Supplementation of antioxidants glutathione and -lipoic acid attenuates oxidative stress and Th2 response in

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1 original article Supplementation of antioxidants glutathione and -lipoic acid attenuates oxidative stress and Th2 response in Prabhanshu Tripathi, Smitha Nair, Naveen Arora Access this article online Website: DOI: / Quick Response Code: ABSTRACT Background: Inflammatory lung diseases such as asthma are characterized by chronic inflammation, oxidative stress, and altered antioxidant defenses. Increased levels of reactive oxygen species (ROS) contribute to asthma exacerbation and cell damage. Exogenous supplementation of antioxidant combinations has shown potential in asthma management. Previously, a hypoallergenic mutated glutathione S-transferase (mgst) singly or in combination with reduced glutathione (GSH) showed reduction in airway inflammation in mice model. Objective: The present study evaluates the effects of antioxidants -lipoic acid and -tocopherol in combination with mgst or GSH in neutralizing the oxidative stress and reducing the inflammatory responses in a mouse model. Materials and Methods: BALB/c mice were immunized and challenged with ovalbumin, treated with different antioxidant combinations by inhalation, and sacrificed to evaluate inflammation and oxidative stress parameters. Result: Treatment with GSH + -lipoic acid showed significantly reduced total cell and eosinophil counts in bronchoalveolar lavage fluid (BALF) and decreased infiltration of cells into lung interstitium, compared to ovalbumin group. Also, the treatment reduced interleukin (IL)-4 and ovalbumin-specific IgE levels possibly through modulation of redox-sensitive transcription factor nuclear factor-kappa B (NF- B) levels. Administration of all antioxidant combinations decreased the ROS compared to ovalbumin group as indicated by reduced thiobarbituric acid reactive substance (TBARS) and 8-isoprostane levels in BALF. Conclusion: All antioxidant combinations of -lipoic acid and -tocopherol in combination with mgst or GSH showed reduction in airway inflammation and oxidative stress in vivo, but maximum therapeutic efficacy was observed in GSH + -lipoic acid group. Therefore, a combination of antioxidants may prevent allergic inflammation in the best possible way. Key words: -tocopherol, asthma, oxidative stress, reactive oxygen species Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi, India Address for correspondence: Dr. Naveen Arora, Scientist, Room - 509, Allergy and Immunology Section, Institute of Genomics and Integrative Biology, (Delhi University North Campus), Mall Road, Delhi , India. naveen@igib.res.in 19

2 INTRODUCTION Asthma is a chronic inflammatory disorder characterized by pulmonary obstruction and airway hyperresponsiveness (AHR). The inflammatory response in the lungs involves the influx and activation of cells such as eosinophils, macrophages, and neutrophils to the site of inflammation and generation of reactive oxygen and nitrogen species (ROS/RNS). [1] The oxidants cause endothelial barrier dysfunction, enhance AHR, and activate redox-sensitive transcription factors. [2] The reactive species cause increased lipid peroxidation, mediating direct tissue damage. Thus, oxidative stress caused by overproduction of reactive oxidants and overwhelming of endogenous antioxidants plays an important role in exacerbating the asthmatic condition. [1] In the airways, balance between normal physiologic function and damage is determined by the redox environment maintained by neutralization of free radicals. For this, the lung maintains an endogenous defense system consisting of both enzymatic and nonenzymatic components. Quenching of free radicals/oxidants is accomplished by enzymes such as superoxide dismutase, catalase, glutathione peroxidase, heme oxygenase, glutathione S-transferase, glutaredoxin, and thioredoxin. The nonenzymatic components include vitamins A and C, uric acid, and glutathione. [2,3] -Tocopherol (vitamin E) present in lung lining is the most important scavenger and inhibitor of lipid peroxidation. Studies with tocopherol