Intonation of Nrf2 and Hif1-α pathway by curcumin prophylaxis: A potential strategy to augment survival signaling under hypoxia

Accepted Manuscript Title: Intonation of Nrf2 and Hif1-␣ pathway by curcumin prophylaxis: A potential strategy to augment survival signaling under hypoxia Authors: Titto Mathew, S.K.S. Sarada PII: DOI: Reference:

S1569-9048(18)30141-1 https://doi.org/10.1016/j.resp.2018.09.008 RESPNB 3151

To appear in:

Respiratory Physiology & Neurobiology

Received date: Revised date: Accepted date:

3-5-2018 22-9-2018 24-9-2018

Please cite this article as: Mathew T, Sarada SKS, Intonation of Nrf2 and Hif1-␣ pathway by curcumin prophylaxis: A potential strategy to augment survival signaling under hypoxia, Respiratory Physiology and Neurobiology (2018), https://doi.org/10.1016/j.resp.2018.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title:

Intonation of Nrf2 and Hif1-α pathway by curcumin prophylaxis: a potential strategy to augment survival signaling under hypoxia.

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Titto Mathewa, Sarada SKS*a

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Title:

Intonation of Nrf2 and Hif1-α pathway by curcumin prophylaxis: a potential strategy to augment survival signaling under hypoxia. Titto Mathewa, Sarada SKS*a

aHaematology Division, Defence

Institute of Physiology and Allied Sciences, Timarpur, Delhi-

54, India. Author: Sarada SKS, Scientist "F", Head of Haematology Division, Defence

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*Corresponding

Institute of Physiology and Allied Sciences, Timarpur, Delhi- 54, India. +91-9811576850,

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[email protected]

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Graphical abstract

High lights. 2

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High altitude induced oxidative stress may contribute to alveolar collapse. Surfactants production is altered under reduced oxygen supply. Nrf2 and HIF 1- α are the major players in surfactant homeostasis. Curcumin enhances Nrf2 and HO-1 thereby augments survival signalling at HA

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Abstract Background: Pulmonary surfactant oxidation leads to alveolar collapse- a condition often noticed in high altitude pulmonary edema (HAPE). The present study was aimed to determine

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the effect of curcumin prophylaxis in augmenting the phase II antioxidant enzymes and

surfactant proteins expression in enabling the pulmonary surfactant homeostasis under

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hypoxia. Methods: A549 cells were exposed to 3% hypoxia for different time durations (1 h,

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3 h, 6 h, 12 h and 24 h). The Cells were pretreated (1 h) with 10 μM curcumin and exposed to

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hypoxia. The in-vivo results were extrapolated into in–vivo system using male Sprague Dawley

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rats, exposed to a stimulated altitude of 7620 m for 6 h. The rats were supplemented with curcumin (50mg/kgBW) 1 h prior to hypoxia exposure. Results: Results showed that, the

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expression of surfactant proteins (SPs) A and B decreased from 3 h of hypoxic exposure, whereas expression of SP-C and SP-D proteins were increased within 1 h of hypoxic exposure

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over control cells. Hypoxic exposure resulted into significant increase in protein and lipid

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peroxidation (p<0.001), reduced levels of antioxidants (GSH, GPx and SOD) (p <0.001) along with significant down regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Heme oxygenase-1 (HO-1) in A549 cells over control. However, the curcumin supplementation both

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in-vitro and in-vivo resulted into increased expressions of HO-1 and Nrf2 significantly (p<0.001), which enabled the cells in balanced expression of SPs with reduced levels of oxidants. Further curcumin significantly enhanced the levels of antioxidant enzymes in BALF along with stabilized expression of hypoxia inducible factor 1(HIF-1α) followed by reduced expression of vascular endothelial growth factor (VEGF) in lungs of rats. The 3

immunohistochemistry observations provided substantial evidence of enhanced surfactant protein expressions in lungs of curcumin administered hypoxia exposed rats. Conclusion: These results indicate that curcumin augment survival signaling by reinforcing the induction of phase II antioxidant enzymes thereby enabling the pulmonary surfactant homeostasis under

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hypoxia. Keywords: Alveolar epithelium, Curcumin, Hypoxia, Nrf2, Phase II antioxidant enzymes,

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Surfactant proteins. List of Abbreviations:

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Active protein 1 (AP-1), Acute Lung Injury (ALI), Acute respiratory distress syndrome

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(ARDS), Alveolar lining fluid (ALF), Analysis of variance (ANOVA), All trans retinoic acid

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(ATRA), Body Weight (BW), Bronchoalveolar lavage (BAL), Bronchoalveolar lavage

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(BALF), Chronic obstructive pulmonary diseases (COPD), Deoxyribonucleic acid (DNA), Diaminobenzidine (DAB), Dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s

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medium (DMEM), Enzyme-linked immune sorbent assay (ELISA), Epithelial lining fluid (ELF), Ethylenediaminetetraacetic acid (EDTA), Fetal bovine serum (FBS), Glutathione-S-

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transferase (GST), GPx (Glutathione peroxidase), GSH (Glutathione), Heme Oxygenase-1

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(HO-1), High altitude (HA), High altitude pulmonary edema (HAPE), Horse radish peroxidase (HRP), Hypoxia -inducible factor 1 alpha (HIF-1α ), Hypoxia-induced mitogenic factor (HIMF), Immunohistochemistry (IHC), lipopolysaccharide (LPS), Lysophosphatidylcholine

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(LPC), Malondialdehyde (MDA), Nicotinamide adenine dinucleotide phosphate (NADP), Nrf2 (nuclear factor (erythroid-derived 2)-like 2), surfactant proteins (SPs), Nuclear factor Kappa B (NFκB), Phosphate-buffered saline (PBS), Phosphatidylcholine (PC), Reactive oxygen species (ROS), Room temperature (RT), SD- Sprague Dawley, Surfactant protein (SP-A),

Surfactant protein B (SP-B),

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Super oxide dismutase (SOD), Surfactant protein C (SP-C),

Surfactant protein D (SP-D), Universities of Federation for Animal Welfare (UFAW), VEGFVascular endothelial growth factor.

1. Introduction:

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Surfactant system is affected by oxygen free radicals, proteases and cytokines released by the inflammatory cells which take place in many pathological conditions such as high

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altitude pulmonary edema (HAPE), acute respiratory distress syndrome (ARDS), chronic

obstructive pulmonary diseases (COPD) (Haagsman 1998) etc. Pulmonary surfactants are

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inactivated by the leakage of serum proteins, secretory phospholipase A2, cholesterol and/or

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other inflammatory molecules into the alveoli (Maggiorini 2006, Hite et al., 2005, Iwanicki et

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al., 2010). Several studies have shown that hypoxia at high altitude increases oxidative stress

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(Vockeroth et al., 2010, Himadri et al., 2010; Sarada et al., 2008), and decreases the surfactant lipids, such as phosphatidylcholine (↓ 20%, PC). Prevest et al., (1980) reported that nearly10-

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fold increase in lysophosphatidylcholine (LPC) under hypoxia leads to increased surface tension. Lyamtsev and Arbuzov (1981) revealed that surfactant surface activity decreased in

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rats after 6 h of hypoxia (6000 m) exposure. It appears that hypoxia alters the surfactant lipid

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metabolism in such a way that the total and individual lipids decreases under hypoxic conditions either as a consequence of increased lipid peroxidation, macrophage activity or decreased lipid synthesis /secretion facilitating the movement of fluid from the interstitium into

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the alveoli (Behn et al., 2007) leading to pulmonary edema. Surfactant protein A (SP-A) is required for the secretion, synthesis and recycling of

surfactant phospholipids and is also involved in the innate immune reactions. Surfactant B (SPB) is required for normal lung function. Surfactant C (SP-C) imparts important surface properties to surfactant phospholipids mixture. Both SP-B and SP-C are essential for reduction 5

of surface tension and stabilization of mammalian surfactant lipids. Surfactant protein D (SPD) regulates the lipid homeostasis. Both SP-A and SP-D acts as opsosnins by binding to a variety of pathogens and therefore enhances the phagocytosis process as well as pulmonary clearance. Besides these immune defense tasks, they divulge in anti-inflammatory effects and play a role in the resolution of pulmonary inflammation (Droma et al., 2012). More importantly

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pulmonary surfactant was responsible for reducing the surface tension in the lungs; promoting alveolar stability and increasing lung compliance hence help in increasing oxygen uptake at

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high altitude (HA). There is a paucity of information regarding how hypoxia alters surfactant

proteins expression leading to mismatched surfactant homeostasis in lungs of animals Moreover, the available literature on the effect of hypoxia on

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(especially in HAPE)?

