats, 8 weeks old, weighting 200 25 g were obtained animal house and research center of Jundishapur University of Medical Sciences, Ahvaz, Iran. Rats were kept in polypropylene cages at a controlled condition of
temperature (25 2 C) with a 12 h light: 12 h dark cycle. The animals were given standard rat chow and drinking water ad libitum. All experimental procedures were conducted according to the ethical standards and protocols approved by the Committee of Animal Experimentation of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 2.3. Extract preparation Berberis vulgaris fruit (BVF) were purchased in Ahvaz, southwest of Iran. Fruit were identified at department of pharmacognosy, School of Pharmacy, Ahvaz, Iran. The fruit (500 g) was soaked in a 70% aqueous ethanol solution in a large container for 3 days with occasional shaking. The extract was filtered through a clean cotton cloth and then was dried by using a rotary evaporator at 40 C. 2.4. HPTLC assay Dried extract was standardized by amount of berberine by HPTLC (Camag, Switzerland) methods by silica gel plate (20 10 cm 60 F 254 E MERCK KGaA co). Mobile phase n.propanol, water, formic acid (80:10:4). Standard of berberine (Sigma chemical Co, USA) was prepared in seven dilutions and calibrate for detection. 2.5. Experimental design The animals were randomly divided into five experimental groups each containing eight rats. Groups 1 and 2, served as a negative and positive control and received a single dose of intratracheal instillation of saline and PQ (20 mg/kg), respectively. Groups 3–5 were treated with different doses of BVFE (100, 200, 400 mg/kg/day, orally) 1 week before the PQ injection and continued for 3 weeks. The rats were sacrificed 21 days after PQ. 2.6. Tissue collection At the end of the treatment course, all rats were weighed and killed with a lethal dose (120 mg/kg, i.p.) of sodium pentobarbitone. After mid-line sternotomy, whole lung (include both lobes) was dissected out, separated from other tissues, and washed free of blood with ice-cold saline, then the whole lung weight was recorded and placed in a sterile plastic petri dish. One part of the right lung was fixed in formalin for histological examination, and the remaining lung tissues were immediately removed and washed in normal saline solution and frozen in liquid nitrogen. 2.7. Body weight and lung index In the course of the experiment, the body weight of rats was measured every 7 days. After sacrifice, the lung index was expressed as the ratio of wet lung weight (mg) to body weight (g). 2.8. Hydroxyproline (HP) assay The HP content of the lung tissue was determined using a colorimetric assay described else ware [29]. Briefly, 100 mg samples were homogenized in 6 molar HCl and hydrolyzed for five hours at 130 C. The pH was adjusted to 6.5–7.0 with NaOH, and the sample volume was adjusted to 30 ml with distilled water. The sample solution (1.0 ml) was mixed with 1.0 ml of chloramine T solution (0.05 mol/L), and then the mixture was incubated at room temperature for 20 min. When 1.0 ml of 20% dimethyl benzaldehyde solution was added, the mixture was incubated at 60 C for 20 min. The absorbance of each sample at 550 nm was measured. The results were calculated as mg HP per g wet lung weight using HP standards.
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2.9. Lipid peroxidation assay The lipid peroxidation was expressed by measuring the amounts of MDA via the TBA color reaction by the method described by Buege and Aust [30]. Briefly, 0.5 ml of homogenate (prepared with 0.1 M Tris–HCl buffer (pH 7.4) at 4 C) was mixed with 2.5 ml of TCA (10%, w/v), the samples were centrifuged at 3000 rpm for 10 min and 2 ml of each sample supernatant was transferred to a test tube containing 1 ml of TBA solution (0.67%, w/v). The mixture was kept in boiling water for 10 min, forming a pink color solution. The mixture was then cooled immediately and the absorbance was measured at 532 nm by spectrophotometer. The concentration of MDA was calculated based on the absorbance coefficient of the TBA–MDA complex (e = 1.56 105 cm1 M1). 2.10. Measurement of TNF-a, IL-6, and TGF-b1 levels The samples were homogenized in Tris–HCl buffer (pH = 7.4) containing protease inhibitors (trypsin and other serine and cysteine proteases). All homogenized samples were centrifuged (20,000g, 4 C) in a refrigerated centrifuge for 20 min and the supernatant was taken and frozen at 80 C. Supernatant samples were thawed and analyzed for murine cytokine TNF-a, IL-6, and TGF-b1 levels using specific ELISA kits (eBioscience). For the expression of results of certain parameters, the protein content was estimated using bovine serum albumin as the standard protein. Cytokine concentrations in the samples were expressed as pg cytokine/mg of protein.