alone or in combination with vitamin C have demonstrated reduction in bronchial hyperresponsiveness and inflammation, low frequency of allergen sensitization, and immunomodulation. [4-6] -Lipoic acid, another free radical scavenger, can recycle other antioxidants and also accelerate the synthesis of reduced glutathione (GSH). [7] The GSH redox buffer serves as one of the most important defense systems of the lung. It is crucial in maintaining intracellular GSH/oxidized glutathione (GSSG) homeostasis. Any alteration in the lung redox potential influences the activation of proinflammatory transcription factors such as nuclear factor-kappa B (NF- B) and activator protein 1 (AP-1). [8] The enzyme glutathione S-transferase catalyzes the conjugation of GSH with the electrophilic reactive compounds resulting from oxidant-mediated lipid peroxidation, thus effectively detoxifying them. Earlier studies showed that supplementation of antioxidants in combination is more efficient in reducing oxidative stress while maintaining cellular redox homeostasis (high GSH/GSSG). [2,6] Our earlier work with the antioxidants, mutated glutathione S-transferase (mgst) and GSH, has also demonstrated a synergistic effect in ameliorating oxidative stress and airway inflammation. [9] Thus, in the present study, we have explored the effect of mgst and GSH in combination with other known antioxidants such as -lipoic acid and -tocopherol (vitamin E) in reducing the oxidative stress and inflammation in mice. MATERIALS AND METHODS Animal study protocol Female BALB/c mice (6-8 weeks old) were randomly divided into seven groups of six mice each. Group 1 mice were sensitized, challenged, and treated with phosphate buffered saline (PBS) as control. Groups 2-7 mice were given i.p. injection of 10 g ovalbumin with 1 mg alum in 100 l of PBS on days 1 and 14. The mice were challenged with aerosolized OVA (4 g/mice) in saline in a Plexiglas chamber using nebulizer (Omron, Tokyo, Japan) for 30 min on days 28, 29, and 30 [Figure 1]. After 1 h of challenge, the mice were anesthetized by briefly exposing them to 3% isoflurane. Then, the mice were held in a nose-up supine position and 50 l of the antioxidant/pbs was released into the nostrils with a micropipette. The rate of release was adjusted so as to allow the mice to inhale without trying to form bubbles. Group 2 mice served as PBS control, whereas groups 3, 4, 5, 6, and 7 mice were treated with 1 g mgst + 1 g -lipoic acid, 1 g mgst + 1 g -tocopherol, 1 g GSH + 1 g -lipoic acid, 1 g GSH + 1 g -tocopherol, and 2 g -lipoic acid, respectively. The study protocol was approved by animal ethics committee of the Institute of Genomics and Integrative Biology, Delhi, following the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, Chennai, India. Collection of bronchoalveolar lavage fluid and blood After inhalation treatment, the mice were sacrificed with an Figure 1: 20

3 overdose of sodium pentobarbital on day 31 and tracheotomy was performed. Ice-cold PBS (0.5 ml) was instilled into the lungs and bronchoalveolar lavage fluid (BALF) was obtained by aspiration three times (total 1.5 ml) by tracheal cannulation. BALF was centrifuged, and the supernatant inflammatory cell number was assessed by hemocytometer after excluding the dead cells by staining with Trypan blue. One hundred microliters of BALF was spread on the slide, fixed, and stained with Leishman for differential cell counts. Blood was collected, and the sera were separated and used for the analysis of serum immunoglobulins. Determination of immunoglobulins by enzyme-linked immunosorbent assay OVA-specific IgE, IgG1, and IgG2a were measured in the serum by enzyme-linked immunosorbent assay (ELISA), as described elsewhere. [10] Briefly, microtiter plates (Nunc, Roskilde, Denmark) were coated with 5 g/ml of OVA. The plates were washed with PBS, blocked with 3% defatted milk, and incubated with mice sera for IgE (1:10), IgG1 (1:50), and IgG2a (1:50), individually. The plates were washed with phosphate buffered saline with Tween-20 (PBST) followed by PBS and incubated with IgG1-peroxidase and IgG2a-peroxidase (1:1000 PBS; BD Pharmingen, San