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pulmonary surfactant proteins have mostly been determined in cultured lung cells, thus

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resulting in contradictory conclusions. For instance, Rehan et al (1977) have reported that SP-

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A levels remained relatively constant when NCI-H441 cells were exposed to 7%, 3% and 1% oxygen for 8 h, while SP-B expression decreased along with decreased percentage of oxygen

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exposure. On the contrary, hypoxia exposure (1% O2) down regulated the expression of surfactant SP-C after 8 h and found nearly abolished protein expression up on 24 h hypoxia

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exposure in cultured alveolar epithelial cells (A549 cells) (Vaporidi et al., 2005). Whereas, Tong et al (2006) have demonstrated that SP-B and SP-C expressions were regulated by

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hypoxia-induced mitogenic factor (HIMF) (Tong et al., 2006). Saxena et al (2005) have reported that mutation in SP-A1 and SP-A2 genes in human subjects contribute to HAPE

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susceptibility. However rats exposed to intermittent hypoxia did not show complete reduction of all the surfactant proteins (Vives et al., 2008). These studies indicate that surfactants proteins expression depends up on the time, duration and severity of hypoxia exposure. Nrf2 is maintained primarily in the cytoplasm where it binds to the BTB-Kelch-like ECH-associated protein 1 (Keap1 or KLHL19). Nrf2-regulated genes can be classified into 6

phase II xenobiotic-metabolizing enzymes such as glutathione S-transferase and UDPglucuronosyl transferase, antioxidants (HO-1, SOD, NADP(H), quinoneoxido reductase, ϒglutamyl cysteine synthetase), molecular chaperones, DNA repair enzymes and antiinflammatory response proteins (Bryan et al., 2013) etc. Some studies have demonstrated that dexamethasone increases the production of pulmonary surfactants (Wang et al., 1996, Young

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and Silbajoris 1986). This could be one of the possible mechanisms through which

dexamethasone endowed with prophylaxis in HAPE susceptible adults. But this steroidal drug

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has various side effects such as bradycardia, hyperglycemia, insomnia etc. Thus, identification of a potent prophylactic agent which can prevent or reduce hypobaric hypoxia-induced

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pulmonary oxidative stress, surfactant surface tension as well as inflammation is required. The

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best example of such molecule is curcumin, a derivative of turmeric, used for centuries to treat

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a wide variety of inflammatory conditions.

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Curcumin (1, 7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a major chemical component of turmeric powder extracted from its rhizome Curcuma longa L (Beevers

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and Huang, 2011). Pharmacologically, curcumin has been used as a traditional medicinal agent in Ayurvedic medicine for ∼6000 years and used for its excellent anti-oxidant, anti-

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inflammatory, anti-microbial, anti-parasitic, anti-mutagenic, anti-cancer properties etc. (Gupta

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et al., 2013, Hewlings and Kalman, 2017). In addition to its inherent ability of attenuating the reactive oxygen species (ROS), it has ability to enhance the activities of detoxifying enzymes such as glutathione-S-transferase (Dubey and Apenten, 2014), inducing antioxidant defense

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mechanism by modulating transcriptional factors such as Nrf2, HIF-1, activator protein 1 (AP1) and nuclear factor Kappa B (NFκB) etc.(Gonzalez-Reyes et al., 2013). Therefore in the present study we were interested in finding out (i) the two most important transcriptional factors viz. Hif1-α and Nrf2, and their association in maintaining downstream proteins in remodeling the surfactant proteins expression leading to oxygen homeostasis via enhancing 7

Phase II antioxidant enzymes in the pulmonary tissue of rats under hypoxia and further we also reasoned that (ii) if the above mentioned condition was true then, prophylactic treatment with the most potent anti-oxidant and anti-inflammatory molecule-curcumin will certainly facilitate the induction of phase II antioxidant enzymes enabling the pulmonary surfactant

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homeostasis under hypoxia. 2. Materials and Methods:

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2.1. Reagents.

Reagents for cell culture media like Dulbecco’s modified Eagle’s medium F-12 (DMEM F-12), trypsin- EDTA, penicillin, streptomycin, fetal bovine serum (FBS) and curcumin

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powder were purchased from Sigma (St. Louis, MO USA).

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2.2. Cell Culture.

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The A549 cell line (American Type Culture Collection, Rockville, MD) is an epitheliallike human lung cell line (gift from Institute of Nuclear Medicine and Allied Sciences, New

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Delhi, India) (Lieber et al., 1976). A549 cells were cultured in DMEM F-12 medium, supplemented with 10% fetal bovine serum, 100 units of penicillin and 50µg/ml streptomycin

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and maintained at 370 C, 5% CO2 and 21% O2..

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2.3 . Experimental animals.

Experiments were carried out on male SD rats, weighing 150-200 gm BW. Rats were

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maintained at 25± 20 C with day and night cycles of 12 h each and given food and water adlibitum. The Institute’s ethics committee approved all the experimental protocols for this study and followed the guidelines of the Universities of Federation for Animal Welfare (UFAW) for animal research. 2.4. Administration of curcumin. 8

The drug curcumin was diluted in a vehicle (DMSO-0.5%). A549 cells were treated with freshly prepared curcumin (10µM) 1 h prior to the hypoxia exposure and subsequently freshly prepared curcumin solution (1ml) was administered orally to the SD rats (50 mg/Kg BW) 1 h prior to hypobaric hypoxia exposure.

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2.5. Study design: The experiment was carried out in two phases. Phase - I was in- vitro study and Phase II was in-vivo study.

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2.5.1. Phase I: In phase I study, we determined the time at which the change in surfactant

proteins expression took place under hypoxia. Cells were plated at a density of 0.1 million cells per well in 6 well plates, let to adherence for over 12 h under normal conditions (Normoxia -

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21% O2 - 5% CO2) and then cultured under hypoxic conditions (3% O2– 5% CO 2– 92% N2).

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Prior to hypoxic exposure, culture media was replaced with thin layer of one equilibrated to

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respective atmosphere. Hypoxic conditions were achieved in a humidified variable aerobic

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incubator (Galaxy 170R, New Brunswick Scientific, CT, USA) for different durations (0 h, 1

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h, 3 h, 6 h, 12 h and 48 h). After different hours of hypoxic exposure the cells were collected for further analysis by trypsinization. From the time dependent studies of hypoxia exposure we

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selected to expose the cells for 6 h at 3 % O2 for further studies and the optimum curcumin dose

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for in-vitro study was found to be 10 µM as described elsewhere (Titto and Sarada, 2015) 2.5. 2. Phase -II: Exposure to hypobaric hypoxia: The rats were exposed to simulated altitude of 7620 m in a hypobaric chamber (Decibel

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Instruments, India) for 6 h. The reason for exposing the rats to hypobaric hypoxia for 6 h was based on our previous studies (Sarada et al., 2008). In brief, the rats were exposed to hypobaric hypoxia for different time durations (3 h, 6 h, 12 h, and 24 h), where the maximum transvascular leakage and edema index was obtained at 6 h of hypoxic exposure. Therefore in the present study, the rats were exposed to hypobaric hypoxia for 6 h and curcumin (50mg/Kg 9

BW) was administered as a prophylactic drug 1 h before the hypoxic exposure. The optimum curcumin dose (50 mg/Kg BW) selected was based on our previous studies (Sarada et al., 2014). Briefly, rats were administered with different doses of curcumin (25, 50, 100 and 200 mg curcumin /Kg BW) 1 h prior to hypobaric hypoxia exposure for 6 h and maximum significant reduction in transvascular leakage and edema index was occurred at dosage of 50

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mg curcumin/Kg BW compared to the other doses tested. In the present in-vivo studies- the rats were divided into 4 groups, each group containing 6 rats. Group 1 served as control or

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normoxia (Nor) that received only vehicle. Group 2 received only vehicle and was exposed to hypoxia (Hypo) for 6 h. Group 3 (Normoxia+ Cur) was supplemented with curcumin 50 mg

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Cur/kg BW and Group 4 (Hypoxia+Cur) was supplemented with curcumin 50 mg Cur/kg BW and was exposed to hypoxic stress (6 h). The temperature of the hypobaric chamber was

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maintained at 25±10C with an air flow rate of 4 l/h with 55% humidity and barometric pressure

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of 280 mmHg. The partial pressure of arterial oxygen was found to be 95.6 ±2 mmHg and 38.6±2 mmHg in control rats and hypobaric hypoxia exposed rats respectively indicating that,

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rats were exposed to reduced levels of partial pressure of oxygen in the hypobaric chamber. However, the animals were provided with adequate quantities of food and water during

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exposure to hypoxia. Three time's higher dose of Ketamine (100 mg/kg BW) and Xylazine

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hydrochloride (20 mg/kg BW) IP solution were used as euthanasia 2.6. Determination of oxidative stress parameters.

