Protective effect of HMG CoA reductase
inhibitors against running wheel activity induced fatigue, anxiety like behavior, oxidative stress and mitochondrial dysfunction in mice
Anil Kumar, Aditi Vashist, Puneet Kumar, Harikesh Kalonia, Jitendriya Mishra
Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study (UGC-CAS), Panjab University, Chandigarh-160014, India
Correspondence: Anil Kumar, e-mail: kumaruips@yahoo.com
Abstract:
Background: Chronic fatigue stress (CFS) is an important health problem with unknown causes and unsatisfactory prevention strategies, often characterized by long-lasting and debilitating fatigue, myalgia, impairment of neuro-cognitive functions along with other common symptoms. The present study has been designed to explore the protective effect of statins against running wheel activ- ity induced fatigue anxiety.
Methods: Male albino Laca mice (20–30 g) were subjected to swim stress induced fatigue in a running wheel activity apparatus.
Atorvastatin (10, 20 mg/kg, po) and fluvastatin (5, 10 mg/kg, po) were administered daily for 21 days, one hour prior to the animals being subjected to running wheel activity test session of 6 min. Various behavioral tests (running wheel activity, locomotor activity and elevated plus maze test), biochemical parameters (lipid peroxidation, nitrite concentration, glutathione levels and catalase activ- ity) and mitochondrial complex enzyme dysfunctions (complex I, II, III and IV) were subsequently assessed.
Results: Animals exposed to 6 min test session on running wheel for 21 days showed a significant decrease in number of wheel rota- tions per 6 min indicating fatigue stress like behavior. Treatment with atorvastatin (10 and 20 mg/kg) and fluvastatin (10 mg/kg) for 21 days significantly improved the behavioral alterations [increased number of wheel rotations and locomotor activity, and anxiety like behavior (decreased number of entries and time spent in open arm)], oxidative defence and mitochondrial complex enzyme ac- tivities in brain.
Conclusion: Present study suggests the protective role of statins against chronic fatigue induced behavioral, biochemical and mito- chondrial dysfunctions.
Key words:
running wheel activity test session (RWATS), chronic fatigue stress, anxiety, atorvastatin, fluvastatin, oxidative stress, mitochondrial enzyme complexes
Introduction
Chronic fatigue stress (CFS) is an important health problem. The causes of CFS are unknown and effec- tive prevention strategies are still unsatisfactory.
A growing literature suggests that early adverse expe- rience increases the risk for a range of negative health outcomes [25]. Anxiety, depression, panic attacks and mood swings are just a few of these symptoms which are associated with chronic fatigue. Anxiety is a gen- eralized mood condition that can often occurs without
Pharmacological Reports 2012, 64, 13261336 ISSN 1734-1140
Copyright © 2012 by Institute of Pharmacology Polish Academy of Sciences
an identifiable triggering stimulus. As such, it can be easily distinguished from fear, which is an emotional response to a perceived threat. Additionally, fear is re- lated to the specific behaviors of escape and avoid- ance, whereas anxiety is related to situations per- ceived as uncontrollable or unavoidable.
Chronic fatigue stress is characterized by long- lasting and debilitating fatigue, myalgia, impairment of neuro-cognitive functions along with some common symptoms like arthralgia, adenopathy, exacerbation of allergic responses, intermittent fever, post-exertional fatigue, which are severely worsened after physical ex- ercise [9, 13, 15]. The pathophysiology of chronic fa- tigue stress is incompletely understood so far and com- monly multifactorial in nature [19]. Previous studies revealed that neuro-inflammation plays a key role in the genesis of chronic fatigue stress [11, 19]. Up- regulation of inducible nitric oxide synthase enzyme has been noticed in patients suffering from chronic stress [24]. Elevated inducible nitric oxide synthase (iNOS) expression can lead to increased production of nitric oxide, which initiates diverse inflammatory cas- cades. Moreover, nitric oxide together with increased expression of cyclooxygenase-2 (COX-2) can further deteriorate the illness [42]. Apart from neuro-inflam- mation, oxidative stress contributes significantly to the chronic fatigue stress pathology and its clinical symp- toms [16–18]. It is still uncertain whether oxidative stress is a main cause or result of this illness.
HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors commonly categorized as statins have long been used in patients with athero- sclerotic disease and hyperlipidemia [43, 57]. Be- sides, their established cholesterol-lowering property, statins exert a number of cholesterol-independent, pleiotropic effects including anti-inflammatory ac- tions [7, 37]. During the past decade, several evi- dences suggested neuroprotective effect of statins in various neurological conditions. Reports clearly docu- mented beneficial effect of statins in lowering the in- cidence of Alzheimer’s disease (AD) [24]. Recent clinical studies also showed unexpected effects of statins on the incidence of Parkinson’s disease (PD) [59]. Besides, animal models also suggest its benefi- cial effect in the treatment of multiple sclerosis (MS) [14] and acute stroke [56]. Statins also reduce circu- lating levels of various inflammatory molecules and restore nitric oxide (NO) bioavailability via several distinct mechanisms, which might contribute to their reduction of clinical events [14]. However, the exact
role of statins in chronic stress pathology is still elu- sive and poorly understood.
Therefore, the present study has been undertaken to explore the potential role of atorvastatin and fluvastatin in chronic fatigue stress induced anxiety like behavior, oxidative stress and mitochondrial dysfunction in mice.
Materials and Methods
Animals
Male albino Laca mice (20–30 g) bred in the Central Animal House facility of Panjab University, Chandi- garh, India were used. The animals were housed in normal temperature (25 ± 1°C) and humidity (45–55%) with alternate 12 h light and dark cycle and had free access to standard rodent food pellets and water. The animals were housed for a minimum of 7 days in the animal house after procurement, to be acclimatized to the laboratory conditions before the start of the experiment. On the day of experiment, the animals were kept in the experimental room for one hour before start of the experimental procedure. All the experiments were conducted between 09:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee and con- ducted according to the National Science Academy Guidelines for the use and care of animals.
Drugs and treatment schedule
Atorvastatin and fluvastatin were suspended in 0.5%
w/v sodium carboxymethyl cellulose (CMC) solution and administered by oral route (po) in a constant vol- ume of 1 ml/100 g of body weight. Animals were ran- domly divided into six (n = 10) groups: (1) naive (ve- hicle treated), (2) control [running wheel activity test session (6 min each) for 21 days], (3–4) [At (10) and At (20)] atorvastatin (10 mg/kg and 20 mg/kg) treat- ment followed by running wheel activity test session (6 min) for 21 days, (5–6) [Fl (5) and Fl (10)] fluvas- tatin (5 mg/kg and 10 mg/kg) treatment followed by running wheel activity test session (6 min) for 21 days. Drugs were administered daily for 21 days, one hour prior to the animals being subjected to running wheel activity test session of 6 min.
Rotating/running wheel activity test session (RWATS)
The swim stress induced fatigue model is useful but lacks objectivity in its evaluation of immobility.
Hence, in our study, we devised our own behavioral screening test which enabled us to quantify the escape behavior. The apparatus used in the present study is a modification of the original apparatus proposed by Nomura et al. [45], Tadokoro et al. [54] and Shimizu et al. [50]. The apparatus consisted of a plastic glass water tank (38 × 30 × 15 cm) with a wheel at a height of 6 cm. The wheel resembles a circular hollow cage with diameter of 28 cm. Water at 15 ± 2°C was put in the tank to a level such that half part of the wheel dips in water (Fig. 1). One hour after oral administration of drugs, the mice were placed individually in the tank at the base of wheel and removed from water after 6 min. As an attempt to escape cold water, the mice would try to climb on the walls of wheel. However, it would fail to escape cold water due to rotation of wheel. However, when attempts to escape were finally abandoned, the wheel would stop turning. In the meantime, the number of rotations of the wheel was counted with the help of a digital counting device attached to the assembly. The wheel rotations during the complete 6 min test for mice receiving drug treatment were compared with those of control group. After com- pletion of 6 min test session, the mice were dried with soft cotton pad and put under a heating lamp to avoid hypothermia. The same procedure (6 min RWATS) was repeated after every 24 h for all the groups except the naive group up to 21 days [45, 50, 54].
BEHAVIORAL ASSESSMENT
Assessment of gross behavioral activity (locomotor activity)
In order to assess the influence of running wheel appa- ratus test session on motor activity, the gross locomotor activity was recorded for a period of 5 min using acto- photometer (IMCORP, Ambala) on 0th, 8th, 15th and 22nd day. Each mouse was kept in actophotometer 3 min habituation period initially before performing ac- tual 5 min recording. Each animal was observed in a square (30 cm) closed arena equipped with infrared light sensitive photocells using digital actophotometer and locomotor activity was expressed in terms of total photo beam counts for 5 min per animal. The apparatus was placed in a darkened, light and sound attenuated and ventilated testing room [35].
