Epigallocatechin gallate potentially abrogates fluoride

International Immunopharmacology 39 (2016) 128–139

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Epigallocatechin gallate potentially abrogates fluoride induced lung oxidative stress, inflammation via Nrf2/Keap1 signaling pathway in rats: An in-vivo and in-silico study Thangapandiyan Shanmugam a,⁎, Miltonprabu Selvaraj a, Senthilraja Poomalai b a b

Department of Zoology, Annamalai University, Annamalainagar 608002, Tamilnadu, India Divisions of Bioinformatics, Annamalai University, Annamalainagar 608002, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 9 April 2016 Received in revised form 4 July 2016 Accepted 20 July 2016 Available online xxxx Keywords: EGCG NaF Lung ROS Nrf2 Rat

a b s t r a c t Background: Since this Nrf2-dependent cellular defense response is able to protect multi-organs, including cancer, neurodegenerative diseases, cardiovascular diseases, inflammation and chronic lung injury. The antioxidant and anti-inflammatory potential of Epigallocatechin gallate (EGCG) and Nrf2/Keap1 signaling mechanisms in pulmonary toxicity have not been clarified. In the present study, we demonstrated that protective efficacy of EGCG against fluoride (Fl) induced oxidative stress mediated lung injury in rats. Methods: The animals were divided in to four groups. Group 1: Control rats received normal saline; Group 2 rats received EGCG (40 mg/kg/bw) alone for four weeks; Group 3 rats received Fl (25 mg/kg/bw) alone for four weeks, Group 4 rats received EGCG (90 min before administration) along with Fl for four weeks. Results: Oral administration of Fl (25 mg/kg/bw) significantly (p b 0.05) increased the ROS, inflammatory cytokines, lung edema, melonaldehyde (MDA) and myeloperoxidase (MPO) in rats. In addition, upon administration of Fl significantly (p b 0.05) decreased the antioxidant status, Nrf2, and HO-1 with increased Keap1 protein. Histological and immunohistochemical (iNOS) study also revealed the Fl induced significant (p b 0.05) changes in the lung tissue of rats. Pre-administration of EGCG significantly (p b 0.05) improved the antioxidant status, and inhibited the oxidative stress, inflammatory cytokines, and Keap1 protein via the activation of Nrf2 translocation in to the nucleus. Moreover, the molecular docking studies also support the antioxidant potential of EGCG and Nrf2 activation. Conclusion: Taken together, our data indicate that EGCG potentially abrogates Fl induced oxidative lung injury by activation of the Nrf2/Keap1 pathway in rats. © 2016 Published by Elsevier B.V.

1. Introduction Fluorine is one of the trace elements in environment when amounts exceeding accumulates in hard and soft tissues, where it disturbs metabolic processes and produces obvious changes in subsequent morphological pictures [1,2]. Fluoride exposure has been associated with asthmatic symptoms among workers in the aluminum industry [3]. Phosphate ore production and aluminum manufacture are the major industrial sources of environmental fluoride pollution. The use of fluoride containing pesticides and combustion of the coal and fuel also contribute to fluoride dispersion. These processes result in accumulation of fluoride compounds in the surface waters and groundwater reserves, air, soils, and in the living organisms [4]. The high affinity of fluoride compounds to numerous chemical elements makes them highly disruptive to metabolic processes conditioned by these elements. The most ⁎ Corresponding author at: Department of Health and Nutrition, Suguna Institute of Poultry Management, Udumalpet 642147, Tamil Nadu, India. E-mail address: [email protected] (T. Shanmugam).

http://dx.doi.org/10.1016/j.intimp.2016.07.022 1567-5769/© 2016 Published by Elsevier B.V.

important adverse effect of fluorides is the inhibition of Kreb's cycle enzymes. Thus, they affect the metabolic pathways of carbohydrates, lipids and proteins. The impact of fluorides upon carbohydrate metabolism occurs via the inhibition of glycolysis, whereas the influence of fluorides upon protein metabolism causes a decrease of protein levels in serum, skeletal muscles, liver and the lung [1,5]. Fluoride can induce the formation of free radicals (ROS) and disturb the battle of antioxidative systems [1,6]. The disturbance of oxidoreductive processes leads to oxidative stress, which damages the metabolism of all compounds found in a cell, even the cell's genome [7]. Lung is a site of major ROS production because it is having a large surface that is constantly contact with air oxygen and pollutants [8]. Furthermore, several studies have shown that the interaction of the lung system with ROS might be associated with the development of several lung diseases [9,10]. The fluoride induced oxidative stress mediated pulmonary disease has become a global epidemic and is likely to become the third largest cause of death worldwide [6]. For a long time it has been contemplated that fluoride induced ROS are involved in the pathogenesis of many diseases and pathologic processes in lungs [5].