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Bronchoalveolar lavage (BAL) was performed on normoxia and hypoxia exposed SD

rats. Rats were anaesthetized and a trimmed sterile 18-guaze micro-cannula (Major surgical India ltd, India) was inserted into the lumen of the exposed trachea. The lungs were lavaged in-situ with two separate washes (1 ml) of sterile normal saline. The BAL fluid was centrifuged at 3000 rpm at 40C for 10 min. The supernatant was stored at -800C until further use. Total protein content in the bronchoalveolar lavage fluid (BALF) and in the lung homogenate of rats 10

was estimated as described by Lowry et al., (1951). Glutathione peroxidase (GPx) and superoxide

dismutase

(SOD)

activities

in

cell

free

BALF

were

measured

spectrophotometrically using kit (RANDOX, UK). Glutathione-S-transferase (GST) activity in cell free BALF samples was measured spectrophotometrically using kit (Sigma, St Lious, MO, USA). The lipid peroxidation in cells and BALF samples were estimated by measuring

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malondialdehyde (MDA) levels in normoxia and hypoxia exposed cells as mentioned earlier

(Okhawa et al., 1979). The reduced GSH levels in BALF were determined as per Kum-Talt

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and Tan method (Kum-Talt and Tan, 1974). Protein oxidation in A549 cells and BALF was

estimated by derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH),

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which led to the formation of a stable 2,4-dinitrophenyl (DNP) hydrazone product as described

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by Levine et al (1990).

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2.7. Identification of Lamellar bodies by quinacrine staining in A549 cells.

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Quinacrine staining was done in control, hypoxia exposed and curcumin treated

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hypoxia exposed cells as described by Chintagari et al (2010). Briefly, A549 cells were plated at a density of 0.5–1×10

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cells in 12 well plates. Following overnight culture in DMEM

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(supplemented with 10% FBS, non-essential amino acids, penicillin and streptomycin) the plates were washed to remove the unattached cells. Freshly made quinacrine was added to

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media at a final concentration of 10 µM. Lamellar body staining was examined at room temperature with dye using Olympus BX51TF (Center valley, PA USA).

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2.8. Protein Expression studies 2.8.1. Western Blotting Rinsed cells / tissues were lysed with radioimmuno precipitation assay (RIPA) buffer at 40 C. Nuclear extracts were obtained by re-suspending the cells in high salt buffer (20 mM Hepes, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and protease inhibitor 11

cocktail) and then centrifuged. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane (Millipore, USA) and probed with primary antibodies to Nrf2, HIF1α, HO-1, VEGF, SP-A, SP-B, SP-C and SP-D and ATRA along with β-actin (for cytoplasmic proteins) and Histone H3 (for nuclear proteins) (Santa Cruz Biotechnology) and again probed with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Later, the

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membranes were thoroughly washed with PBST (five to six times) and the bands were developed on X-ray film (Kodak, Rochester, NY) using chemiluminescent peroxidase substrate

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(Sigma, LO, USA).

2.9. mRNA expression studies: The design of each primer was based on the published sequences Table 1. The total cellular RNA was extracted from 0.1 g weight of lung tissue using

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trizol reagent method following the manufacturer’s recommended protocol (Invitrogen). The concentration of RNA was measured by absorbance at 260 nm. Reverse transcription was

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performed with Invitrogen cDNA synthesis kit as per manufacturers’ instructions. Briefly, 5μg

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total RNA, 0.1 μg oligo (dT)18, 5 U RNase OUTMoloney murine leukaemia reverse

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transcriptase, 5 mM dNTP mixture, 10 mM dithiothreitol dissolved in reverse transcription buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl and 3 mM MgCl2) at 250C for 10 min, 420C for

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50 min and 700C for 15 min to yield cDNA. The first strand cDNA was amplified in 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 100 μM dNTP mixture, 0.75 μM specific

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primers and 0.75 units' high fidelity Taq DNA polymerase (Invitrogen, Singapore). The PCR reaction was performed in PCR system 9700 (Biorad, USA) under the following conditions-

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940C for 5 min (pre-PCR), 30 cycles of 940 C for 30s, 55-650C for 45s, 720C for 30s and 720C for 5 min. The optimal cycle number (35 cycles) for each primer was determined by sequentially performed PCR amplification of 26, 28, 30, 32, and 35 cycles. The PCR products were separated onto 1.2% agarose gel by electrophoresis. After ethidium bromide staining all the DNA bands were photographed using Gel Doc System (Innotech CA, USA).

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2.10. Immunohistochemistry: As described by Sati et al., (2010) immunohistochemistry was performed in normoxia or hypoxia exposed rat lung tissue with some modifications. Briefly, thin (20 µm) cryosections (Leica CM 1950, Microtome, Leica Wtzlar, Germany) of alveolar tissues were prepared from normoxic/hypoxic rat lungs perfused with saline. The paraformaldehyde fixed tissues were further immersed in gradient sucrose and thin sections

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were made. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, in distilled water for 30 min, then repeatedly washed with phosphate-buffered saline (PBS, pH

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7.2) and subsequently placed in 0.05% trypsin- EDTA for 20 min for antigen retrieval. After washing with PBS- Triton100 (PBST), these sections were again incubated with 5% normal

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goat serum for 60 min at room temperature (RT). The sections were then incubated with each of the primary antibodies (SP-A, SP-B, SP-C, SP-D, hypoxia inducible factor 1 alpha (HIF-1α)

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and vascular endothelial growth factor (VEGF) (Santa Cruz Biotechnology) for overnight at 40

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C followed by incubation with HRP conjugated goat anti-rabbit IgG blocking buffer for 60 min at RT. Secondary antibodies were supplied by Sigma, MO, USA. Labelling was

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‘‘visualized’’with 3, 3’-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole as the

USA).

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chromogen. The images were then captured by using Olympus BX51TF (Centre valley, PA,

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2.11. Nrf2 blockade studies: In order to know, whether blocking of Nrf2 leads to altered surfactant homeostasis, we used all trans retinoic acid (ATRA) which is a known Nrf2 inhibitor. A549 cells treated with different concentrations of ATRA ranging from 0.25 µM to

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4 µM concentration and exposed them to hypoxia at 3 % O2 for 6 h. The best optimum concentration was found to be 1.5 µM as this concentration showed more than 80 % inhibition of Nrf2 protein expression compared to control (0 h) in A549 cells under hypoxia (data not shown), therefore the rest of the experiments mentioned in this study was conducted using this concentration. 13

2.12 Enzyme-linked immuno sorbent assay. An enzyme linked immunosorbent assay was performed qualitatively. Briefly, 50 µg of BALF with an equal volume of coating buffer (0.5 M carbonate buffer; pH 9.6) was incubated overnight in an assay plate at 4 °C. Nonspecific binding was blocked by 5% BSA in the same buffer. The samples were then washed with PBS containing 0.05% Tween 20 and incubated with diluted rabbit anti SP-A, rabbit anti SP- B,

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rabbit anti SP-C and rabbit anti SP-D primary antibodies in blocking buffer (1:500) for 2 h.

The samples were then washed and incubated with diluted goat anti-rabbit IgG-alkaline

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phosphatase (1:2000) in the same buffer for 2 h. Further, the samples were incubated with p-

nitrophenyl phosphate (1mg/mL) in carbonate buffer containing 10 mM MgCl2. The

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development of color was assessed at 450 nm. The reaction was stopped by adding 50 µL of 1

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M NaOH. The final value was represented as normalized against the normoxia samples.

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2.13 Statistical analysis.

