Pharmacological Reports Copyright © 2012 2012, 64, 11051115 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Potentialroleoflicofelone,minocyclineandtheir combination against chronic fatigue stress induced behavioral,biochemicalandmitochondrial alterationsinmice Anil Kumar, Aditi Vashist, Puneet Kumar, Harikesh Kalonia, Jitendriya Mishra PharmacologyDivision,UniversityInstituteofPharmaceuticalSciences,UGCCentreofAdvancedStudy (UGC-CAS),PanjabUniversity, Chandigarh-160014,India Correspondence: AnilKumar,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- protectiveeffect. 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- drialenzymecomplexesactivity. 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- cantascomparedtotheireffect perse. Conclusion: The present study highlights the therapeutic potential of licofelone, minocycline and their combination against CFS in mice. Keywords: chronicfatiguestress,licofelone,oxidativestress,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 relatedbehavioralcomplications. 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 drialenzymecomplexactivityinmice. 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]. MaterialsandMethods In addition, licofelone has good oral bioavailability, longhalflife(11h)withnogenotoxicity[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 AcademyGuidelinesfortheuseandcareofanimals. 1106 Pharmacological Reports, 2012, 64, 11051115 Licofelone,minocyclineandtheir combinationagainstCFS Anil Kumar et al. Drugsandtreatmentschedule 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 naivegroupupto21days. (50 and 100 mg/kg, po). All the drugs were suspended in 0.5% w/v sodium carboxymethyl cellulose (CMC) Behavioralassessments solution and administered by oral route in a constant volumeof1ml/100gofbodyweight. Measurementoflocomotoractivity 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