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Tamarind seed extract mitigates the liver oxidative stress in arthritic rats Cite this: DOI: 10.1039/c3fo60381d
Mahalingam Shanmuga Sundaram,a Mahadevappa Hemshekhar,a Ram M. Thushara,a Martin Sebastin Santhosh,a Somanathapura K. Naveen Kumar,a Manoj Paul,a Sannaningaiah Devaraja,b Kempaiah Kemparaju,a Kanchugarakoppal S. Rangappac and Kesturu S. Girish*ab Although arthritis is primarily a joint disorder that mainly targets the articular cartilage and subchondral bone, several recent investigations have reported oxidative burst and vital organ damage that are being considered as secondary complications of arthritis. The continuous generation of free radicals like reactive oxygen and nitrogen species is considered as a key culprit in the initiation and propagation of oxidative damage. In addition, activation of T and B cells, macrophages, inflammatory mediators such as TNF-a, IL-1b and IL-6 aggravates the oxidative damage of the vital organs, particularly the liver. The current piece of work demonstrates oxidative stress in the liver of arthritic rats and its amelioration by the procyanidin-rich tamarind seed extract (TSE). The arthritic liver homogenate, mitochondrial and cytosolic fractions were found with increased levels of oxidative stress markers including free radicals. As a consequence, depletion in the levels of glutathione, total thiols, glutathione peroxidase and reductase was evident. Furthermore, the activities of endogenous antioxidant enzymes like superoxide dismutase, catalase and glutathione-S-transferase were found to be significantly altered. The increased and decreased activity of transaminases respectively in serum and liver, along with histological observations, further confirms the liver damage. Unfortunately, the commonly used drugs like NSAIDs and DMARDs have failed to prevent oxidative damage, rather they were found to be the inducers themselves. Received 2nd September 2013 Accepted 23rd December 2013
Interestingly, TSE supplementation was found to significantly inhibit oxidative burst in the liver and maintain homeostasis. Thus, the study clearly demonstrates the protective efficacy of TSE against
DOI: 10.1039/c3fo60381d
arthritis-associated oxidative liver damage, including mitochondrial oxidative burst and its associated
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secondary complications.
Introduction Arthritis is a degenerative joint disease that targets articular cartilage and subchondral bone. Articular cartilage is constituted by collagen and proteoglycans linked to hyaluronan (HA) and aggrecan. The aggravated and uncontrolled activity of proteases and glycosidases is being referred to as a primary culprit in the cartilage damage. In addition to cartilage degeneration, oxidative stress has been considered a major threat in the management of arthritis that largely contributes to oxidative damage of vital organs.1,2 Several individuals with arthritis are reported to experience severe oxidative stress leading to damage of vital organs like the liver, kidney, lungs and spleen.3 These pathological events might also trigger cardio vascular diseases a
Department of Studies in Biochemistry, University of Mysore, Mysore-570 006, Karnataka, India. E-mail: [email protected]; Tel: +91-9964080540
b
Department of Studies and Research in Biochemistry, Tumkur University, Tumkur-572 103, Karnataka, India
c Department of Studies in Chemistry, University of Mysore, Mysore-570 006, Karnataka, India
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(CVDs) and diabetes that are known as secondary complications of arthritis. This might be attributed to the increased production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), and activation of T and B-cells, macrophages, inammatory mediators in particular tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6. The highly reactive free radicals like superoxide (O2) predominantly, hydroxyl radical (OH), hydrogen peroxide (H2O2) and peroxynitrite (ONOO) can effectively induce tissue injury causing oxidative damage to macromolecules of vital organs including proteins, lipids and nucleic acids.1,4 Liver is one of the major organs which experience severe alterations due to increase in the steady concentration of ROS and RNS. The ROS indeed act on the fatty acid side chains of lipids present in different cellular membranes, in particular, mitochondrial membranes that are exposed directly and frequently to superoxide radicals generated upon cellular respiration. Evidently, mitochondria from arthritic rats were consistently detected with elevated rates of oxygen uptake in the absence or presence of exogenous ADP. The oxidative damage of
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mitochondrial macromolecules such as mtDNA, proteins and lipids, along with protein synthesis machinery in liver cells, induces mitochondrial dysfunction. Recent studies have demonstrated that the oxidative lesions like 8-oxo-dG are found more in mtDNA than in nuclear DNA suggesting the fact that mtDNA is more susceptible to oxidative damage.5 These molecular proceedings trigger further increase in the steady concentration of ROS resulting in energy depletion leading to cell death, which eventually damages the organ and its function.5–8 Bearing in mind that the ROS actively participate in the pathogenesis of arthritis, it is apparent that the biochemical alterations in the liver are greatly inuenced by its oscillating oxidative state, specically mitochondria and peroxisomes that are directly associated with oxidative damage. Unfortunately, prolonged use of well-known anti-arthritic drugs like non-steroidal anti-inammatory drugs (NSAIDs) and disease modifying anti-rheumatic drugs (DMARDs) are also known to induce liver damage and aggravate oxidative stress.9,10 Based on these interesting facts, the present study sheds light on the oxidative status of the adjuvant-induced arthritic rat liver and its amelioration by orally supplemented procyanidin rich tamarind (Tamarindus indica) seed extract (TSE). TSE has received vast attention by pharmacologists due to the presence of therapeutic agents like antioxidants, triterpenes, polyphenolic bioactive compounds, procyanidins, polysaccharides and other unknown factors, which have healing activities for various human pathophysiological disorders.11–13 The present study aims to investigate the various facets of oxidative stress in arthritic rat liver mitochondria in detail and its suppression using TSE by assessing various oxidative stress markers and antioxidant status.
Materials and methods Chemicals Freund's complete adjuvant (FCA), dihydrodichlorouorescein diacetate (DCFDA), O-phthalaldehyde (OPT), dichlorouorescein (DCF), HEPES, 1,10 ,3,30 -tetramethoxypropane (TMP), reduced glutathione (GSH) and oxidized glutathione (GSSG) were obtained from Sigma chemicals, St. Louis, USA. Glutathione peroxidase kit was purchased from Randox laboratories, Northern Ireland (United Kingdom). Commercial ALT and AST kits were from Agappe Diagnostics Limited, Ernakulam (India). Micro-titer plates were purchased from Tarsons product Pvt. Ltd., India. All other chemicals were of analytical grade purchased from Sisco research laboratories, India. Tamarind seeds were obtained from local grocer, Mysore (India). Experimental animals Adult Wistar rats (12 weeks old) weighing around 140–170 g were used in the experiment. Animals were collected from University Central Animal Facility and housed under a controlled environment. All experiments were approved by the Animal Ethical Committee (order no: MGZ/637/2011-12 dated 12-07-2011; UOM/IAEC/04/2011), Department of Zoology, University of Mysore, Mysore and were in accordance with the
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guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Preparation of procyanidin rich tamarind seed extract (TSE) Tamarind seeds were collected from a grocer, Mysore. Dried whole seeds were nely powdered and 25 g of the powder was subjected to Soxhlet extraction using 100 mL of 95% ethanol. The solvent was removed in vacuo and the dried extract was dissolved in saline and is known as TSE and is used for further experiments.12,14 Experimental design and induction of arthritis The experimental rats were randomly divided into 6 groups each consisting of 6 rats. Group I – saline control; group II – arthritic; group III – arthritic rats treated with ibuprofen (10 mg kg1 body weight); groups IV and V – arthritic rats treated with TSE (25 and 50 mg kg1 body weight respectively); group VI – TSE (50 mg kg1 body weight) alone, non-arthritic rats. Arthritis was induced by a single subcutaneous injection (100 mL) of Freund's complete adjuvant (FCA) containing 10 mg mL1 heatkilled Mycobacterium tuberculosis at the right hind palm surface of all rats except groups I and VI. The FCA-induced rats were le for disease development for 10 days and the respective treatment was given from the 11th day to the 25th day. Aqueous solution of TSE, ibuprofen syrup and saline were administered orally to the respective experimental groups using a intragastric tube (gavage) once in a day in the morning. Ibuprofen was used as a standard NSAID treatment control and saline as a negative control. Preparation of liver homogenate, mitochondria and cytosolic fractions On the nal day of the experiment, animals were fasted for 18 h and sacriced and the livers were removed immediately and blood was collected through cardiac puncture. Serum was separated and stored at 20 C. The excised liver tissues were blotted free of blood, rinsed with ice-cold saline and homogenized (10% w/v) in ice-cold phosphate-buffered saline (PBS, 100 mM, pH 7.4). The liver homogenates were centrifuged at 5000 g at 4 C for 10 min and the supernatant obtained was known as total liver homogenate and was stored at 20 C for the assessment of different biochemical parameters. Mitochondria were isolated according to the method reported by Chandrashekar and Muralidhara.15 Briey, liver was homogenized in ice-cold Tris–sucrose buffer (2 mM Tris–HCl, 250 mM sucrose, pH 7.4) and subjected to differential centrifugation to separate mitochondria and cytosol. The mitochondrial pellet obtained was washed and suspended in ice-cold mannitol– sucrose–HEPES buffer (200 mM mannitol, 70 mM sucrose, 0.1 mM EDTA, 10 mM HEPES, pH 7.4). The post-mitochondrial fraction obtained in the above step was further centrifuged at 20 000 g and the obtained supernatant was used as cytosolic fractions. All the operations were performed at 4 C. Disrupted mitochondria were obtained by repeated freeze-thawing procedures.
