Quercetin: a savior of alveolar barrier integrity under hypoxic microenvironment

ABSTRACT

High altitude pulmonary edema (HAPE) is generally characterized by the loss of alveolar epithelial barrier integrity. The current study was undertaken to assess the noninvasive approaches of HAPE diagnosis and to evaluate the prophylactic potential of quercetin in preventing alveolar junction impairments. Male SD rats fed with quercetin 1 h prior to hypoxia (7,620 m, for 6 h) were selected. PET/CT imaging was performed to visualize the lung uptake of ¹⁸F-FDG in animals under hypoxia. Further, oxidant status, catalase activity, hematological & blood gas parameters were evaluated. Moreover, tight junction (TJ) proteins (ZO-1, JAM-C, Claudin-4, and occludin) expression analysis was accomplished using immune-blotting. The structural differences in lung epithelia were noted by TEM imaging. Quercetin prophylaxis has significantly reduced the FDG uptake in rat lungs under hypoxia. It has also dramatically alleviated the protein oxidation followed by an elevation in catalase activity in the lungs under hypoxia. The TJ protein expression in the lungs has also been restored to normal upon quercetin pre-treatment. Concomitantly, the quercetin preconditioning has elicited the stable blood gas and hematological parameters under hypoxia. The observations from TEM imaging have also implicated the normal lung epithelial structures in the quercetin pretreated animals under hypoxia. Quercetin prophylaxis has significantly restored alveolar epithelium integrity by abating oxidative stress in the lungs under hypoxia.

Abbreviations: CT- Computed Tomography¹⁸F-FDG- Fluorodeoxyglucose (¹⁸FHAPE- High Altitude Pulmonary EdemaHb- HemoglobinHCT- HematocritHCO₃^– BicarbonateJAM- Junctional Adhesion MoleculeKBq- Killo BecquerelPaO₂- Partial pressure of arterial oxygenPaCO₂- Partial pressure of arterial carbon di-oxidePET- Positron Emission TomographyRBC- Red Blood CorpusclesSD- Sprague DawleyTJ- Tight JunctionsTEM- Transmission Electron MicroscopyWBC- White Blood CorpusclesZO- Zona Occludin.

1. Introduction

High altitude pulmonary edema (HAPE) is generally characterized by extravascular fluid accumulation into alveolar spaces of the individuals ascending at an altitude of 2,500 m or above, due to aberrations in the structure & function of the tight junction (TJ) proteins assembly resulting in loss of alveolar barrier function¹. HAPE is known to be completely reversed if diagnosed and treated at an early stage¹. HAPE typically occurs into two forms- in the first form, it influences non-acclimatized, healthy individuals ascending to HA rapidly; while in the second form or commonly referred to as ‘reentry HAPE, it is sought to affect the acclimatized individuals ascending at HA after a short stay at low altitude regions, depending on the speed, time, and mode of ascent.² Therefore, studies have substantiated mostly on the time of acclimatization, the pace of ascent, and appropriate planning prior to ascending at HA.^2,3 HAPE is a multi-factorial pathological condition arising due to reduced partial pressure of oxygen as a consequence of lower barometric pressure at HA regime.¹

Many of the recent studies have indicated that enhanced production of reactive oxygen and nitrogen species (ROS & RNS) generated are mainly responsible for the exacerbation of vascular permeability.^4,5 In addition to this, exposure to a low barometer has demonstrated dramatic alterations in Hif-1α levels followed by hypersensitivity of NFĸB signaling leading to the deregulation of essential genes involved in the development of- inflammation, oxidative stress, apoptosis, erythropoiesis, etc. which altogether contributes to fluid influx within the lungs.^6,7 All these factors cumulatively result in the loss of tight junctions (ZO-1, ZO-2, JAM-C, claudin-4, etc.) integrity operating within the lung vasculature. This loss of tight junction integrity will lead to the onset of HAPE via fluid buildup in the alveolar epithelium.^8,9 TJ serves as the backbone of the cellular epithelium which maintains the structural integrity of alveolar epithelial barriers and enables the physical basis for solutes and ions permeability.¹⁰ Not only this but these TJ proteins also functions as a fence by preventing the diffusion of lipids apical and basolateral membranes and hence commonly known as “fence-function”.¹⁰ These TJs are reported to be composed of the peripheral membrane and transmembrane proteins associated firmly with the actin-based cytoskeletal.¹¹ This assembly of TJ protein structure and function inside the membrane is controlled by various physiological and pathophysiological stimuli.¹¹ Despite an extensive recollection of the natural history and clinical features of HA- induced illnesses, the exact pathophysiology of HAPE is not clear. Therefore, in the present study, efforts were made to outline the diagnostic approaches to distinguish HAPE from other HAI (high altitude illnesses) under reduced oxygen environment at an early stage using noninvasive nuclear medicine based procedures and further we strived to prevent the extravasation of plasma proteins from pulmonary vascular bed to alveolar spaces, a major manifestation of HAPE, by attenuating inflammation and oxidative stress under hypoxic condition.