Measurement of anxiety (elevated plus maze test)
The elevated plus maze apparatus is used to evaluate the anxiety. The apparatus consists of two open arms (16 × 5 cm) and two enclosed arms (16 × 5 × 12 cm).
The arm extended from a central platform (5 × 5 cm).
Each mouse was placed individually at the centre of the arms facing either of the open arms. During a 5 min session, the following parameters were ob- served: a) number of entries in open and closed arm, b) time spent in open and closed arm. An anxiogenic response was observed when number of entries and time spent in the closed arm increased [34].
Preparation of brain homogenate
On the 22nd day, animals were randomized into two groups, one group was used for the biochemical assays and another for the estimation of mitochondrial enzyme complex activities. Immediately after the behavioral quantification, the animals were decapi- tated by cervical dislocation under light ether anesthe- sia. Brains were dissected and rinsed in ice cold saline. Cerebellum was discarded and cerebrum was used for biochemical estimations. For biochemical estimations, a 10% (W/V) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g for 15 min and aliquots of supernatants were separated and further used for estimating lipid peroxidation, ni- trite, reduced glutathione and catalase assay.
Fig. 1. Running wheel activity apparatus
MEASUREMENT OF OXIDATIVE STRESS PARAMETERS
Measurement of lipid peroxidation
The quantitative measurement of lipid peroxidation in brain was performed according to the method of Wills [52]. The amount of malondialdehyde (MDA), a meas- ure of lipid peroxidation was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA).
The values were calculated using molar extinction co- efficient of chromophore (1.56 × 105 M1 cm1) and expressed as nanomoles of MDA per milligram pro- tein [62].
Estimation of nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of NO, was determined with a colorimetric assay with Griess reagent (0.1%
N-(1-naphthyl)ethylenediamine dihydrochloride, 1%
sulfanilamide and 2.5% phosphoric acid) as described by Green et al. [22]. Equal volumes of supernatant and Griess reagent were mixed, and this mixture was incubated for 10 min at room temperature in the dark.
Absorbance was recorded at 540 nm with Perkin Elmer lambda 20 UV-VIS spectrophotometer. The concentration of nitrite in the supernatant was deter- mined from a sodium nitrite standard curve and ex- pressed as micromoles per milligram protein [22].
Estimation of reduced glutathione
Reduced glutathione in brain was estimated according to the method described by Ellman [11]. Supernatant (1 ml) was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4°C for 1 h. The sample was centrifuged at 1,200 × g for 15 min at 4°C. To 1 ml of this supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) were added. The yellow color developed was read immediately at 412 nm using Perkin Elmer lambda 20 UV-VIS spectrophotometer. Results were calculated using molar extinction coefficient of chro- mophore (1.36 × 104 M1 cm1) and expressed as nanomoles of GSH per milligram protein [11].
Catalase estimation
Catalase activity was assayed by the method of Luck [41], where the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, assay mixture consisted of 3 ml of H2O2phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), and change in absorbance was recorded at 240 nm. The results were expressed as micromoles of H2O2decom- posed per milligram of protein per min [41].
Protein estimation
The protein content was measured by biuret method using bovine serum albumin as standard [21].
MITOCHONDRIAL COMPLEX ENZYMES ESTIMATION
Isolation of mice brain mitochondria
Second group of animals were used for mitochondrial isolation as described in the method of Berman and Hastings [4]. The brain regions were homogenized in isolated buffer. Homogenate was centrifuged at 13,000 × g for 5 min at 4°C. Pellet was resuspended in isolation buffer with ethylene glycol tetraacetic acid (EGTA) and spun again at 13,000 × g at 4°C for 5 min. The resulting supernatant was transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 × g at 4°C for 10 min.
Pellet containing pure mitochondria was resuspended in isolation buffer without EGTA [4].
NADH dehydrogenase activity
NADH dehydrogenase activity was measured spec- trophotometrically by the method of King and How- ard [33]. The method involves catalytic oxidation of NADH to NAD+with subsequent reduction of cyto- chrome C. The reaction mixture contained 0.2 M glycyl glycine buffer pH 8.5, 6 mM NADH in 2 mM glycyl glycine buffer and 10.5 mM cytochrome C.
