Pharmacological Reports Copyright © 2012 2012, 64, 11051115 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Potential role of licofelone, minocycline and their combination against chronic fatigue stress induced behavioral, biochemical and mitochondrial alterations 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: [email protected]
Abstract: Background: Chronic fatigue stress (CFS) is a common complaint among general population. Persistent and debilitating fatigue se- verely impairs daily functioning and is usually accompanied by combination of several physical and psychiatric problems. It is now well established fact that oxidative stress and neuroinflammation are involved in the pathophysiology of chronic fatigue and related disorders. Targeting both COX (cyclooxygenase) and 5-LOX (lipoxygenase) pathways have been proposed to be involved in neuro- protective effect. Methods: In the present study, mice were put on the running wheel apparatus for 6 min test session daily for 21 days, what produced fatigue like condition. The locomotor activity and anxiety like behavior were measured on 0, 8th, 15th and 22nd day. The brains were isolated on 22nd day immediately after the behavioral assessments for the estimation of oxidative stress parameters and mitochon- drial enzyme complexes activity. Results: Pre-treatment with licofelone (2.5, 5 and 10 mg/kg, po) and minocycline (50 and 100 mg/kg, po) for 21 days, significantly attenuated fatigue like behavior as compared to the control (rotating wheel activity test session, RWATS) group. Further, licofelone (5 and 10 mg/kg, po) and minocycline (50 and 100 mg/kg, po) drug treatments for 21 days significantly attenuated behavioral altera- tions, oxidative damage and restored mitochondrial enzyme complex activities (I, II, III and IV) as compared to control, whereas combination of licofelone (5 mg/kg) with minocycline (50 mg/kg) significantly potentiated their protective effect which was signifi- cant as compared to their effect per se. Conclusion: The present study highlights the therapeutic potential of licofelone, minocycline and their combination against CFS in mice.
Key words: chronic fatigue stress, licofelone, oxidative stress, RWATS
Introduction and related complications. Therefore, treatment con- tinues to be directed towards limiting the effects of Chronic fatigue stress (CFS) is predominantly a medi- the symptoms rather than treating its root cause. The cal problem, with complex pathology with inadequate clinical symptoms of chronic fatigue stress also re- diagnosis and treatment profile. There are no firmly semble various central nervous system (CNS) disor- established treatment recommendations for fatigue ders [15, 21]. Researchers have investigated a CNS
Pharmacological Reports, 2012, 64, 11051115 1105 link to CFS by means of structural and functional neu- (MMPs) [50]. Pro-inflammatory cytokines, such as roimaging, cognitive testing, neuropeptide assays, and TNF-a, IL-1b and IL-6 are produced by microglial autonomic assessment [44]. Therefore, the use of vari- cells, astrocytes, neutrophils and macrophages and ous drugs which have been shown to have a neuropro- augment both inflammation and subsequent immune tective effect against CFS seems to be clinically rele- responses [22, 51, 52]. vant. The exercise-induced stress response is an adap- Studies have clearly demonstrated that chronic stress tational homeostatic shift intended to facilitate the de- plays a key role in the activation of microglia, the resident mand put on the body by physical exertion [9, 10]. immune cells of brain, which results in an increase in CFS up-regulates cyclooxygenase-2 (COX-2) and pro-inflammatory cytokine levels such as TNF-a, reac- tive oxygen and nitrogen species (ROS and RNS) secre- 5-lipooxygenase (5-LOX) expression in endothelial tion leading to pathological alterations [6, 32, 40]. Keep- cells, which results