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changes in each histological section of the lung was assessed as the mean score of severity from observing microscopic fields. Twenty five fields in each rat lung section were analyzed. After examination of the whole fields of the section, the mean of the scores from all fields was considered as the fibrotic score. The entire lung section was reviewed at a magnification of 400. A score ranging from zero (normal lung) to eight (total fibrosis) was assigned. The mean score of all fields was taken as a fibrosis score of that lung section. Criteria for grading pulmonary fibrosis were as follows: A grade 0 normal lung; 1 = minimal fibrosis thickening of alveolar or bronchial walls; 2–3 = moderate thickening of the walls without obvious damage to the lung architecture; 4–5 = increased fibrosis with definite damage to lung architecture and formation of fibrotic bands or small fibrotic mass; 6–7 = severe distortion of structure and large fibrotic areas; “honeycomb lung” was placed in this category and grade 8 indicates a total fibrotic change of the field. The mean score of all fields was taken as the fibrosis score of that lung section [31]. 2.12. Statistical analysis The results are reported as mean SD. The statistical analyses were performed using one-way analysis of variance (ANOVA) by SPSS (v.20). Group differences were calculated by post hoc analysis using Tukey test. For all tests, differences with values of P < 0.05 were considered significant. 3. Results
2.11. Histological studies 3.1. HPTLC results Rat lungs on day 21 after PQ administration were inflated with a buffered 10% formalin solution for 24 h and embedded in paraffin. Sections (3 mm) were stained with hematoxylin and eosin for histological examination. The Ashcroft score was used for semiquantitative assessment of fibrotic changes. The severity of fibrotic
Determination amount of berberine in Berberis vulgaris hydroalcoholic extract (BVHE)showed many ingredients in BVHE that amount of berberine was measured as 0.763 percent of dried extract contains berberine (Fig. 1).
Fig. 1. HPTLC of amount of berberine. HPTLC chromatogram scanned at UV 254 nm. Regression via height: Polynomial Y = 276.6 + 10.03 X+(0.0476) X2 r = 0.93988, sdv = 17.32 Regression via area: Polynomial Y = 4189 + 337.4 X +(1.129) X2, r = 0.99275, sdv = 10.14.
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3.2. Effects of BVFE and PQ on lung indices The lung index (wet lung weight/body weight) in the PQ group was significantly increased compared with the control group on day 28 (p < 0.001). However, the rats received BVFE (200 and 400 mg/kg) significantly lower lung index compared with the PQ group (p < 0.01 and p < 0.001, respectively) (Fig. 2). 3.3. Effects of BVFE and PQ on HP content of lung tissue HP is the main component of collagen protein in the body. We measured the HP content as a marker of fibrosis in the lung tissue. As shown in Fig. 3, the HP content in lung of the PQ-treated mice significantly increased compared with the control group (p < 0.001). Administration of BVFE (200 and 400 mg/kg) significantly reduced the content of HP in lung tissue (p < 0.05 and p < 0.001, respectively). These findings were consistent with the histological results. 3.4. Effects of BVFE and PQ on MDA levels Results showed that a significant rise in MDA level in lung of rats exposed to PQ which is an index of lipid peroxidation, when compared with the control group (p < 0.001). As shown in Fig. 4, the decrease in MDA level was observed in pretreated rats by BVFE (200 and 400 mg/kg) (p < 0.001).
Fig. 3. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on hydroxyproline content in the lungs of rats with pulmonary fibrosis. Each column represents mean SEM. ### P < 0.001 compared to the control group; *p < 0.05, ***p < 0.001 compared to the PQ group.
3.5. Effect of BVFE and PQ on the levels of TNF-a, IL-6 and TGF-b1 in lung tissues To evaluate the effect of BVFE on PQ-induced lung injury, we measured the levels of important proinflammatory and profibrotic cytokines TNF-a, IL-6 and TGF-b1 in lung tissues (Fig. 5). Administration of PQ caused significant elevation of tissue levels of TNF-a, IL-6 and TGF-b1 as compared with those in the control group. Treatment of rats with BVFE in three doses of 100,200 and 50 mg/kg significantly reduced PQ-induced production of TNF-a, IL-6 and TGF-b1 compared to the PQ group in a dose-dependent manner. 3.6. The light microscopic findings Photomicrographic evaluation of pulmonary fibrosis by infiltration of fibroblast, inflammatory cells and extracellular matrix
Fig. 4. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on MDA in the lungs of rats with pulmonary fibrosis. Each column represents mean SEM. ## P < 0.01, ###P < 0.001 compared to the control group; ***p < 0.001 compared to the PQ group.