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Statistical analysis was performed using SPSS (15.0) for windows software (SPSS Inc.,

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Chicago, IL). Comparison between experimental groups and curcumin treated groups were made by using two ways ANOVA with student Newman-Keuls test for comparison between

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groups. Differences were considered statistically significant for p<0.05. Results were

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expressed as mean ± SD. 3. RESULTS:

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3.1. Hypoxia alters surfactant proteins expression pattern in A549 cells. A549 cells exposed to hypoxia showed SP-A (Figure 1a) protein expression decreased

from 1 h to 3 h but showed a slightly increased expression in 6 h of hypoxic exposure, however, further exposure to longer durations ie. 12 h and 24 h reduced the expression and almost remained constant. On the contrary, A549 cells within 1 h of hypoxic stress, did not modify

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SP-B protein expression, but virtually no SP-B protein expression were detected on further hypoxic exposures (from 3 h - 48 h) (Figure 1b). Whereas SP-C and SP-D expressions in A549 cells were increased within the 1 h of hypoxic exposure and remained constant till 24 h of hypoxic stress over control (1c and d respectively). In order to determine the association of altered expression of SPs with oxidative stress, we examined protein oxidation and lipid

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peroxidation in hypoxia exposed A549 cells. The results showed no significant changes in

protein oxidation within the 1 h of hypoxic exposure, but further exposures of cells from 3 h

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onwards showed an increased protein oxidation as compared to control; and this upsurge remained constant up to 24 h of hypoxic exposures (Figure 2 a; p<0.001). Similarly, MDA

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levels in A549 cells showed anon significant increase in 1 h of hypoxia exposure but further

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compared to the control (Figure 2 b; p<0.001).

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showed a gradual and sustained significant increase from 3 h to 24 h of hypoxic exposures as

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3.2. Curcumin refurbishes oxidant and antioxidant levels in BALF of rat lungs.

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The functional site of pulmonary surfactant is the alveolar epithelium which is lined by thin lining fluid called alveolar lining fluid (ALF). ALF is enriched with antioxidants to combat

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any oxidant insult. Therefore, we further investigated whether hypoxia augmented oxidation can be abated by curcumin prophylaxis or not? Table 2 represents the effect of curcumin on

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lipid peroxidation (measured as malondialdehyde-MDA), Protein oxidation, GSH, GPx and SOD values in A549 cells. Hypoxia exposure resulted into increased lipid peroxidation (MDA) and protein oxidation (↑ 3.13 fold; ↑ 4.25 % respectively; p<0.001) along with decreased GSH,

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GPx and SOD levels (↓ 1.88 fold, ↓ 1.90 fold; ↓ 2.68 fold respectively; p<0.001) in A549 cells compared to control (0 h). Curcumin administered hypoxia exposed A549 cells showed decreased lipid peroxidation (↓ 6.01 fold; p<0.001) and protein oxidation (↓ 2.93 fold; p<0.001) as compared to control (hypoxia-6h) (Table 2). Similarly curcumin preconditioning restored

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the antioxidant levels (↑1.80 fold GSH, ↑1.75 fold GPx and ↑ 2.21 fold SOD) in A549 cells as compared to control (hypoxia (6h); p<0.001). Table 3 represents the effect of curcumin on lipid peroxidation (MDA), protein oxidation, GSH and GST values in BALF of the rat lungs under hypoxia. Rats exposed to hypoxia for 6 h at 7620 m, showed a significant increase in lipid peroxidation (MDA) and

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protein oxidation (↑6.51 fold; ↑5.58 % fold respectively; p<0.001) with concomitant decrease in GSH and GST levels (↓2.07 fold; ↓3.50 fold respectively; p<0.001) in BALF, as compared

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to control indicating the increased oxidation in BALF. Prior treatment of rats with curcumin and exposed to hypobaric hypoxia resulted into significant reduction (p<0.001) in lipid

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peroxidation (↓3.59 fold; p<0.01) and protein oxidation (↓2.34 fold; p<0.001) followed by

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appreciable increase in GSH and GST levels (↑1.73 fold and ↑3.16 fold respectively; p<0.001)

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in BALF compared to control (hypoxia (6h); p<0.001) (Table 3).

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3.3. Curcumin activates Phase II antioxidant defense mechanism (Nrf2, HO-1 and GSH)

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and stabilizes HIF -1α both in-vitro and in-vivo upon hypoxia exposure. HO-1 is activated by Nrf2, a major transcription factor regulating antioxidant response

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element (ARE)-driven phase-II gene expressions (Turpaev, 2013). We, therefore, attempted to

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determine whether curcumin is able to activate Nrf2 up-regulation in cultured A549 cells and in rat lung? Activation of Nrf2 was determined by western blot using nuclear extracts from the cultured A549 cells and rat lung tissues. The effect of curcumin on A549 cells on Nrf2 and

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HO-1 protein expression is depicted in Figure 3 a and 3 c respectively. This data was extrapolated into an in-vivo system using SD male rats which were exposed to 7620 m for 6 h. The protein expression analysis of Nrf2 and HO-1 in rat lung tissue is displayed in Figure 4 a and 4 c respectively. The activated Nrf2 might have trans-located itself into nucleus and stimulated one of its target genes HO-1. The increased expression of HO-1 in A549 cells and

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in rat lungs was a clear confirmation of the up-regulated Nrf2 activation in curcumin treated cells and rat lungs extract as compared to their respective hypoxia controls. Gene expression pattern of Nrf2 and HO-1 coincided with our western blotting data which is depicted in Figure. 4 e and 4 g. We further examined, whether curcumin treatment can stabilize the transcriptional factor HIF-1α under hypoxic conditions? The fresh nuclear extracts were isolated from A549

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cells as well as from rat lung homogenate and subjected to HIF-1α expression levels by western

blot analysis. Curcumin treatment facilitated stabilization of HIF-1α (Figure 3 b) but one of its

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regulatory proteins VEGF expression was slightly reduced (Figure 3 d) in A549 cells. We also observed curcumin induced stabilization of HIF-1α in hypoxia exposed rat lung tissues (Figure 4 b). HIF-1α stabilization was further confirmed by mRNA expression (Figure 4 f) and by IHC

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(Figure 5 i). The up-regulated HIF-1α expression ultimately resulted into enhanced VEGF

A

N

levels under hypoxia in both in-vitro and in-vivo. Even though HIF-1α was stabilized in

M

curcumin treated hypoxia exposed cells and animals, curcumin abolished the enhanced expression of VEGF in lungs of rat under hypoxia (Figure 4 d). This was further confirmed at

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gene level (Figure 4 h) and by IHC analysis of VEGF in lung tissues (Figure 5 ii). This data clearly indicated that, curcumin was effective in inhibiting HIF-1α mediated higher expression

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of VEGF in both in-vitro and in-vivo. To ensure that equal concentration of proteins have been loaded, β-actin (for cytoplasmic protein expressions) proteins were determined in A549 cells

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and lung homogenates by western blotting. Similarly, histone protein H3, was used as a loading control for representing the nuclear fraction proteins. For mRNA expression studies, β-actin

A

gene expression was used as loading control. Since curcumin can induce Nrf2 both at gene and protein level, experiments were set to estimate the capacity of curcumin in induction of Glutathione-S-transferases (GSTs) at gene level. GST belongs to the family of Phase II detoxification enzymes that catalyzes the conjugation of GSH to wide variety of oxidants. We

17

observed that, curcumin was able to induce the GST gene expression in rat lung tissues, both at normoxia and hypoxia (Figure 4 i). 3.4. Effect of hypoxia and curcumin treatment on pulmonary surfactant proteins expression.

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We further analyzed the expression of pulmonary surfactant proteins in response to hypoxia and modulatory role of curcumin in amelioration of hypoxia induced surfactant protein

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inactivation. Figure 6 depicts the lamellar bodies containing surfactant proteins which were stained by quinacrine under normoxia or hypoxia treated with/without curcumin. However, it

should be noted that, quinacrine staining does not indicate the individual surfactant protein's

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expression in A549 cells, but represents the presence of all four surfactants in the alveoli. The

N

green fluorescence showed the appearance of lamellar bodies, indicating the overall expression

A

of all four surfactant proteins. Under normoxia (Figure 6 a), the florescence intensity was very

M

high indicating the higher expression of all four surfactant proteins which were packed into the

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lamellar bodies. Hypoxia exposed A549 cells (Figure 6 b) showed minimal appearance of stained lamellar bodies indicating decrease in the surfactant production under hypoxia. On the

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other hand A549 cells treated with curcumin 1 h prior to hypoxia showed an appreciable increase in the amount of lamellar bodies expression in relation to hypoxia exposed cells

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(Figure 6 c).