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Measurement of oxidative stress markers
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Estimation of ROS The endogenous generation of ROS was quantied in liver homogenate, disrupted mitochondria and cytosolic fractions using dihydrodichlorouorescein diacetate (DCFDA). DCFDA is a non-polar uorescent probe commonly employed to estimate total ROS in general including hydrogen peroxide, hydroxyl radicals and peroxynitrite. The cell-permeant DCFDA passively diffuses into cells and is retained in the intracellular level aer cleavage by intracellular esterases. Upon oxidation by ROS, the non-uorescent DCFDA is converted to the highly uorescent 20 ,70 -dichlorouorescein (DCF). Briey, an aliquot (0.2 mg protein) of the incubation mixture (200 mL) was dispensed into a 96 well micro-titer plate containing Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5 mM HEPES, 2 mM CaCl2, 10 mM glucose, pH 7.4) to which 10 mL of DCFDA was added to a nal concentration of 10 mM and incubated for 30 min at room temperature to allow the cleavage of DCFDA by esterases and further conversion into the uorescent product dichlorouorescein. The uorescence was measured using a multimode plate reader (Thermo scientic, USA) with excitation at 480 nm and emission at 530 nm. Background uorescence was corrected by the inclusion of parallel blanks and levels of ROS were quantied from a dichlorouorescein standard curve and data were expressed as pmol DCF formed per mg protein.16
Estimation of hydrogen peroxide levels The hydrogen peroxide levels were quantied in liver homogenate, disrupted mitochondria and cytosolic fractions using homovanillic acid (HVA), a specic H2O2-sensitive uorescent probe used to detect H2O2 production in the tissues and cells. Briey an aliquot (0.2 mg protein) of the incubation mixture (200 mL) was dispensed into a 96 well micro-titer plate containing HEPES buffered saline (HBS, 10 mM HEPES, 145 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgSO4, pH 7.45) incubated with 100 mM HVA for 30 min at room temperature. Fluorescence was recorded at 420 nm by exciting the samples at 310 nm. The H2O2 levels were expressed as nmol hydrogen peroxide (HP) per mg protein.17
Estimation of MDA and protein carbonyls Lipid peroxidation (LPO) status was assessed in liver homogenate, disrupted mitochondria and cytosolic fractions by estimating thiobarbituric acid reactive substances (TBARS).18 Aliquots of the samples (1 mg protein) were added to tubes containing 1.5 mL acetic acid (20% v/v, pH 3.5), SDS (8% w/v, 0.2 mL) and thiobarbituric acid (0.8% w/v, 1.5 mL). The mixture was heated on a boiling water bath for 45 min. Adducts formed were extracted into 1-butanol (3 mL) and the TBARS formed was read at 532 nm and quantied using TMP as the standard. Values are expressed in terms of malondialdehyde (MDA) equivalents as nmol MDA formed per mg protein. Precautions were taken to
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avoid over estimation of MDA formation by carrying out incubation in the presence of 0.17% (v/v) butylated hydroxyl toluene. Protein carbonyl content (PCC) was measured using dinitrophenylhydrazine (DNPH) in liver homogenate, disrupted mitochondria and cytosolic fractions.19 To 0.5 mL of sample (1– 2 mg protein), an equal volume of 10 mM DNPH in 2 N HCl was added and incubated for 1 h, shaking intermittently at room temperature. Corresponding blank was carried out by adding only 2 N HCl to the sample. Aer incubation, the mixture was precipitated with 20% TCA and centrifuged. The precipitate was washed twice with acetone and nally dissolved in 1 mL of Tris buffer (20 mM, pH 7.4 containing 140 mM NaCl, 2% SDS-w/v) and the absorbance was read at 360 nm. The difference in absorbance is determined and expressed as nmol of carbonyl groups per mg protein, using an extinction coefficient of 22 mM1 cm1. Measurement of GSH and thiols The levels of GSH in liver homogenate, disrupted mitochondria and cytosolic fractions were measured as described by Mokrasch and Teschke20 with slight modications. The assay mixture consisted of 1 mg protein made up to 100 mL with buffered formaldehyde (1 : 4 v/v, formaldehyde: 100 mM Na2HPO4) on a micro-titer plate and kept at 37 C for 5 min. Then 150 mL of 100 mM phosphate buffer (pH 8.0 containing 5 mM EDTA) and 25 mg of O-phthalaldehyde were added and kept at 37 C for 45 min, and the uorescence was recorded at 425 nm by exciting the sample at 340 nm. The GSH level was expressed as mg GSH per mg protein. Further, to estimate the total thiol content in liver homogenate, disrupted mitochondria and cytosolic fractions, an aliquot of sample (0.05 mg protein) was added to 0.375 mL of Tris–HCl buffer (200 mM, pH 8.2) containing di-thiobis-nitrobenzoic acid (DTNB, 10 mM) and 1.975 mL of methanol. Following incubation for 30 min at room temperature, the tubes were centrifuged at 3000 g for 10 min.21 The absorbance of the supernatant was measured at 410 nm and expressed as mmol DTNB oxidized per mg protein, using an extinction coefficient of 13.6 mM1 cm1. Determination of antioxidant enzymes The superoxide dismutase (SOD) activity was measured in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by monitoring the inhibition of quercetin autoxidation.22 Briey, an aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.01 mg protein) was added to the reaction mixture (1 mL) consisting of phosphate buffer (16 mM, pH 7.8) containing TEMED–EDTA (8 mM/0.08 mM) mixture and quercetin (0.15% w/v). The decrease in absorbance was monitored for 3 min at 406 nm. The amount of protein that inhibits quercetin autoxidation by 50% is dened as one unit. The catalase (CAT) activity was measured in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by measuring the rate of hydrolysis of H2O2 at 240 nm.23 To the reaction mixture (1 mL) containing sodium phosphate buffer (100 mM, pH 7.4), H2O2 (8.8 mM), an aliquot
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of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added. The decrease in absorbance was monitored for 3 min at 240 nm and the activity was expressed as mmol H2O2 decomposed per min per mg protein (3 ¼ 43.6 mM1 cm1). The glutathione-S-transferase (GST) activity was measured in liver homogenate; supernatant of disrupted mitochondria and cytosolic fractions by monitoring the enzyme catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm.24 An aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added to the reaction mixture (1 mL) containing phosphate buffer (100 mM, pH 6.5, 0.5 mM EDTA), CDNB (1.5 mM) and GSH (1 mM). The increase in absorbance was monitored for 3 min at 340 nm and the activity was expressed as mmol GS-DNB conjugate formed per min per mg protein (3 ¼ 9.6 mM1 cm1). The glutathione reductase (GRdx) activity was measured in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by monitoring the enzyme catalysed reduction of GSSG (oxidized glutathione) in the presence of NADPH which is then oxidised to NADP+.25 Briey, an aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added to the reaction mixture (1 mL) containing phosphate buffer (100 mM, pH 7.0 containing 2 mM EDTA), 20 mM GSSG and 2 mM NADPH (in 0.1% NaHCO3 (w/v)). The decrease in absorbance was monitored at 340 nm for 3 min and the activity was expressed as mmol GSSG reduced per min per mg protein. The glutathione peroxidase (GPx) activity in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions were measured spectrophotometrically by the standard enzymatic method using a commercial kit (Randox laboratories, Northern Ireland, UK) at 340 nm and expressed as mmol NADPH oxidized per min per mg protein. Estimation of glucose-6-phosphate dehydrogenase enzyme activity The glucose-6-phosphate dehydrogenase (G6PDH) activity was estimated in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by monitoring the increase in absorbance at 340 nm for 3 min due to NADP+ dependent glucose 6-phosphate transformation.26 Briey, an aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added to the reaction mixture (1 mL) containing Tris–HCl buffer (0.05 mM, pH 7.5), 3.8 mM NADP, 3.3 mM glucose-6-phosphate and 6.3 mM MgCl2. The activity was expressed as nmol NADPH formed per min per mg protein. Determination of serum and liver ALT and AST activity The serum and liver alanine transaminase (ALT) and aspartate transaminase (AST) activities were measured spectrophotometrically by the standard enzymatic method using commercial kits (Agappe Diagnostics Ltd., Kerala, India). The reaction mixture containing the serum/liver homogenate (10 mL) and AST/ALT reagent (1 mL) was mixed and incubated at 37 C for 1 min and read at 340 nm for 3 min and expressed as unit L1.
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Histopathology The liver tissues were dissected out and blotted free of blood, rinsed in ice-cold saline and xed in Bouin's solution overnight. The tissue samples were subjected to dehydration by processing with different grades of alcohol and chloroform mixture. The processed tissues were embedded in paraffin wax, and sections (5 mm thickness) were prepared, stained with hematoxylin– eosin dye and observed under an Axio imager A2 microscope and photographed. Protein estimation The protein estimation was done according to the method reported by Lowry27 using bovine serum albumin (BSA) as standard. Statistical analysis All the experimental data are presented as the mean SEM of three independent experiments. The differences between groups were assessed by repeated analysis of variance (ANOVA), followed by the Tukey “honestly signicantly different” (HSD) post hoc analysis. p value of < 0.05 was considered as statistically signicant. a* – signicant compared to the saline control group and b* – signicant compared to the arthritis induced group.