in the release of pro-inflammatory ing in mind the therapeutic potential of both the drugs, the cytokines with subsequent induction of synthesis of present study has been further extended to explore the various prostaglandins and leukotrienes, respectively, combination of licofelone and minocycline against CFS which influences the central neuroendocrine regula- induced behavioral and biochemical alterations. tory mechanisms [38]. These inflammatory media- Further, it has been observed that, chronic stress tors, in turn, contribute to the progression of CFS and also leads to mitochondrial dysfunction and oxidative related behavioral complications. stress [36] and neuroprotective effect of minocycline Licofelone {2-[6-(4-chlorophenyl)-2,2-dimethyl-7- and licofelone has been demonstrated in different ex- phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid}, perimental systems [16–19]. As their exact mecha- is a potent, well-balanced inhibitor of the 5-LOX and nisms of neuroprotective action are not fully under- COX pathways, which binds with the active sites due stood so far, this makes it prerequisite to assess their to their conformational similarity with arachidonic effect on oxidative stress and mitochondrial enzyme acid, thus blocking the catalytic activity [5]. Licofe- complexes activities in the chronic fatigued animals. lone inhibits the synthesis of prostaglandins as well as Therefore, the present study is an attempt to explore leukotrienes, responsible for the various behavioral the possible neuroprotective and antioxidant like (anxiety or depression like behaviors) and biochemi- mechanisms of minocycline and its interaction with cal alterations. Experimental reports suggest that lico- licofelone (dual COX and LOX inhibitor) against ro- felone has neuroprotective, anti-inflammatory, anal- tating wheel activity test session (RWATS) induced gesic, antipyretic, and anti-platelet activities [18, 42, behavioral alterations, oxidative stress and mitochon- 47]. General toxicological studies on animals have drial enzyme complex activity in mice. also confirmed that licofelone is safe in the therapeu- tic doses far above pharmacologically active doses and devoid of deleterious effects on the autonomic nervous system, CNS, and cardiovascular system [1]. Materials and Methods In addition, licofelone has good oral bioavailability, long half life (11 h) with no genotoxicity [47, 48]. Minocycline is a semi-synthetic tetracycline that Animals readily crosses blood brain barrier and has been re- ported to have anti-inflammatory and neuroprotective Male albino Laca mice (20–30 g) bred in Central Ani- properties [2, 14, 19]. Besides, its antimicrobial ac- mal House facility of the Panjab University, Chandi- tivities and neuroprotective potential have been well garh, India were used for the study. The animals were documented in experimental models of cerebral housed in normal temperature and humidity with al- ischemic injury, traumatic brain injury, amyotrophic ternate 12 h light and dark cycle and had free access lateral sclerosis, Parkinson’s disease, Huntington’s to standard rodent food pellets and water. They were disease, multiple sclerosis, Alzheimer’s disease and acclimatized to the laboratory conditions before the other brain injuries [16, 17, 19, 22, 37, 54]. Mino- experiment. All the experiments were conducted be- cycline exerts its anti-inflammatory actions by modu- tween 9:00 and 17:00. The experimental protocol was lating microglia, immune cell activation and subse- approved by the Institutional Animal Ethics Commit- quent release of cytokines, chemokines, lipid media- tee and conducted according to the National Science tors of inflammation and matrix metalloproteases Academy Guidelines for the use and care of animals.
1106 Pharmacological Reports, 2012, 64, 11051115 Licofelone, minocycline and their combination against CFS Anil Kumar et al.