Hypoxia exposure (6 h) resulted in to decreased SP-A protein expression levels in A549

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cells as compared to control, whereas curcumin mitigated these changes under hypoxia (Figure 7 a). The SP-B expression was diminished beyond detectable levels upon hypoxia exposure in A549 cells, whereas curcumin treatment prevented this diminish (Figure 7 b). SP-C is considered as marker for the alveolar type II cells (AT II). In the present study, we found that

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hypoxia increased the expression of SP-C (Figure 7 c) and SP-D (Figure 7 d) however, curcumin supplementation attenuated these changes under hypoxia. Intriguingly, hypoxia exposure enhanced SP-A protein expression in the rat lung tissues (Figure 8 a). However, curcumin supplementation had aided balanced expression of SP-A in

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rat lung tissue homogenates (Figure 8 a). We carried out experiments to determine whether SPA gene transcript levels were affected by hypoxia and curcumin treatment in the rat lung tissues? To our surprise, gene expression levels in rat lung tissues were unaffected in hypoxia

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and curcumin treatment (Figure 8 e). This suggests the fact that SP-A protein expression was

regulated post transcriptionally. These results were further verified by IHC (Figure 9 i). Protein

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expression analysis of SP-B in rat lungs under hypoxia showed diminished levels (Figure 8 b),

N

whereas curcumin treatment appreciably enhanced its expression under hypoxia. However,

A

curcumin did not affect the protein expression pattern of SP-B in the unexposed lung samples

M

(ie. Normoxia animals treated with curcumin). This was further affirmed by mRNA transcript analysis (Figure 8 f) and IHC of paraformaldehyde embedded lung tissue sections (Figure 9 ii).

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The SP-C considered as alveolar type II marker showed augmented expression under hypoxia (Figure 8 c). This was further confirmed at gene transcript levels (Figure 8 g) and IHC (Figure

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10 i). The primary role of SP-D is in host defense against any invading pathogens or allergens.

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The present study revealed that there was a significant increase in SP-D levels upon hypoxia exposure in both in- vitro and in-vivo (Figure 7 d and 8 d). Therefore, we examined whether these changes were at gene level or not? The results revealed that the curcumin abated the

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elevated SP-D mRNA transcripts levels under hypoxia, but didn’t alter SP-D protein expression levels in curcumin supplemented control animals (Figure 8 h). These findings were further confirmed by the immunohistochemistry analysis of SP-D in rat lung tissues (Figure 10 (ii)). We then qualitatively estimated the matured surfactant proteins secreted into BAL fluid by ELISA. The ELISA data (Figure 12) was further affirmed by western blotting, 19

immunohistochemistry and gene expression studies. We regret to inform that, in the present study, we were unable to perform gene expression studies (mRNA studies) in A549 cells due to some experimental restrictions. 3.5. Nrf2 blockade by ATRA: The Nrf2 inhibitory studies revealed that cells treated with

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ATRA and exposed to hypoxia inhibited the Nrf2 expression levels compared to control (Normoxia) and hypoxia exposed cells (6 h). Further, interestingly, we observed that Nrf2

inhibition by ATRA showed reduced expression of SP-A and SP-B with enhanced expression

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of SP-C and SP-D compared to normoxia exposed cells, but showed more or less similar

expression to that of hypoxia exposed cells (Figure 11). When both drugs (curcumin and

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ATRA) were compared, we observed that, curcumin is able to stabilize the Nrf2 expression

N

compared to ATRA treated cells indicating that curcumin has ability to stimulate the up

A

regulation of Nrf2 protein expression and thereby maintained the surfactants proteins

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production under hypoxia. The schematic representation of the study has been clearly

4. DISCUSSION

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represented in Figure 13 which is discussed in the ensuing paragraphs.

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The loss or inactivation of surfactant is associated with number of lung diseases

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such as acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pulmonary edema (Haagsman 1998, Creuwels et al., 1997) etc. Till date, the precise role of oxidative stress induced lung surfactant proteins dysfunction has not been addressed in high altitude

A

pulmonary edema. In our current study, we reported that prophylactic administration of curcumin (both in-vitro and in-vivo) reduced the oxidative stress by enhancing the expression of Phase II antioxidant enzymes mediated through Nrf2 and HIF-1α pathway leading to balanced expression of pulmonary surfactant homeostasis there by reinstated survival signaling under hypoxia.

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Exposure to hypoxia has been reported to be associated with increased pulmonary surfactant's oxidation leading to changes in structural integrity, lungs collapse and edema formation (Haagsman 1998). The increased levels of MDA (a marker of lipid peroxidation) and protein oxidation in hypoxia exposed A549 cells along with BALF of animals exposed to hypoxia indicated the increased oxidative stress in lungs. However, prior

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supplementation of curcumin to rats and exposed to hypoxia significantly (p<0.001)

reduced MDA and protein oxidation levels in A549 cells as well as in the BALF under

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hypoxia. GSH (L-c-glutamyl-L-cysteinyl-glycine) is an important protective antioxidant against free radicals and other oxidants and thus is implicated in immune modulations and

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inflammatory responses (Kuzmenko et al., 2005). The depleted levels of GSH in hypoxia

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exposed cells and BALF samples in the present study underlined the fact in contributing

A

the increased oxidative stress in lungs of rats. The reduced GSH levels in BALF were

M

restored by curcumin supplementation under hypoxia. Our previous studies have revealed the importance of curcumin prophylaxis which was capable of increasing the levels of other

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antioxidant enzymes like GPx and SOD in rat lungs (Sarada et al., 2014). There are various enzymatic and non-enzymatic mechanisms that protect the cells and tissues from oxidants.

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GST and SODs are amongst such mechanisms that are thought to have a key protective role especially in the lungs (Cho and Kleeberger 2009, Dubey and Apenten 2014). Thus, in

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the present study, increased levels of SOD and GST might have favored the reduced lipid peroxidation in the surfactant system.

A

It is well known that, oxidative stress under hypoxia can modulate activation of

distinct signaling pathways. A key molecule regulating the cellular antioxidant response is the basic region leucine-zipper transcription factor Nrf2. Nrf2 is a ubiquitous key modulator of cellular defense against oxidative stress and inflammation. Nrf2 binds to a cis-acting ARE to induce transcription of multiple cytoprotective proteins, including phase II

21

detoxifying enzymes, drug efflux pumps and ROS scavengers (Cho and Kleeberger 2009). Nrf2-ARE binding regulates the expression of more than 200 genes, involved in the cellular antioxidant and anti-inflammatory defense (Dubey and Apenten 2014, Khalak et al., 1999). In the present study, the increased expression Nrf2 under hypoxia must have stimulated its target protein, HO-1. However, HO-1 might have also been induced by a variety of non

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heme products such as ROS, endotoxins, inflammatory cytokines and nitric oxide (Putman

et al., 1997). Several researchers have shown the over expression of HO-1 in human

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pulmonary epithelial cells which conferred resistance to oxidant mediated lung injury

(Yano et al., 2000). Rats pre-treated with haemoglobin (a potent inducer of HO-1) were

U

less susceptible to oxidant-mediated endotoxin shock and to hyperoxic lung injury (Griese

N

1999). In our present study, we showed that curcumin prophylaxis enhanced the HO-1

A

mRNA followed by protein expression of HO-1, both under normoxia and hypoxia. Our

M

results were in accordance with the earlier studies (Pastva et al., 2007). The enhanced Nrf2 followed by increased HO-1 under curcumin administration indicated the enhanced cellular

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resistance to oxidative damage in the lungs, but could not able to enhance the antioxidant enzymes and all 4 surfactants. This infers that the cells must have tried to enhance the Nrf2

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and HO-1 to cope up with the increased oxidative stress under hypoxia, but due to some unknown mechanisms there must be some changes taking place at translation level of these

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proteins under low oxygen availability (Hypoxia); which needs further investigation. These results inferred that, the curcumin prophylaxis under hypoxia activated the Nrf2 which

A

further triggered the up-regulation of antioxidants (GSH, GPx, SOD and GST) and HO-1 leading to maintenance of lung's oxidant and antioxidant balance. The alveolar walls are very delicate and alveolar stability is dependent on an intact surfactant system. Generally, it was reported earlier that, oxidized surfactant was very fragile and less surface active (Khalak et al., 1999). Therefore, any impairment of the

22

surfactant system will contribute to lung injury and fluid accumulation. Surfactant proteins predominantly include hydrophilic surfactant proteins like SP-A and SP-D, and the hydrophobic surfactant proteins like SP-B and SP-C (Putman et al., 1997). Initially it was thought that all four proteins were important in facilitating the reduction in surface tension. However, subsequent studies have shown that, only SP-B is essential for this function

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(Haagsman 1998). Erika et al., (2000) have reported that SP-A and SP-D protein expressions by pulmonary cells were increased in many forms of pulmonary diseases and

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therefore play a pivotal role in surfactant homeostasis and pulmonary immune responses.