Results Modulatory effect of TSE on endogenous generation of ROS The result of the present study evidenced increased levels of ROS 67%, 74% and 46% respectively in liver homogenate, mitochondria and cytosolic fractions of the arthritic group when compared to the saline control group (Fig. 1A). TSE (50 mg kg1) administration signicantly reduced the endogenous generation of ROS to an extent of 97%, 99% and 98% correspondingly. While the ibuprofen-treated group showed 84%, 81% and 56% reduction in liver homogenate, mitochondria and cytosolic fractions respectively when compared to the arthritic group. Modulatory effect of TSE on endogenous generation of hydroperoxide Endogenous H2O2 levels were also elevated by 70%, 77% and 65% in liver homogenate, mitochondria and cytosolic fractions of the arthritic group when compared to the saline control group (Fig. 1B). TSE (50 mg kg1) administration reduced the endogenously generated H2O2 levels by 95%, 98% and 94% correspondingly when compared to the arthritic group which is highly signicant whereas the ibuprofen-treated group was effective by 61%, 54% and 52% in reducing the H2O2 levels in comparison with the arthritic group. Modulatory effect of TSE on lipid peroxidation and protein oxidation A substantial elevation of LPO and PCC in the arthritic liver was observed. Mitochondria of the arthritic liver exhibited a signicant increase in LPO by 90%, whereas homogenate and cytosolic fractions showed 77% and 48% respectively when
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Effect of TSE on arthritis-induced: (A) ROS and (B) hydroperoxide levels in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 1
compared to the saline control group (Fig. 2A). The TSE (50 mg kg1) treated group reverted the altered LPO signicantly by 90%, 90% and 93% correspondingly with respect to the arthritic group, while the ibuprofen-treated group showed moderate protection by 70%, 77% and 48% in mitochondria, homogenate and cytosolic fractions respectively when compared to the arthritic group. Furthermore, a signicant elevation of PCC was observed in cytosolic fractions of arthritic liver by 140% followed by mitochondria and homogenate by 137% and 61% respectively when compared to saline control (Fig. 2B). Conversely the TSE (50 mg kg1)-fed group re-established the altered PCC effectively by 93%, 92% and 94% correspondingly with respect to the arthritic group. While the ibuprofen-treated group showed moderate protection against the arthritic group by 60%, 70% and 77% with respect to cytosolic fraction, mitochondria and homogenate.
Modulatory effect of TSE on the levels of GSH and total thiols The arthritic group showed a signicant decrease in GSH content in homogenate, mitochondria and cytosolic fractions of the liver to an extent of 54%, 42% and 40% respectively with reference to the saline control group (Fig. 3A). The TSE (50 mg kg1) treated group reversed the altered GSH content by 90%, 98% and 95% correspondingly when compared to the arthritic group, while the ibuprofen treated group showed a protection by 66%, 77% and 75% with respect to homogenate,
mitochondria and cytosolic fractions when compared to the arthritic group. Furthermore, the liver of arthritic rats showed a signicant decline in total protein thiol content to an extent of 75%, 70% and 64% in mitochondria, cytosolic fractions and homogenate respectively when compared to the saline control group, whereas the TSE (50 mg kg1) treated group reverted the declined levels of protein thiols to an extent of 94%, 93% and 95% correspondingly (Fig. 3B). On the other hand, the ibuprofen-treated group showed moderate protection against the declined total protein thiol levels by 72%, 69% and 60% in homogenate, mitochondria and cytosolic fractions respectively.
Modulatory effect of TSE on antioxidant enzyme activities To recompense the oxidative damage of the liver that occurred during the course of arthritis, the host system has to rejuvenate the antioxidant enzymes. As a result, a severe alteration in the homeostasis of antioxidant enzymes viz., SOD, CAT, GST, GPx and GRdx occurred in the due course. Thus, the present study evaluated these antioxidant enzyme activities in the homogenate, supernatant of disrupted mitochondria and cytosolic fractions of each group (Table 1). The present study evidenced the drastically altered activities of these enzymes in the arthritic group in comparison with the saline control group. The SOD activity was signicantly augmented to 80%, 95% and 79% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the saline control group. The TSE (50 mg
Effect of TSE on arthritis-induced: (A) lipid peroxidation and (B) protein carbonyl content in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 2
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Effect of TSE on arthritis-induced: (A) GSH and (B) total thiol levels in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 3
kg1)-administered group recovered the altered SOD activity by 92%, 96% and 99% correspondingly, whereas the ibuprofentreated group showed protection to an extent of 51%, 67% and 76% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. The CAT activity was also signicantly augmented to 65%, 87% and 76% in homogenate, mitochondria and cytosolic fractions respectively in comparison with saline control rats. The TSE (50 mg kg1)-administered group re-established the augmented CAT activity by 98%, 98% and 97% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 72%, 75% and 63% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. Similarly, the GST activity was also signicantly augmented to 75%, 87% and 78% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the saline control group. The TSE (50 mg kg1)-administered group refurbished the augmented GST activity by 97%, 94% and 94% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 52%, 64% and 44% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. There was a signicant diminution of GPx activity to an extent of 61%, 53% and 45% in homogenate, mitochondria and
Table 1
cytosolic fractions respectively in comparison with the saline control group (Fig. 4A). The TSE (50 mg kg1)-administered group rejuvenated the augmented GPx activity by 97%, 96% and 96% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 71%, 73% and 74% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. Similarly, diminution of the GRdx activity was also observed to an extent of 68%, 70% and 63% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the saline control group (Fig. 4B). The TSE (50 mg kg1)-administered group restored the augmented GRdx activity by 94%, 97% and 93% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 75%, 73% and 74% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group.
Modulatory effect of TSE on G6PDH activity Several reports have demonstrated that adjuvant-induced arthritic rats present altered activities of the hepatic G6PDH. The current study evidenced a signicant augmented activity of G6PDH to an extent of 85% and 68% in cytosolic fractions and homogenate respectively when compared to the saline control
Activities of antioxidant enzymes in liver homogenate, mitochondria and cytosolic fractions of control and experimental ratsa Groups
Parameter CATb
SODc
GSTd
Liver homogenate Mitochondria Cytosol Liver homogenate Mitochondria Cytosol Liver homogenate Mitochondria Cytosol
Control
Arthritic
0.20 0.007 1.58 0.275 0.81 0.009 0.83 0.013 1.54 0.106 3.38 0.167 0.88 0.035 1.87 0.016 1.18 0.074
0.34 2.95 1.43 1.54 3.01 6.03 1.56 3.57 2.17
0.012* 0.322* 0.142* 0.103* 0.407* 0.175* 0.089* 0.075* 0.052*
Ibuprofen (10 mg kg1) 0.26 1.97 1.11 1.23 2.04 4.18 1.31 2.54 1.93
0.009 0.177 0.126 0.168 0.215 0.375 0.095 0.083 0.068
TSE (25 mg kg1)
TSE (50 mg kg1)
TSE alone (50 mg kg1)
0.004† 0.218† 0.115† 0.017† 0.173† 0.361† 0.073† 0.062† 0.027†
0.21 0.003† 1.61 0.142† 0.83 0.013† 0.89 0.024† 1.59 0.158† 3.41 0.183† 0.91 0.064† 1.98 0.052† 1.24 0.031†
0.20 1.59 0.82 0.81 1.56 3.41 0.89 1.91 1.22
0.22 1.74 1.01 1.03 1.76 3.76 1.12 2.34 1.81
0.010 0.335 0.025 0.032 0.162 0.259 0.058 0.085 0.063
a
Effect of TSE on activities of antioxidant enzymes such as Catalase, superoxide dismutase and glutathione-S-transferase. Data are represented as mean SEM. * – signicant when compared to the saline control (p < 0.05, n ¼ 6);† – signicant when compared to the arthritic group (p < 0.05, n ¼ 6). b nmol NADPH formed per min per mg protein c Units per mg protein. d mmol GS-DNB conjugate formed per min per mg protein.
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Fig. 4 Effect of TSE on arthritis-induced antioxidant enzyme activities: (A) glutathione peroxidase and (B) glutathione reductase in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
group (Table 2). Interestingly, the TSE (50 mg kg1) administered group reverted the augmented activity of G6PDH to an extent of 95% and 96% correspondingly, while the ibuprofentreated group showed moderate protection up to 62% and 74% in cytosolic fractions and homogenate respectively in comparison with the arthritic group. Modulatory effect of TSE on liver toxicity Liver toxicity is supported by the activities of liver and serum ALT and AST. A remarkable augmentation was observed in both the ALT and AST activities by 66% and 73%, respectively in the serum of the arthritic group in comparison with the saline control group (Fig. 5A). In contrast, both ALT and AST activities were found to be reduced by 51% and 52% in the arthritic liver, respectively, when compared to the saline control group (Fig. 5B). The concomitant supplementation of TSE (50 mg kg1) signicantly restored the altered activities of ALT and AST respectively by 98% and 98% in serum and 98% and 99% in liver, whereas the ibuprofen-treated group was able to protect the altered ALT and AST activities by 70% and 83% in serum
Table 2 Glucose-6-phosphate dehydrogenase activity in liver homogenate and cytosolic fractionsa
Parameter Glucose-6-phosphate dehydrogenase (units per mg protein) Groups
Liver homogenate
Cytosolic fractions
Control Arthritic Ibuprofen (10 mg kg1) TSE (25 mg kg1) TSE (50 mg kg1) TSE alone (50 mg kg1)
107.84 2.90 58.72 1.89* 80.57 2.51 92.59 2.35# 103.81 2.92# 106.63 2.71
0.041 0.009 0.074 0.012* 0.055 0.007 0.048 0.011# 0.042 0.006# 0.039 0.013
a
Effect of TSE on glucose-6-phosphate dehydrogenase activity in liver homogenate and cytosolic fractions of arthritic and control rats. The experiment was carried out as described in the Materials and methods section. Data are represented as mean SEM. * – signicant when compared to the saline control (p < 0.05, n ¼ 6); # – signicant when compared to the arthritic group (p < 0.05, n ¼ 6).