showed grade 0 for control and grade 8 more prominent for PQ group. The rats pretreated by Berberis vulgaris showed grade 6– 7 for the dose of 100 mg/kg of Berberis vulgaris and grade 4–5 more evident for the lung photomicrographs in the dose of 200 and 400 mg/kg of BVFE (Fig. 6). 4. Discussion Fig. 2. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on Lung Index of rats with pulmonary fibrosis. Each column represents mean S.E.M. # P < 0.05, ###P < 0.001 compared to the control group; **p < 0.01, *** p < 0.001 compared to the PQ group.
Acute respiratory distress syndrome (ARDS) is the major cause of death after PQ poisoning, which clinical course of it divided into three phases: (1) an early inflammatory phase characterized by
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Fig. 5. Effect of pretreatment with BVFE at the doses of 100, 200 and 400 mg/kg on PQ-induced production of proinflammatory cytokines TNF-a, IL-6 and TGF-b1 in lung tissues of rats. Each column represents mean SEM. # P < 0.05, ###P < 0.001 compared to the control group; *P < 0.05, **P < 0.01, ***p < 0.001 compared to the PQ group.
damage and destruction of alveolar epithelial, pulmonary edema, and infiltration of inflammatory cells, (2) proliferative phase with a process of pneumocyte and fibroblast proliferation, and (3) phase of collagen deposition and massive pulmonary interstitial fibrosis (PIF) [32,33]. It is clear that inflammation response is a major component in the pathogenesis of interstitial lung fibrosis and inflammatory cytokines, particularly TNF-a, TGF-b1, interferon (IFN)-c, platelet-derived growth factor (PDGF), interleukin (IL)-1, and IL-6 are found to be involved in the initiation and development of this process [34,35]. TGF-b1 is a multifunction cytokine, which not only stimulates the proliferation and differentiation of fibroblasts, synthesizes extracellular matrix, suppresses collagen degradation, enhances collagen deposition but also promotes inflammatory responses by releasing related inflammatory cells during the development of lung fibrosis [36]. It is well known that abundant oxygen free radicals such as HO, O2, H2O2, nitrite oxide (NO) and ONOO may participate in sophisticated process of PQ toxicity [18,37,38]. PQ is metabolized by several enzyme systems (NADPH-cytochrome P450 reductase, xanthine oxidase, NADHubiquinone oxidoreductase and nitric oxide synthase), which leads to generation of PQ mono-cation radical (PQ+) that Inside the cell, it is rapidly reoxidized by oxygen to PQ2+ with the concomitant production of O2. O2 anion could be transformed by various reactions, for example SOD can catalyze the dismutation of the highly reactive O2 anion to O2 and to the less reactive species H2O2. H2O2 may then be reduced to HO in the presence of iron via the Fenton reaction. PQ also directly or indirectly induces NOS (NO synthase)-mediated NO production and NO may react with O2 to generate ONOO, a very strong oxidant and a nitrating agent, which react with several biomolecules leading to enormous implications in biological process. It is well known that reactive oxygen species can activate nuclear factor kappa B (NF-kB) that in normal condition it is bound to an inhibitory protein (IkBa). The active form of NF-kappa B rapidly translocates into the nucleus where it binds to specific sequences of DNA called response elements (RE) and induces target genes involved in inflammation, such as inflammatory enzymes, cytokines and chemokines leading to platelet adhesion and aggregation, fibrogenesis and attraction of inflammatory cells. Free radicals directly or indirectly damage membrane components of the cells and it is one of the production of reactive oxygen species, which monitored by measuring MDA level. It is shown that PQ causes increased contents of MDA, an index of lipid peroxidation, and decreased activities of SOD and GSH-PX, three oxidant stress markers that are considered to reflect the injured levels of oxygen radicals [39–44]. The results of our study were consistent with these reports. Our data confirmed that all rats received a single dose of intratracheal PQ (20 mg/kg)