T. Shanmugam et al. / International Immunopharmacology 39 (2016) 128–139

However, recently there is growing evidence that fluoride induced ROS play an important part in the complex physiological processes such as cell signaling, apoptosis, etc. [5]. In the cytoplasmic membrane of lung contain enzymes such as NADPH oxidase, which generates large amounts of superoxide radicals (O2) during oxidative stress. Another enzyme contributing to the complex defense mechanism is myeloperoxidase, which catalyzes the reaction of H2O2 radicals [11]. Uncontrolled elucidation of fluoride induced ROS drastically damage the antioxidant defense system in the lungs and leads to cell death [12]. Natural antioxidants can help to conquer the oxidative stress and free radical-induced disorders. Numerous side effects of synthetic antioxidants have been reported previously. Therefore, attention has been recently paid to find natural antioxidants with lower side effects. Green tea catechin, especially epigallocatechin gallate (EGCG), is known to be the most potent antioxidant among all catechins [13]. Epidemiological and intervention studies indicate that consumption of 5– 6 or more cups of green tea, containing 200–300 mg EGCG, per day may be beneficial for maintaining normal health status [14]. EGCG acts as a scavenger of many reactive oxygen/nitrogen species (ROS/RNS) such as superoxide radical anion, peroxyl and hydroxyl radicals, singlet oxygen, nitric oxide and peroxynitrite. EGCG may trap peroxyl radicals and thus break the chain reaction of free radicals and terminate lipid peroxidation. An electron paramagnetic resonance (EPR) study on tea catechins indicated that each molecule of EGCG has the ability to trap six superoxide anion or hydroxyl radicals [15]. The NrF2/Keap-1 signaling pathway plays a key role in the regulation cell's survivability by improving natural antioxidant levels in the body under stress condition. In addition, several recent studies have shown that polyphenols from green tea, especially EGCG significantly improved the quality of wound healing and remarkably suppressed A549 cancer cells proliferation in human lungs [16]. Therefore, the principle objective of this study is to investigate the protective effects of EGCG on fluoride-induced oxidative injuries, by assessing biochemical and histological aspect in lungs. Further, this study attempted to identify the possible mechanism of Nrf2-Keap1 signaling pathway underlying the protective effects of EGCG against Fl induced oxidative changes in lungs.

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the animals were cared in accordance with the “Guide for the care and use of laboratory animals” and “Committee for the purpose of control and supervision on experimental animals.” 2.3. Experimental design In the present study, fluoride (Fl) was administered orally (gastric intubation) as sodium fluoride (NaF) at a dose of 25 mg/kg body weight/day for 4 weeks, which was 1/10 of the oral LD50 values in rats [17]. Control rats (Normal group 1) received the vehicles (saline) only. The experimental rats were subdivided into (Group 3 and Group 4). Drug control group (EGCG group 2) received only EGCG (40 mg/ kg. b.w/day) (dissolved in 10% of Tween 80) alone. In the experiment, a total of 24 rats were used. The rats were randomly divided into 4 groups of 6 animals in each.

2. Materials and methods 2.1. Chemicals Sodium fluoride (NaF), Epigallocatechin gallate (EGCG), bovine serum albumin were purchased from Sigma Chemical Co., St. Louis, MO, USA. Bax, Bcl-2, Nrf2, HO1, and Keap1 antibodies were purchased from Santa-cruz Biotechnology, Inc., USA. All other chemicals and solvents were of certified analytical grade and purchased from S.D. Fine Chemicals, Mumbai or Himedia Laboratories Pvt. Ltd., Mumbai, India. Reagent kits were obtained from span Diagnostics, Mumbai, India. 2.2. Animals and diet Healthy adult male albino rats of Wistar strain, bred and reared in Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, and Annamalai University were used for the experiment. Males were preferred to avoid complications of the estrous cycle. Animals of equal weight (160–180 g) were selected and housed in polypropylene cages lined with husk and kept in a semi natural light/dark condition (12 h light/12 h dark). The animals had free access to water and were supplied with standard pellet diet (Amrut Laboratory Animal Feed, Pranav Agro Industries Ltd., Bangalore, India), constitution of protein (22.21%), fat (3.32%), fiber (3.11%), balanced with carbohydrates (N67%), vitamins and minerals. Animal handling and experimental procedures were approved by the Institutional Animal Ethics Committee, Annamalai University (Registration Number: 952/2012/CPCSEA) and

Fig. 1. Effect of EGCG on Fl induced lung superoxide radicals (1A), hydroxyl radical (1B) and hydrogen peroxide (1C) of control and experimental rats. Values are mean ± SD for 6 rats in each group; a, b and c Values are not sharing a common superscript letter (a, b and c) differ significantly at p b 0.05 (DMRT).