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and 71% and 70% in liver respectively. Hepatic damage is further supported by the histological study in which hepatocellular damage was clearly observed with marked vacuolization, cell death and a large number of inammatory cells immigrated to the sinusoid of the arthritic liver (Fig. 6B), which is indicative of congestion and the same was observed with the ibuprofen-treated group in which congestion in liver sinusoids was signicant with scattered inltration of inammatory cells (Fig. 6C) when compared to the saline control liver (Fig. 6A). However, in TSE-treated groups (Fig. 6D) the extent of inammatory cell immigration, cell death and hepatic congestion were attenuated. Differences were not found in the liver sections of the TSE alone-treated group in comparison with the saline control group.
Discussion The disease arthritis is no more constrained to joint degeneration but also damage other vital organs of the body through the oxidative burst. The present study evidenced for the oxidative burst in the arthritic rat liver total homogenate, cytosolic and mitochondrial fractions. The experimental results demonstrated the augmented levels of ROS specically, H2O2 and oxidative markers like LPO and PCC. A large amount of ROS are released from mitochondria in a cell through electron transport chain (ETC). The leaked electrons from the ETC release highly active free radicals like O2 anion that live for a short time. Besides, ROS can also be secreted by the cell as a response to inammatory cytokines, growth factors, chemical oxidants, chemotherapeutics and radiations. The released ROS can directly act on DNA, specically mtDNA, and form a variety of DNA lesions resulting in oxidized DNA bases, basic sites and DNA strand breaks, and ending in genomic instability. Thus, elevated ROS is critical in the induction of oxidative damage of cells. Further, it was also shown that the peroxynitrite species (RNS) is potent enough to initiate mitochondrial membrane lipid peroxidation and protein thiol oxidation.1,28,29 The incessant release of ROS and RNS stimulates signicant oxidation of lipids by generating lipid radicals from unsaturated fatty acids of the membrane. The membrane lipid
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Effect of TSE on arthritis-induced liver toxicity: serum (A) and liver (B) AST and ALT activities. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 5
The hematoxylin–eosin stained histological pictures of control and experimental rat livers (with/without TSE and ibuprofen) 5 and 10 magnifications. (A) Control rat, (B) arthritic rat – foci of the hepatocellular damage are evident and a large number of inflammatory cells in the sinusoid indicative of congestion (arrows), (C) arthritic rat treated with ibuprofen – foci of hepatocellular damage are evident with significant congestion in liver sinusoids and scattered infiltration of inflammatory cells (arrows) and (D) arthritic rat treated with TSE – hepatic congestion was mild and almost similar to the control rat liver.
Fig. 6
peroxidation of mitochondria leads to irreversible dysfunction of mitochondrial respiration, oxidative phosphorylation and membrane uidity, permeability and ion transport. Besides, lipid peroxidation also induces alterations in membrane proteins, formation of lipid protein adducts might be through Schiff's base formation between lipid peroxidation products and amine group of proteins. The mitochondrial inner membrane proteins are considered as primary targets for ROS generated in mitochondria. Consistently, it was shown that during mitochondrial stress conditions, the membrane protein thiol groups experience extensive oxidation. The protein modication results in malfunctioning of a variety of regulatory, structural and functional proteins like cell receptors, signal transducers and enzymes.28–30 The present study demonstrated the signicantly elevated levels of ROS, LPO and PCC in liver homogenate, cytosolic and mitochondrial fractions of arthritic rats. Interestingly, these augmented oxidative elements were potentially scavenged in TSE-supplemented rat liver fractions, which might be attributed to its anti-oxidant ROS scavenging abilities.
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Furthermore, the stress condition is all the more aggravated when GSH and thiol pool begins to deplete. The endogenous levels of GSH and thiol directly reect the damage caused by ROS and organic peroxides as they act as an intracellular reductant in oxidation–reduction processes. Decreased levels of GSH and thiol in the liver fractions of arthritic rats might be due to the excessive consumption of GSH and thiols by the system to defend oxidative damage. In addition, depleted thiol groups might also be due to increased membrane protein aggregation, as the oxidized groups may involve in the formation of disulphide bridges. The TSE administration was found to help in maintaining the homeostasis of GSH and thiol depletion in liver fractions might be acting as an auxiliary antioxidant. Decreased activities of GPx and GRdx in all the tested fractions of arthritic liver further evidenced for the GSH depletion and increased LPO. GPx is basically a seleno-protein that catalyzes the reduction of lipid hydroperoxide with the help of GSH. The selenol of selenocysteine is oxidized by hydroperoxide forming selenic acid, which is then converted back to selenol by using GSH releasing GSSG as a byproduct. The GSSG is then reduced back to GSH by GRdx using NADPH.1,31–33
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Further, increased activities of endogenous antioxidant enzymes like SOD, CAT and GST in all the fractions of arthritic rat liver were observed, which might be due to increased ROS generation. The observed results were in the same line of previous studies reporting the oxidative stress in arthritis. 1,2,28,34 Both SOD and CAT are considered to be the primary line of defense against free radical stress that catalyses the conversion of superoxide radicals to hydrogen peroxide and then to water respectively.1,28,34 Further, GST catalyzes the conjugation of the reduced GSH to xenobiotic substrates to get rid of lipid peroxides and toxic electrophiles releasing harmless byproducts. The augmented enzyme activities can be as a response to the severe oxidative stress due to FCA and also an increased defense of the body against the effects of toxic electrophilic chemicals and hydrophobic compounds.33 In addition, the severe stress causing GSH depletion might be responsible for rise in the activities of these enzymes to greatly suppress the excess superoxides and peroxides in arthritic liver fractions, which was brought back to the normal state in TSE administered rats. Thus, TSE maintains the homeostasis of endogenous GSH and antioxidant enzymes, which might be by free radical scavenging and antioxidant properties. On the other hand, elevated G6PDH activity in liver fractions was evident from the results. G6PDH is the rst rate-limiting enzyme of the pentose phosphate pathway, which plays a vital role in the maintenance of the cytosolic pool of NADPH and thus the cellular redox balance as an antioxidant. Increased glucokinase and decreased glucose 6-phosphatase activity leading to elevation in glucose 6 phosphate level and higher rates of glycolysis has been reported previously.35,36 Increase in the glucose 6-phosphate activity results in the increased activity of G6PDH. The higher activity of G6PDH was also observed in the TNF-a-induced cachexia rat liver suggesting aggravated inammation and oxidative stress.1,2,33 Moreover, the overall liver damage was evident by the increased activity of serum AST and ALT, which was found to be reduced in the liver. All these altered parameters were brought back to the normal levels in the liver fractions of TSE-administered rats. In addition, the histopathological observations further supported the hepatocyte damage due to oxidative stress. Increased hepatic hypertrophy with marked vacuolization, necrosis and sinusoid congestion along with inltration of inammatory cells were evident in the liver of arthritic animals. Subsequent treatment with TSE completely restored the stress-induced hepatic damage and morphological features. Interestingly, the obtained experimental results demonstrated TSE as a potent anti-arthritic agent than ibuprofen. Although ibuprofen is known to reduce the swelling and pain, it failed to block oxidative stress in the liver. None of the altered oxidative parameters were signicantly re-established in ibuprofen treated rats; in contrast, TSE treatment was able to maintain the endogenous antioxidant homeostasis (p < 0.05). However, further studies can keep up the hope that TSE could emerge as an alternative to existing drug therapy.
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Conclusion To conclude, the present study demonstrated that adjuvantinduced arthritic rats experience severe oxidative stress, in particular mitochondrial oxidative burst in the liver apart from joint pain. As a consequence, hepatic proteins, lipids and nucleic acids are oxidatively modied and damaged due to the increased production of ROS. Further, it was also evident from the results that arthritic rat liver fractions were depleted in glutathione and thiol levels, which are primary defense of oxidative damage. The elevated activities of antioxidant enzymes like SOD, CAT and GST and the reduced activities of GPx and GRdx reect the severity of oxidative burst. In addition, the histopathological observations of the arthritic rat liver further supported the hepatocyte damage due to oxidative stress. The study certainly shows the protective efficacy of TSE against arthritis-associated oxidative damage particularly towards mitochondrial oxidative burst. Besides, the study also hints over the stimulation of secondary complications like CVDs, CHDs and diabetes due to increased oxidative stress during arthritis.
Declaration of interest The authors declare that they have no conicts of interest to disclose.
Acknowledgements MSS thanks UGC BSR-SAP for the research fellowship. MSS also thanks Dr Naveen S, Scientist, DFRL, Mysore, Mr Raghunandan P and Mr Manjunath MJ, CFTRI, Mysore for their timely help. The authors thank Central instrumentation facility, Institute of excellence (IOE), University of Mysore.