The reaction was initiated by addition of requisite amount of solubilized mitochondrial sample and fol- lowed by absorbance change at 550 nm for 2 min. The results were expressed as nanomoles of NADH oxi- dized per min per milligram of protein [33].
Succinate dehydrogenase (SDH) activity
SDH was measured spectrophotometrically according to King [32]. The method involves oxidation of succi- nate by an artificial electron acceptor, potassium ferri- cyanide. The reaction mixture contained 0.2 M phos- phate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of mitochondrial sample and absorbance change was followed at 420 nm for 2 min.
The results were expressed as nanomoles per min per milligram of protein [32].
Mitochondrial redox activity
The MTT assay is based on the reduction of (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-H-tetrazolium bro- mide, MTT) by hydrogenase activity in functionally intact mitochondria. The MTT reduction rate was used to assess the activity of the mitochondrial respi- ratory chain in isolated mitochondria by the method of Liu et al. [40]. Briefly, 100 µl mitochondrial sam- ples were incubated with 10 µl MTT for 3 h at 37°C.
The blue formazan crystals were solubilized with dimethyl sulfoxide and measured by an ELISA reader at 580 nm filter [40].
Cytochrome oxidase assay
Cytochrome oxidase activity was assayed in brain mi- tochondria according to the method of Sotocassa et al.
[52]. The assay mixture contained 0.3 mM reduced cytochrome C in 75 mM phosphate buffer. The reac- tion was started by the addition of solubilized mito-
chondrial sample and absorbance change was recorded at 550 nm for 2 min. The results were ex- pressed as nanomoles of cytochrome C oxidized per min per milligram of protein [52].
Statistical analysis
Values are expressed as the mean ± SEM. The behav- ioral assessment data were analyzed by a repeated measures two-way analysis of variance (ANOVA) and the biochemical estimations were analyzed by one-way ANOVA. Post-hoc comparisons between groups were made using Bonferroni’s test and Tuk- ey’s test, respectively; p < 0.05 was considered to be statistically significant.
Results
Effect of atorvastatin and fluvastatin on running wheel activity
Animals exposed to 6 min test session on running wheel for 21 days showed a significantly decreased number of wheel rotations per 6 min as compared to the naive animals, indicating fatigue stress like behav- ior in mice. Atorvastatin (10 and 20 mg/kg) and flu- vastatin (5 mg/kg and 10 mg/kg) treatment for 21 days significantly improved the number of wheel ro- tations per 6 min as compared to the control group [Two way ANOVA, p < 0.001, (DF = 5, 3; DFd = 96;
F = 103.26, 123.73 for interaction of days and treat- ment)] (Fig. 2).
Fig. 2. Effect of atorvastatin and fluvas- tatin on running wheel activity. Values are expressed as the mean ± SEM;
ap < 0.05 as compared to naive,bp <
0.05 as compared to RWATS (control).
Data were analyzed using two-way ANOVA followed by Bonferroni’s t-test
Effect of atorvastatin and fluvastatin on locomotor activity
Locomotor activity was recorded in order to detect the association of decreased wheel running activity with the motor activity. Interestingly, locomotor activity of the control group (animals exposed to 6 min test ses- sion on running wheel for 21 days) gradually de- creased significantly as compared to the naive group.
Atorvastatin (10 and 20 mg/kg) and fluvastatin (10 mg/kg) treatment for 21 days significantly im- proved locomotor on 21st day. Fluvastatin (5 mg/kg) treatment did not produce any significant effect as compared to the control group [Two way ANOVA, p < 0.001, (DF = 5, 3; DFd = 96; F = 35.66, 47.81 for interaction of days and treatment)] (Tab. 1).
Effect of atorvastatin and fluvastatin on anxiety like behavior
Each 6 min test session on running wheel apparatus for 21 days significantly caused anxiety like behavior (decreased number of entries and time spent in open arm) as compared to naive animals. Twenty one days treatment with atorvastatin (10 and 20 mg/kg) and flu- vastatin (10 mg/kg) significantly increased number of entries and time spent in open arm showing anti- anxiety like behavior as compared to control group.
Fluvastatin (5 mg/kg) treatment did not produce any significant effect on plus maze performance task as compared to the control group [Two way ANOVA, p < 0.001, (DF = 5, 3; DFd = 96; F = 76.47, 106.37 for interaction of days and treatment)] (Tab. 2).