Diego, CA, USA). For IgE, biotinylated anti-mouse IgE (2 g/ml, BD Pharmingen) was used; the plate was washed and incubated with streptavidin-peroxidase (1:1000; BD Pharmingen). Color was developed using ortho-phenylenediamine (OPD) and absorbance read at 492 nm. Determination of cytokines by ELISA Interleukin (IL)-4, IL-10, and interferon-gamma (IFN- ) levels were determined in BALF by ELISA using paired antibodies according to manufacturer s instruction (BD Pharmingen). Briefly, capture antibody (1:250 v/v) for each cytokine was coated separately on microtiter plates, incubated overnight, and blocked with 10% fetal calf serum (FCS). BALF samples (1:2 v/v) were added to wells and incubated for 2 h at 30 C. Biotinylated anti-il-4 or anti-il-10 or anti-ifn- was used to detect specific cytokine levels. The detection limit for IL-4 was 7.8 pg/ml, and for IL-10 and IFN-, it was 31.3 pg/ml. Histopathology Lungs were fixed with 10% neutral buffered formalin (ph 7.0) and embedded in blocks containing paraffin. Sections of 4 m were cut and stained with hematoxylin and eosin (H and E) or periodic acid Schiff (PAS). Twelve slides were made for each type of staining (two slides per mice) from every group and were analyzed using light microscope for antigen-induced peribronchial and perivascular inflammation. Semi-quantification of inflammation score was made on the scale of 0-4 for perivascular accumulation of inflammatory cells, followed by statistical analysis. [11] Lung inflammation score was calculated in terms of eosinophil infiltration and mucus secretion. Score 0 was assigned to no or occasional inflammatory cells, score 1 for a few inflammatory cells, score 2 for scattered aggregates of inflammatory cells, score 3 for thin layer of inflammatory cells surrounding the airways and vessels, and score 4 was given for a thick layer of inflammatory cells surrounding the airways and vessels. Oxidative stress markers in BALF Oxidative stress was determined by measuring the concentration of thiobarbituric acid reactive substance (TBARS) spectrophotometrically. [12] Malondialdehyde (MDA) and thiobarbituric acid (TBA) react to form a product with maximum absorption at 532 nm. BALF (200 l) was mixed with 500 l of 10% w/v trichloroacetic acid to precipitate the protein. The precipitated protein was pelleted by centrifugation at 10,000 g at 4 C for 15 min and the supernatant was treated with 0.67% TBA in boiling water for 15 min. Different concentrations of MDA (Sigma, St. Louis, USA) diluted in saline were taken as the standard and incubated with 0.67% TBA in boiling water. The mixture was cooled and centrifuged at 1500 g at 25 C for 10 min. The supernatant was collected and absorbance recorded at 532 nm. The MDA equivalents in the samples were calculated from the MDA standards. The concentration of 8-isoprostanes was measured using 8-isoprostane EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA) as per manufacturer s instruction. Briefly, plates were pre-coated with mouse monoclonal antibody and blocked with defatted milk (Bio-Rad, Hercules, USA). After washing, 50 l of standard/balf sample was added to the corresponding wells and incubated for 18 h at 4 C. The wells were washed five times with wash buffer. Ellman s reagent (200 l) was added to each well and kept in dark for min for color development. The plates were read at 420 nm and the blank was subtracted. The concentration of 8-isoprostanes was calculated from the standard curve plotted with standard samples. Nuclear extract preparation and NF- B (p65) estimation Nuclear extract of lung tissues was prepared using NuCLEAR Extraction Kit (Sigma, USA) following manufacturer s instruction. PBS-rinsed tissue (100 mg) was suspended in 1 ml of lysis buffer containing dithiothreitol (DTT) and protease inhibitors. The tissue was homogenized and the disrupted cell suspension was centrifuged at 10,000 g for 20 min. The pellet was resuspended in 150 l of extraction buffer with DTT and protease inhibitor for 30 min and centrifuged for 5 min at 20,000 g. The supernatant was NF- B (p65) transcription factor was determined in the above fraction using an immunoassay kit (Cayman Chemical Company). Ninety microliters of complete transcription 21