presented a typical pattern of pulmonary fibrosis, which was correlated with alveolar thickening associated with fibroblasts and myofibroblasts proliferation that this histopathological examination was accompanied by an increase in lung weight, MDA level and hydroxyproline as an index of the amount of collagen. However, our data showed that the BVHE induced a significant decrease in lung weight, MDA and collagen amounts in a dosedependent manner in rats pulmonary fibrosis induced by PQ. Since ancient times, the extracts of various Berberidaceae (Berberis aquifolium, Berberis vulgaris and Berberis aristata) have been administered as a valuable natural resource of traditional remedy for the treatment of chronic inflammatory disorders while Berberis vulgaris consumption has been approved by FDA due to its great amount of phenolic and flavonoids compounds, which have significant antioxidant and cytoprotective activities [21,44–46]. These components were found to possess significant reductive ability and radicals scavenging activity and are able to absorb and neutralize free radicals, quench singlet and triplet oxygen, or decompose peroxides. In previous studies it was shown that Berberis vulgaris extract is able to increase CAT, SOD and GPX activities and is able to increase GSH content probably by stimulation of g-glutamyl cycle and/or enzymatic regeneration of GSH from oxidized glutathione [47,48]. Also, El-Wahab et al. have shown that Berberis vulgaris extract is a potent inhibitor for hepatocytes' lipid peroxidation induced by Fe2+ and H2O2 and can reduce the generation of NO in a concentration dependent manner [49]. In this way its ability to scavenge free radicals and reduce NO production leads to inhibition of ONOO generation. It was shown Berberis vulgaris ethanolic extract induced apoptosis in MCF-7 cells that it might be due to its antioxidant and free radical scavenging activities [50]. Minaiyan et al. investigated the protective effect of Berberis vulgaris fruit extract (orally and rectally) on acute colitis induced by acetic acid and they saw that Berberis vulgaris fruit extract, which administered orally, was effective to reduce total colitis index and they suggested that this effect may be attributed to the its anthocyanin constituents or may be mediated by its berberine alkaloid content, which could be attributed to this alkaloid possible potential in activation of mechanism that resembles glucocorticoides and/or raising blood corticosterone levels [51]. In another study, Majeed et al. evaluated gastro protective activity of B. vulgaris seed powder. They saw B. vulgaris was efficacious to reduce aspirin induced gastro toxicity in male adult albino mice. They suggested this effect of B. vulgaris mainly is due to its antioxidant potential [52]. Also, it has shown that Berberis vulgaris has anti-inflammatory effect that it may be attributed to the presence of phenolic compounds in Berberis vulgaris extract [53].
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Fig. 6. The figures are representatives of the lungs from animals in each treatment group. Lung tissues were stained 21 days after administration of PQ in saline with H&E. Appearance of rat lungs in different groups: (A) control (B) PQ (C) PQ treated with 100 mg/kg Berberis vulgaris fruit extract (D) PQ treated with 200 mg/kg Berberis vulgaris fruit extract and (E) PQ treated with 400 mg/kg Berberis vulgaris fruit extract. Lung parenchyma of control is well preserved and intact. Extensive interstitial infiltration and fibrosis showing in group (B). A marked prevention of the PQ-induced histological changes was seen in the rat lungs in groups (D) and (E).
Hermenean et al. showed that Berberis vulgaris can reduce infiltration of inflammatory cells induced by carbon tetrachloride (CCl4) in the liver and can protect hepatocytes against liver injuries induced by CCl4 [54].