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Tamarind seed extract mitigates the liver oxidative stress in arthritic rats Cite this: DOI: 10.1039/c3fo60381d
Mahalingam Shanmuga Sundaram,a Mahadevappa Hemshekhar,a Ram M. Thushara,a Martin Sebastin Santhosh,a Somanathapura K. Naveen Kumar,a Manoj Paul,a Sannaningaiah Devaraja,b Kempaiah Kemparaju,a Kanchugarakoppal S. Rangappac and Kesturu S. Girish*ab Although arthritis is primarily a joint disorder that mainly targets the articular cartilage and subchondral bone, several recent investigations have reported oxidative burst and vital organ damage that are being considered as secondary complications of arthritis. The continuous generation of free radicals like reactive oxygen and nitrogen species is considered as a key culprit in the initiation and propagation of oxidative damage. In addition, activation of T and B cells, macrophages, inflammatory mediators such as TNF-a, IL-1b and IL-6 aggravates the oxidative damage of the vital organs, particularly the liver. The current piece of work demonstrates oxidative stress in the liver of arthritic rats and its amelioration by the procyanidin-rich tamarind seed extract (TSE). The arthritic liver homogenate, mitochondrial and cytosolic fractions were found with increased levels of oxidative stress markers including free radicals. As a consequence, depletion in the levels of glutathione, total thiols, glutathione peroxidase and reductase was evident. Furthermore, the activities of endogenous antioxidant enzymes like superoxide dismutase, catalase and glutathione-S-transferase were found to be significantly altered. The increased and decreased activity of transaminases respectively in serum and liver, along with histological observations, further confirms the liver damage. Unfortunately, the commonly used drugs like NSAIDs and DMARDs have failed to prevent oxidative damage, rather they were found to be the inducers themselves. Received 2nd September 2013 Accepted 23rd December 2013
Interestingly, TSE supplementation was found to significantly inhibit oxidative burst in the liver and maintain homeostasis. Thus, the study clearly demonstrates the protective efficacy of TSE against
DOI: 10.1039/c3fo60381d
arthritis-associated oxidative liver damage, including mitochondrial oxidative burst and its associated
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secondary complications.
Introduction Arthritis is a degenerative joint disease that targets articular cartilage and subchondral bone. Articular cartilage is constituted by collagen and proteoglycans linked to hyaluronan (HA) and aggrecan. The aggravated and uncontrolled activity of proteases and glycosidases is being referred to as a primary culprit in the cartilage damage. In addition to cartilage degeneration, oxidative stress has been considered a major threat in the management of arthritis that largely contributes to oxidative damage of vital organs.1,2 Several individuals with arthritis are reported to experience severe oxidative stress leading to damage of vital organs like the liver, kidney, lungs and spleen.3 These pathological events might also trigger cardio vascular diseases a
Department of Studies in Biochemistry, University of Mysore, Mysore-570 006, Karnataka, India. E-mail: [email protected]; Tel: +91-9964080540
b
Department of Studies and Research in Biochemistry, Tumkur University, Tumkur-572 103, Karnataka, India
c Department of Studies in Chemistry, University of Mysore, Mysore-570 006, Karnataka, India
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(CVDs) and diabetes that are known as secondary complications of arthritis. This might be attributed to the increased production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), and activation of T and B-cells, macrophages, inammatory mediators in particular tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6. The highly reactive free radicals like superoxide (O2) predominantly, hydroxyl radical (OH), hydrogen peroxide (H2O2) and peroxynitrite (ONOO) can effectively induce tissue injury causing oxidative damage to macromolecules of vital organs including proteins, lipids and nucleic acids.1,4 Liver is one of the major organs which experience severe alterations due to increase in the steady concentration of ROS and RNS. The ROS indeed act on the fatty acid side chains of lipids present in different cellular membranes, in particular, mitochondrial membranes that are exposed directly and frequently to superoxide radicals generated upon cellular respiration. Evidently, mitochondria from arthritic rats were consistently detected with elevated rates of oxygen uptake in the absence or presence of exogenous ADP. The oxidative damage of
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mitochondrial macromolecules such as mtDNA, proteins and lipids, along with protein synthesis machinery in liver cells, induces mitochondrial dysfunction. Recent studies have demonstrated that the oxidative lesions like 8-oxo-dG are found more in mtDNA than in nuclear DNA suggesting the fact that mtDNA is more susceptible to oxidative damage.5 These molecular proceedings trigger further increase in the steady concentration of ROS resulting in energy depletion leading to cell death, which eventually damages the organ and its function.5–8 Bearing in mind that the ROS actively participate in the pathogenesis of arthritis, it is apparent that the biochemical alterations in the liver are greatly inuenced by its oscillating oxidative state, specically mitochondria and peroxisomes that are directly associated with oxidative damage. Unfortunately, prolonged use of well-known anti-arthritic drugs like non-steroidal anti-inammatory drugs (NSAIDs) and disease modifying anti-rheumatic drugs (DMARDs) are also known to induce liver damage and aggravate oxidative stress.9,10 Based on these interesting facts, the present study sheds light on the oxidative status of the adjuvant-induced arthritic rat liver and its amelioration by orally supplemented procyanidin rich tamarind (Tamarindus indica) seed extract (TSE). TSE has received vast attention by pharmacologists due to the presence of therapeutic agents like antioxidants, triterpenes, polyphenolic bioactive compounds, procyanidins, polysaccharides and other unknown factors, which have healing activities for various human pathophysiological disorders.11–13 The present study aims to investigate the various facets of oxidative stress in arthritic rat liver mitochondria in detail and its suppression using TSE by assessing various oxidative stress markers and antioxidant status.
Materials and methods Chemicals Freund's complete adjuvant (FCA), dihydrodichlorouorescein diacetate (DCFDA), O-phthalaldehyde (OPT), dichlorouorescein (DCF), HEPES, 1,10 ,3,30 -tetramethoxypropane (TMP), reduced glutathione (GSH) and oxidized glutathione (GSSG) were obtained from Sigma chemicals, St. Louis, USA. Glutathione peroxidase kit was purchased from Randox laboratories, Northern Ireland (United Kingdom). Commercial ALT and AST kits were from Agappe Diagnostics Limited, Ernakulam (India). Micro-titer plates were purchased from Tarsons product Pvt. Ltd., India. All other chemicals were of analytical grade purchased from Sisco research laboratories, India. Tamarind seeds were obtained from local grocer, Mysore (India). Experimental animals Adult Wistar rats (12 weeks old) weighing around 140–170 g were used in the experiment. Animals were collected from University Central Animal Facility and housed under a controlled environment. All experiments were approved by the Animal Ethical Committee (order no: MGZ/637/2011-12 dated 12-07-2011; UOM/IAEC/04/2011), Department of Zoology, University of Mysore, Mysore and were in accordance with the
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guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Preparation of procyanidin rich tamarind seed extract (TSE) Tamarind seeds were collected from a grocer, Mysore. Dried whole seeds were nely powdered and 25 g of the powder was subjected to Soxhlet extraction using 100 mL of 95% ethanol. The solvent was removed in vacuo and the dried extract was dissolved in saline and is known as TSE and is used for further experiments.12,14 Experimental design and induction of arthritis The experimental rats were randomly divided into 6 groups each consisting of 6 rats. Group I – saline control; group II – arthritic; group III – arthritic rats treated with ibuprofen (10 mg kg1 body weight); groups IV and V – arthritic rats treated with TSE (25 and 50 mg kg1 body weight respectively); group VI – TSE (50 mg kg1 body weight) alone, non-arthritic rats. Arthritis was induced by a single subcutaneous injection (100 mL) of Freund's complete adjuvant (FCA) containing 10 mg mL1 heatkilled Mycobacterium tuberculosis at the right hind palm surface of all rats except groups I and VI. The FCA-induced rats were le for disease development for 10 days and the respective treatment was given from the 11th day to the 25th day. Aqueous solution of TSE, ibuprofen syrup and saline were administered orally to the respective experimental groups using a intragastric tube (gavage) once in a day in the morning. Ibuprofen was used as a standard NSAID treatment control and saline as a negative control. Preparation of liver homogenate, mitochondria and cytosolic fractions On the nal day of the experiment, animals were fasted for 18 h and sacriced and the livers were removed immediately and blood was collected through cardiac puncture. Serum was separated and stored at 20 C. The excised liver tissues were blotted free of blood, rinsed with ice-cold saline and homogenized (10% w/v) in ice-cold phosphate-buffered saline (PBS, 100 mM, pH 7.4). The liver homogenates were centrifuged at 5000 g at 4 C for 10 min and the supernatant obtained was known as total liver homogenate and was stored at 20 C for the assessment of different biochemical parameters. Mitochondria were isolated according to the method reported by Chandrashekar and Muralidhara.15 Briey, liver was homogenized in ice-cold Tris–sucrose buffer (2 mM Tris–HCl, 250 mM sucrose, pH 7.4) and subjected to differential centrifugation to separate mitochondria and cytosol. The mitochondrial pellet obtained was washed and suspended in ice-cold mannitol– sucrose–HEPES buffer (200 mM mannitol, 70 mM sucrose, 0.1 mM EDTA, 10 mM HEPES, pH 7.4). The post-mitochondrial fraction obtained in the above step was further centrifuged at 20 000 g and the obtained supernatant was used as cytosolic fractions. All the operations were performed at 4 C. Disrupted mitochondria were obtained by repeated freeze-thawing procedures.