4 factor binding buffer (CTFB) was added to each well of the microtiter plate pre-coated with dsdna consensus sequence containing the NF- B response sequence. Ten microliters of each sample was added to the wells and incubated overnight at 4ºC without shaking. Wells were washed five times with wash buffer, followed by addition of 100 l of diluted NF- B (p65) antibody, and incubated for 1 h at 25 C. After washing for five times with wash buffer, 100 l of secondary antibody was added per well and incubated for 1 h at 25 C. One hundred microliters of developing solution was added to each well, and the reaction was stopped after 30 min and the absorbance read at 450 nm. The blank and positive controls were taken as supplied with the kit. Statistical analysis Data were expressed as mean ± standard deviation. The data were analyzed using GraphPad Prism 5.00 (GraphPad software Inc., La Jolla, USA). The sample size for each parameter tested was six mice. Statistically significant difference was determined using one-way analysis of variance (ANOVA) followed by Dunnett s multiple comparison tests between ovalbumin-challenged and PBS-treated mice and the different treatment groups. P <0.05 was considered significant in all cases. RESULTS Intranasal treatment with antioxidant combinations suppresses allergen-induced inflammatory cells in BALF BALF analysis showed increased number of total cells [(40.67 ± 2.52) 10 4 ] and eosinophils [(15.50 ± 2.50) 10 4 ] in ovalbumin-challenged mice than PBS control [(2.87 ± 0.31) 10 4 and (1.31 ± 0.34) 10 4, respectively] [Figure 2]. Antioxidants administration through intranasal route significantly reduced the cellular infiltration in all the treatment groups (P < 0.01). Administration of GSH + -lipoic acid reduced the total cell [(13.37 ± 1.47) 10 4 ] and eosinophil [(4.61 ± 0.56) 10 4 ] count to one-third of the ovalbumin group. These values are comparable to the -lipoic acid group [(8.42 ± 0.92) 10 4, (5.08 ± 1.18) 10 4 ], which is considered as a standard antioxidant in the present study. Administration of antioxidant combinations reduces ovalbumin-specific IgE in sera Ovalbumin immunization and challenge elevated specific IgE levels (0.380 ± 0.03 OD 492 nm ) than in PBS control (0.033 ± OD 492nm ) [Figure 3]. IgE levels significantly decreased (from ± 0.01 to ± OD 492 nm ) in different treatment groups as compared to ovalbumin-immunized PBS-treated mice. Treatment with GSH or mgst with -lipoic acid showed IgE values (0.228 ± and ± OD 492 nm, respectively) similar to those of -lipoic acid group. IgG1 levels reduced marginally in GSH + -lipoic acid group (0.385 ± 0.02 OD 492nm ) as compared to ovalbumin (0.43 ± OD 49 ) group. However, the change was not pronounced in the 2nm other treatment groups. Specific IgG2a levels were below detectable level in all treatment groups (data not shown). Antioxidant treatment reduces Th2 cytokines in BALF Ovalbumin-immunized PBS-treated mice showed ± 6.00 pg/ml IL-4 levels in BALF, whereas the level in PBS control was below the detection limit (7.8 pg/ ml). Intranasal administration of GSH + -lipoic acid and GSH + -tocopherol decreased the IL-4 levels to approximately half (52.77 ± 4.9 pg/ml and ± 2.52 pg/ml, respectively) of the ovalbumin-challenged PBS-treated mice [Figure 4]. IL-10 and IFN- levels did not change much post-treatment with any of the antioxidant combinations. Antioxidant combinations reduce airway inflammation Ovalbumin-challenged mice showed perivascular and peribronchial infiltration of numerous eosinophils into the lung interstitium, along with narrowing of airway lumen (score 4) [Figure 5b]. Mice treated with antioxidants showed substantial reduction in cellular infiltration in airways as compared to OVA-challenged PBS-treated mice [Figure 5c-f]. Reduction in cellular infiltration in GSH + -lipoic Figure 2: Data are presented as mean ± SD (n Figure 3: -challenged mice after intranasal administration of antioxidants. n 22