Drugs and treatment schedule were put on cotton bedding at room temperature con- dition. The same procedure (6 min RWATS) was re- The following drugs were used in the study protocol: peated after every 24 h for all the groups except the licofelone (2.5, 5 and 10 mg/kg, po) and minocycline naive group up to 21 days. (50 and 100 mg/kg, po). All the drugs were suspended in 0.5% w/v sodium carboxymethyl cellulose (CMC) Behavioral assessments solution and administered by oral route in a constant volume of 1 ml/100 g of body weight. Measurement of locomotor activity Animals were randomly divided into nine groups. Group 1 was designated as naive animals, received In order to detect the association of decreased activity vehicle (0.5 % w/v sodium CMC); Group 2 consid- in RWATS apparatus with changes in motor activity, ered as control (RWATS) group – exposed to RWATS the locomotor activity was recorded for a period of for 21 days; Groups 3 to 5 received licofelone (2.5, 5 5 min using actophotometer (IMCORP, Ambala) on and 10 mg/kg po), respectively) followed by RWATS 0th, 8th, 15th, and 22nd day. Each animal was observed for 6 min test session for 21 days; similarly, Groups 6 in a square (30 × 30 cm) closed arena equipped with and 7 received minocycline (50 and 100 mg/kg po), infrared light sensitive photocells using digital acto- respectively) followed by RWATS for 6 min test ses- photometer and locomotor activity was then ex- sion for 21 days. Further, Group 8 received combina- pressed in terms of total photo beam counts for 5 min tion of licofelone (5 mg/kg po) with minocycline per animal. Animals were placed individually in the (50 mg/kg po), followed by RWATS for 6 min test activity chamber for a 3 min habituation period before session for 21 days. actual start of activity tasks. The apparatus was placed Drugs (licofelone and minocycline) were administered in a darkened, light and sound attenuated and venti- daily one hour prior to the overnight fasted animals being lated testing room [28, 30]. subjected to RWATS for 6 min test session for 21 days. Doses were selected on the basis of previous reported Measurement of anxiety like behavior (elevated studies and our preliminary laboratory data [18, 37]. plus maze test)
RWATS The elevated plus maze apparatus was used to evalu- ate the anxiety like behavior. The apparatus consisted The apparatus used in the present study is a modifica- of two open arms (16 × 5 cm) and two closed arms tion of the original apparatus proposed by Nomura et (16 × 5 × 12 cm). The arms extended from a central al. [40, 45]. The apparatus consisted of a plastic glass platform (5 × 5 cm). The apparatus was elevated to water tank (38 × 30 × 15 cm) with a wheel at a height a height of 25 cm above the floor. The mice were of 6 cm. The wheel resembles a circular hollow cage placed individually at the center of the arms facing ei- with diameter of 28 cm. Water at 15 ± 2°C was put in ther of the open arms. During a 5 min session, the fol- the tank to a level such that the inner base of wheel lowing parameters were noted: a) number of entries to just dips in water. One hour after oral administration open arm c) time spent in open arm. An anxiogenic of drugs, the mice were placed individually in the response was observed when number of entries and tank at the base of wheel and removed from water af- time spent in the open arm were decreased. Mice were ter 6 min test session. As an attempt to escape from allowed to explore the maze for 30 s after recording cold water, the mice tried to climb on the walls of the initial transfer latency (ITL) and returned to the wheel, but failed to escape due to free rotation of home cage [29]. wheel. However, when attempts to escape were fi- nally abandoned, the wheel would stop turning. In the Preparation of brain homogenate meantime, the number of rotations of the wheel was counted with the help of a digital counting device at- On the 22nd day, animals were randomized into two tached to the assembly. The wheel rotations during the groups; one was used for the biochemical assays and complete 6 min test session for mice receiving drug second group for the estimation of mitochondrial en- treatment were compared with those of control group. zyme complex activities, immediately after the behav- After the completion of 6 min test session, the mice ioral quantification. Brains were dissected and rinsed