This study employed the use of PET/CT- imaging techniques using 2-deoxy-2-[fluorine-18] fluoro-D-glucose^12F-FDG as a radiotracer of PET for visualizing the leakage of plasma proteins into the pulmonary vasculature of the hypoxia exposed animals. Positron emission tomography (PET), is reported to be a breakthrough in the field of nuclear medicine, which works on the principle of quantifying positrons emitting out of radiotracer molecules and allows the visualization of the abnormal site within the body.^13,14 This radiotracer measures biological processes and the resultant images of the sites where these tracers accumulate are provided by PET acquisition.¹⁴ The commonest of all in today’s nuclear medicine use is¹²F-FDG, a radio-labeled glucose molecule with a half-life of nearly 110 min.¹⁵ The enormous amount of data available in the literature stipulating the applications of¹²F-FDG in accurately ascertaining the sites and stage of indispositions such as- various types of cancers, inflammation borne- diseases, pulmonary edema, lung fibrosis, muscular atrophy, kidney dysfunctioning, and neurological disorders^{12,16, 17}F-FDG-PET imaging is mainly applied to determine the sites where unusual glucose metabolism occurs.¹⁵ Initially, computed tomography (CT), was being carried out for determining the structural changes in the tissues, but the functional imaging remained unseen, therefore, the use of PET/CT co-registered imaging came into existence to ascertain both structural and functional changes in the tissues of the body due to any diseased condition.¹⁸

The second aspect of the study dealt with the prevention of HAPE causing factors especially- transvascular leakage, oxidative stress, and inflammation, by bioflavonoid therapy. However, the administration of commercially synthesized drugs for HAPE prevention especially- nifedipine, acetazolamide, salmeterol, and aminophylline, have been known to be associated with several ill-effects on mammalian system.¹² Direct exposure to nifedipine is associated to cause nausea, headache, dizziness, and cardiac issues, whereas acetazolamide and salmeterol are reported to cause respiratory failure, hyperglycemia, and frequent mood swings when taken in excess.^19,20 Aminophylline is associated with cardiotoxicity and chest tightness.²⁰ Therefore, to overcome these shortcomings arising out of these commercially available drugs for preventing HAPE, we have used quercetin (Quer;2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4 H-chromen-4-one), a phyto-flavonoid as a prophylactic agent that tends to reduce hypoxia-induced inflammation, oxidative stress, and increased capillary permeabilization resulting in HAPE onset with minimum or no side-effects upon direct exposure to the drug. Quercetin is a naturally occurring dietary flavonol, which consists of 2- benzene rings associated with heterocyclic pyran or pyrone ring.²¹ It is widely distributed in edible parts of all phyto-products such as- bulbs, tubers, leafy vegetables, cocoa, tea, fruits, etc.²² It is reported to have potent anti-inflammatory, anti-oxidant, anti-apoptotic, anti-cancerous, and nitric oxide induction properties.²² Studies performed both on in vivo (mice and rats) and in vitro (B. subtilis strain) systems by taking quercetin as an anti-toxicity agent has elicited its anti-carcinogenic and anti-mutagenic characteristics as well.²³ Besides this, various recent studies have also proved that quercetin is acting most suitably when fed orally compared to other routes of administration such as- intravenous.^23,24 The LD₅₀ range of quercetin when supplemented orally to the rats is reported to be nearly 160 mg/Kg BW.^23,24 In addition to this, literature also suggests, that quercetin uptake from 2000–5000 mg/day did not show any toxicity or deleterious effects.²⁵ Thus it can be interpreted, that quercetin prophylaxis is devoid of any side-effects when supplemented orally and within the quantifiable range under hypoxic conditions to prevent transvascular leakage in the lungs.

Thus, the major objectives of the current study are: (1) To ascertain the effects of acute hypoxia on the TJ proteins within the lungs of rats using molecular biology approaches and nuclear medicine modalities; (2) To govern the effectiveness of quercetin prophylaxis in abrogating the impairment of alveolar barrier integrity in lungs of animals exposed to hypoxia by attenuating oxidative stress and restoring the basal expressions of TJ proteins.