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Measurement of oxidative stress markers
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Estimation of ROS The endogenous generation of ROS was quantied in liver homogenate, disrupted mitochondria and cytosolic fractions using dihydrodichlorouorescein diacetate (DCFDA). DCFDA is a non-polar uorescent probe commonly employed to estimate total ROS in general including hydrogen peroxide, hydroxyl radicals and peroxynitrite. The cell-permeant DCFDA passively diffuses into cells and is retained in the intracellular level aer cleavage by intracellular esterases. Upon oxidation by ROS, the non-uorescent DCFDA is converted to the highly uorescent 20 ,70 -dichlorouorescein (DCF). Briey, an aliquot (0.2 mg protein) of the incubation mixture (200 mL) was dispensed into a 96 well micro-titer plate containing Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5 mM HEPES, 2 mM CaCl2, 10 mM glucose, pH 7.4) to which 10 mL of DCFDA was added to a nal concentration of 10 mM and incubated for 30 min at room temperature to allow the cleavage of DCFDA by esterases and further conversion into the uorescent product dichlorouorescein. The uorescence was measured using a multimode plate reader (Thermo scientic, USA) with excitation at 480 nm and emission at 530 nm. Background uorescence was corrected by the inclusion of parallel blanks and levels of ROS were quantied from a dichlorouorescein standard curve and data were expressed as pmol DCF formed per mg protein.16
Estimation of hydrogen peroxide levels The hydrogen peroxide levels were quantied in liver homogenate, disrupted mitochondria and cytosolic fractions using homovanillic acid (HVA), a specic H2O2-sensitive uorescent probe used to detect H2O2 production in the tissues and cells. Briey an aliquot (0.2 mg protein) of the incubation mixture (200 mL) was dispensed into a 96 well micro-titer plate containing HEPES buffered saline (HBS, 10 mM HEPES, 145 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgSO4, pH 7.45) incubated with 100 mM HVA for 30 min at room temperature. Fluorescence was recorded at 420 nm by exciting the samples at 310 nm. The H2O2 levels were expressed as nmol hydrogen peroxide (HP) per mg protein.17
Estimation of MDA and protein carbonyls Lipid peroxidation (LPO) status was assessed in liver homogenate, disrupted mitochondria and cytosolic fractions by estimating thiobarbituric acid reactive substances (TBARS).18 Aliquots of the samples (1 mg protein) were added to tubes containing 1.5 mL acetic acid (20% v/v, pH 3.5), SDS (8% w/v, 0.2 mL) and thiobarbituric acid (0.8% w/v, 1.5 mL). The mixture was heated on a boiling water bath for 45 min. Adducts formed were extracted into 1-butanol (3 mL) and the TBARS formed was read at 532 nm and quantied using TMP as the standard. Values are expressed in terms of malondialdehyde (MDA) equivalents as nmol MDA formed per mg protein. Precautions were taken to
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avoid over estimation of MDA formation by carrying out incubation in the presence of 0.17% (v/v) butylated hydroxyl toluene. Protein carbonyl content (PCC) was measured using dinitrophenylhydrazine (DNPH) in liver homogenate, disrupted mitochondria and cytosolic fractions.19 To 0.5 mL of sample (1– 2 mg protein), an equal volume of 10 mM DNPH in 2 N HCl was added and incubated for 1 h, shaking intermittently at room temperature. Corresponding blank was carried out by adding only 2 N HCl to the sample. Aer incubation, the mixture was precipitated with 20% TCA and centrifuged. The precipitate was washed twice with acetone and nally dissolved in 1 mL of Tris buffer (20 mM, pH 7.4 containing 140 mM NaCl, 2% SDS-w/v) and the absorbance was read at 360 nm. The difference in absorbance is determined and expressed as nmol of carbonyl groups per mg protein, using an extinction coefficient of 22 mM1 cm1. Measurement of GSH and thiols The levels of GSH in liver homogenate, disrupted mitochondria and cytosolic fractions were measured as described by Mokrasch and Teschke20 with slight modications. The assay mixture consisted of 1 mg protein made up to 100 mL with buffered formaldehyde (1 : 4 v/v, formaldehyde: 100 mM Na2HPO4) on a micro-titer plate and kept at 37 C for 5 min. Then 150 mL of 100 mM phosphate buffer (pH 8.0 containing 5 mM EDTA) and 25 mg of O-phthalaldehyde were added and kept at 37 C for 45 min, and the uorescence was recorded at 425 nm by exciting the sample at 340 nm. The GSH level was expressed as mg GSH per mg protein. Further, to estimate the total thiol content in liver homogenate, disrupted mitochondria and cytosolic fractions, an aliquot of sample (0.05 mg protein) was added to 0.375 mL of Tris–HCl buffer (200 mM, pH 8.2) containing di-thiobis-nitrobenzoic acid (DTNB, 10 mM) and 1.975 mL of methanol. Following incubation for 30 min at room temperature, the tubes were centrifuged at 3000 g for 10 min.21 The absorbance of the supernatant was measured at 410 nm and expressed as mmol DTNB oxidized per mg protein, using an extinction coefficient of 13.6 mM1 cm1. Determination of antioxidant enzymes The superoxide dismutase (SOD) activity was measured in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by monitoring the inhibition of quercetin autoxidation.22 Briey, an aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.01 mg protein) was added to the reaction mixture (1 mL) consisting of phosphate buffer (16 mM, pH 7.8) containing TEMED–EDTA (8 mM/0.08 mM) mixture and quercetin (0.15% w/v). The decrease in absorbance was monitored for 3 min at 406 nm. The amount of protein that inhibits quercetin autoxidation by 50% is dened as one unit. The catalase (CAT) activity was measured in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by measuring the rate of hydrolysis of H2O2 at 240 nm.23 To the reaction mixture (1 mL) containing sodium phosphate buffer (100 mM, pH 7.4), H2O2 (8.8 mM), an aliquot
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of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added. The decrease in absorbance was monitored for 3 min at 240 nm and the activity was expressed as mmol H2O2 decomposed per min per mg protein (3 ¼ 43.6 mM1 cm1). The glutathione-S-transferase (GST) activity was measured in liver homogenate; supernatant of disrupted mitochondria and cytosolic fractions by monitoring the enzyme catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm.24 An aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added to the reaction mixture (1 mL) containing phosphate buffer (100 mM, pH 6.5, 0.5 mM EDTA), CDNB (1.5 mM) and GSH (1 mM). The increase in absorbance was monitored for 3 min at 340 nm and the activity was expressed as mmol GS-DNB conjugate formed per min per mg protein (3 ¼ 9.6 mM1 cm1). The glutathione reductase (GRdx) activity was measured in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by monitoring the enzyme catalysed reduction of GSSG (oxidized glutathione) in the presence of NADPH which is then oxidised to NADP+.25 Briey, an aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added to the reaction mixture (1 mL) containing phosphate buffer (100 mM, pH 7.0 containing 2 mM EDTA), 20 mM GSSG and 2 mM NADPH (in 0.1% NaHCO3 (w/v)). The decrease in absorbance was monitored at 340 nm for 3 min and the activity was expressed as mmol GSSG reduced per min per mg protein. The glutathione peroxidase (GPx) activity in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions were measured spectrophotometrically by the standard enzymatic method using a commercial kit (Randox laboratories, Northern Ireland, UK) at 340 nm and expressed as mmol NADPH oxidized per min per mg protein. Estimation of glucose-6-phosphate dehydrogenase enzyme activity The glucose-6-phosphate dehydrogenase (G6PDH) activity was estimated in liver homogenate, supernatant of disrupted mitochondria and cytosolic fractions by monitoring the increase in absorbance at 340 nm for 3 min due to NADP+ dependent glucose 6-phosphate transformation.26 Briey, an aliquot of liver homogenate or supernatant of disrupted mitochondria or cytosolic fractions (0.05 mg protein) was added to the reaction mixture (1 mL) containing Tris–HCl buffer (0.05 mM, pH 7.5), 3.8 mM NADP, 3.3 mM glucose-6-phosphate and 6.3 mM MgCl2. The activity was expressed as nmol NADPH formed per min per mg protein. Determination of serum and liver ALT and AST activity The serum and liver alanine transaminase (ALT) and aspartate transaminase (AST) activities were measured spectrophotometrically by the standard enzymatic method using commercial kits (Agappe Diagnostics Ltd., Kerala, India). The reaction mixture containing the serum/liver homogenate (10 mL) and AST/ALT reagent (1 mL) was mixed and incubated at 37 C for 1 min and read at 340 nm for 3 min and expressed as unit L1.
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Histopathology The liver tissues were dissected out and blotted free of blood, rinsed in ice-cold saline and xed in Bouin's solution overnight. The tissue samples were subjected to dehydration by processing with different grades of alcohol and chloroform mixture. The processed tissues were embedded in paraffin wax, and sections (5 mm thickness) were prepared, stained with hematoxylin– eosin dye and observed under an Axio imager A2 microscope and photographed. Protein estimation The protein estimation was done according to the method reported by Lowry27 using bovine serum albumin (BSA) as standard. Statistical analysis All the experimental data are presented as the mean SEM of three independent experiments. The differences between groups were assessed by repeated analysis of variance (ANOVA), followed by the Tukey “honestly signicantly different” (HSD) post hoc analysis. p value of < 0.05 was considered as statistically signicant. a* – signicant compared to the saline control group and b* – signicant compared to the arthritis induced group.
Results Modulatory effect of TSE on endogenous generation of ROS The result of the present study evidenced increased levels of ROS 67%, 74% and 46% respectively in liver homogenate, mitochondria and cytosolic fractions of the arthritic group when compared to the saline control group (Fig. 1A). TSE (50 mg kg1) administration signicantly reduced the endogenous generation of ROS to an extent of 97%, 99% and 98% correspondingly. While the ibuprofen-treated group showed 84%, 81% and 56% reduction in liver homogenate, mitochondria and cytosolic fractions respectively when compared to the arthritic group. Modulatory effect of TSE on endogenous generation of hydroperoxide Endogenous H2O2 levels were also elevated by 70%, 77% and 65% in liver homogenate, mitochondria and cytosolic fractions of the arthritic group when compared to the saline control group (Fig. 1B). TSE (50 mg kg1) administration reduced the endogenously generated H2O2 levels by 95%, 98% and 94% correspondingly when compared to the arthritic group which is highly signicant whereas the ibuprofen-treated group was effective by 61%, 54% and 52% in reducing the H2O2 levels in comparison with the arthritic group. Modulatory effect of TSE on lipid peroxidation and protein oxidation A substantial elevation of LPO and PCC in the arthritic liver was observed. Mitochondria of the arthritic liver exhibited a signicant increase in LPO by 90%, whereas homogenate and cytosolic fractions showed 77% and 48% respectively when
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Effect of TSE on arthritis-induced: (A) ROS and (B) hydroperoxide levels in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 1
compared to the saline control group (Fig. 2A). The TSE (50 mg kg1) treated group reverted the altered LPO signicantly by 90%, 90% and 93% correspondingly with respect to the arthritic group, while the ibuprofen-treated group showed moderate protection by 70%, 77% and 48% in mitochondria, homogenate and cytosolic fractions respectively when compared to the arthritic group. Furthermore, a signicant elevation of PCC was observed in cytosolic fractions of arthritic liver by 140% followed by mitochondria and homogenate by 137% and 61% respectively when compared to saline control (Fig. 2B). Conversely the TSE (50 mg kg1)-fed group re-established the altered PCC effectively by 93%, 92% and 94% correspondingly with respect to the arthritic group. While the ibuprofen-treated group showed moderate protection against the arthritic group by 60%, 70% and 77% with respect to cytosolic fraction, mitochondria and homogenate.