Pharmacological Reports, 2012, 64, 11051115 1107 in ice cold saline. Cerebellum was discarded. For bio- tion coefficient of chromophore (1.36 × 104 M1cm1) chemical estimations, a 10% (w/v) tissue homogen- and expressed as percentage of control. ates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g Catalase estimation for 15 min and aliquots of supernatants were sepa- rated and further used for estimating lipid peroxida- Catalase activity was assayed by the method of Luck, tion, nitrite, reduced glutathione and catalase assay. where the breakdown of hydrogen peroxides (H2O2) is measured at 240 nm [35]. Briefly, each assay mix- Measurement of oxidative stress parameters ture consisted of 3 ml of H2O2, phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), Measurement of lipid peroxidation and change in absorbance was recorded at 240 nm. The results were expressed as micromol of H2O2 de- The quantitative measurement of lipid peroxidation composed per milligram of protein/min. was performed according to the method of Wills [55]. The amount of malondialdehyde (MDA), a measure of lipid peroxidation, was measured by reaction with Protein estimation thiobarbituric acid at 532 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The protein content was measured by biuret method The values were calculated using molar extinction co- using bovine serum albumin (BSA) as standard [12]. efficient of chromophore (1.56 × 105 M1cm1) and expressed as percentage of vehicle treated group. Mitochondrial enzyme complexes estimations
Estimation of nitrite Isolation of mice brain mitochondria The accumulation of nitrite in the supernatant, an in- dicator of the production of nitric oxide (NO), was de- Second group of animals were used for mitochondrial termined with a colorimetric assay with Griess rea- isolation as described in the method of Berman and gent (0.1% N-(1-naphthyl)ethylenediamine dihydro- Hastings [3]. The brain regions were homogenized in chloride, 1% sulfanilamide and 2.5% phosphoric isolated buffer. Homogenate was centrifuged at acid) as described by Green et al. [13]. Equal volumes 13,000 × g for 5 min at 4°C. Pellets were resuspended of supernatant and Griess reagent were mixed, and in isolation buffer with ethylene glycol tetraacetic this mixture was incubated for 10 min at room tem- acid (EGTA) and spun again at 13,000 × g at 4°C for perature in the dark. Absorbance was noted at 540 nm 5 min. The resulting supernatants were transferred to using Perkin Elmer lambda 20 spectrophotometer. new tubes and topped off with isolation buffer with The concentration of nitrite in the supernatant was EGTA and again spun at 13,000 g at 4°C for 10 min. determined from a sodium nitrite standard curve and Pellets containing pure mitochondria were resus- expressed as percentage of vehicle treated group. pended in isolation buffer without EGTA.
Estimation of reduced glutathione NADH dehydrogenase activity (Complex-I)
Reduced glutathione in the brain was estimated ac- Complex-I was measured spectrophotometrically by cording to the method described by Ellman [7]. Su- the method of King and Howard [24]. The method pernatants (1 ml) were precipitated with 1 ml of 4% involves catalytic oxidation of NADH to NAD+ with sulfosalicylic acid and cold digested at 4°C for 1 h. subsequent reduction of cytochrome C. The reaction The samples were centrifuged at 1200 × g for 15 min mixture contained 0.2 M glycylglycine buffer pH 8.5, at 4°C. To each 1 ml of this supernatant, 2.7 ml of 6 mM NADH in 2 mM glycyl glycine buffer and phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5- 10.5 mM cytochrome C. The reaction was initiated by dithiobis(2-nitrobenzoic acid) (DTNB) were added. addition of requisite amount of solubilized mitochon- The yellow color developed was read immediately at drial sample and followed absorbance change at 412 nm. Results were calculated using molar extinc- 550 nm for 2 min.
1108 Pharmacological Reports, 2012, 64, 11051115 Licofelone, minocycline and their combination against CFS Anil Kumar et al.
Succinate dehydrogenase (SDH) activity (Complex-II) Statistical analysis
SDH was measured spectrophotometrically according The behavioral data were analyzed by using two-way to King [23]. The method involves oxidation of succi- analysis of variance (ANOVA) followed by Bonfer- nate by an artificial electron acceptor, potassium ferri- roni’s test. The biochemical data were analyzed by cyanide. The reaction mixture contained 0.2 M phos- one-way analysis of variance (ANOVA) followed by phate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, Bonferroni’s test. All the values are expressed as the and 0.03 M potassium ferricyanide. The reaction was mean ± SEM. In all tests, the criterion for statistical initiated by the addition of mitochondrial sample and significance was p < 0.05. absorbance change was followed at 420 nm for 2 min.