2. Materials and methods

2.1. Chemicals and reagents

Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4 H-chromen-4-one) was procured from Sigma Aldrich (St. Louis MO, USA), Dimethylsulphoxide (DMSO) from Sisco Research Laboratory (SRL, Maharashtra) and Flourscein sodium salt from sigma Aldrich (St. Louis MO, USA), DNPH (sigma Aldrich (St. Louis MO, USA) and rest all the other chemicals and reagents were of analytical grade.

2.2. Equipment

PET/CT imaging was performed using a micro-PET/CT scanner (TriFoil Imaging, Northridge, CA, United States).

2.3. Drug preparation

Quercetin (50 mg/Kg BW) was prepared freshly by dissolving in a vehicle (0.5% DMSO) and administered orally to the animals 1 h prior to the hypoxia exposure. 50 mg/Kg BW dose of quercetin was found to have significantly reduced the transvascular leakage in the lung of rats exposed to hypoxia for 6 h.²⁶

2.4. Experimental animals and ethical guidelines

Male Sprague Dawley (SD) rats weighing between 180–200 g were obtained from the central animal facility of DIPAS-DRDO, Delhi, India. Animals were housed in experimentally designed polypropylene cages of 32in$.\times 24in.\times 16in$. dimension provided with standard conditions (25 ± 2°C temperature, 55 ± 5% relative humidity, and 12 h light/dark cycle) and free retrieval to standard laboratory food and water ad libitum. All protocols involving animal studies were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), DIPAS, Delhi, India, accredited to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. We have followed the standards outlined in the guide for the Care and Use of Laboratory Animals (National Academy of Sciences, Washington, DC).

2.5. Experimental procedure

Phase I: Phase I study was carried out to ascertain the effect of hypoxia-induced alterations in oxidative stress levels &TJ proteins expressions leading to the loss of alveolar barrier integrity in the lungs of rats exposed to hypoxia, and to determine the prophylactic potential of quercetin in reverting these changes in the animals exposed to hypoxia (6 h). The study involved 24 healthy, male SD rats divided into 4 groups (n = 3). Where group 1 served as normoxia control (0 h) received the only vehicle, group 2 animals, considered as hypoxia control (6 h), were primarily fed with a vehicle and then exposed to hypobaric hypoxia for 6 h. Group 3 animals were supplemented with quercetin (50 mg/kg BW) without exposure and group 4 animals were preconditioned with quercetin (50 mg/kg BW) 1 h prior to hypoxia exposure (6 h).

Phase II: The findings from the Phase I study were confirmed later by conducting this second phase (Phase II) of the study by using 24 male SD rats divided into the same 4 groups (n = 6) as that of Phase I, to evaluate/diagnose the hypoxia-induced fluid accumulation in the lungs of rats using PET-CT imaging studies.

2.6. Exposure to hypoxia

Animals were exposed to a simulated hypobaric hypoxia chamber (Matrix, India) for 6 h at an altitude of 7,620 m (280 mm Hg) with a sustained temperature of 25 ± 2°C. This 6 h of hypoxia exposure has been opted based on previous studies by our lab demonstrating the elevated transvascular leakage at 6 h of hypoxia exposure time at 7620 m.⁴ Fresh air was flushed at the rate of 4 l/h along with the relative humidity of 55 ± 5% inside the hypoxia chamber. Furthermore, the arterial partial pressure of oxygen (PaO₂) in control rats was observed to be 95.2 ± 3.1 mm Hg whereas, in hypoxic rats PaO₂ was 17.9 ± 6.8 mm Hg, indicating that the rats were exposed to low barometric pressure at high altitude. The animals were provided with food and water ad libitum during hypoxia exposure. Utmost care was taken to minimize animal sufferings while performing the experiments.

2.7. Biochemical parameters estimation

2.7.1. Sample processing

After 6 h of hypoxia exposure, rats were sacrificed by injecting Ketamine hydrochloride (80 mg/kg BW) and Xylazine (20 mg/kg BW) intraperitoneally. Lungs were perfused using chilled PBS (1X), washed with saline (0.9% NaCl), and homogenized (10%) using 0.154 M KCL consisting of DTT, PMSF, and protease inhibitor cocktail (PIC) for further performing biochemical parameters.

2.7.2. Protein oxidation

Protein oxidation in the lungs of hypoxia (6 h) exposed animals was evaluated by spectrophotometrical assessment of carbonyl groups (Levine et al., 1990). In this procedure, 2ʹ4’-dinitrophenylhydrazine (DNPH) interacts with protein carbonyls resulting in Schiff’s base formation to generate the corresponding hydrazine, then analyzed spectrophotometrically (Synergy H1, Biotek, Germany).²⁷

2.7.3. Catalase activity

To ascertain the effect of hypoxia and quercetin supplementation on the catalytic activity of catalase expressing in the lung homogenate of rats, the commercially available standard catalase diagnostic kit (Sunlong biotech Co. Pvt. Ltd., China) was used following the manufacturer’s instructions.

