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Protective effects of carnosol against oxidative stress induced brain damage by chronic stress in rats
© The Author(s). 2017
Received: 19 January 2017
Accepted: 25 April 2017
Published: 4 May 2017
Oxidative stress through chronic stress destroys the brain function. There are many documents have shown that carnosol may have a therapeutic effect versus free radical induced diseases. The current research focused the protective effect of carnosol against the brain injury induced by the restraint stress.
The restraint stress induced by keeping animals in restrainers for 21 consecutive days. Thereafter, the rats were injected carnosol or vehicle for 21 consecutive days. At the end of experiment, all the rats were subjected to his open field test and forced swimming test. Afterwards, the rats were sacrificed for measuring their oxidative stress parameters. To measure the modifications in the biochemical aspects after the experiment, the activities of malondialdehyde (MDA), reduced glutathione (GSH), as well as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) were evaluated in the whole brain.
Our data showed that the animals received chronic stress had a raised immobility time versus the non-stressed animals (p < 0.01). Furthermore, chronic stress diminished the number of crossing in the animals that were subjected to the chronic stress versus the non-stressed rats (p < 0.01). Carnosol ameliorated this alteration versus the non-treated rats (p < 0.05). In the vehicle treated rats that submitted to the stress, the level of MDA levels was significantly increased (P < 0.001), and the levels of GSH and antioxidant enzymes were significantly decreased versus the non-stressed animals (P < 0.001). Carnosol treatment reduced the modifications in the stressed animals as compared with the control groups (P < 0.001). All of these carnosol effects were nearly similar to those observed with fluoxetine.
The current research shows that the protective effects of carnosol may be accompanied with enhanced antioxidant defenses and decreased oxidative injury.
Stress is an uncontrolled psychological or emotional condition that disturbs cellular homeostasis . Among the many types of tissues in the body, the brain is more susceptible to stress because of its high levels of intracellular mediators that are involved in the stress response (glucocorticoid receptors) . Stress may induce a variety of central nervous system abnormalities, including depression, anxiety, locomotor activity, and cognitive function . The main mechanism(s) associated with stress-induced behavioral disorders is the activation of the hypothalamo-hypophyseal-adrenal (HPA) axis, which involves elevation of the brain corticosterone level . The increased level of corticosterone results in a rapid depolarization that evoked glutamate release in the cortical and limbic regions . The over-production of glutamate can lead to a mitochondrial dysfunction and an increase in metabolic rate . The increased metabolic rate produces extra free radicals, leading to an imbalance between reactive oxygen species (ROS) generation and anti-oxidant system . These free radical species result in oxidative damage to different molecules in cells, such as proteins, lipids, and nucleic acids . Brain is especially sensitive to oxidative stress due to its high content of readily oxidizable fatty acids, high consumption of oxygen, and low levels of antioxidants. The increased ROS and also lowered levels of antioxidants have been associated with the pathogenesis of neurodegenerative diseases such as depression and cognitive impairment . However, the enhancement of antioxidant system may be effective to combat against the excessive ROS production [9–11]. According to strong evidences, the several natural antioxidants may be effective against stress-induced mental health complications . Carnosol (CS) is a natural polyphenol (dietary diterpene) seen in plants belonging to Lamiaceae family (Mint Family) such as sage, rosemary, lavenders, and oregano . Studies indicated that CS has many pharmacological effects, including antioxidant, anti-inflammatory, anti-microbial, and anti-cancer activities [14, 15]. The antioxidant activity of CS is activated via the catechol hydroxyl and is changed to a carnosol quinine. This quinone derivative is the main antioxidant products of CS, and has very weak antioxidant activity, but, under proper conditions the antioxidant activity can be ameliorated . Previous studies indicated that culinary herbs such as rosemary may be effective for treating the neurodegenerative disorders in animal models. Recently, it was observed that the hydroalcoholic crude extract of R. officinalis as well as essential oil, the isolated compounds CS and betulinic acid caused an antidepressant-like effect in mice by evaluating the forced swimming test (FST) and tail suspension test (TST), that are the predictive tests of antidepressant activity, through a mechanism dependent on the interaction with the monoaminergic systems . However, the involvement of this effect on brain and its anti−/pro-oxidative aspect has not yet been studied. Considering the protective effect of CS on the function of free radicals, the study was designed to investigate the protective effect of CS on the behavioral changes in the FST, open field test (OFT) and hippocampal antioxidant imbalance induced by the chronic restraint stress. We chose the antidepressant, fluoxetine, as a positive control in our experiment.