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Fig. 2. Effect of EGCG on Fl induced lung inflammatory cytokine TNF-α (2A), IL-β1 (2B), IL-6 (2C), and CINC-3 (2D) in control and treated rats. Values are mean ± SD for 6 rats in each group; a, b and c Values are not sharing a common superscript letter (a, b and c) differ significantly at p b 0.05 (DMRT).

Fig. 3. Effect of EGCG on Fl induced lung oxidative stress marker MDA (3A) and MPO (3B) and non-enzymatic antioxidant GSH (3C) and Vit. E (3D) and Lung edema (wet-to-dry lung weight ratio) (3E) in control and experimental rats. Values are mean ± SD for 6 rats in each group; a, b and c Values are not sharing a common superscript letter (a, b and c) differ significantly at p b 0.05 (DMRT).


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Group 1: (n = 6) Control rats received the normal saline and the vehicle for 4 weeks. Group 2: (n = 6) Rats administered with EGCG (40 mg/kg. b.w/day) dissolved in Tween 80. for 4 weeks. Group 3: (n = 6) Rats administered with NaF (25 mg/kg. b.w/day) dissolved in normal saline for 4 weeks. Group 4: (n = 6) EGCG (40 mg/kg. b.w/day) dissolved in Tween 80 was administrated 90 min before administration of NaF (25 mg/kg. b.w/ day) for 4 weeks. After the last treatment, rats were fasted overnight and anesthetized with pentobarbital sodium (35 mg/kg, IP) and euthanized by cervical decapitation. Blood was collected in unheparinized tubes. The lung tissues were fixed in 10% formalin for histological examination. A small portion of lung tissue was homogenized in 5.0 ml of 0.1 M Tris-HCl buffer (pH 7.4) solution. The homogenate was centrifuged and the supernatant was used for the estimation of various biochemical parameters.

2.5. Measurement of pro-inflammatory cytokines in broncho alveolar lavage fluid (BALF)

2.4. Determination of reactive oxygen species (ROS)

2.7. Determination of oxidative stress markers

The superoxide radicals (O2) formation in lung tissues was determined a previously described [18]. The results were expressed as reactive luminescence units (RLU) per 1 min per milligram dry weight (RLU/min/mg dry weight). Hydrogen peroxide (H2O2) generation was assessed by the spectrophotometric method of Pick and Keisari [19]. The H2O2 content of the sample was expressed as μmol/min/mg protein. Hydroxyl radical (OH) production was quantified by the method of Puntarulo and Cederbaum [20]. The hydroxyl radical content of the samples was expressed as μmol/min/mg protein.

Analysis of tissue malondialdehyde (MDA) level as an indicator of lipid peroxidation was performed by the spectrophotometry method [21]. This method was used to obtain a spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with MDA at 535 nm. The MDA level is expressed as nmol/g-tissue protein. Myeloperoxidase (MPO) activity, an index of the degree of neutrophil accumulation, was measured in tissues with commercially available ELISA kit (Bioxytech MPO-EIA, USA). The absorbance was read at 405 nm using Multi-Detection Micro Plate Reader.

At the end of the experiment, the lungs tissues were placed with 1 ml of autoclaved PBS for five times. The recovery ratio of the fluid was about 80% (4 ml) and the BALF was immediately centrifuged at 300g for 10 min at 4 °C, and the supernatants were stored at − 70 °C until required for subsequent tests. The levels of TNF-α, IL-1β, IL-6, and CINC-3 in BALF were determined by enzyme-linked immunosorbent (ELISA) kits. 2.6. Wet-to-dry lung weight ratio (W/D ratio) After euthanasia of rats, the lungs tissue were immediately weighed to get the wet weight, and then placed in an oven at 60 °C for 48 h and weighed to obtain the dry weight. The ratio of the wet lung to the dry lung was calculated to assess the lung edema.

Fig. 4. Effect of EGCG on Fl induced lung tissue enzymatic antioxidant SOD (4A), CAT (4B), GPx (4C), GR (4D), and GST (4E) of control and experimental rats. Values are mean ± SD for 6 rats in each group; a, b and c Values are not sharing a common superscript letter (a, b and c) differ significantly at p b 0.05 (DMRT).