2.7.4. Assesment of hematological parameters and blood gases

The blood was withdrawn from the left ventricle of anesthetized animals from both normoxia and hypoxia exposed groups. The blood was collected and equally distributed in EDTA containing and heparinized vials for assessing the effect of hypoxia on hematological parameters by using Sysmex XT 2000i (Lincolnshire, USA) and blood gases using i-STAT-7 from Abott Pvt.Ltd. (USA).

2.7.5. Protein expression studies

The protein content in the lung homogenates from both normoxia and hypoxia exposed animals was quantified using Lowery’s method (1951).²⁸ Further, the western blotting analysis was done, to evaluate the effect of quercetin on altered expressions of ZO-1, JAM-C, claudin-4, and occludin in the lung of hypoxia exposed rats. The proteins present in the lung homogenates were separated by using 10% SDS-PAGE (ZO-1, JAM-C, claudin-4, occludin, and β-actin). Further, the separated proteins were electro-blotted onto the nitrocellulose membranes (0.45 μm thickness) and later, blocked by using 5% bovine serum albumin (BSA) dissolved in phosphate-buffered saline (PBS, 1X) (pH ~7.4) overnight. Later, the membranes were washed & probed with primary antibodies (Santa Cruz biotechnology, 1:5000 dilution) and incubated at room temperature for 2 h. Thereafter, followed by the 4–5 washings with PBST the membranes were probed by HRP-conjugated, enzyme-linked secondary antibodies (Santa Cruz, 1:15000 dilution) and incubated for 1 h at RT. After 6–7 thorough washings using PBST, the membranes were developed using chemiluminescent peroxidase substrate (Luminata forte, Millipore, U.S.A.) and bands were visualized in Chemi-Doc (UVITEC, Cambridge, U.K.). The optical density of bands was further measured using lab works software (UVP-Bio-imaging systems, CA).

2.8. Transmission electron microscopy (TEM)

To investigate the occurrence of distinct TJs in the lungs of rats from all groups, the TEM was being performed with few modifications in Turi et.al. (2011) method.²⁹ Quickly the lungs were fixed via 4% paraformaldehyde (PFA) at 40°C overnight. After 24 h, the fixed tissues were washed repeatedly with 1XPBS and underwent post-fixation using 1% osmium tetroxide in phosphate buffer saline (pH ~ 7.2–7.4) for an hour. Further, the tissues were treated with graded ethanol series for dehydration and then fixed firmly in an epoxy resin media. Later, these blocks were fragmented into the ultra-thin sections using a diamond knife and stained with lead citrate and uranyl acetate. The resultant stained sections were then viewed under TEM at different magnifications (1,500 X-3,000 X) (JEOL- 2100 F, USA).

2.9. In-vivo imaging studies PET/CT imaging

After 6 h of hypobaric hypoxia exposure both normoxia and hypoxia exposed animals were anesthetized using intraperitoneal administration of ketamine (80 mg/Kg BW) and xylazine (20 mg/Kg BW). Then animals were fixed in a prone position on the animal bed. Once the animals were anesthetized, 2-deoxy-2-[fluorine-18] fluoro-D-glucose^12F-FDG (80MBq/Kg BW) was injected intravenously via an indwelling catheter in the tail vein of each rat. The uptake of¹²F-FDG and its widespread into the body via circulation takes around 60 minutes. The animals were housed in dark before the PET acquisition.

At 20 min. prior to PET acquisition, a non-contrasted micro-CT focussed on the lungs (area of interest) of rats was performed and the images were acquired in FLY mode on the micro-PET/CT scanner using the following parameters: 65kV; 170 mA; focal spot size: 50 microns; magnification 2.0; 512 projections, resulting in a total acquisition time of approximately 4 min.

After 60 min of¹²F-FDG administration, PET acquisition was carried out to quantify the uptake of¹²F-FDG in the lungs of rats, using the same PET modality of micro PET/CT scanner.^30,31 The data was reconstructed using MLEM (50 iterations).

2.10. Statistical analysis

Statistical analysis was performed using SPSS for Windows (15.0) software (SPSS Inc., Chicago, IL). Comparisons between experimental groups and quercetin pretreated groups were made by using one-way ANOVA with Student-Newman-Keuls test for multiple comparisons between groups. Whereas, comparisons between normoxia-exposed (0 hrs), hypoxia-exposed (6 h) animals, and hypoxia + quercetin treated groups were made using Student’s t-test. Differences were considered statistically significant for p < .001. Results were expressed as mean ± SD.