All chemicals were purchased from Sigma-Aldrich Chemical (St. Louis, USA). The corticosterone ELISA kit was purchased from Cusabio (Cusabio Biotech Co., Ltd).
Wistar albino rats (213 ± 10.2 g) were bred at the university experimental animal care center. Animals were maintained under standard environmental conditions and had free access to standard rodent feed and water.
Rats were randomly divided into the nine experimental groups (8 rats per group) as follows: 1) Vehicle (Veh) + no-stress (NS) (Veh-NS); 2) Vehicle + stress (Veh-S), 3) CS (1 mg/kg, IP) + no-stress (CS1-NS), 4) CS (5 mg/kg, IP) + no-stress (CS5-NS); 5) CS (10 mg/kg, IP) + no-stress (CS10-NS), 6) CS (1 mg/kg, IP) + stress (CS1-S), 7) CS (5 mg/kg, IP) + stress (CS5-S), 8) CS (10 mg/kg, IP) + stress (CS10-S), 9) Fluoxetine (Flu) (10 ml/kg, IP) + stress (Flu-S). Protocols were approved by the Ethical Committee (The Ethical Research Committee of Mashhad University of Medical Sciences). The restraint stress was performed using a rodent restrainer made of plexiglass that closely fit the rats’ body. For the chronic restraint stress, the rats were kept in the restrainers for 1 h per day for 21 consecutive days. The animals received systemic administrations of the vehicle (3% dimethyl sulfoxide-DMSO) or CS daily for 21 days . After the experimental period, all the rats were released from their enclosure and 40 min post-release were submitted to the FST, OFT and finally were sacrificed for evaluation the biochemical parameters.
Forced swimming test (FST)
Briefly, the rats were individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm), containing 19 cm of water (depth) at 25 ± 1 °C; the total duration of immobility was measured during a 6-min test period by observers blind to the treatment conditions. Each rat was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water .
Open field test (OFT)
To assess possible interferences on locomotor activity, the rats were evaluated in the open field paradigm as previously described [19, 20]. Rats were individually placed in a wooden box (40 × 60 × 50 cm) with the floor divided into 12 equal squares and the number of crossings with all paws was manually counted in a 6 min period. The light was maintained at the minimum to avoid anxiety behavior and the apparatus was cleaned with a solution of 10% ethanol between tests in order to hide animal clues [19, 20].
For biochemical tests, the animals were anesthetized with ether and blood was subsequently collected from the retro-orbital sinus. Blood and sera were separated by centrifugation at 5000 RPM for 5 min for corticosterone measurement. Then, the hippocampi were removed and homogenized in 50 mM phosphate buffer, pH 7.4 and centrifuged at 16,000×g, at 4 °C for 20 min. The homogenate and supernatant were used for the assays.
Under deep anesthesia, blood was collected from the retro-orbital sinus of the rats. Blood was allowed to clot and the sera was separated using centrifugation at 5000 RPM for 5 min and stored at −80 °C until use. The total serum level of corticosterone was measured by ELISA kits (CORT ELISA Kit CSB-E07014r).
Measurement of MDA
Malondialdehyde (MDA) results from degradation of polyunsaturated lipids. The production of this substance is used as a biomarker to measure the level of lipid peroxidation. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) to form a 1:2 MDA-TBA adduct, which absorbs at 532 nm. Thus, the quantity of TBARS is proportionate to the amount of MDA. The concentration of TBARS is determined according to a method of Mihara and Uchiyama. The concentration of TBARS was calculated using the MDA standard curve and was expressed as nmol/mg of protein .
Estimation of GSH
The GSH level was measured by the method of Beutler et al. (1963) . Briefly, 0.1 ml of sample was added to 0.9 ml distilled water and 1.5 ml of precipitating reagent (3.34 g meta-phosphoric acid, 0.4 g EDTA and 60.0 g sodium chloride). Tubes were shaken and allowed to stand for 5 min at the room temperature (25 ± 1 °C). The mixture was centrifuged for 15 min at 4000 RPM at 4 °C. In the 1.0 ml supernatant, 4.0 ml of phosphate solution (0.3 M disodium hydrogen phosphate) and 0.5 ml 5–50-dithiobis-(2-nitrobenzoic acid) (DTNB) (80 mg in 1% sodium citrate) were added. The development of yellow color complex was read immediately at 412 nm on a spectrophotometer. A standard curve using the GSH level was prepared and GSH concentration in the experimental samples was extrapolated from the standard curve. The GSH concentration was calculated and expressed as μmol of GSH/mg protein.