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protein/lane) were loaded and separated on 10% sodium dodecylsulfate (SDS)-polyacrylamide (PAGE) followed by transferring to polyvinylidene fluoride membranes and blocking. Then, the membranes were incubated overnight at 4 °C with specific primary antibody against target genes followed by addition of horse radish peroxidasecoupled secondary antibody (Abcam, Cambridge,UK). The immune-relative bands were visible after development with a chemiluminescence (ECL) reagent (Amersham International Plc., Buckinghamshire, UK) and were quantified by densitometry and normalized with respective β-actin. 2.11. RT-PCR analysis RNA isolation and RT-PCR The total RNA was isolated by using Tri Reagent (Sigma). Total RNA (2 μg) from each sample was subjected to reverse transcription using a Superscript first strand cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol. PCR reactions were then carried out by mixing 1 μl of cDNA, 10 μl of KAPA Fast PCR Master mix, 1 μl of specific gene primer pair, and glyceraldehyde-3phosphate dehydrogenase primer pair (Internal control) and made up to 20 μl with sterile water and then amplified for 35 cycles. Each cycle

Fig. 5. (A) Effect of EGCG on Nrf2, HO1, and Keap1 protein expressions in the lung tissue of control and Fl treated rats by western blot. Lane1.Control, Lane2.Control ± EGCG (40 mg/ kg BW), Lane3.Fl-control (25 mg/kg BW), Lane4.EGCG (40 mg/kg BW) ± Fl (25 mg/kg BW). (B). Effect of EGCG on Nrf2, HO1, and Keap1 protein band intensities scanned by densitometer in control and experimental rats. Values are mean ± SD for 6 rats in each group; a, b and c Values are not sharing a common superscript letter (a, b and c) differ significantly at p b 0.05 (DMRT).

Quantifications were achieved by the construction of standard curve using known concentrations of MPO. Results were expressed as ng/mg tissue protein [22]. 2.8. Measurement of non-enzymatic antioxidant Glutathione (GSH) level was measured using the method of Moron et al. [23]. The level of glutathione was expressed as 1 g/mg protein. Vitamin E (a-tocopherol) level was estimated by the method of Desai [24] and was expressed as 1 g/mg protein. 2.9. Estimation of enzymatic antioxidants Total superoxide dismutase activity was determined from its ability to inhibit the auto-oxidation of pyrogallol according to the method of Marklund and Marklund [25]. The enzymic activity was expressed as U/mg protein. Catalase activity was assayed using the method of Sinha [26] and its activity was expressed as U/mg protein (1 U is the amount of enzyme that utilizes 1 μmol of hydrogen peroxide/min). Glutathione peroxidase activity was determined by the method of Rotruck et al. [27]. The enzyme activity was expressed as U/mg protein (1 U is the amount of enzyme that converts 1 1 mol of GSH to GSSG in the presence of H2O2/ min). Glutathione reductase activity was assayed by the method of Stall et al. [28]. Activity of GR was expressed as nmol of NADPH oxidised/ min/mg protein of cell extract. Glutathione-S-transferase was assayed by the method of Habig et al. [29]. GST activity was expressed as U/mg protein (1 U is the amount of enzyme that conjugates 1 μmol of CDNB with GSH/min). 2.10. Western blot analysis Lung tissues were homogenized in RIPA lysis buffer (50 mM Tris– HCl, pH 7.4;150 mM NaCl,0.25%deoxycholicacid,1%NP-40, and 1 mM EDTA) containing EDTA-free protease inhibitor cocktail (Thermo scientific, USA). After extraction of protein, the protein samples (100 μg

Fig. 6. Effect of EGCG on Nrf2 (6A), HO1 (6B), and Keap1 (6C) mRNA expressions in the lung tissue of control and Fl treated rats by RT-PCR analysis. Values are mean ± SD for 6 rats in each group; a, b and c Values are not sharing a common superscript letter (a, b and c) differ significantly at p b 0.05 (DMRT).


consisted of denaturation for 5 min at 94 °C, annealing for 30 s at appropriate annealing temperature and polymerization for 30 s at 72 °C. The PCR products were resolved by electrophoresis through a 2% agarose gel and stained with ethidium bromide. The densities of PCR products in the agarose gel were scanned with a Gel Doc image scanner (BioRad), and quantified by Quantity One Software (Bio-Rad).

2.12. Preparation of protein structure The 3D co-ordinates of the crystal structure of Keap1 like growth factor 1β (PDB ID: 1ZGK) was downloaded from the protein data bank (http://www.rcsb.org/pdb/) established by Brookhaven National Laboratories (BNL) in 1971. It contains the structural information of the macromolecules determined by X-ray crystallographic and NMR methods. Before docking, water molecules were removed from protein file 1ZGK. The drug ability site of the protein (1K3A) was defined where the ligand can bind and interact after energy minimization.

2.13. Preparation of ligand The structure was drawn using ChemSketch. ACD/ChemSketchTM software is an integrated software pack-age from Advanced Chemistry Development, Inc., Toronto, Canada, (http://www.acdlabs.com/com), for generating chemical structures of bioactive compounds, 2D structure cleaning, 3D optimization etc. After ligand preparation, hydrogen bonds were added and energy minimization was done using CHARMM force field an Accelrys software package. The chemical properties and molecular structures, was used for the calculation of Lipinski's Rule of Five.