Modulatory effect of TSE on the levels of GSH and total thiols The arthritic group showed a signicant decrease in GSH content in homogenate, mitochondria and cytosolic fractions of the liver to an extent of 54%, 42% and 40% respectively with reference to the saline control group (Fig. 3A). The TSE (50 mg kg1) treated group reversed the altered GSH content by 90%, 98% and 95% correspondingly when compared to the arthritic group, while the ibuprofen treated group showed a protection by 66%, 77% and 75% with respect to homogenate,
mitochondria and cytosolic fractions when compared to the arthritic group. Furthermore, the liver of arthritic rats showed a signicant decline in total protein thiol content to an extent of 75%, 70% and 64% in mitochondria, cytosolic fractions and homogenate respectively when compared to the saline control group, whereas the TSE (50 mg kg1) treated group reverted the declined levels of protein thiols to an extent of 94%, 93% and 95% correspondingly (Fig. 3B). On the other hand, the ibuprofen-treated group showed moderate protection against the declined total protein thiol levels by 72%, 69% and 60% in homogenate, mitochondria and cytosolic fractions respectively.
Modulatory effect of TSE on antioxidant enzyme activities To recompense the oxidative damage of the liver that occurred during the course of arthritis, the host system has to rejuvenate the antioxidant enzymes. As a result, a severe alteration in the homeostasis of antioxidant enzymes viz., SOD, CAT, GST, GPx and GRdx occurred in the due course. Thus, the present study evaluated these antioxidant enzyme activities in the homogenate, supernatant of disrupted mitochondria and cytosolic fractions of each group (Table 1). The present study evidenced the drastically altered activities of these enzymes in the arthritic group in comparison with the saline control group. The SOD activity was signicantly augmented to 80%, 95% and 79% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the saline control group. The TSE (50 mg
Effect of TSE on arthritis-induced: (A) lipid peroxidation and (B) protein carbonyl content in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 2
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Effect of TSE on arthritis-induced: (A) GSH and (B) total thiol levels in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 3
kg1)-administered group recovered the altered SOD activity by 92%, 96% and 99% correspondingly, whereas the ibuprofentreated group showed protection to an extent of 51%, 67% and 76% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. The CAT activity was also signicantly augmented to 65%, 87% and 76% in homogenate, mitochondria and cytosolic fractions respectively in comparison with saline control rats. The TSE (50 mg kg1)-administered group re-established the augmented CAT activity by 98%, 98% and 97% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 72%, 75% and 63% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. Similarly, the GST activity was also signicantly augmented to 75%, 87% and 78% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the saline control group. The TSE (50 mg kg1)-administered group refurbished the augmented GST activity by 97%, 94% and 94% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 52%, 64% and 44% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. There was a signicant diminution of GPx activity to an extent of 61%, 53% and 45% in homogenate, mitochondria and
Table 1
cytosolic fractions respectively in comparison with the saline control group (Fig. 4A). The TSE (50 mg kg1)-administered group rejuvenated the augmented GPx activity by 97%, 96% and 96% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 71%, 73% and 74% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group. Similarly, diminution of the GRdx activity was also observed to an extent of 68%, 70% and 63% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the saline control group (Fig. 4B). The TSE (50 mg kg1)-administered group restored the augmented GRdx activity by 94%, 97% and 93% correspondingly, whereas the ibuprofen-treated group showed protection to an extent of 75%, 73% and 74% in homogenate, mitochondria and cytosolic fractions respectively in comparison with the arthritic group.
Modulatory effect of TSE on G6PDH activity Several reports have demonstrated that adjuvant-induced arthritic rats present altered activities of the hepatic G6PDH. The current study evidenced a signicant augmented activity of G6PDH to an extent of 85% and 68% in cytosolic fractions and homogenate respectively when compared to the saline control
Activities of antioxidant enzymes in liver homogenate, mitochondria and cytosolic fractions of control and experimental ratsa Groups
Parameter CATb
SODc
GSTd
Liver homogenate Mitochondria Cytosol Liver homogenate Mitochondria Cytosol Liver homogenate Mitochondria Cytosol
Control
Arthritic
0.20 0.007 1.58 0.275 0.81 0.009 0.83 0.013 1.54 0.106 3.38 0.167 0.88 0.035 1.87 0.016 1.18 0.074
0.34 2.95 1.43 1.54 3.01 6.03 1.56 3.57 2.17
0.012* 0.322* 0.142* 0.103* 0.407* 0.175* 0.089* 0.075* 0.052*
Ibuprofen (10 mg kg1) 0.26 1.97 1.11 1.23 2.04 4.18 1.31 2.54 1.93
0.009 0.177 0.126 0.168 0.215 0.375 0.095 0.083 0.068
TSE (25 mg kg1)
TSE (50 mg kg1)
TSE alone (50 mg kg1)
0.004† 0.218† 0.115† 0.017† 0.173† 0.361† 0.073† 0.062† 0.027†
0.21 0.003† 1.61 0.142† 0.83 0.013† 0.89 0.024† 1.59 0.158† 3.41 0.183† 0.91 0.064† 1.98 0.052† 1.24 0.031†
0.20 1.59 0.82 0.81 1.56 3.41 0.89 1.91 1.22
0.22 1.74 1.01 1.03 1.76 3.76 1.12 2.34 1.81
0.010 0.335 0.025 0.032 0.162 0.259 0.058 0.085 0.063
a
Effect of TSE on activities of antioxidant enzymes such as Catalase, superoxide dismutase and glutathione-S-transferase. Data are represented as mean SEM. * – signicant when compared to the saline control (p < 0.05, n ¼ 6);† – signicant when compared to the arthritic group (p < 0.05, n ¼ 6). b nmol NADPH formed per min per mg protein c Units per mg protein. d mmol GS-DNB conjugate formed per min per mg protein.
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Fig. 4 Effect of TSE on arthritis-induced antioxidant enzyme activities: (A) glutathione peroxidase and (B) glutathione reductase in liver homogenate, mitochondria and cytosolic fractions of control and experimental rats. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
group (Table 2). Interestingly, the TSE (50 mg kg1) administered group reverted the augmented activity of G6PDH to an extent of 95% and 96% correspondingly, while the ibuprofentreated group showed moderate protection up to 62% and 74% in cytosolic fractions and homogenate respectively in comparison with the arthritic group. Modulatory effect of TSE on liver toxicity Liver toxicity is supported by the activities of liver and serum ALT and AST. A remarkable augmentation was observed in both the ALT and AST activities by 66% and 73%, respectively in the serum of the arthritic group in comparison with the saline control group (Fig. 5A). In contrast, both ALT and AST activities were found to be reduced by 51% and 52% in the arthritic liver, respectively, when compared to the saline control group (Fig. 5B). The concomitant supplementation of TSE (50 mg kg1) signicantly restored the altered activities of ALT and AST respectively by 98% and 98% in serum and 98% and 99% in liver, whereas the ibuprofen-treated group was able to protect the altered ALT and AST activities by 70% and 83% in serum
Table 2 Glucose-6-phosphate dehydrogenase activity in liver homogenate and cytosolic fractionsa
Parameter Glucose-6-phosphate dehydrogenase (units per mg protein) Groups
Liver homogenate
Cytosolic fractions
Control Arthritic Ibuprofen (10 mg kg1) TSE (25 mg kg1) TSE (50 mg kg1) TSE alone (50 mg kg1)
107.84 2.90 58.72 1.89* 80.57 2.51 92.59 2.35# 103.81 2.92# 106.63 2.71
0.041 0.009 0.074 0.012* 0.055 0.007 0.048 0.011# 0.042 0.006# 0.039 0.013
a
Effect of TSE on glucose-6-phosphate dehydrogenase activity in liver homogenate and cytosolic fractions of arthritic and control rats. The experiment was carried out as described in the Materials and methods section. Data are represented as mean SEM. * – signicant when compared to the saline control (p < 0.05, n ¼ 6); # – signicant when compared to the arthritic group (p < 0.05, n ¼ 6).
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and 71% and 70% in liver respectively. Hepatic damage is further supported by the histological study in which hepatocellular damage was clearly observed with marked vacuolization, cell death and a large number of inammatory cells immigrated to the sinusoid of the arthritic liver (Fig. 6B), which is indicative of congestion and the same was observed with the ibuprofen-treated group in which congestion in liver sinusoids was signicant with scattered inltration of inammatory cells (Fig. 6C) when compared to the saline control liver (Fig. 6A). However, in TSE-treated groups (Fig. 6D) the extent of inammatory cell immigration, cell death and hepatic congestion were attenuated. Differences were not found in the liver sections of the TSE alone-treated group in comparison with the saline control group.