Measurements of enzymes
The activity of SOD was determined by the method of Marklund and Marklund 1979 , using inhibition of pyrogallol autoxidation at pH 8. The specific activity of SOD is expressed as units per mg protein per minute. The activity of GPx was measured by the method of Paglia and Valentine . GPx catalysis the oxidation of glutathione by Cumene hydroperoxide. In the presence of glutathione reductase (GR) and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP. The decrease in absorbance is measured at 340 nm. GR catalyzes the reduction of glutathione in the presence of NADPH, which is oxidized to NADP. The decrease in absorbance is measured at 340 nm. The levels of GPx and GR were expressed as U/mg protein. The CAT activity was assayed by H2O2 consumption, following Aebi’s method  and modified by Pieper et al. (1995) .
Protein was estimated in the subcellular fractions by the method of Bradford (1976) using bovine serum albumin (BSA) as standard .
All experiments were carried out at least in duplicate. The each group consisted of eight rats. One way analysis of variance (ANOVA) was performed and Tukey post-hoc test was used for multiple comparisons. Statistical analyses were performed using the InStat 3.0 program. The results are expressed as mean ± SEM. Differences of p < 0.05 were considered significant.
Effect of CS and Flu (positive control) on the MDA (nmol/mgp), GSH (μmol/mgp), SOD (U/mgp), GPx (U/mgp), GR (U/mgp), and CAT (U/mgp) levels in brain of immobilization stress and control groups (n = 8, for each group)
4.02 ± 0.74
6.13 ± 0.69
1.32 ± 0.09
0.69 ± 0.07
0.51 ± 0.05
3.42 ± 0.96
8.97 ± 0.88***
2.87 ± 0.71**
0.61 ± 0.11**
0.35 ± 0.10**
0.26 ± 0.02*
1.08 ± 0.21*
4.12 ± 0.31++
5.89 ± 0.31+
1.01 ± 0.13
0.59 ± 0.10
0.50 ± 0.06+
3.00 ± 0.34
3.89 ± 0.49+++
6.23 ± 0.29++
1.13 ± 0.10
0.63 ± 0.11+
0.48 ± 0.08+
3.12 ± 0.41+
4.06 ± 0.97+++
6.04 ± 0.97++
1.28 ± 0.07++
0.71 ± 0.06++
0.53 ± 0.04++
3.47 ± 0.38++
7.88 ± 0.65*++
3.07 ± 0.55*
0.73 ± 0.15*
0.41 ± 0.05
0.32 ± 0.01
1.26 ± 0.25
6.43 ± 0.75***+++
4.12 ± 0.19
0.96 ± 0.20
0.52 ± 0.02
0.39 ± 0.03
1.77 ± 0.30
5.06 ± 1.08+++
5.67 ± 0.61+
1.17 ± 0.09+
0.61 ± 0.07
0.45 ± 0.07
3.10 ± 0.14+
4.66 ± 0.62+++
5.88 ± 0.73+
1.28 ± 0.19+
0.66 ± 0.10
0.49 ± 0.09
3.31 ± 0.39+
The GSH level of the Veh-S group was significantly lower than those of the Veh-NS group (P < 0.01). The GSH level in the CS10-S group was significantly higher than those of the Veh-S group (P < 0.05). The activities of SOD, GPx, GR, and CAT in the Veh-S group were significantly lower than those of the Veh-NS group (P < 0.01, P < 0.01, P < 0.05, P < 0.05, respectively). The SOD and CAT activities in the CS10-S groups were significantly higher than those of the Veh-S groups. The increases were obtained with the fluoxetine (Flu) (10 mg/kg) injection in the stressed rats (Flu-S) (P < 0.05).
In conclusion, the present study showed that the CS administration prevented behavioral alterations via the modulating hippocampal oxidative response in the animals subjected to the chronic restraint stress procedure.
The authors would like to thank Research Affairs of Neyshabur University of Medical Sciences for financially supporting this work. The authors also would like to thank of Department of Neurosurgery, Mashhad University of Medical Sciences for their assistance for the current project.
Research Affairs of Neyshabur University of Medical Sciences.
Availability of data and materials
The data sets supporting the results of this article are included within the article (and its additional files).
Study conception and design: SS, MA-N. Acquisition of data: TF, MS, AB. Analysis and interpretation of data: TF, MS, SS, MA-N, AB. Drafting of manuscript: SS, MA-N. Critical revision: SS. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The Authors hereby consent to publication of the Work in the Journal of BMC Complementary and Alternative Medicine.
The protocol for the research project has been approved by the constituted Ethics Committee of Mashhad University of Medical Sciences.
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