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2.14. Docking analysis To explore the interaction and accurate binding model for the active site of Keap1 like growth factor the molecular docking analysis was carried out by using ligand fit of Discovery Studio (http://www. accelrys. com/product/discover studio/) Accelrys® software corporation, San Diego, USA. The mechanism of Fitting points are added to hydrogen bonding groups on the protein and ligand. Scoring functions implemented in docking programs includes terms of hydrogen bonds employed by Discovery Studio to rank the docked bases and to assess the binding site bonds present. 2.15. Immunohistochemistry of iNOS Immunohistochemical detection of iNOS was performed using iNOS monoclonal antibody from Lab vision Inc., Fremont, CA, USA. The avidin–biotin complex technique was used. Quantitative measurements were carried out using an image analysis system (Leica Qwin 500 C Imaging System Ltd., Cambridge, England) at National Research Centre in Cairo, Egypt to measure the mean of optical density of iNOS reaction at magnification of 400× in 10 non-overlapping fields from each animal in all groups. 2.16. Histological examination of lung For histopathology examination, lung tissues were dissected, fixed in Bouin's solution for 14–18 h, processed in a series of graded ethanol and embedded in paraffin. Paraffin sections were cut at 5-μm thickness and stained with hematoxylin and eosin for light microscopy examination (400 ×). The sections were viewed and photographed on an

Fig. 7. Protective role of EGCG on Fl induced iNOS expression in lung tissue by immunohistochemistry analysis in control and experimental rats. Control rats (Fig. 7A) show no expression of iNOS in the lung tissue which was similar to that of the EGCG alone treated rats (Fig. 7B). A significant increase of iNOS expression was observed in the lung tissue of Fl treated rats (Fig. 7C) when compared to the control rats. Pre-administration of EGCG shows significant reduction in the over expression of iNOS in the lung tissue (Fig. 7D).

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Olympus light microscope (Olympus BX51, Tokyo, Japan) with attached camera (Olympus C-5050, Olympus Optical Co. Ltd.; Tokyo, Japan).

these free radicals (ROS) further production when compared with the Fl alone treated rats.

2.17. Statistical analysis

3.2. Effect of EGCG on Fl induced pro-inflammatory cytokines

All the data were expressed as mean ± SD of a number of experiments. The statistical significance was evaluated by one-way analysis of variance using SPSS version 16.0 (SPSS, Cary, NC, USA) and the individual comparisons were obtained by Duncan's multiple range test (DMRT). Followed by the post hoc test, least significant difference (LSD). Values were considered statistically significant when p b 0.05.

The effect of EGCG on Fl induced pro-inflammatory cytokines in control and experimental rats showed on Fig. 2. Fl administration significantly (p b 0.05) increased the levels of pro-inflammatory cytokines such as TNF-α (2A), IL-β1 (2B), IL-6 (2C), and CINC-3 (2D) in lungs treated with fluoride. Pre administration of EGCG significantly (p b 0.05) decreased the pro-inflammatory cytokines remarkably when compared to that of control. EGCG alone administrated rats also showed some significant inhibitory effect on inflammation.

3. Results 3.1. Effect of EGCG on free radical scavenges Fig. 1 shows the free radical scavenging activity of EGCG on fluoride induced oxidative stress in control and treated rats. The levels of superoxide radicals (1A), hydroxyl radical (1B) and hydrogen peroxide (1C), were significantly (p b 0.05) increased in lung exposed to Fl. However, pre oral supplementation of EGCG significantly (p b 0.05) decreased

3.3. Effect of EGCG on lung edema (g/g), oxidative stress markers and nonenzymatic Fig. 3 shows the Lung edema, oxidative stress markers (MDA and MPO) and non enzymatic antioxidants (GSH and Vit. E) levels in the control and experimental rats. A significant (p b 0.05) increase in the levels of melonaldehyde (MDA) (Fig. 3A) and myeloperoxidase (MPO)

Fig. 8. Protective effect of EGCG on Fl altered lung histoarchitecture of control and experimental rats. Control and EGCG rats did not show changes in the structure of lung tissue (Fig. 8A&B). Fl treated rats showed extensive inflammation and fibrosis in the lung tissue (Fig. 8C&D). Pre-administration of EGCG significantly recouped all the changes elicited by Fl (Fig. 8E).