Tab. 1. Effect of atorvastatin and fluvastatin on locomotor activity
Treatment group (mg/kg) Locomotor activity/5 min (mean ± SEM)
Day 0 Day 8 Day 15 Day 22
Naive 213.0 ± 3.0 215.0 ± 1.0 215.0 ± 3.0 214.0 ± 4.0
Control (RWATS) 212.0 ± 6.0 192.0 ± 7.0 165.0 ± 9.0a 140.0 ± 6.0a
At (10) 208.0 ± 5.0 193.0 ± 4.0 172.0 ± 4.0 158.0 ± 2.0
At (20) 210.0 ± 3.0 195.0 ± 3.0 182.0 ± 3.0b 174.0 ± 3.0b
FL (5) 211.0 ± 4.0 192.0 ± 4.0 169.0 ± 3.0 149.0 ± 3.0
FL (10) 210.0 ± 3.0 194.0 ± 3.0 179.0 ± 3.0b 166.0 ± 3.0b
Data expressed as the mean ± SEM;ap < 0.05 as compared to naive,bp < 0.05 as compared to control (RWATS)
Tab. 2. Effect of atorvastatin and fluvastatin on anxiety like behavior in elevated plus maze test
Treatment group (mg/kg)
Day 8 Day 15 Day 22
No. of entries in open arm mean ± SEM
Time spent in open arm (s) mean ± SEM
No. of entries in open arm mean ± SEM
Time spent in open arm (s) mean ± SEM
No. of entries in open arm mean ± SEM
Time spent in open arm (s)
mean ± SEM
Naive 11.0 ± 1.0 81.0 ± 8.0 10.0 ± 1.0 79.0 ± 6.0 10.2 ± 1.2 80.3 ± 5.0
Control (RWATS) 8.3 ± 1.0 65.0 ± 9.2 5.3 ± 2.0a 30.0 ± 3.2a 3.3 ± 1.2a 17.2 ± 4.2a
At (10) 9.0 ± 0.5 71.2 ± 3.0 7.1 ± 0.3 50.0 ± 4.1b 6.3 ± 0.3b 35.3 ± 2.3b
At (20) 10. ± 0.5 72.2 ± 4.2 6.1 ± 0.2 59.2 ± 4.2b 7.2 ± 0.5b 48.2 ± 4.4b
FL (5) 8.5 ± 1.1 67.0 ± 5.2 6.0 ± 1.1 43.2 ± 6.3 5.3 ± 1.2 19.2 ± 3.3
FL (10) 9.3 ± 1.2 74.1 ± 4.1 5.0 ± 0.2 53.2 ± 4.5 6.2 ± 0.3b 42.3 ± 3.2b
Data expressed as the mean ± SEM;ap < 0.05 as compared to naive,bp < 0.05 as compared to control
Effect of atorvastatin and fluvastatin on the brain lipid peroxidation, nitrite level, and cata- lase enzyme and GSH levels
Six minutes test session on running wheel apparatus for 21 days significantly caused oxidative stress (elevated lipid peroxidation and nitrite levels, as well as decreased catalase levels and reduced glutathione activity) as com- pared to naive animals. Twenty one days treatment with
atorvastatin (10 and 20 mg/kg) and fluvastatin (10 mg/
kg) significantly attenuated oxidative stress (decreased lipid peroxidation and nitrite levels [(p < 0.001; DFn = 5; DFd = 24; F = 58.06)], as well as restored reduced glutathione and catalase enzyme activity [(p < 0.001, DFn = 5; DFd = 30; F = 22.84)] as compared to the con- trol group. Fluvastatin (5 mg/kg) treatment did not pro- duce significant effect on these oxidative stress parame- ters as compared to the control group (Fig. 3).
Fig. 3. Effect of atorvastatin and fluvas- tatin on biochemical parameters.