Mitochondrial redox activity (Complex-III)
The MTT assay is based on the reduction of 3-(4,5- Results dimethylthiazol-2-yl)-2,5-diphenyl-H-tetrazolium bro- mide (MTT) by hydrogenase activity in functionally in- tact mitochondria. The MTT reduction rate was used to Effects of licofelone, minocycline and their assess the activity of the mitochondrial respiratory chain combination on RWATS in isolated mitochondria by the method of Liu and co- workers [34]. Briefly, 100 µl mitochondrial samples were Animals exposed to a 6 min RWATS test session incubated with 10 µl MTT for 3 h at 37°C. The blue for- daily, for 21 days showed a persistent decline in the mazan crystals were solubilized with dimethyl sulfoxide wheel rotation count per 6 min, indicating fatigue like and measured by an ELISA reader at 580 nm filter. behavior in animals. Twenty one days drug treatment with licofelone (5, 10 mg/kg) and minocycline (50, Cytochrome oxidase assay (Complex-IV) 100 mg/kg) significantly improved number of counts (wheel rotation per 6 min) on 14th and 21nd day as Cytochrome oxidase activity was assayed in brain mi- compared to control (RWATS), whereas treatment tochondria according to the method of Sotocassa and with licofelone (2.5 mg/kg) was found to be non- its group [49]. The assay mixture contained 0.3 mM significant as compared to control. Further, combina- reduced cytochrome C in 75 mM phosphate buffer. tion of licofelone (5 mg/kg) with minocycline The reaction was started by the addition of solubilized (50 mg/kg) significantly improved wheel rotation per mitochondrial sample and absorbance change was re- 6 min, which was significant on 7th , 14th and 21st days corded at 550 nm for 2 min. as compared to their effect per se (Tab. 1).
Tab. 1. Effect of licofelone, minocycline and their combination on RWATS
Treatment Wheel rotation count per 6 min (mg/kg) Day 1 Day 7 Day 14 Day 21 Naive 23 ± 1.1 22 ± 0.9 23 ± 1.3 22 ± 1.1 a a a RWATS 21 ± 1.1 15 ± 0.8 10 ± 0.9 6 ± 0.6 Li (2.5) 22 ± 0.9 15 ± 1.0 11 ± 0.6 7 ± 0.7 b b Li (5) 22 ± 0.7 18 ± 0.7 13 ± 0.7 9 ± 0.9 b c,d c Li (10) 23 ± 1.2 20 ±1.2 16 ± 0.9 12 ± 0.9 b b Me (50) 22 ± 0.5 16 ± 0.5 13 ± 0.7 10 ± 0.6 d d d Me (100) 22 ± 0.6 19 ± 1.0 17 ± 0.8 13 ± 0.7 c,d c,d c,d Me (50) + Li (5) 20 ± 0.6 22 ± 0.8 16 ± 0.5 14 ± 0.6
Data expressed as the mean ± SEM; a p < 0.05 as compared to naive, b p < 0.05 as compared to RWATS (control), c p < 0.05 as compared to Li (5), d p < 0.05 as compared to Me (50). Two-way ANOVA followed by Bonferroni’s test. Li – licofelone, Me – minocycline
Pharmacological Reports, 2012, 64, 11051115 1109 a Fig. 1. Effect of licofelone, minocycline and their combination on locomotor activity. Data expressed as the mean ± SEM; p < 0.05 as com- b c d pared to naive, p < 0.05 as compared to RWATS (control), p < 0.05 as compared to Li (5), p < 0.05 as compared to Me (50). Two-way ANOVA followed by Bonferroni’s test. Li – licofelone, Me – minocycline
Effect of licofelone, minocycline and their Effects of licofelone, minocycline and their combination on the locomotor activity combination on anxiety like behavior on elevated plus maze test performance RWATS significantly impaired locomotor activity af- nd rd th nd Animals exposed to RWATS (6 min test session) signifi- ter 2 and 3 week (on 15 and 22 day) as com- st cantly caused anxiety like behavior after 1 week (de- pared to naive animals. Further, licofelone (2.5, 5 and creased number of entries and time spent in open arm) 10 mg/kg) and minocycline (50 and 100 mg/kg) treat- as compared to naive animals. Treatment with licofelone