3. Results

3.1. Effect of quercetin supplementation on protein oxidation contents

The results from protein oxidation analysis revealed that hypoxia exposure (6 h) has abruptly elevated the protein oxidation in the lungs of rats compared to control (0 h). Whereas, animals preconditioned with quercetin prior to hypoxia exposure (6 h) exhibited a significant downregulation in the protein oxidation levels compared to control (hypoxia, 6 h). However, there was no modification in the protein oxidation levels observed in the lungs of rats in the normoxia and normoxia+quercetin group. (Figure 1).

Figure 1.

Effect of quercetin prophylaxis on oxidant status in the lungs of rats exposed to hypoxia at 7,620 m for 6 h. Values are mean±SD (n = 6). *P < .001 normoxia vs hypoxia, #P < .001 hypoxia vs hypoxia+quercetin. Nor- normoxia, Hypo- hypoxia and Nor+Qct- normoxia+quercetin and Hypo+Qct- hypoxia+quercetin

3.2. Effect of quercetin administration on catalase activity

There was a significant (p < .001) down-regulation (1.8-folds↓) observed in the catalytic activity of catalase enzyme expressing in the lung of rats under hypoxia compared to normoxia control (0 h). Whereas, animals fed with quercetin prior to hypoxia exposure demonstrated a dramatic elevation in catalase activity (p < .001) (1.5-folds↑) compared to control hypoxia (6 h). However, a similar pattern of catalase activity has been noted in the case of normoxia and quercetin pre-treated animals under normoxic conditions (Figure 2).

Figure 2.

Effect of quercetin prophylaxis on catalases activity in the lungs of rats exposed to hypoxia at 7,620 m for 6 h. Values are mean±SD (n = 6). *P < .001 normoxia vs hypoxia, #P < .001 hypoxia vs hypoxia+quercetin. Nor- normoxia, Hypo- hypoxia and Nor+Qct- normoxia+quercetin and Hypo+Qct- hypoxia+quercetin

3.3. Prophylactic potential of quercetin on hematological and blood gas parameters

The findings from the hematological analysis have indicated a significant elevation (p < .001) in the levels of red blood cells (RBC), hemoglobin (Hb), white blood cells (WBC), monocytes, lymphocytes, and hematocrit (HCT) respectively in the blood of animals exposed to hypoxia compared to normoxia. Whereas, the supplementation of quercetin to the animals subjected to hypoxia exhibited significantly (p < .001) alleviated levels of WBC, monocytes, and lymphocytes in the animals under hypoxia. On the other hand, the counts of Hb, RBC, and HCT were found to be consistently higher in the blood of rats exposed to hypoxia [Table 1].

Table 1.

Effect of quercetin on hematological parameters of the animals exposed to 7,620 m for 6 h

Parameters	Nor	Hypo	Nor+Qct	Hypo+Qct
RBC (million/mm3)	5.6 ± 0.18	7.1 ± 0.2	5.5 ± 0.55	7.0 ± 0.69
Hb (million/mm3)	11.8 ± 0.57	14.02 ± 2.01	10.7 ± 0.48	13.42 ± 0.64
WBC (million/mm3)	8.8 ± 0.48	14.2 ± 0.99	8.0 ± 0.56	10.04 ± 0.88
Monocytes (%)	3.0 ± 0.081	7.29 ± 0.88	2.89 ± 0.66	4.82 ± 0.54
Lymphocytes (%)	66.08 ± 2.66	81.6 ± 6.6	58.5 ± 3.2	70.7 ± 4.8
HCT (%)	38.4 ± 1.5	50.02 ± 0.91	35.4 ± 1.89	49.8 ± 2.0

The results of the hematological analysis were further validated by a blood gas composition study, demonstrating the significant fall in the levels of PaO₂, PaCO₂, and SaO₂ in the blood samples of animals exposed to hypoxia compared to control (0 h). Apart from this an elevation in the levels of HCO₃⁻ has also been observed in the blood of hypoxia exposed animals compared to normoxia. Whereas, quercetin preconditioning to the rats before the hypoxia exposure contributed to the restoration of PaO₂, PaCO₂, SaO₂, and HCO₃⁻ levels to the normal [Table 2].

Table 2.