5 Figure 4: are presented as mean ± SD (n a b c d e f g h Figure 5: -lipoic acid [Figure 5e] and -lipoic acid [Figure 5g] treated mice was similar with an inflammation score of 1.5. Antioxidant combinations reduced oxidative stress significantly Oxidative stress was assessed by measuring the markers of lipid peroxidation, such as MDA (TBARS assay) and 8-isoprostanes levels in the BALF of mice. Ovalbumin-challenged mice showed enhanced TBARS level of 2.92 ± 0.21 pm/ l. All the groups given antioxidants showed a slight decrease in TBARS levels in the range from 2.13 ± 0.21 pm/ l to 1.86 ± 0.13 pm/ l. GSH + -tocopherol group showed the lowest TBARS level (1.86 ± 0.13 pm/ l) in BALF compared to other combinations of antioxidant(s). However, none of the antioxidant groups could achieve a reduction similar to that of -lipoic acid group (1.39 ± 0.17 pm/ l) [Figure 6a]. 23

6 Ovalbumin-immunized and -challenged mice showed increased lipid peroxidation as demonstrated by high 8-isoprostanes (potent pulmonary vasoconstrictor) level (82.75 ± 4.95 pg/ml) as compared to that of PBS control (22.87 ± 3.07 pg/ml) [Figure 6b]. The level of 8-isoprostanes reduced significantly in GSH + -lipoic acid group (57.68 ± 5.37 pg/ml) than OVA-immunized and -challenged mice, whereas GSH + -tocopherol group P value (47.42 ± 4.86 pg/ml) was similar to that of -lipoic acid treated group (48.95 ± 4.63 pg/ml). Antioxidant combinations reduced NF- B (p65) level in lung s nuclear extract Lung nuclear extract in PBS control showed NF- B level of ± (OD 490 nm ); OVA immunization and challenge significantly raised the level to ± (OD 490 nm ). Intranasal administration of antioxidant combinations of GSH + -lipoic acid (0.255 ± 0.053) and GSH + -tocopherol (0.251 ± 0.055) significantly reduced the NF- B (p65) level in the nuclear extract of lung tissues, as compared to the ovalbumin group [Figure 7]. However, reduction in the levels comparable to -lipoic acid group could not be achieved with any of the antioxidant combination groups. oxidative stress in the pathogenesis of allergic rhinitis and asthma. In the present study, the authors have intranasally administered antioxidant combinations to reduce oxidative stress in a mice model of allergic airway inflammation. Increased ROS levels in asthmatics have been associated with causing bronchial hyperreactivity, histamine release, and mucus secretion. [2] The oxidant/antioxidant imbalance is characterized by alteration of endogenous antioxidant levels including GSH and -tocopherol in the extracellular lining fluid. Studies with combination of antioxidants such as vitamins C and E or GSH with mgst have shown therapeutic potential in reducing oxidative stress and associated cellular inflammation. [6,9,10] Cho et al. have suggested the use of -lipoic acid as an adjunct therapy in asthma, which suppressed allergic inflammation and AHR. [15] Since oxidative stress results in alteration to the cellular GSH/GSSG levels, exogenous supplementation of GSH or its redox enzymes like GST can repair the oxidant/antioxidant balance in vivo. In earlier experiments carried out in our lab, GST allergen from Alternaria alternata was mutated at residues 21 and 27 with no effect on enzymatic activity. Further studies showed that inhalation of mgst alone had DISCUSSION Recent understanding of the mechanisms of allergic rhinitis and asthma and their pathophysiology have led to the concept of united airway disease, which proposes that upper and lower respiratory tract diseases are manifestations of a single inflammatory process. The same patterns of inflammatory cells and similar cytokines and chemokines are found at both the sites. Therefore, the clinical, epidemiological, and biological data recommend an integrated management of respiratory diseases. [13] A study which assessed the inflammation and oxidative stress in upper and lower respiratory tracts showed oxidative stress markers in case of persistent allergic rhinitis, intermittent asthma, mild and moderate asthma. [14] Several other studies have implicated Figure 7: NF- B level in lung nuclear extract of different treatment groups, A and PBS control. NF- B (p 5) levels were determined by E ISA kit. Data are presented as mean ± SD (n mice per group) a Figure 6: (a) xidative stress in BA F of different antioxidant treated groups, A and PBS control. xidative stress was determined by measuring thiobarbituric acid reactive substance (TBARS) concentration spectrophotometrically. Data are presented as mean ± SD (n mice per group). (b) 8-isoprostanes in BA F of different treatment groups, A and PBS control. 8-Isoprostane was determined as a potent pulmonary vasoconstrictor using EIA kit. Data are presented as mean ± SD (n mice per group) b 24