ment failed to produce any significant effects on loco- th th (5 and 10 mg/kg), minocycline (50 and 100 mg/kg) and motion only on 8 and 15 day as compared to con- combination of licofelone (5 mg/kg) with minocycline trol, but the combination of licofelone (5 mg/kg) with (50 mg/kg) significantly attenuated the anxiety like be- minocycline (50 mg/kg) significantly improved loco- nd th havior after 2 week as compared to RWATS group. motor activity on 15 day as compared to the RWATS Lower dose of licofelone (2.5 mg/ kg) was found to be group. However, licofelone (5 and 10 mg/kg), mino- non-significant throughout the treatment period (Tab. 2). cycline (100 mg/kg) and combination of licofelone (5 mg/kg) with minocycline (50 mg/kg) significantly rd Effects of licofelone, minocycline and their restored the wheel running activity after 3 week (on nd combination on the brain lipid peroxidation, 22 day) as compared to the RWATS group. Treat- nitrite level, catalase enzyme and GSH levels ment with licofelone (2.5 mg/kg) did not show any significant effect on locomotor activity as compared RWATS (6 min test session) for 21 days significantly to the RWATS group. Further, the combination of increased lipid peroxidation and nitrite concentration, licofelone (5 mg/kg) with minocycline (50 mg/kg) depleted glutathione and catalase activity as com- significantly potentiated the effect as compared to pared to naive group. Twenty one days drug treatment their effects per se (Fig. 1). with licofelone (5 and 10 mg/kg) as well as mino-
1110 Pharmacological Reports, 2012, 64, 11051115 Licofelone, minocycline and their combination against CFS Anil Kumar et al.
Tab. 2. Effect of licofelone, minocycline and their combination on anxiety like behavior in elevated plus maze test
Day 0 Day 8 Day 15 Day 22 Treatment Average no. Average Average no. Average Average no. Average Average no. Average (mg/kg) of entries time spent of entries time spent of entries time spent of entries time spent in open arm in open arm in open arm in open arm in open arm in open arm in open arm in open arm (s) (s) (s) (s) Naive 10 ± 0.6 69 ± 7.7 13 ± 0.9 62.6 ± 8.1 12 ± 0.9 64 ± 6.3 13 ± 0.9 62 ± 5.2 a a a a a a RWATS 11 ± 0.8 65.5 ± 8.8 7 ± 0.5 39.7 ± 0.46 5 ± 0.7 19.5 ± 0.56 2 ± 0.4 8.5 ± 0.55 Li (2.5) 10 ± 0.7 60 ± 1.7 8 ± 0.9 41 ± 1.3 5 ± 0.5 20 ± 0.7 4 ± 0.3 11 ± 0.3 b b b Li (5) 10 ± 1.0 63 ± 1.4 10 ± 1.3 42 ± 0.6 6 ± 0.4 23 ± 0.7 5 ± 0.4 14 ± 0.4 b c c c Li (10) 12 ± 0.5 66 ± 1.1 11 ± 1.2 43 ± 0.8 7 ± 0.6 30 ± 0.9 8 ± 0.2 16 ± 0.4 b b Me (50) 11 ± 0.9 66 ± 1.9 9 ± 1.2 39 ± 0.5 6 ± 0.7 23 ± 0.7 5 ± 0.6 13 ± 0.5 b d c,d d Me (100) 12 ± 0.8 67 ± 1.8 11 ± 1.1 43 ± 0.7 10 ± 0.5 32 ± 0.6 9 ± 0.7 17 ± 0.3 c,d c,d c,d c,d Li (5) + Me (50) 11 ± 0.6 68 ± 1.3 12 ± 0.3 41 ± 0.7 10 ± 0.3 31 ± 0.5 8 ± 0.2 18 ± 0.8
Data expressed as the mean ± SEM; a p < 0.05 as compared to naive, b p < 0.05 as compared to control (RWATS), c p < 0.05 as compared to Li (5), d p < 0.05 as compared to Me (50). Two-way ANOVA followed by Bonferroni’s test. Li – licofelone, Me – minocycline
cycline (50, 100 mg/kg) significantly caused antioxi- minocycline (50 mg/kg) produced significant antioxi- dant like effect in terms of lipid peroxidation, nitrite dant like effect as compared to their effect per se. Li- concentration, restored depleted glutathione and cata- cofelone (2.5 mg/kg) treatment did not show any lase activity as compared to RWATS group. Further, significant effects on any of the oxidative stress pa- combined treatment of licofelone (5 mg/kg) with rameters as compared to RWATS group (Fig. 2).