Blood gas profile of the animals fed with quercetin prior to hypobaric hypoxia (7,620 m for 6 h)

Parameters	Nor	Hypo	Nor+Qct	Hypo+Qct
pH	7.14 ± 0.02	7.58 ± 0.08	7.01 ± 0.018	7.24 ± 0.06
PaO2 (mmHg)	95.2 ± 3.18	47.9 ± 6.8	94.8 ± 5.11	92.6 ± 4.88
PaCO2 (mmHg)	48.7 ± 2.98	26.8 ± 5.98	46.4 ± 4.11	46.13 ± 5.9
SaO2 (%)	94.4 ± 4.18	69.4 ± 5.92	96.18 ± 8.8	92.8 ± 6.8
HCO3− (mmol/L)	23.8 ± 2.14	31.0 ± 1.8	22.10 ± 2.2	26.0 ± 3.09

Nonetheless, there were no modifications in the counts of hematological and blood gas parameters observed in the case of animals under the normoxia+quercetin pretreated and normoxia group.

3.4. Validation of the quercetin prophylaxis in ameliorating the expressions of TJ proteins- ZO-1, JAM-C, Claudin-4 and Occludin proteins in lung of rats exposed to hypoxia by western blotting analysis

The levels of ZO-1, Claudin-4, and occludin in the lung homogenates of hypoxia exposed animals were found to be significantly (p < .001) down-regulated (2-folds ↓, 2.5-folds ↓and 2.5-folds ↓) compared to normoxia control (normoxia). On contrary, the protein expression of JAM-C in the lung homogenate of hypoxia exposed animals has been observed to be up-regulated (2.2- folds ↑) significantly (p < .001) compared to normoxia. Whereas, quercetin pre-conditioning has resulted in the elevation in the levels of ZO-1, Claudin-4, and occludin (2-folds ↑, 2-folds ↑ and 2-folds ↑) significantly (p < .001) compared to hypoxia control (6 h) and on the other hand JAM-C elicited the pattern of significant reduction (2.5- folds↓) in the lung homogenate of animals exposed to hypoxia compared to control (hypoxia, 6 h) (Figure 3 (i), (ii), (iii), and (iv)) respectively). The densitometric analyses of ZO-1, JAM-C, Claudin-4, and occludin have been represented adjacent to each blot.

Figure 3.

Prophylactic potential of quercetin on the protein expression: 3(i), (ii), (iii), and (iv). ZO-1, JAM-C, Claudin-4, and Occludin in the lung of rats exposed to hypoxia at 7,620 m for 6 h. Values are mean±SD (n = 6). *P < .001 normoxia vs hypoxia, #P < .05 hypoxia vs hypoxia+quercetin. Nor- normoxia, Hypo- hypoxia and Nor+Qct- normoxia+quercetin and Hypo+Qct- hypoxia+quercetin

3.5. Effect of quercetin pre-conditioning on preventing the impairment of TJs integrity in lungs of rat under hypoxia

The results from TEM observations have unraveled the normal and intact TJ structures in the lung sections of control animals (normoxia, 0 h) (Figure 4(a)(i) & (ii)). However, the TEM images of the lung sections from the group exposed to hypoxia (6 h) exhibited widening up of the cells indicating the loss of TJs integrity (Figure 4(b)(i) & (ii)). Whereas, the animals pre-treated with quercetin under normoxia represented a normal TJs distribution throughout the lungs (Figure 4(c)(i) & (ii)). On contrary, the animals supplemented with quercetin prior to hypoxia demonstrated the reduction in the widening of TJs present in the lungs (Figure 4(d)(i) & (ii)).

Figure 4.

Effect of quercetin prophylaxis on the TJs integrity in the lung sections of rat exposed to 7,620 m for 6 h. Nor- normoxia, Hypo-hypoxia, Nor+qct- normoxia+quercetin, and Hypo+qct- hypoxia+quercetin. Scale 100–500 nm; image (i) 1,500X magnification and image (ii) 3,000X magnification

3.6. Determination of transvascular leakage in the lungs of rats exposed to hypoxia using noninvasive imaging approach

The count of¹²F-FDG in the lungs of hypoxia (6 h) exposed animals was found to be significantly (p < .001) up-surged (2-folds↑) compared to the normoxia control. Whereas, the ratio of¹²F-FDG activity in the lungs of animals supplemented with quercetin prior to hypoxia exposure (6 h) was found to be significantly attenuated (1.8- folds↓) compared to hypoxia control (6 h). However, there were no such modifications observed in the radioactivity uptake of normoxia+quercetin group and normoxia hence it is not presented (Figure 5 (b)). Similarly, in Figure 5 (a), the similar effects of hypoxia and the quercetin pre-conditioning under hypoxia exposure on the lungs of rat have been demonstrated with the help of PET ((i), (ii) and (iii)) and PET-CT co-registered imaging ((iv), (v) and (vi) respectively).