Values are expressed as the mean
± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s test;ap < 0.05 as compared to naive,
bp < 0.05 as compared to RWATS (control)
Fig. 4. Effect of atorvastatin and fluvas- tatin on mitochondrial enzyme com- plexes. Values are expressed as the mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s test;ap < 0.05 as compared to naive, b p < 0.05 as compared to RWATS (control). At (10) – atorvastatin 10 mg/kg, At (20) – atorvastatin 20 mg/
kg, Fl (5) – fluvastatin 5 mg/kg, Fl (10) – fluvastatin 10 mg/kg
Effect of atorvastatin and fluvastatin on the mitochondrial enzyme complexes
Six minute test session on running wheel apparatus for 21 days significantly impaired the mitochondrial enzyme complexes (I, II and IV) and mitochondrial redox activity (MTT assay) as compared to naive group. Atorvastatin (10 and 20 mg/kg) and fluvastatin (10 mg/kg) drug treatment for 21 days significantly restored mitochondrial enzyme complex I, II, IV and mitochondrial redox activity (MTT assay) in the respective treatment groups as compared to control.
Fluvastatin (5 mg/kg) treatment did not produce any significant effect on these mitochondrial enzyme complexes activities as compared to the control group [(p < 0.001; DFn = 5; DFd = 30; F = 218.61)] (Fig. 4).
Discussion
Although various drugs have been studied for their ef- fectiveness in CFS, no specific standard treatment is available till date. Reason being, the exact mechanism behind complex CFS pathology is still elusive. It is possible that chronic fatigue stress is a heterogeneous problem with different pathophysiological anomalies manifesting with the same or similar symptoms [1, 48]. Many theories have been suggested to explain the neurobiology of chronic fatigue. The fatigue and re- lated symptoms must be due to the wastage of energy by the mental and physical processes like stress, anxi- ety, tension and depression. The metabolic defects may contribute to the reduced physical endurance of fatigue patients [16].
In the present study, animals subjected to running wheel test session for 6 min daily for 21 days pro- duced fatigue like behavior. These results are consis- tent with previous studies reporting that exercise in- duced rodent models of chronic fatigue are associated with greater fibre damage, soreness, inflammation, fa- tigue, and other functional deficits [5]. The swim stress induced fatigue model is useful but lacks objec- tivity in its evaluation of immobility, because each ex- perimenter evaluates it subjectively. Hence, in our study, we devised our own behavioral screening test which enabled us to quantify the escape behavior. The apparatus used in the present study is a modification of the original apparatus proposed by Nomura et al. in 1982 [45, 50]. Chronic exposure to running wheel ac-
tivity produced a fatigue like state with gradual in- crease in anxiety like behavior, impaired locomotor activity as observed on 8th, 15thand 22nd day. Daily 6 min test session for 21 days in the running wheel ap- paratus filled with water maintained at a temperature of (15 ± 2°C) produced fatigue like condition in ani- mals. Exposure to cold for extended periods of time can cause a significant drop of core body temperature, which can be responsible for ataxia, hypovolemia, atrial dysrhythmias, cold diuresis and mental confusion [46]. Some studies found that swim stress in cold water increases the permeability of the blood-brain barrier in mice [29]. In our study, we observed an increase in anxiety like behavior and reduced locomotor activity in the stressed animals after 14 and 21 days stress expo- sure. No significant behavioral changes were observed in 7 days in stressed animals. These behavioral altera- tions are a hallmark of fatigue disorders which might appear due to cumulative stress [30].
At cellular level fatigue is involved with cellular energy systems that for the most part are found in mi- tochondria. Recent studies suggest that oxidative stress also contributes in the pathology and clinical symptoms of CFS [31]. The role of oxidative stress in CFS, suggests the use of antioxidants in its manage- ment [28]. Theoretically, oxidative stress can be caused by an increase in the generation of reactive oxygen species of which mitochondrial dysfunction is believed to be a main source, or it can be caused by a decline in the efficiency of antioxidant enzyme sys- tems [17]. The source of excessive free radical gen- eration in CFS patients, which involves oxidation of lipids and proteins may be associated with a variety of altered biological processes. We have observed in- creased activity of antioxidant enzyme systems including reduced glutathione and catalase enzyme activity, which is believed to be the compensatory measure in response to oxidative stress. Elevated per- oxynitrite levels have also been reported in the CFS patients [47], which causes mitochondrial dysfunc- tion, lipid peroxidation, and, by way of positive feed- back, elevated cytokine levels. The cytokines, in turn, cause the formation of nitric oxide that combines with superoxide to form the potent oxidant peroxynitrite, thus continuing the cycle. As a support of the above peroxynitrite theory, decreased succinate dehydroge- nase activity has been found in CFS patients which are apparently the consequence of inactivation of these enzyme systems by peroxynitrite [12, 51]. The results of the present study are very similar to previ-
ous studies where chronic stress significantly induced oxidative damage in the brain [31]. Further, the esti- mation of mitochondrial complex enzymes levels in the whole brain revealed a significant decrease in the activity of the complex enzymes (I, II and IV) as esti- mated on 22nd day. These results are in accordance with a recent report stating that in the brain mitochon- dria, the activities of enzymes critical for mitochon- drial function decrease during high-intensity exercise of continuous or intermittent frequency [2].