a Fig. 2. Effect of licofelone and minocycline on biochemical parameters. Data expressed as the mean ± SEM; p < 0.05 as compared to naive, b c d p < 0.05 as compared to RWATS (control), p < 0.05 as compared to Li (5), p < 0.05 as compared to Me (50). One-way ANOVA followed by Bonferroni’s test. Li – licofelone, Me – minocycline
Pharmacological Reports, 2012, 64, 11051115 1111 Effects of licofelone, minocycline and their axis [43]. These alterations in the HPA and sympa- combination on the on mitochondrial enzyme thetic nervous system cause various psychiatric as well complexes as pathological alteration in discrete areas of brain. The forced swim stress induced fatigue model is useful, but Six min RWATS on running wheel apparatus for 21 it lacks objectivity in the evaluation of immobility, as it days significantly impaired the mitochondrial enzyme evaluates subjectively, which varies from investigator complexes (I, II and IV) and mitochondrial redox ac- to investigator. Therefore, in the present study, we de- tivity (MTT assay) as compared to naive group. Mino- vised our own behavioral screening test model which cycline (50, 100 mg/kg), licofelone (10 mg/kg) and enabled us to quantify the escape behavior. It is more combination of licofelone (5 mg/kg) with minocycline specific as activity can be measured directly in the (50 mg/kg) significantly restored the mitochondrial en- form of number of wheel rotations, ruling out the sub- zyme complexes (I, II and IV) and mitochondrial redox jective variation in measuring the immobility. activity (MTT assay) as compared to RWATS group. In the present study, animals exposed to RWATS On the other hand, licofelone (5 mg/kg) treatment was for 21 days showed a gradual decrease in the running found to have significant effect on restoration of mito- wheel activity (number of wheel rotations/6 min) and chondrial enzyme complex I and II activities as com- anxiety like behavior, suggesting chronic fatigue pared to RWATS group, whereas licofelone (2.5 mg/kg) stress in these animals. The observed impairment in treatment did not produce any significant effect on res- locomotor activity and anxiety like behavior, demon- toration of mitochondrial enzyme complex activities as strated behavioral alterations, which is consistent with compared to control (Fig. 3). the previous reports of Kumar et al. [29], Short et al. [46] and Kroenke et al. [27]. Increased cortisol level also has been linked with anxiety-like behavior and painful response in humans [4]. Exercise-induced Discussion stress stimulates prostaglandin synthesis by releasing pro-inflammatory cytokines that in turn, initiates and Stress has been well known to trigger various hemo- modulates many aspects of the stress response, which static disturbances in the neuronal cells, which ulti- lead to the development of the behavioral symptoms mately lead to the activation of sympathetic nervous associated with CFS problems such as anxiety and de- system and hypothalamic-pituitary-adrenal (HPA) pression [8, 26]. Involvements of the above factors
a Fig. 3. Effect of licofelone and minocycline on mitochondrial enzyme complexes. Data expressed as the mean ± SEM; p < 0.05 as compared b c d to naive, p < 0.05 as compared to RWATS (control), p < 0.05 as compared to Li (5), p < 0.05 as compared to Me (50). One-way ANOVA fol- lowed by Bonferroni’s test. Li – licofelone, Me – minocycline
1112 Pharmacological Reports, 2012, 64, 11051115 Licofelone, minocycline and their combination against CFS Anil Kumar et al.