Figure 5.

Effect of quercetin prophylaxis on the uptake of¹²F-FDG in the lungs of rat exposed to 7,620 m for 6 h, represented by: (a) PET imaging ((i), (ii), & (iii)) and PET-CT co-registered ((iv), (v), & (vi)) and (b) Ratios (KBq/mm³). Values are mean±SD (n = 6). *P < .001 normoxia vs hypoxia, #P < .05 hypoxia vs hypoxia+quercetin. L- left lung, R- right lung, Nor- normoxia, Hypo- hypoxia and Nor+Qct- normoxia+quercetin and Hypo+Qct- hypoxia+quercetin

4. Discussion

The present study was undertaken to investigate the protective role of quercetin prophylaxis in ameliorating the high altitude-induced impairment in the alveolar barrier integrity within the lungs due to hypoxia exposure which can further progress to HAPE onset. HAPE, a non-cardiogenic PE is a life-threatening condition affecting non-acclimatized individuals ascending to high altitude and can result in fatalities, if left untreated.³² Thus, the early diagnosis, preventive measures, and on-site treatment of HAPE are essential to confirm the safety of high landers and people ascending to high altitude frequently.³³ HAPE can be markedly characterized by exacerbated oxidative stress, increased inflammation, impaired alveolar junction’s integrity, and enhanced in-flux of concentrated plasma proteins (Albumin and LDH) into the lung microvasculature.^32,33 Therefore the investigations were made in the present study to delineate the molecular mechanism involved in the modifications of tight junction proteins under a reducing environment and the beneficial effects of quercetin in counter-acting those hypoxia-induced alterations TJ proteins expressions using molecular biology approaches and PET- imaging.

Literature has revealed, that the exaggerated ROS- mediated protein oxidation at high altitude is a noteworthy factor causing oxidant injury.³⁴ Elevated oxidative stress results in the emergence of free radicals, a leading cause of damage to the biological membranes, and proceeds to disrupt cellular integrity and function.³⁵ As per the recent research, the increased production of free radicals is associated with the attenuation in anti-oxidants synthesis namely- catalase, GSH, GPx, SOD, etc. which are reported to be the major manifestations of inflammatory processes triggered due to hypoxic condition.³⁶ Among these anti-oxidant systems, catalases are the major anti-oxidant enzymes secreted in the liver, kidneys, and lungs are also reported to be dramatically subsided by the elevated levels of free radicals which in turn are reported to be involved in the release of NFĸB, a master regulator of inflammation.^36,37 NFĸB is responsible for regulating the expressions of numerous genes such as- IL-1, IL-6, IL-8, TNF- α, which contributes to causing inflammation upon activation leading to affect the TJs protein integrity and ultimately leading to the onset of pulmonary vascular remodeling and enhanced vascular leakage.^38,39 Whereas, the oral supplementation of quercetin to the rats 1 h prior to hypoxia demonstrated a dramatic reduction in protein oxidation followed by elevated catalase levels. This perhaps demonstrated the stabilization in TJs protein integrity leading to curtailed transvascular leakage and edema index in the lung of animals. In the present study, we have observed the altered expressions of ZO-1, JAM-C, claudin-4, and occludin TJ proteins in the lungs of animals under hypoxia. This indicates that hypoxia directly leads to impaired alveolar barrier integrity. Especially, ZO-1 in association with claudin-4 and occludin are considered to be directly involved in the up-regulation of transepithelial resistance (TER) in the alveolar barrier.⁴⁰ Apart from this, JAM-C is reported to be involved in controlling leukocyte migration, maintaining cellular polarity, angiogenesis, and vascular permeability.⁴¹ Due to the onset of HAPE, these aforementioned roles of TJ proteins are reported to get de-regulated directly or indirectly. Whereas, the supplementation of quercetin prior to hypoxia exposure has demonstrated the stable and normal expressions of TJ proteins (ZO-1, JAM-C, claudin-4, and occludin) in the lung of rats presenting the augmented alveolar barrier integrity. These results were further confirmed by carrying out transmission electron microscopy (TEM) indicating the hypoxia-mediated differences in the structural assembly of TJs within the lung architecture of rats compared to normal, however, preconditioning with quercetin exhibited almost normal TJs morphology in the lungs of rats exposed to hypoxia.