Recent studies revealed that SP-C binds to lipopolysaccharide (LPS) (Griese 1999)

U

however, the function of this protein in-vivo is not fully understood. In the present study,

N

we found that hypoxia significantly reduced the expression of SP-A protein at 1 h and 3 h

A

of hypoxia exposure in A549 cells compared to control. However further increase in

M

hypoxia duration up to 6 h showed a significant increase in SP-A production compared to 1 h and 3 h of hypoxia exposure but still significantly reduced compared to control. Later

ED

when these cells were exposed to higher hypoxia durations (12 h and 24 h) showed diminished expression. The slight increase in 6 h hypoxia exposure could be attributed to

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fact that the cells might have tried to enhance the SP-A protein expression in order to maintain the cell survival (under hypoxia) but could not sustain the hypoxic insult at these

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time points. Moreover, in contrast to these findings, surprisingly, we observed an interesting phenomenon in the present study was that, the SP-A levels were significantly

A

enhanced in lung tissues, under hypoxia. The increased expression of SP-A in the lungs of rats in present study might be due to the triggering of the non alveolar (other than type II cells) cells upon hypoxic exposure to cope up with increased oxidative stress in lungs. The same results were confirmed by the immunohistochemical analysis of SP-A (Fig 9 i). In order to further strengthen our results, we measured the levels of SP-A secreted into the

23

BALF by ELISA (Fig 12). The results showed 200 % increase in SP-A proteins in BALF, as compared to the control. This indicated that SP-A was secreted from ATII cells in the lungs. Apart from ATII cells, SP-A is also produced by Clara and conducting airway cells (Pastva et al., 2007). Therefore, it seems that SP-A expression was also triggered in the non ATII cells, contributing in causing increased levels of SP-A in the lung tissue. Recent

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studies have revealed that SP-A protects surfactant phospholipids from oxidative stresses due to hypoxia, hyperoxia, air pollutants and lung inflammation (Kuzmenko et al., 2005).

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SP-B is the only surfactant protein strictly required for breathing. Indeed, the

absence of SP-B is associated with a lethal respiratory failure. SP-B along with SP-A and

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calcium is involved in formation of tubular myelin (Parra et al., 2013). Tong et al (2006)

N

have reported that hypoxia induced mitogenic factor (HIMF) modulates SP-B and SP-C in

A

mouse lungs. Likewise, Ito et al., (2011) have also reported that HIF modulates SP-B and SP-C expression under hypoxia. It is clear that under hypoxia, the expression of SP-B and

M

SP-C seems to be regulated through HIF-1α. However, elevated levels of VEGF might have

ED

induced the lung injury and therefore resulted into decreased production of SP-B (Digeronimo et al., 2007). Boggaram et al., (2010) have reported that the expression of SP-

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B can be decreased by the oxidants. In the present study, this could be the reason for the diminished SP-B levels in hypoxic exposure even though HIF-1α was expressed

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abundantly. Vives et al., (2008) have reported that the decreased levels of SP-B for 4 h under hypobaric hypoxia remained same upon further prolonged extension of intermittent

A

hypobaric hypoxia exposure time. Our mRNA expression data was in disagreement with them, as we observed reduced SP-B mRNA expression by 6 h of hypoxia. The contradicting results may be due to the difference in exposure to altitudes and animals used in these experiments. We have exposed the rats to hypobaric hypoxia (282 mm Hg) because the smaller animals have higher capillary density in tissues, making them more resistant to

24

hypoxia. The loss of SP-B under hypoxia must have resulted into increased surface tension, thereby leading to collapsed alveoli. Supplementation of curcumin, stabilized the expression of HIF-1α, but reduced its target gene, VEGF protein expression, both in A549 cells and lungs of rats. The stabilized expression of HIF-1α might have favored the augmented expression of SP-B in curcumin supplemented hypoxia exposed cells and

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animals.

SP-C is exclusively synthesized by ATII cells. SP-C stabilizes alveolar surfactant

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film, enhances the adsorption rate of phospholipids and also facilitates reduction in alveolar surface tension. In case of edema formation, SP-C prevents surfactant inactivation from

U

invading of plasma proteins (Chander et al., 1975). In the present study, in order to combat

N

with the increased fluid accumulation, the ATII cells might have tried to increase the SP-C

A

levels to resist surfactant inactivation and also withstand the increased oxidative stress

M

under hypoxia, but could not sustain as evidenced by increase MDA and protein oxidation levels under hypoxia. However, cells under hypoxia in presence of curcumin showed

ED

reduced expressions of SP-C.

SP-D is mainly synthesized in alveolar type II cells of the lung and it is released

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into the blood during lung injury. SP-D appears to have both pro- and anti-inflammatory signaling functions. SP-D multimerization is a critical feature of its function and plays an

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important role in efficient innate host defense. Recent studies have highlighted the capability of NO to modify SP-D through S-nitrosylation, causing the activation of a pro-

A

inflammatory role for SP-D (Guo et al., 2008). The exposed S-nitrosylated tail domain binds to the calreticulin/CD91 receptor complex and initiates a pro-inflammatory response through phosphorylation of p38 and NF-κB activation (Atochina 2012). Earlier, we have reported that hypoxia enhances the NF-κB expression, leading to increased proinflammatory cytokines and cell adhesion molecules (Sarada et al., 2008) in lungs of rats.

25

Hence it was reasonable to hypothesize that increased expression of SP-D must have triggered an increased pro-inflammatory milieu in BALF leading to lung injury. In contrast to our present results, Vives et al., (2008) have noted a significant increase in SP-D levels in 4 h of hypobaric hypoxic exposure at 380 mmHg. Supplementation of curcumin attenuated the lung injury as well as reduced the oxidized environment in lungs by

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augmenting the expression of Nrf2 and HO-1 thus, modulating the expression of SP-D in both in-vitro and in-vivo. The ATRA significantly inhibited the expression of Nrf2 protein

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levels under hypoxia leading to reduced SP-A, SP-B with increased expression of SP-C and SP-D, indicating that under hypoxia inhibition of Nrf2 is able to alter the surfactant

U

proteins production. Whereas curcumin on the other hand is able to increase the Nrf2

N

expression both at mRNA and protein level with balanced expressions of surfactants

A

making it as a potential molecule in maintaining the surfactant homeostasis under hypoxia.

M

5. CONCLUSION:

These results clearly indicate that hypoxia environment at high altitudes lead to

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collapse of the lungs due to change in the oxygen paradox through altered functions of all four types of surfactant proteins. However, curcumin prophylaxis austerely mitigated these

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changes and maintained the alveolar membrane by bringing adaptive mechanisms leading to pulmonary surfactant homeostasis involving up-regulation of Nrf2 and HIF1-α

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dependent pathway. Therefore, the present study opens a new area for developing curcumin

A

prophylaxis as potential strategy for the prevention of HAPE.

6. Conflict of interest: We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

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9. DECLARATIONS: 1. Consent for publication: Not Applicable. 2. Availability of data and Materials: The data supporting our results in the manuscript

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has been clearly mentioned in the Materials and Methods section of the main paper. No supporting files have been uploaded with this manuscript. All the data presented here

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is in machine readable format.

3. Authors’ contributions: Sarada SKS conceived and designed the experiments. Titto

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M & Sarada SKS performed the experiments. Titto M did the hypoxia exposure

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experiments. Sarada SKS and Titto M prepared the manuscript. Titto M and Sarada

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SKS prepared the graphs and tables. All the authors read and approved the final

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manuscript.

4. Funding / Grants: The study was conducted, under the project “Facilitation of

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acclimatization to hypobaric hypoxia and improvement of physical performance: Role of hypoxia mimetics” funded by the Defence Research and Development Organisation,

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Government of India.