Also, it was shown that in patients with non-alcoholic fatty liver disease, Berberis vulgaris extract can reduces liver enzymes, triglycerides and cholesterol [55]. The results obtained in the present study have shown that the hydroalcolic extract of Berberis vulgaris has beneficial effects in rat
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pulmonary fibrosis induced by PQ in a dose-dependent manner. However, future researches will be needed to clarify the mechanism(s) and the other active compound(s) involved in this effect. 5. Conclusion The results revealed that hydroalcoholic extract of Berberis vulgaris might be able to diminish the fibrogenic effects of PQ on lung. This effect of Berberis vulgaris can be attributed to active ingredients of the plant with anti-oxidant and anti-inflammatory properties. Conflict of interest There is no conflict of interest to be reported. Acknowledgment This paper financially supported by a grant of Student Research Committee of Ahvaz Jundishapur University of Medical Sciences. Grant Number (92s74). References [1] M.I. Schwarz, T.E. King, Interstitial Lung Dis., PMPH, USA, 2003. [2] M. Wilson, T. Wynn, Pulmonary fibrosis: pathogenesis, etiology and regulation, Mucosal Immunol. 2 (2) (2009) 103–121. [3] T.J. Gross, G.W. Hunninghake, Idiopathic pulmonary fibrosis, N. Engl. J. Med. 345 (7) (2001) 517–525. [4] C. Kroegel, B. Mock, U. Hengst, A. Reissig, Interferon-g-1b: therapeutic option in advanced idiopathic pulmonary fibrosis? Respiration 71 (6) (2004) 656–657. [5] N.W. Todd, I.G. Luzina, S.P. Atamas, Molecular and cellular mechanisms of pulmonary fibrosis, Fibrogenesis Tissue Rep. 5 (1) (2012) 11. [6] M.J. Khodayar, M. Kiani, A.A. Hemmati, A. Rezaie, M.R. Zerafatfard, M.R. Rashidi Nooshabadi, M. Goudarzi, The preventive effect of atorvastatin on paraquatinduced pulmonary fibrosis in the rats, Adv. Pharm. Bull. 4 (4) (2014) 345–349. [7] I. Adamson, D. Bowden, M. Cote, H. Witschi, Lung injury induced by butylated hydroxytoluene: cytodynamic and biochemical studies in mice, Lab. Investig. 36 (1) (1977) 26–32. [8] A. Hemmati, N. Zaeemzadeh, A. Arzi, T. Jalali, I. Rashidi, Protective effect of caffeic acid phenethyl ester (CAPE) on amiodarone-Induced pulmonary fibrosis in rat, Iran. J. Pharm. Res. (2011) 321–328. [9] I. Javadi, M. Rashidi Nooshabadi, M. Goudarzi, R. Roudbari, Protective effects of celery (Apium graveloens) seed extract on bleomycin-Induced pulmonary fibrosis in rats, J. Babol Univ. Med. Sci. 17 (2015) 70–76. [10] A.A. Hemmati, A. Rezaie, P. Darabpour, Preventive effects of pomegranate seed extract on bleomycin-induced pulmonary fibrosis in rat, Jundishapur, J. Nat. Pharm. Prod. 8 (2013) 76. [11] A.A. Hemmati, N. Aghel, Z. Nazari, B. Mohammadian, N. Hasanvand, Protective effect of grape seed extract against the fibrogenic effect of bleomycin in rat lung, Iran. J. Pharm. Sci. 2 (2006) 143–150. [12] V.L. Kinnula, J.D. Crapo, K.O. Raivio, Generation and disposal of reactive oxygen metabolites in the lung, Lab. Investig. 73 (1) (1995) 3. [13] H. Forman, T.K. Aldrich, M.A. Posner, A.B. Fisher, Differential paraquat uptake and redox kinetics of rat granular pneumocytes and alveolar macrophages, J. Pharmacol. Exp. Ther. 221 (2) (1982) 428–433. [14] H. Naito, M. Yamashita, Epidemiology of paraquat in Japan and a new safe formulation of paraquat, Hum. Exp. Toxicol. 6 (1) (1987) 87–88. [15] M.S. Rose, L.L. Smith, I. Wyatt, Evidence for energy-dependent accumulation of paraquat into rat lung, Nature (1974). [16] N.A. van der Wal, J.F. van Oirschot, A. van Dijk, J. Verhoef, B.S. Van Asbeck, Mechanism of protection of alveolar type II cells against paraquat-induced cytotoxicity by deferoxamine, Biochem. Pharmacol. 39 (11) (1990) 1665–1671. [17] D. Bonneh-Barkay, S.H. Reaney, W.J. Langston, D.A. Di Monte, Redox cycling of the herbicide paraquat in microglial cultures, Mol. Brain Res. 134 (1) (2005) 52–56. [18] Z.E. Suntres, Role of antioxidants in paraquat toxicity, Toxicology 180 (1) (2002) 65–77. [19] M. Fatehi, T.M. Saleh, Z. Fatehi-Hassanabad, K. Farrokhfal, M. Jafarzadeh, S. Davodi, A pharmacological study on Berberis vulgaris fruit extract, J. Ethnopharmacol. 102 (1) (2005) 46–52. [20] M. Imanshahidi, H. Hosseinzadeh, Pharmacological and therapeutic effects of Berberis vulgaris and its active constituent, berberine, Phytother. Res. 22 (8) (2008) 999–1012. [21] N. Ivanovska, S. Philipov, Study on the anti-inflammatory action of Berberis vulgaris root extract, alkaloid fractions and pure alkaloids, Int. J. Immunopharmacol. 18 (10) (1996) 553–561.
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