Animals treated with atorvastatin (10 and 20 mg/
kg) and fluvastatin (5 and 10 mg/kg) has significantly recovered all the behavioral, biochemical and mito- chondrial alterations in the chronically fatigued ani- mals, thus showing its protective potential against chronic fatigue induced manifestations and showing antioxidant like effect of statins. Statins (3-hydroxy- 3-methylglutaryl A reductase inhibitors) are well known to be used in lowering serum cholesterol con- centrations. Recently, statins have also been reported to possess intrinsic antioxidant activity by protecting cells and tissues from oxidative damage [6, 27, 61].
The anti-oxidative properties of statins may be of value in the fight against oxidative DNA damage. In neurons, they are reported to reduce lipid peroxida- tion following oxygen and glucose deprivation and reperfusion [39]. Statins can reduce the production of reactive oxygen species by inhibiting the assembly and activation of the NADPH-complex [53, 55, 59].
In addition, they reduce oxidative damage by control- ling nitric oxide production and possibly reducing the inflammatory response [23]. Statins have significant antioxidant activity against both peroxyl and hydroxyl radicals. In particular, fluvastatin is among the most active antioxidants towards peroxyl radicals. Atorvas- tatin, on the other hand, is known to possess the anti- oxidant property against both hydroxyl as well as per- oxyl radicals. Metabolites of atorvastatin reduce lipo- protein oxidation in a number of oxidative systems [3]. Statins attenuate the inflammatory cytokine re- sponses that accompany cerebral ischemia, and they possess antioxidant properties that likely ameliorate ischemic oxidative stress in the brain. Clinical trials report that the class of drugs known as statins may be neuroprotective in Alzheimer’s and Parkinson’s dis- eases [57]. Ghosh and his co-workers demonstrated that statins (simvastatin and pravastatin) protect against MPTP-induced nigrostriatal degeneration in a mouse model [20]. Statins inhibit oxidant enzymes activity such as that of reduced nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase and myelo- peroxidase and up-regulate the activity of antioxidant enzymes such as catalase and paraoxonase. They re- duce endothelial dysfunction mainly by their ability to enhance endothelial nitric oxide bioavailability, which is achieved by several mechanisms [8]. In our study, we proposed the antioxidant like effect of statins against chronic fatigue stress, which is supported by the above mentioned research findings by different authors.
Reports suggest that fluvastatin enhanced transcrip- tional activating function within peroxisome proliferator- activated receptor g coactivator-1a (PGC-1a), which serves as an inducible coactivator for a number of transcription factors to control energy metabolism [38, 49, 60]. The activity of PGC-1a is down- regulated by PI3K/Akt pathway in the liver. Evi- dences indicate that statins increase the activity of PGC-1a through decreasing the amount of activated Akt. Recent study has also demonstrated that statins may stimulate mitochondrial biogenesis [60]. Thus, there is every possibility that prolonged administra- tion of statins (atorvastatin and simvastatin) may alter the mitochondrial density/biogenesis, cytochrome C concentration, citrate synthase activity, PGC-1a ex- pression, etc. Further, the present study could not rule out the possibility of the role of statins in the mito- chondrial biogenesis.
In conclusion, the running wheel filled with water significantly induced chronic fatigue stress in mice.
Pre-treatment with statins (atorvastatin and fluvasta- tin) significantly protects against chronic fatigue in- duced behavioral, biochemical and mitochondrial dysfunctions.
Acknowledgment:
Authors gratefully acknowledged the financial support of Council of Scientific and Industrial Research (CSIR), New Delhi, provided to Professor Anil Kumar for carrying out this research work.
Conflict of interest:
The authors declare that they have no competing financial interests. There is no conflict of interest for any of the authors.
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Received: December 6, 2011; in the revised form: July 14, 2012;
accepted: August 9, 2012.