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Fig. 9. The antioxidant EGCG in lung tissue was docked with human Keap1 (PDB ID: 1ZGK) protein and showed the interaction bond distance with Gly 343(2.00A), Thr 595 (3.21A), Lue 578 (1.73A), and Asp 579 (2.52A). The binding affinities of EGCG with Keap1 were calculated with the LigScore Docking value of 126.155. (Discover studio, Version 4.0, Accelry's Software Inc. USA).

(Fig. 3B) in the fluoride intoxicated rats when compared to the control rats. However, there was a significant (p b 0.05) decrease in the levels of GSH (Fig. 3C) and Vit. E (Fig. 3D) with significant (p b 0.05) increase lung edema (Fig. 3 E) were also observed in Fl intoxicated rats when compared to the control and EGCG groups. Pre-treatment with EGCG significantly (p b 0.05) decreased the Fl induced edema, oxidative stress markers and increased the antioxidant levels when compared to that of

Fl alone treated rats. EGCG alone administrated rats did not exhibit any changes in the lungs. 3.4. Effect of EGCG on enzymatic antioxidants The levels of enzymatic antioxidant such as SOD, CAT, GPx, GR and GST were significantly (p b 0.05) decreased in Fl intoxicated rats were

Fig. 10. Effect of EGCG on Keap1 interaction in lung tissue with hydrogen bond (A), hydrophobic bond (B), favorable (C) and charge (D) of control and experimental rats.

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Fig. 11. The electrostatic surface representation of EGCG that interacts with Keap1 protein in lung tissue of experimental rats.

shown in Fig. 4. Pre-oral administration of EGCG significantly (p b 0.05) increased SOD (Fig. 4A), CAT (Fig. 4B), GPx (Fig. 4C), GR (Fig. 4D), and GST (Fig. 4E) through its strong antioxidant and hydrogen donating ability. 3.5. Effect of EGCG on Nrf2, HO-1 and Keap-1expression in lung Western blot analysis of Fig. 5 showed the expression pattern of Nrf2, HO-1 and Keap-1 proteins in the lung tissues of control and experimental rats. The level of Keap-1 protein expression significantly (p b 0.05) increased with a significant (p b 0.05) decrease in the

expression level of Nrf2 and HO-1 protein were observed in the Fl treated lung tissue when compared with the control rats. Pre-treatment of EGCG to Fl intoxicated rats showed a significant (p b 0.05) decrease in the level of Keap-1 with significant (P b 0.05) increase in the level of Nrf2, and HO-1 in the lung tissue when compared to the Fl alone treated rats. 3.6. Effect of EGCG on RT-PCR analysis The protective role of EGCG on Nrf2, HO-1, and Keap1 appearance in control and experimental rats were shown in Fig. 6. A significant

Fig. 12. Graphical abstract shows the protective mechanism of EGCG against Fl induced oxidative stress via Nrf2/Keap 1 signaling pathway.


(p b 0.05) decrease in the levels of Nrf2 (Fig. 6A) and HO-1 (Fig. 6B) mRNA with significant (p b 0.05) increase Keap-1 (6C) levels was observed in Fl intoxicated rats when compared with the control. Rats pre-administrated with EGCG significantly (p b 0.05) increased Nrf2 and HO-1 levels with significant (p b 0.05) decrease level of Keap-1 in the lung tissue when compare to Fl alone treated rats. 3.7. Effect of EGCG on iNOS expression in lung tissue Immunohistochemical examinations of iNOS in rat lung of control and experimental rats were shown in Fig. 7. A significant increase of iNOS expression was observed in the lung tissue of Fl treated rats when compared to the control rats (Fig. 7C). On the other hand, pre administration of EGCG to Fl intoxicated rats shows significant reductions in the over expression of iNOS in the lung tissue when compared with Fl alone treated group (Fig. 7D). Rats received EGCG alone (Fig. 7B) show negative immunostaining of iNOS in the lung tissue and are similar to that of the control group (Fig. 7A). 3.8. Effect of EGCG on lung histology Fig. 8 represented the photomicrograph of lung histoarchitecture from control and experimental rats. Control and EGCG group rats showed normal appearance of lung histology (Fig. 8 A&B). Extensive inflammation and fibrosis showing Fl intoxicated group (Fig. 8 C&D). Preadministration of EGCG prevented the histological changes induced by Fl (Fig. 8E). 3.9. Docking analysis The antioxidant EGCG docked with binding site of the protein of Keap1 responsible for Nrf2 deactivation. The ligand molecule of EGCG was docked with human Keap1 (PDB ID: 1ZGK) protein. The EGCG interaction with Keap1 distance (Fig. 9) was indicated with Gly 343(2.00A), Thr 595 (3.21A), Lue 578 (1.73A), and Asp 579 (2.52A) through Discover studio, Version 4.0, Accelry's Software Inc. USA. The binding affinities of EGCG calculated with the LigScore Docking value of 126.155. The hydrogen bond, hydrophobic, favorable and charge count interaction of receptor protein Keap1 with EGCG ligand molecule as showed in Fig. 10 (A-D). The electrostatic surface cavity of Keap1 protein has binding with the EGCG ligand molecule was shown in Fig. 11. The surface was created based on the hydrogen donor and acceptor of EGCG that interacts with Keap1 protein in lung tissue (Fig. 11A&B). 4. Discussion The reactive oxygen species (ROS) play a significant role in living organisms. For a long time, it has been ventured that ROS involved in the many pathogenic diseases associated to inflammatory processes [30]. One of the organs commonly affected by Fl induced ROS generation is the lungs [5]. In the present study a significant increase of ROS such as O2, OH, and H2O2 in Fl intoxicated lung tissue observed, which are corroborated the earlier report [6]. This may be due to the Fl induced complex chain of one-electron reduction of molecular oxygen, leading to the production of superoxide radicals (O2) in the lungs. The O2 is unstable and quickly undergoes another reduction to form H2O2 either spontaneously or in a much faster reaction catalyzed by superoxide dismutase (SOD). The H2O2 is relatively stable and can migrate from its site of origin and capable of affecting a large scale of an important cellular process. Pre-administration of EGCG significantly decreased these free radicals into normal in Fl intoxicated rats. This might be due to the effective free radicals scavenging ability and strong hydrogen donating property of EGCG [31]. Fl induced ROS causes additional damage through induction of inflammatory responses via activation of different transcription factors including activation of inflammatory cytokines [32,33]. Our results