Discussion The disease arthritis is no more constrained to joint degeneration but also damage other vital organs of the body through the oxidative burst. The present study evidenced for the oxidative burst in the arthritic rat liver total homogenate, cytosolic and mitochondrial fractions. The experimental results demonstrated the augmented levels of ROS specically, H2O2 and oxidative markers like LPO and PCC. A large amount of ROS are released from mitochondria in a cell through electron transport chain (ETC). The leaked electrons from the ETC release highly active free radicals like O2 anion that live for a short time. Besides, ROS can also be secreted by the cell as a response to inammatory cytokines, growth factors, chemical oxidants, chemotherapeutics and radiations. The released ROS can directly act on DNA, specically mtDNA, and form a variety of DNA lesions resulting in oxidized DNA bases, basic sites and DNA strand breaks, and ending in genomic instability. Thus, elevated ROS is critical in the induction of oxidative damage of cells. Further, it was also shown that the peroxynitrite species (RNS) is potent enough to initiate mitochondrial membrane lipid peroxidation and protein thiol oxidation.1,28,29 The incessant release of ROS and RNS stimulates signicant oxidation of lipids by generating lipid radicals from unsaturated fatty acids of the membrane. The membrane lipid
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Effect of TSE on arthritis-induced liver toxicity: serum (A) and liver (B) AST and ALT activities. Data are represented as mean SEM. a* – significant when compared to the saline control (p < 0.05, n ¼ 6); b* – significant when compared to the arthritic group (p < 0.05, n ¼ 6).
Fig. 5
The hematoxylin–eosin stained histological pictures of control and experimental rat livers (with/without TSE and ibuprofen) 5 and 10 magnifications. (A) Control rat, (B) arthritic rat – foci of the hepatocellular damage are evident and a large number of inflammatory cells in the sinusoid indicative of congestion (arrows), (C) arthritic rat treated with ibuprofen – foci of hepatocellular damage are evident with significant congestion in liver sinusoids and scattered infiltration of inflammatory cells (arrows) and (D) arthritic rat treated with TSE – hepatic congestion was mild and almost similar to the control rat liver.
Fig. 6
peroxidation of mitochondria leads to irreversible dysfunction of mitochondrial respiration, oxidative phosphorylation and membrane uidity, permeability and ion transport. Besides, lipid peroxidation also induces alterations in membrane proteins, formation of lipid protein adducts might be through Schiff's base formation between lipid peroxidation products and amine group of proteins. The mitochondrial inner membrane proteins are considered as primary targets for ROS generated in mitochondria. Consistently, it was shown that during mitochondrial stress conditions, the membrane protein thiol groups experience extensive oxidation. The protein modication results in malfunctioning of a variety of regulatory, structural and functional proteins like cell receptors, signal transducers and enzymes.28–30 The present study demonstrated the signicantly elevated levels of ROS, LPO and PCC in liver homogenate, cytosolic and mitochondrial fractions of arthritic rats. Interestingly, these augmented oxidative elements were potentially scavenged in TSE-supplemented rat liver fractions, which might be attributed to its anti-oxidant ROS scavenging abilities.
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Furthermore, the stress condition is all the more aggravated when GSH and thiol pool begins to deplete. The endogenous levels of GSH and thiol directly reect the damage caused by ROS and organic peroxides as they act as an intracellular reductant in oxidation–reduction processes. Decreased levels of GSH and thiol in the liver fractions of arthritic rats might be due to the excessive consumption of GSH and thiols by the system to defend oxidative damage. In addition, depleted thiol groups might also be due to increased membrane protein aggregation, as the oxidized groups may involve in the formation of disulphide bridges. The TSE administration was found to help in maintaining the homeostasis of GSH and thiol depletion in liver fractions might be acting as an auxiliary antioxidant. Decreased activities of GPx and GRdx in all the tested fractions of arthritic liver further evidenced for the GSH depletion and increased LPO. GPx is basically a seleno-protein that catalyzes the reduction of lipid hydroperoxide with the help of GSH. The selenol of selenocysteine is oxidized by hydroperoxide forming selenic acid, which is then converted back to selenol by using GSH releasing GSSG as a byproduct. The GSSG is then reduced back to GSH by GRdx using NADPH.1,31–33
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Further, increased activities of endogenous antioxidant enzymes like SOD, CAT and GST in all the fractions of arthritic rat liver were observed, which might be due to increased ROS generation. The observed results were in the same line of previous studies reporting the oxidative stress in arthritis. 1,2,28,34 Both SOD and CAT are considered to be the primary line of defense against free radical stress that catalyses the conversion of superoxide radicals to hydrogen peroxide and then to water respectively.1,28,34 Further, GST catalyzes the conjugation of the reduced GSH to xenobiotic substrates to get rid of lipid peroxides and toxic electrophiles releasing harmless byproducts. The augmented enzyme activities can be as a response to the severe oxidative stress due to FCA and also an increased defense of the body against the effects of toxic electrophilic chemicals and hydrophobic compounds.33 In addition, the severe stress causing GSH depletion might be responsible for rise in the activities of these enzymes to greatly suppress the excess superoxides and peroxides in arthritic liver fractions, which was brought back to the normal state in TSE administered rats. Thus, TSE maintains the homeostasis of endogenous GSH and antioxidant enzymes, which might be by free radical scavenging and antioxidant properties. On the other hand, elevated G6PDH activity in liver fractions was evident from the results. G6PDH is the rst rate-limiting enzyme of the pentose phosphate pathway, which plays a vital role in the maintenance of the cytosolic pool of NADPH and thus the cellular redox balance as an antioxidant. Increased glucokinase and decreased glucose 6-phosphatase activity leading to elevation in glucose 6 phosphate level and higher rates of glycolysis has been reported previously.35,36 Increase in the glucose 6-phosphate activity results in the increased activity of G6PDH. The higher activity of G6PDH was also observed in the TNF-a-induced cachexia rat liver suggesting aggravated inammation and oxidative stress.1,2,33 Moreover, the overall liver damage was evident by the increased activity of serum AST and ALT, which was found to be reduced in the liver. All these altered parameters were brought back to the normal levels in the liver fractions of TSE-administered rats. In addition, the histopathological observations further supported the hepatocyte damage due to oxidative stress. Increased hepatic hypertrophy with marked vacuolization, necrosis and sinusoid congestion along with inltration of inammatory cells were evident in the liver of arthritic animals. Subsequent treatment with TSE completely restored the stress-induced hepatic damage and morphological features. Interestingly, the obtained experimental results demonstrated TSE as a potent anti-arthritic agent than ibuprofen. Although ibuprofen is known to reduce the swelling and pain, it failed to block oxidative stress in the liver. None of the altered oxidative parameters were signicantly re-established in ibuprofen treated rats; in contrast, TSE treatment was able to maintain the endogenous antioxidant homeostasis (p < 0.05). However, further studies can keep up the hope that TSE could emerge as an alternative to existing drug therapy.
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Conclusion To conclude, the present study demonstrated that adjuvantinduced arthritic rats experience severe oxidative stress, in particular mitochondrial oxidative burst in the liver apart from joint pain. As a consequence, hepatic proteins, lipids and nucleic acids are oxidatively modied and damaged due to the increased production of ROS. Further, it was also evident from the results that arthritic rat liver fractions were depleted in glutathione and thiol levels, which are primary defense of oxidative damage. The elevated activities of antioxidant enzymes like SOD, CAT and GST and the reduced activities of GPx and GRdx reect the severity of oxidative burst. In addition, the histopathological observations of the arthritic rat liver further supported the hepatocyte damage due to oxidative stress. The study certainly shows the protective efficacy of TSE against arthritis-associated oxidative damage particularly towards mitochondrial oxidative burst. Besides, the study also hints over the stimulation of secondary complications like CVDs, CHDs and diabetes due to increased oxidative stress during arthritis.
Declaration of interest The authors declare that they have no conicts of interest to disclose.
Acknowledgements MSS thanks UGC BSR-SAP for the research fellowship. MSS also thanks Dr Naveen S, Scientist, DFRL, Mysore, Mr Raghunandan P and Mr Manjunath MJ, CFTRI, Mysore for their timely help. The authors thank Central instrumentation facility, Institute of excellence (IOE), University of Mysore.
References 1 J. F. Comar, A. Babeto de S´ akNakanishi and A. L. de Oliveira, Marques Nogueira Wendt M, Bersani Amado CA, Ishii Iwamoto EL, Peralta RM, Bracht A. Oxidative state of the liver of rats with adjuvant-induced arthritis, Free Radicals Biol. Med., 2013, 58, 144–153. 2 M. Hemshekhar, M. Sebastin Santhosh, K. Sunitha, R. M. Thushara, K. Kemparaju, K. S. Rangappa and K. S. Girish, A dietary colorant crocin mitigates arthritis and associated secondary complications by modulating cartilage deteriorating enzymes, inammatory mediators and antioxidant status, Biochimie, 2012, 94(12), 2723–2733. 3 M. S. Chandankhede and M. M. Gupta, Oxidative stress and antioxidant status in patients with rheumatoid arthritis, Int. J. Biol. Med. Res., 2013, 4(2), 3088–3090. 4 S. Hajizadeh, J. DeGroot, J. M. TeKoppele, A. Tarkowski and L. V. Collins, Extracellular mitochondrial DNA and oxidatively damaged DNA in synovial uid of patients with rheumatoid arthritis, Arthritis Res. Ther., 2003, 5(5), R234– R240.
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