suggest the involvement of neuroinflammatory mecha- sistent with the previous studies which claimed the nism in disease pathology of CFS. Stress also plays antioxidant like effect of licofelone and minocycline a key role in the activation of microglia, and repeated re- [18, 19, 25]. Further, combination of licofelone with straint stresses induce proliferation of microglial cells in minocycline significantly potentiated their antioxi- the mice brain [40]. In addition, repeated immobilization dant like effect as well as restored mitochondrial en- stress induces morphological microglial activation in the zyme complex activities. Stress itself stimulates nu- hippocampus and striatum in mice [32, 33]. merous pathways which generate free radicals. Mito- In this study, licofelone and minocycline treatment chondrial inhibition, increased excitatory amino acid for 21 days, significantly improved locomotor activity release, activation of second messenger systems and as compared to the control group, suggesting their decreased efficiency of antioxidant defense mecha- role in the improvement of locomotor activity against nisms are the factors responsible for increased lipid CFS like behavior. Further, these results are also in peroxidation in stress [41]. Hence, it is uncertain that accordance with the previous study reports of our oxidative stress is either a cause of the CFS or a result laboratory [29]. Furthermore, licofelone and mino- of this illness. Oxidative stress can be caused by an cycline treatment significantly attenuated anxiety like increase in the generation of ROS. Among the differ- behavior in RWATS treated animals suggesting their ent sources, mitochondrial dysfunction is believed to protective effect in neurobehavioral alterations, be the major source. Further, it can be caused by a de- whereas their combination further potentiated the pro- cline in the efficiency of antioxidant enzyme systems tective effect as compared to their effect per se sug- [10]. Mitochondrial dysfunction may be implicated in gesting their common mechanism of action. However, the genesis of chronic fatigue pathology. In the early the exact mechanism of their potentiation is far from 1990s, Kuratsune et al. showed that the low acylcar- our elucidation. Besides, protective effects of these nitine content in the serum of CFS patients is related drugs in combination further suggest their therapeutic to reduced energy production by muscle mitochondria effect against CFS induced behavioral alterations. In [31]. In 2000, Pall and its co-workers reported that many of the recent reports, the neuroprotective activi- mitochondrial enzymes succinate dehydrogenase and ties of licofelone and minocycline have been high- cis-aconitase are inactivated by peroxynitrite [41]. The lighted [18, 19], which was attributed due to their results of the presents study are very similar with pre- anti-inflammatory, anti-apoptotic, anti-microglial and vious studies which demonstrated that chronic stress anti-oxidant properties [22, 42, 47]. The above find- significantly induced oxidative damage in the brain ings were further supported by the evidences that [19, 20, 41]. Present study results are very similar with minocycline inhibits the activation of microglia, the previous preclinical and clinical reports that chronic expression of inducible nitric oxide synthase (iNOS), stress significantly inhibited the mitochondrial en- IL-1b converting enzyme (ICE), and COX-2, the zymes activity, which ultimately lead to increased lipid caspase-dependent and independent apoptotic path- peroxidation [31, 36, 41]. ways, and oxidative stress, whereas licofelone is a dual As CFS is a result of stimulation of numerous path- COX-LOX inhibitor and possesses antioxidant and ways (oxidative stress, neuro-inflammation and apop- antiapoptotic activity [25, 37, 54]. tosis), a muti-pathway targeting approach is needed RWATS for 21 days significantly induced oxidative for the effective management of the disease. Further, damage in brain as shown by increased level of lipid treatment with licofelone and minocycline combina- tion is found to be a promising approach for the man- peroxidation, nitrite concentration, decreased levels agement of CFS. of glutathione and catalase. Recent reports have dem- onstrated that oxidative stress and, more specifically, lipid peroxidation contributes to the disease process and to some of the symptoms in the chronic stress ill- Conclusion ness [20]. Pretreatment with licofelone and mino- cycline significantly attenuated oxidative stress, as well as restored mitochondrial enzyme complex ac- The study highlights the neuroprotective effect of li- tivities suggesting their potential antioxidant like ef- cofelone and minocycline and further demonstrates fect, as well as their role in restoration of mitochon- their protective effect against CFS induced behavioral drial enzyme complex activities. The results are con- alteration and oxidative damage.
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