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showed that the levels of the pro-inflammatory cytokines, TNF-α, IL1β, IL-6, and CINC-3, are significantly increased in the lung tissues in response to Fl administration when compared to the normal control group which are in line with the earlier report [34]. It has been shown that ROS activates alveolar and interstitial macrophages to express the early response pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α and chemokines CINC-3 [35]. Excessive release of early response proinflammatory cytokines triggers and intensifies the pulmonary inflammatory cascade [36]. The inflammatory cascade can activate the lung endothelial cells, and epithelial cells to produce chemokines which, in turn, attract inflammatory cells such as activated neutrophils (a major source of ROS), exacerbating tissue injury [35]. Although Fl induced pro-inflammatory cytokines intensify the inflammatory cascade and formulate tissue injury (IL-1β, IL-6, and TNF-α) through which decreasing the availability of antioxidant in lung tissue for scavenge the ROS in cells participate in the redox chemistry. However, the pre-administration of EGCG in the present research significantly decreased the levels of pro-inflammatory cytokines in lung tissue. This is in accordance with the results of previous investigator who have shown that EGCG inhibits the induction of inflammatory signals through its strong antioxidant activity in lung tissue [37]. Fl induced ROS caused the release of pro-inflammatory substances and the formation of free radicals as oxygen-derived reactive oxygen species in lungs. In our study, the pulmonary edema (Wet-to-dry lung weight ratio) was significantly increased in Fl intoxicated rats when compared to the normal control rats which corroborated with the previous report [38]. This may be mainly due to the Fl induced free radicals lead to lipid peroxidation within the cell membranes reacting directly with polyunsaturated fatty acids which are believed to cause pulmonary edema in rats. Failure to get rid of excessive ROS leads to subsequent oxidative damage to the pathogenesis of lung injury [5]. However, pretreatment with EGCG significantly decreased the lung edema when compared to that of Fl intoxicated rats. This is probably due to the reduced inflammatory mediated lipid peroxidation in the lung tissue through HAT (hydrogen atom transfer) mechanism of EGCG to maintain paired electrons in the normal cells. In this study, malondialdehyde (MDA) and myeloperoxidase (MPO) activity was used to measure the extent of lipid peroxidation and inflammation caused by Fl in lung tissue. We found that an elevation of MDA activity, indicating the presence of enhanced lipid peroxidation by Fl induced ROS in the lung tissue of rats. The increased levels of ROS degrade polyunsaturated lipids, forming malondialdehyde [39]. The results obtained in this study correlate with earlier report [40]. However, a significant decrease in MDA level was seen in the EGCG pre-treated group. This normalization may be accomplished by the potent antioxidant, free radical scavenging and anti-lipoperoxidative nature of EGCG, because it has been reported to protect the cells from oxidative stress mediated cell death by lipid peroxidation in rats [13]. Another biochemical parameter was also investigated in this study, the lung tissue content of myeloperoxidase (MPO), an index of lung inflammation which was significantly increased in the Fl intoxicated rats due to over accumulation of ROS. Furthermore, Fl induced ROS (especially H2O2) oxidizes tyrosine to tyrosyl radical using hydrogen peroxide as an oxidizing agent during the neutrophil's respiratory burst [41]. Fl has been well proved trace element that induce lipid peroxidation and inflammatory cytokines in lungs via increased ROS levels [33,40]. Preadministration with EGCG significantly decreased the MPO in Fl treated rats as compared with the control rats. This is mainly due to the anti-inflammatory efficacy, potent antioxidant, and major free radicals scavenging ability of EGCG that reduces the chance of inflammation mediated lung damage. In this study, the non-enzymatic antioxidant GSH and Vit. E was significantly decreased in Fl intoxicated rats which fairly consistent with the previously published studies [42]. GSH, which has a sulfhydryl the group in its peptide is an important antioxidant largely present in biological systems. The sulfhydryl group and GSH interact and form a