Interestingly, the rats exposed to acute hypoxia demonstrated an elevated level of HCT which might be considered as a major manifestation of either high altitude-induced dehydration or the reduced intake of water by the animals due to hypoxic stress, resulting in the reduction in total plasma volume. On contrary, the consistently higher levels of Hb in both hypoxia and hypoxia+quercetin group exhibited normal physiology to maintain cellular homeostasis. This is in corroboration with the study conducted by Leo-Velarde et.al., (2000), reporting elevated levels of HCT and Hb during chronic intermittent hypoxia.⁴² Another interesting finding by Patricia et.al. (2018)⁴³ has also unraveled a persistently increased level of RBCs and WBCs in the HAPE patients, which was in relation with our study wherein, the blood profile of the animals under both hypoxia and hypoxia+quercetin group demonstrated an increased level of RBCs and WBCs. We have also assessed the blood gas composition of the animals under all the selected groups. Wherein, we observed an elevated PaO_2, PaCO₂, and SaO₂ in the animals fed with quercetin under hypoxia, which is in support with the recent studies by Chawla et.al. (2014) and Powell et.al (1998) suggesting the occurrence of hyper-ventilation as a major manifestation of hypoxia in order to perpetuate an optimal alveolar pO₂, which further causes a dramatic decline in pCO₂ to below the threshold level resulting in the development of hypercapnia.^44,45 However, quercetin supplementation has resulted in the amelioration and stabilization of blood gas and hematological profiles of the animals under hypoxia, which is a clear indication of elevated TJs protein expression integrity leading to improved acclimatization and enhanced fluid clearing capacity of lungs.

The findings from the biochemical studies were further strengthened by carrying out noninvasive methods employing the use of a specific radionuclide.^12F-FDG For this, we employed PET and PET/CT co-registered imaging, the noninvasive modalities that can yield high-resolution anatomical depictions of small animals efficiently. In addition to this, the use of noninvasive imaging techniques also reduces the number of animals used in a study by minimizing the animal-to-animal variation and providing recurrent measurements in an individual animal, which is not possible in invasive techniques.⁴⁶ The potential to evaluate hypoxia-driven changes in the lung parenchyma noninvasively in experimental animals is of great interest. It facilitates the examination of issues concerning the onset, progression, and remedy of the disease at the early stages.⁴⁷

FDG-PET is a presumptive imaging approach for ascertaining the redox-mediated fluid-flux into the lung parenchyma.⁴⁷ In corroboration to these studies, PET imaging in the present study has indicated an appreciably increased FDG uptake within the lungs of animals exposed to hypoxia whereas, the prophylactic supplementation of quercetin showed a reduction in lung FDG uptake under hypoxia. To confirm this, we have further performed PET/CT co-registered analysis unraveling the oxidative stress-induced fluid-flux in the lung parenchyma of animals under hypoxia. Therefore, the results obtained from PET/CT-imaging have evidenced the attenuation in the hypoxia-mediated increase in vascular permeability of lungs by quercetin prophylaxis, as also indicated by the reduction in¹²F-FDG count in the lungs of animals fed with quercetin prior to hypoxia exposure compared to control (hypoxia (6 h)).

Thus, based on these findings, it can be deciphered that, hypoxia-induced impairments in the alveolar barrier integrity within the lungs of animals can be easily, early, and efficiently diagnosed by using noninvasive techniques by making use of specific radionuclide in the rat model and the prophylactic administration of quercetin resulted into the restoration of TJ proteins integrity in the alveolar epithelium of animals exposed to hypoxia by attenuating oxidative stress followed by elevation in anti-oxidants production under hypoxia.

Conclusion

The conclusion drawn from the present unravels that the prophylactic supplementation of quercetin not only prevents the altercations in TJ proteins expression into the lungs of animals exposed to hypoxia but it also appreciably maintained the Hb & RBC counts leading to regulate PaO₂ & PaCO₂ levels in the blood resulting into the minimization of HAPE severity. Thus it can also be interpreted that quercetin pre-treatment can provide similar protection in humans ascending to high altitude regimes.

Funding Statement

The study was conducted under the project entitled “Improving performance under different operational environments using suitable interventions” funded by the Defence Research and Development Organization, Government of India. Grant No.: DIP-265;DIPAS [DIP-265];

Consent for publication

Not applicable.

Data availability

The data supporting the results in our manuscript have been clearly stated in the materials and method section. All the data presented in the manuscript is in a machine-readable format.

Disclosure statement

The manuscript has not been published and is not under consideration for publication elsewhere. All authors have given their consent for its publication. The authors have declared that no competing interests exist.

Ethical approval for animal studies

All rat experiments in this study were carried out following the recommendations and approval of the institutional ethical committee (IEC). We followed the guidelines from the Universities of Federation for Animal Welfare (UFAW) for animal-based research work.