7 limited therapeutic effect, whereas intranasal administration of mgst along with its substrate GSH demonstrated greater efficacy in reducing oxidative stress and airway inflammation in mice. [9,10] Thus, based on its therapeutic effect in allergic airway model, mgst was selected for further studies. The antioxidant -lipoic acid is capable of maintaining GSH levels, thereby assisting in the maintenance of cellular redox homeostasis. [16] Vitamin E, a powerful antioxidant capable of direct free radical scavenging, was selected for its role in improving the allergic state both as an antioxidant and an immune modulator. [17] Thus, in the present study, the antioxidants -lipoic acid and -tocopherol were tested in combination with GSH or mgst for their therapeutic efficacy in mice model of airway inflammation. Cellular infiltration in asthma is characterized by airway eosinophilia. Previous studies with -lipoic acid significantly reduced eosinophils among BAL cells and also improved pathologic lesion scores of the lungs. [15] Our earlier study showed that intranasal administration of mgst + GSH in airway inflammation caused a significant reduction in inflammatory cell recruitment to the lungs. [9] In the present study, total and eosinophil cell infiltration increased after ovalbumin challenge, but treatment with all antioxidant combinations was effective in reducing inflammatory cell recruitment into the airways. The lung histopathology also showed a low score, indicating decrease in airway inflammation upon treatment. Both airway and intravascular inflammatory cells from asthmatics are capable of producing more ROS than healthy subjects. This association between inflammation and ROS can generate a positive feedback loop, thus mediating tissue damage. [18] The presence of oxidative stress has been demonstrated by higher GSSG levels in bronchial wash and BAL in asthma patients. [19] In the present study, ovalbumin-challenged PBS-treated group showed elevated lipid peroxidation indicated by high levels of oxidative stress markers, 8-isoprostanes and TBARS. Mice treated with various antioxidant combinations showed reduction in oxidative stress, as compared to ovalbumin control. The redox-sensitive transcription factor NF B regulates the production of IL-4, IgE, and allergen-induced eosinophil influx. Previously, both N-acetylcysteine and a catalytic antioxidant were shown to suppress NF B DNA binding activity and inhibit cytokines such as IL-4, IL-5, and tumor necrosis factor (TNF)-. [20,21] In the present study, all antioxidant treatment groups showed decreased NF B levels in the lung nuclear extract, which correlated with a similar decrease in ovalbumin-specific IgE and IL-4 levels. Antioxidants can mediate their immunomodulatory effects via different mechanisms. Since the cellular redox homeostasis (GSH/GSSG) regulates signaling mechanism for many pro- and anti-inflammatory genes, external supplementation of GSH or its redox enzymes can repair the oxidant/antioxidant balance in vivo. -Lipoic acid plays a crucial role in maintaining GSH redox buffer by recycling back GSH from GSSG and also activating phase II antioxidant enzymes involved in GSH synthesis. [22,23] Also, -lipoic acid is known to inhibit activation of NF- B independent of its antioxidant property by inhibition of I B kinase (IKK). This prevents IL-18 mediated increase in Th2 cytokines and serum IgE. [23,24] Oxidative stress in allergic inflammation is responsible for inhibition of macrophage NF-E2-related Factor 2 (NRF2). -Tocopherol can mitigate this inhibition and activate downstream antioxidant target genes like SOD1. 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