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complex with Fl and thereby alter the GSH [43]. This pathophysiology, probably caused by increased cellular demand of GSH, leads to impaired cell function because of the disturbed redox status in the lung tissue. Vit. E is the most effective chain breaking lipid soluble antioxidant present in cell membranes that play a major role in maintaining cell membrane integrity [44]. There is a decline in the level of Vit. E in Fl exposed rats, which may be due to the enhanced lipid peroxidation, induced by ROS and decreased GSH content in the lungs [40]. However, Pre-administration of EGCG to Fl intoxicated rats significantly increased the Vit. E level as compared to the control. This could be mainly due to the presence of more hydroxyl group (8 OH) in the chemical structure which scavenge the maximum ROS and thus elevate the antioxidant levels in the body. SOD catalyzes the dismutation of superoxide into oxygen and hydroperoxides, thereby acting as a potent antioxidant. A decline in the activity of SOD was evident in Fl administered animals, which is in concordance with a previous study [14]. Catalase catalyzes the conversion of hydrogen peroxide to water and molecular oxygen. GPx reduces lipid hydroperoxides to their corresponding alcohols and free hydrogen peroxide to water, eventually protecting the cells from oxidative damage. A notable descend in the activities of these enzymes was observed in Fl treated animals, which might be due to overproduction of ROS that exerts an inhibitory effect on these enzymes [6,42]. However, to remove excess free radicals from the system, GST and GPx make use of GSH during their course of reactions. Decrease in GSH content due to Fl induced ROS toxicity simultaneously decreased the activities of GST as well as GPx with a simultaneous decline in the activity of GSH regenerating enzyme, GR [45]. Pre-administration of EGCG restored the activities of these enzymic antioxidants close to normal values. This might be due to the inhibitory action of EGCG on ROS and LPO, consequently decreasing the oxidative stress produced during pulmonary fibrosis [46]. In the present study the Nrf2 activation was confirmed by the molecular docking analysis was performed with EGCG to assess its possible binding sites to Keap1 protein. The inactivation of Keap1 is suggested to be the major mechanism for activation of Nrf2 and its downstream proteins [47]. EGCG is a potent antioxidant Nrf2 activator that functions by modifying Keap1 residues [48,49]. Molecular docking analysis was performed with EGCG to assess possible binding sites to Keap1 protein in this experiment. The results revealed that EGCG form connections with the aminoacids Gly 343(2.00A), Thr 595 (3.21A), Lue 578 (1.73A), and Asp 579 (2.52A) of Keap1 protein. This bonding could contribute to the activation of Nrf2 by EGCG through inhibiting the Keap1 repressor activity [49]. The immunohistochemistry and histological findings also strongly support our biochemical findings that EGCG protects the Fl induced oxidative stress mediated lung injury. Our study shows the altered lung histoarchitecture and increased iNOS expression in Fl treated rats, such as extensive inflammation and fibrosis. The histological findings of EGCG pre-treated Fl intoxicated rats showed no necrosis, inflammatory cells and edema and less iNOS expression. Thus, EGCG protected the myocardium against Fl induced cardiac damage. This is mainly due to the antioxidant membrane stabilizing and antilipoperoxidative properties of EGCG [50]. In conclusion, we demonstrated that EGCG attenuates the pathological symptoms of Fl treated rats by up-regulating Nrf2 expression, and in turn suppresses pulmonary inflammatory cytokines evoked responses. Moreover, these findings recommended a potential application of EGCG in pulmonary inflammatory disease therapy. Since, activating this pathway (Nrf2/Keap1) leads to decrease the chances of lung cancer progression, and this will be very useful for the reduction of free radicals induced organs damage by phytochemical therapy (Fig. 12). This study not only provides more evidence that EGCG exerts anti-inflammatory, scavenging of ROS activity in Fl treated rats, but also sheds light on the potential use of phytochemicals like EGCG as a therapeutic drug for every one of heavy metals mediated sepsis.

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