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5. Acknowledgment: We are thankful to the Director,

DIPAS, DRDO, India, for

providing all the support and facilities for conducting this experiment.

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6. Ethics Committee Approval: The Institute’s ethics committee (IEC) approved all the experimental protocols for this study and followed the guidelines of Universities of Federation for Animal Welfare (UFAW) for animal research.

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A

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Sarada S.K.S., Mathew T., PatirH., 2014. Prophylactic Administration of curcumin abates the incidence of hypobaric hypoxia induced pulmonary edema in rats: a molecular

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approach. J PulmRespir Med. 4(1),164, https://doi:10.4172/2161-105X.1000164

Sati L., Celik Y.S., Demir R., 2010. Lung surfactant proteins in the early human placenta.Histochem Cell Biol. 133(1), 85–93. 0642-9

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Saxena S., Kumar R., Madan T., Gupta V., Muralidhar K., Sarma P.U., 2005. Association of polymorphisms in pulmonary surfactant protein A1 and A2 genes with high-altitude pulmonary edema. Chest. 128(3),1611-19. https://doi.org/10.1378/chest.128.3.1611 Titto M., and Sarada S.K.S., 2015. Attenuation of NFkB activation augments alveolar transport proteins expression and activity under hypoxia. Intern. J. Sci. Res. 14 (3),

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Tong Q., Zheng L., Dodd O.J., Langer J., Wang D., Li D., 2006. Hypoxia-induced

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mitogenic factor modulates surfactant protein B and C expression in mouse lung. Am. J. Respir. Cell Mol. Biol. 34, 28-38. https://doi.org/10.1165/rcmb.2005-0172OC

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Vaporidi K., Tsatsanis C., Georgopoulos D., Tsichlis P.N., 2005. Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549

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Wang J.Y., Yeh T.F., Lin Y.C., Miyamura K., Holmskov U., Reid K.B., 1996. Measurement of pulmonary status and surfactant protein levels during dexamethasone treatment of neonatal respiratory distress syndrome. Thorax. 51, 907–913. Yano T., Mason R.J., Pan T., Deterding R.R., Nielsen L.D., Shannon J.M., 2000. KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in rat

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8. Legends Figure 1: Effect of hypoxia on surfactant proteins expression pattern in A549 cells. Time course expression of surfactant proteins (a) SP-A (b) SP-B (c) SP-C and (d) SP-D were investigated in A549 cells upon exposure to normoxia (21% O2) or hypoxia (3%) for different

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durations viz. 0h (Normoxia=control), 1 h, 3 h, 6 h, 12 h and 24 h. (a) SP-A transcript expressions were decreased from 1 h-3 h and at thereafter slightly increased but further

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increased hypoxic exposures lead to diminished expression of SP-A. While hypoxia down

regulated SP-B expression from 1 h onwards, but from 3 h exposure onwards virtually no SPB expression was observed by increasing the hypoxia exposure up to 24 h. The expressions of

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SP-C and SP-D were increased in time depended manner. Cells from at least 4-6 independent

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experiments were used for each time point. 0 h-Normoxia or control.

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Figure 2: Hypoxia induced oxidative stress imbalance in A549 cells. A549 Cells were exposed to hypoxia for different durations viz. 0 h, 1 h, 3 h, 6 h, 12 h and 24 h . (a) Protein oxidation and (b) Lipid peroxidation (MDA) in A549 cells. Data is representative results from 4 independent experiments performed in triplicate, values are mean ± S.D. * P,<0.05 Normoxia vs Hypoxia (3 h and 6 h);

#

P <0.001 Normoxia vs Hypoxia (3 h, 6 h, 12 h, and 24 h), Nor:

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normoxia.

Figure 3: Protective effect of curcumin on protein expressions of Nrf2, HIF-1α and their

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regulatory genes HO-1 and VEGF respectively in A549 cells under hypoxia. (a) Curcumin treated A549 cells showed stabilized expression of Nrf2 (arrow indicating the Nrf2 expression) (b) Curcumin treated A549 cells and exposed to hypoxia for 6 h showed increased translocation of HIF-1α protein in nuclear extract. (c) Activated Nrf2 further triggered HO-1 expression in A549 cells and curcumin stabilized its expression. However, (d) pre-treatment with curcumin 37

abolished the enhanced expression of VEGF in A549 cells (arrow indicating the VEGF

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expression). Nor-Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin.

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Figure 4: Prophylactic efficacy of curcumin on Phase II antioxidant enzymes in rat lungs under hypoxia. Representative photographs of Western blot analysis of (a) Nrf2 protein (b)

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HIF-1α (c) HO-1(d) VEGF along with actin /Histone H3 controls in the rat's lung after 6 h

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hypobaric hypoxia treated with and without curcumin. The representative photographs of RTPCR gene sexpression analysis (e) Nrf2 (f) HIF-1α, (g) HO-1 (h) VEGF and (i) GST in rat lung tissue. Prophylactic administration of curcumin increased nuclear translocation of Nrf2 thereby

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also elicited expression of HO-1. Rats treated with curcumin and exposed to hypoxia showed stabilized HIF-1 α expression followed by reduced levels of VEGF expression compared to control (hypoxia). Nor- Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin.

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Figure 5: Immunohistochemical staining of (i) HIF-1α and (ii) VEGF expression in lungs of

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rats exposed to hypobaric hypoxia (7620 m, 6 h). It was noticed that, Hypoxia resulted into significant increase in transcriptional factor HIF-1α and one of its regulatory molecule VEGF

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in rat pulmonary tissue. However, prior treatment of rats with curcumin (50 mg/Kg BW) and exposed to hypobaric hypoxia stabilised the HIF-1α with appreciable reduction in VEGF expression in pulmonary tissues. VEGF was further counter stained with H&E. The signals

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were detected by DAB staining. Magnification: x 400. Nor- Normoxia; Hypo-Hypoxia (6 h) and Cur- curcumin

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Figure 6: Quinacrine staining showing lamellar bodies representing pulmonary surfactants production and their modulation by curcumin prophylaxis. Cells were treated with 10µM

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curcumin for 1 h exposed to hypoxia (3%) and stained with quinacrine. The cells were observed under fluorescence microscope to monitor quinacrine staining of lamellar bodies (Scale bar: 10

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µm). Images are representative of 3 independent cell preparations.

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Figure 7: Effect of curcumin administration on pulmonary surfactant proteins expression in

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A549 cells under hypoxia. Hypoxia exposure showed decreased expressions of SP-A and SPB (arrow indicating the SP-B expression) with increased expressions of SP-C and SP-D.

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Curcumin supplementation increased the expression of (a) SP-A and (b) SP-B expressions in A549 cells under. Curcumin obliterated the enhanced expression of (c) SP-C and (d) SP-D in A549 cells under hypoxia. Data is representative of 3-4 independent experiment. Nor-

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Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin.

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Figure 8: Effect of curcumin administration on pulmonary surfactant proteins expression in lungs of rats under hypoxia. Curcumin supplementation abolished the increased expression of

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(a) SP-A protein but (e) gene expression analysis showed had no change in pulmonary tissue upon hypoxia exposure. (b) SP-B expression was augmented by curcumin administration in

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hypobaric hypoxia exposed rat lung tissues which was also confirmed by (f) RT-PCR gene expression analysis. Curcumin ameliorated the increased protein expression and gene

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expression of SP-C (c and g respectively) and SP-D (d and h respectively) upon hypoxia exposure in lungs of rats. Photographs are representative of 3-4 independent experiment. NorNormoxia; Hypo- Hypoxia (6 h) and Cur- curcumin

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Figure 9: Immunohistochemical analysis showing the prophylactic effect of curcumin (50 mg/kg BW) on the expression of (i) SP-A and (ii) SP-B proteins in the lungs of rats exposed

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to hypobaric hypoxia at 7620 m, for 6 h. Exposure to hypobaric hypoxia showed increased expression of SP-A and reduced expression of SP-B under hypoxia. The non alveolar

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expression of SP-A proteins was vividly seen. Whereas, preconditioning with curcumin significantly abolished the elicited expression of SP-A protein with augmented expression of SP-B protein in lungs of rats under hypoxia. Surprisingly, curcumin did not alter the SP-A and

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SP-B expression under normoxia compared to control. The figure is representative of at least 4–5 animals from each group. The signals were detected by DAB staining. Magnification: 400 X. Nor- Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin.

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Figure 10: Immunohistochemical analysis showing the prophylactic effect of curcumin (50 mg/kg BW) on the expression of (i) SP-C and (ii) SP-D proteins in the lungs of rats exposed to

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hypobaric hypoxia at 7620 m, for 6 h. Exposure to hypobaric hypoxia showed increased

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expression of SP-C and SP-D under hypoxia. Whereas, preconditioning with curcumin significantly abolished the elicited expression of SP-C and SP-D protein in lungs of rats under hypoxia. Surprisingly, Normoxia animals receiving curcumin showed unmodified SP-C and

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SP-D expression as compared to control animals. The figure is representative of at least 4–5 animals from each group. The signals were detected by DAB staining. Magnification: 400 X. Nor- Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin.

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Figure 11: Determination of Nrf2 and surfactant proteins expression in presence of ATRA and

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curcumin in A549 cells under hypoxia. (a) Nrf2 (b) SP-A (c) SP-B (d) SP-C and (e) SP-D. A549 cells treated with ATRA (1.5 µM) and curcumin (10 µM) and exposed to hypoxia (3 %

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for 6h). ATRA significantly attenuated the Nrf2 expression and altered the surfactant proteins. On the other hand curcumin administration under hypoxia up regulated the Nrf2 expression

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along with appreciable production of surfactants. Data is representative of 3-4 independent experiments. Nor- Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin, ATRA- All trans retinoic acid.

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Figure 12: Protective prophylactic efficacy of curcumin on surfactant proteins (SP-A, SP-B, SP-C and SP-D) estimation in BALF of rats exposed to hypobaric hypoxia at 7620m for 6h.

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The BAL fluid was collected from control, hypoxia exposed (7620m, 6h), curcumin treated unexposed and curcumin treated hypoxia exposed animals (n=6 animals per group). The cell

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free BAL fluid was assayed for the secreted pulmonary surfactant proteins qualitatively using

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specific primary antibodies. Hypoxia exposure resulted into significant increase in SP-A, SPC and SP-D with reduced levels of SP-B in BALF of rats indicating the altered surfactant homeostasis. However, preconditioning with curcumin completely abrogate these changes

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under hypoxia. The image is representative of 4 independent assays performed. Nor-Normoxia; Hypo- Hypoxia (6 h) and Cur- curcumin.

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Figure 13: Graphical Abstract: Schematic depiction of possible mechanism of curcumin in

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regulating the pulmonary surfactant homeostasis under hypoxia. Hypoxia exposure enhanced

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the reactive oxygen species (ROS), total protein oxidation and lipid peroxidation (MDA) levels followed by reduced anti oxidant enzymes (GSH, GPx, SOD and GST) along with diminished

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expression of SP-B and increased expression of SP-A, SP-C an SP-D in BALF and in A549 cells, indicating the increased oxidation in epithelial lining fluid (ELF). The administration of curcumin facilitated the induction of ARE/Nrf2 pathway. Our data provide information

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regarding the possible use of curcumin as a potential prophylactic drug as it effectively inhibited the oxidative stress (↓ROS, ↓ MDA and ↓ protein oxidation), maintained surface tension by up regulating surfactant proteins expression (↓SPA, ↑SPB, ↓ SPC and ↓ SPD) via induction of ↑Nrf2, ↑HO-1 along with stabilized HIF1-α followed by reduced VEGF release

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leading to pulmonary surfactant homeostasis under hypoxia. ↑ - Up regulation, ↓ - Down

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regulation.

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Table 1: List of primers used for the reverse transcriptase PCR.

Table 2: Effect of curcumin administration on lipid peroxidation (MDA), protein oxidation, GSH, GPx and SOD in A549 cells exposed to 3 % hypoxia. Hypoxia significantly increased

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the oxidant levels in cells as compared to control. On the other hand curcumin treatment reduced the MDA and protein oxidation levels with significant increase in GSH, GPx and SOD

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levels in A549 cells under hypoxia, indicating the potent antioxidant activity of curcumin under hypoxic conditions. Cells from at least 4-6 independent experiments were used for each time point. Values are mean ±SD. † P < 0.001 compared with control; ¤ P<0.001 compared with

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hypoxia. Cur= Curcumin.

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Table 3 : Effect of curcumin administration on lipid peroxidation (MDA), protein oxidation,

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GSH and GST in BALF of rats, exposed to hypobaric hypoxia at 7620 m for 6 h. It was observed that, hypoxia resulted into significant increase in total protein oxidation and lipid

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peroxidation followed by significant reduction in GSH and GST in BALF indicating the increased oxidation in ELF. However, prior treatment of rats with curcumin and exposed to

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hypoxia resulted into significant decrease in oxidation of ELF (↓ MDA and ↓ protein oxidation) with significant increase in GSH and GST levels in lungs of rats. Values are mean ±SD (n=6).

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†

P<0.001 compared with Normoxia (0h) ¤ P< 0.001 compared with hypoxia group and §P<0.05

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compared with hypoxia (6h) group. Cur= curcumin.

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Table 1:

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Transcript

Primer sequence (F= Forward; R= Reverse) in 5’-3’ F GCCAGCTGAACTCCTTAGAC R

230

NM002133.2

146

Yano et al 2000

176

Do

200

Do

245

Do

350

Gen bank V01217

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A β-Actin

NM 001110334.1

R GGAATTCTCTGGAGCCATCTTCATGATG F CGGATCCCGGAAGAGCCTTTTGAGGATG

SP-D

196

R GGAATTCTGGTCCTTTGGTACAGGTTGC F CGGATCCCATACTGAGATGGTCCTTGAG

SP C

AF057308

R GGAATTCCGTTCTCCTCAGGAGTCCTCG F CGGATCCGAGCAGTTTGTGGAACAGCAC

SP-B

200

R CAGGGCAGCCTAGCCTGGGA

F CGGATCCAGTCCTCAGCTTGCAAGGATC SP-A

NM 012504

A

N

R GCAGTAGCTGCGCTGGTAGAC F TGGCCAGCCGTACGGAGGAA

GST

129

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R ATT TGA TGG GTG AGG AAT GGG TT F GGCTCTGAAACCATGAACTTTCT

VEGF

Rn00477784

R GTACAAGGAGGCCATCACCAGA F TGCTTGGTGCTGATTTGTGA

HIF-1α

200

GATTCGTGCACAGCAGCA

F CAGTCGCCTCCAGAGTTTCC H0-1

Accession numbers or Refrence

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Nrf2

Product size (in bp)

R GGAATTCACAGTTCTCTGCCCCTCCATTG F GAGGATATCGCTGCGCTGG R ATCTTTTCACGGTTGGCCT

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Table 2: Normoxia

Hypoxia

Normoxia+Cur

Hypoxia + Cur

Lipid Peroxidation (MDA) (ηmol/mg rotein) Protein Oxidation (% / mg of protein)

0.98±0.20

3.07±0.30†

0.87±0.20

0.51±0.10¤

100.00 ± 1.50

425.00 ± 7.40†

99.80±2.80

145.00±12.60 ¤

GSH (ηmoles/mg protein)

263.30 ± 10.80

139.70 ± 9.50†

285.60 ± 11.90

250.20 ± 8.03¤

GPx (U/mg proein)

12.69 ± 0.50

6.67 ± 1.00†

14.70± 1.80

11.70± 1.50 ¤

SOD (U/mg protein)

0.25 ± 0.07

0.094 ± 0.05†

0.27 ± 0.06

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Table 3 :

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Parameter

0.208 ± 0.04¤

Normoxia

Hypoxia

Normoxia+Cur

Hypoxia + Cur

Lipid Peroxidation (MDA) (ηmoles/ml of BALF)

2.70 ± 0.09

17.60 ± 1.20†

2.60 ± 0.56

4.90 ± 0.59§

Protein Oxidation (% / 98.50 ± 5.40 ml of BALF)

550.03 ± 41.20†

102.50 ± 8.10

234.80 ± 16.90¤

GSH (ηmoles/ml of BALF)

62.30 ± 3.70

30.08 ± 2.70†

69.60 ± 2.90

GST(μmoles/ml of BALF )

0.42 ± 0.14

0.12 ± 0.07†

0.48 ± 0.10

52.00 ± 1.80¤

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Parameter

0.38 ± 0.09¤